Vol. 57 Issue No. 05 (Pages: 80) MAY 2020 Monthly Publication Price per Copy Rs. 75/- LICENSED TO POST WITHOUT PREPAYMENT LICENCE NO. MR/Tech/WPP-338/West/2018-20 RNI REGN. NO. 11547/1963 REGN.NO.MCW/98/2018-20 ISSN 0019-462X ALL SUBMISSIONS OF REVIEW ARTICLES, ORIGINAL RESEARCH PAPERS, SHORT NOTES ETC FOR INDIAN DRUGS WILL ONLY BE ACCEPTED VIA OUR ONLINE PORTAL www.indiandrugsonline.org The online process will speed up the review and publication process. Authors can submit their article for peer scrutiny by registering and creating an account. The account can be used to submit any number of original papers for publication in ‘INDIAN DRUGS’ Journal. In case of any difficulty in registering, please contact by email: [email protected] or call: 022-24944624 / 24974308. journal is online. INDIAN DRUGS ATTENTION ALL AUTHORS! Monthly Publication Price per Copy Rs. 75/- LICENSED TO POST WITHOUT PREPAYMENT LICENCE NO. MR/Tech/WPP-338/West/2018-20 RNI REGN. NO. 11547/1963 REGN.NO.MCW/98/2018-20 ISSN 0019-462X
Transcript
INDIAN DRUGS 57 (05) MAY 2020 1
INDIAN DRUGS 57 (03) MARCH 2020 1
Vol. 57 Issue No. 05 (Pages: 80) MAY 2020
Monthly PublicationPrice per Copy Rs. 75/- LICENSED TO POST WITHOUT PREPAYMENT LICENCE NO. MR/Tech/WPP-338/West/2018-20RNI REGN. NO. 11547/1963REGN.NO.MCW/98/2018-20ISSN 0019-462X
ALL SUBMISSIONS OF REVIEW ARTICLES, ORIGINAL RESEARCH PAPERS, SHORT NOTES ETC FOR INDIAN DRUGS
WILL ONLY BE ACCEPTED VIA OUR ONLINE PORTAL
www.indiandrugsonline.orgThe online process will speed up the review and publication process.
Authors can submit their article for peer scrutiny by registering and creating an account. The account can be used to submit any number of original papers for publication in
‘INDIAN DRUGS’ Journal. In case of any difficulty in registering, please contact by
Monthly PublicationPrice per Copy Rs. 75/- LICENSED TO POST WITHOUT PREPAYMENT LICENCE NO. MR/Tech/WPP-338/West/2018-20RNI REGN. NO. 11547/1963REGN.NO.MCW/98/2018-20ISSN 0019-462X
abStRaCted by:Scopus, eMbaSe, International Pharmaceuticals abstracts, genamics JournalSeek, ebSCo, Citefactor, oClC Worldcat, Scimago, Journal guide, tdNet, Science library Index, dRJI, CCC (Infotrieve), Index Copernicus, electronic Journals library, Sherpa/Romeo, Researchbib, Indian Citation Index, i-Journals, i-focus, i-future, Researchgate.CoSMoS (germany), MIaR (universitat de barcelona) listed in Journals approved by ugC for CaS & appointment of university teachers
(C) Copyright: No part of this publication may be reproduced by any means without prior written permission of the publisher.- Annual Subscription (India) - For members: ` 1000/-. For bonafide students: ` 1000/-. For Govt Research/Educational Institutions:`2000/-.
For non-members: ` 4000/-. Price per copy: ` 75/- only. (Foreign US$ 25) Annual Subscription (Foreign): US$ 400.- All payments to be made in favour of Indian drug Manufacturers' association, Payable at Mumbai.
- A Review on Ethno-medicinal Uses, Phyto-Chemical Constituents and Pharmacological Evidence of Apium Graveolens Linn. Waseem m., Rauf A., Rehman S. ................................................................................................................ 7oRIgINal ReSeaRCh aRtICleS
- Pharmacognostical and Physico-Chemical Screening of Euphorbia Tirucalli Stem-Bark mali P. y. and Goyal S. ............................................................................................................................... 20- Assessment of Phytoconstituents of medicinal importance from Millettia peguensis Ali (Syn. Millettia ovalifolia Kurz) Kaur Arshpreet and Sidhu m.C .................................................................................................................. 31- A Novel Validated Lc-ms/ms Analytical method For The Estimation of midodrine Hydrochloride In Pharmaceutical Formulation Narenderan S.T., meyyanathan S.N., Babu B and Karthik y. ................................................................... 39- Stability Indicating Uplc method For Estimation of metformin Hydrochloride and Nateglinide Simultaneously in the Presence of Stress Degradation Products Prameela Rani A., madhavi S., Tirumaleswara Rao B. And Sudheer Reddy Ch. ...................................... 44- Exploring The Potential of Orodispersible Tablet of Enalapril maleate and Hydrochlorothia Zide Drug Combination Ammanage Anand Shripal, Patil yojana Dadasaheb, Saboji Jagadish Kamalanath, Kempwade Amolkumar Ashok and Hiremath Ravindra Durdundayya. ....................................................... 53- Hepatoprotective Activity of Whole Flower of Hibiscus Rosa-Sinensis Linn Extracts In Wistar Rats Krishn Kumar Agrawal................................................................................................................................. 61
ShoRt Note
- Simultaneous Determination of Domperidone and Some Preservatives in Oral Formulation: RP-HPLC method Patel Aasha., Firke Sandip D., Patil Ravindra R., Kalaskar mohan G., Bari Sanjay B. and Surana Sanjay J., ........................................................................................................ 67- Determination Of Bioactive Contents and In Vitro Antioxidant Activity of Poly Herbal Formulations Srivastava AK, Kaushik D and Lal VK ......................................................................................................... 70
obituary: Dr. V.K.Parameswaran .............................................................................................................. 76 Recent Publication From Indian Patent Office Journal with PCT/WIPO Number ....................................... 77
abStRaCted by:Scopus, eMbaSe, International Pharmaceuticals abstracts, genamics JournalSeek, ebSCo, Citefactor, oClC Worldcat, Scimago, Journal guide, tdNet, Science library Index, dRJI, CCC (Infotrieve), Index Copernicus, electronic Journals library, Sherpa/Romeo, Researchbib, Indian Citation Index, i-Journals, i-focus, i-future, Researchgate.CoSMoS (germany), MIaR (universitat de barcelona) listed in Journals approved by ugC for CaS & appointment of university teachers
(C) Copyright: No part of this publication may be reproduced by any means without prior written permission of the publisher.- Annual Subscription (India) - For members: ` 1000/-. For bonafide students: ` 1000/-. For Govt Research/Educational Institutions:`2000/-.
For non-members: ` 4000/-. Price per copy: ` 75/- only. (Foreign US$ 25) Annual Subscription (Foreign): US$ 400.- All payments to be made in favour of Indian drug Manufacturers' association, Payable at Mumbai.
- A Review on Ethno-medicinal Uses, Phyto-Chemical Constituents and Pharmacological Evidence of Apium Graveolens Linn. Waseem m., Rauf A., Rehman S. ................................................................................................................ 7oRIgINal ReSeaRCh aRtICleS
- Pharmacognostical and Physico-Chemical Screening of Euphorbia Tirucalli Stem-Bark mali P. y. and Goyal S. ............................................................................................................................... 20- Assessment of Phytoconstituents of medicinal importance from Millettia peguensis Ali (Syn. Millettia ovalifolia Kurz) Kaur Arshpreet and Sidhu m.C .................................................................................................................. 31- A Novel Validated Lc-ms/ms Analytical method For The Estimation of midodrine Hydrochloride In Pharmaceutical Formulation Narenderan S.T., meyyanathan S.N., Babu B and Karthik y. ................................................................... 39- Stability Indicating Uplc method For Estimation of metformin Hydrochloride and Nateglinide Simultaneously in the Presence of Stress Degradation Products Prameela Rani A., madhavi S., Tirumaleswara Rao B. And Sudheer Reddy Ch. ...................................... 44- Exploring The Potential of Orodispersible Tablet of Enalapril maleate and Hydrochlorothia Zide Drug Combination Ammanage Anand Shripal, Patil yojana Dadasaheb, Saboji Jagadish Kamalanath, Kempwade Amolkumar Ashok and Hiremath Ravindra Durdundayya. ....................................................... 53- Hepatoprotective Activity of Whole Flower of Hibiscus Rosa-Sinensis Linn Extracts In Wistar Rats Krishn Kumar Agrawal................................................................................................................................. 61
ShoRt Note
- Simultaneous Determination of Domperidone and Some Preservatives in Oral Formulation: RP-HPLC method Patel Aasha., Firke Sandip D., Patil Ravindra R., Kalaskar mohan G., Bari Sanjay B. and Surana Sanjay J., ........................................................................................................ 67- Determination Of Bioactive Contents and In Vitro Antioxidant Activity of Poly Herbal Formulations Srivastava AK, Kaushik D and Lal VK ......................................................................................................... 70
obituary: Dr. V.K.Parameswaran .............................................................................................................. 76 Recent Publication From Indian Patent Office Journal with PCT/WIPO Number ....................................... 77
ABSTRACTED BY:Scopus, EMBASE, International Pharmaceuticals Abstracts, Genamics JournalSeek, EBSCO, CiteFactor, OCLC Worldcat, Scimago, Journal Guide, TDNet, Science Library Index, DRJI, CCC (Infotrieve), Index Copernicus, Electronic Journals Library, Sherpa/Romeo, ResearchBib, Indian Citation Index, i-Journals, i-Focus, i-Future, ResearchGate.COSMOS (Germany), MIAR (Universitat de Barcelona) Listed in Journals approved by UGC for CAS & Appointment of University Teachers
(C) Copyright: No part of this publication may be reproduced by any means without prior written permission of the publisher.
- Annual Subscription (India) - For members: ` 1000/-. For bonafide students: ` 1000/-. For Govt Research/Educational Institutions:`2000/-. For non-members: ` 4000/-. Price per copy: ` 75/- only. (Foreign US$ 25) Annual Subscription (Foreign): US$ 400.
- All payments to be made in favour of Indian Drug Manufacturers' Association, Payable at Mumbai.
- Biodegradable Nanospheres - Current Status Yarraguntla Srinivasa Rao, Kamala Kumari P.V. .......................................................................................... 7
ORIGINAL RESEARCH ARTICLES
- Synthesis Characterization and Docking study of HCV NS3/4A protease inhibitor molecules Kumar Satish, Santra P. K. and Aryan R. C. ...................................................................................................................................................................19
- Pharmacognostical and Physico-Chemical Screening of Euphorbia Tirucalli Stem-Bark Mali P. Y. and Goyal S. .............................................................................................................................. 32
- Assessment of Phytoconstituents of medicinal importance from Millettia peguensis Ali (Syn. Millettia ovalifolia Kurz) Kaur Arshpreet and Sidhu M. C. ................................................................................................................ 43
- A Novel Validated Lc-Ms/Ms Analytical Method For The Estimation of Midodrine Hydrochloride In Pharmaceutical Formulation Narenderan S. T., Meyyanathan S. N., Babu B. and Karthik Y. ................................................................ 51
- Stability Indicating Uplc Method For Estimation of Metformin Hydrochloride and Nateglinide Simultaneously in the Presence of Stress Degradation Products Prameela Rani A., Madhavi S., Tirumaleswara Rao B. and Sudheer Reddy Ch. ...................................... 56
- Hepatoprotective Activity of Whole Flower of Hibiscus rosa-sinensis Linn Extracts In Wistar Rats Agrawal Krishn Kumar ................................................................................................................................ 65
SHORT NOTE
- Simultaneous Determination of Domperidone and Some Preservatives in Oral Formulation: RP-HPLC Method Patel Aasha., Firke Sandip D., Patil Ravindra R., Kalaskar Mohan G., Bari Sanjay B. and Surana Sanjay J., ........................................................................................................ 71
- Determination Of Bioactive Contents and In vitro Antioxidant Activity of Poly Herbal Formulations Srivastava A. K, Kaushik D. and Lal V. K. .................................................................................................. 74
Obituary: Dr. V. K. Parameswaran ........................................................................................................... 80
Recent Publication From Indian Patent Office Journal with PCT/WIPO Number ....................................... 81
4 INDIAN DRUGS 57 (05) MAY 2020
4 INDIAN DRUGS 57 (05) mAy 2020
founder editor*Dr. A. Patani, D.Sc. (Germany)edItoRIal CoMMItteeeditorDr. Gopakumar G. Nair, Ph.D.associate editorsmr. J. L. Sipahimalani, B. Pharm. Hons. (London), mRCS, FRPharmS Dr. Nagaraj Rao, D.Sc. (Germany) Dr. George Patani, Ph.D. (USA)Consulting editorDr. S. G. Deshpande, m.Sc. (Tech.), Ph.D.Content editorms. meena Shah, B. Pharm (ICT)
editorial advisory boardProf. y. K. Agrawal, Ph.D., F.I.C., F.R.m.S.Dr. Bhupinder Singh Bhoop, Ph.D.Dr. Ganesh Deshpande, Ph.D.Prof. S. S. Handa, Ph.D.Dr. S. P. S. Khanuja, Ph.D.Dr. Chandrakant Kokate, Ph.D. Dr. D. B. Anantha Narayana, Ph.D.Dr. Nitya Anand, Ph.D.Dr. Harish Padh, Ph.D.Dr. Vandana Patravale, Ph.D.Dr. m. K. Raina, Ph.D.Dr. A. V. Rama Rao, Ph.D. (Tech.), D.Sc.Prof. m. N. Saraf, m.Pharm., Ph.D.Dr. G. N. Singh, m.Pharm., Ph.D.Dr. Raman mohan Singh, Ph.D.Dr. Ashok Vaidya, m.D., Ph.D., F.A.I.m.
editorial boardProf. K. G. Akamanchi, Ph.D. (Tech.)Dr. Evans Coutinho, Ph.D. (Tech.)Prof. Padma Devarajan, m.Pharm., Ph.D. (Tech.)Dr. Prashant m. Dikshit, Ph.D.Prof. A. K. Gadad, m.Pharm., Ph.D.Dr. K. N. Ganesh, Ph.D.Dr. (mrs.) Gopa Ghosh, Ph.D.Dr. Parthajyoti Gogoi, Ph.D.Dr. Nirmala D. Grampurohit, Ph.D.Prof. Shreerang Joshi, Ph.D. Dr. (mrs.) S. S. mahajan, m.Sc. (Tech.), Ph.D. Dr. A. A. Natu, Ph.D.Dr. Laxman Parthiban, Ph.D.Prof. Bhushan Patwardhan, Ph.D.Dr. m. N. A. Rao, Ph.D.Dr. Ashwinikumar Raut, m.D.Dr. Sanjay Singh, m.Pharm., Ph. D.Dr. Saranjit Singh, m.Pharm., Ph.D.Dr. N. G. N. Swamy, Ph.D.Prof. N. Udupa, m.Pharm., Ph.D.
*Inga laboratories P. ltd., Inga house, Mahakali Road, andheri (east), Mumbai 400 093, INdIa
Founder Editor*Dr. A. Patani, D.Sc. (Germany)EDITORIAL COMMITTEEEditorDr. Gopakumar G. Nair, Ph.D.Associate EditorsMr. J. L. Sipahimalani, B. Pharm. Hons. (London), MRCS, FRPharmS Dr. Nagaraj Rao, D.Sc. (Germany) Dr. George Patani, Ph.D. (USA)Consulting EditorDr. S. G. Deshpande, M.Sc. (Tech.), Ph.D.Content EditorMs. Meena Shah, B. Pharm (ICT)
Editorial Advisory BoardProf. Y. K. Agrawal, Ph.D., F.I.C., F.R.M.S.Dr. Bhupinder Singh Bhoop, Ph.D.Dr. Ganesh Deshpande, Ph.D.Prof. S. S. Handa, Ph.D.Dr. S. P. S. Khanuja, Ph.D.Dr. Chandrakant Kokate, Ph.D. Dr. D. B. Anantha Narayana, Ph.D.Dr. Nitya Anand, Ph.D.Dr. Harish Padh, Ph.D.Dr. Vandana Patravale, Ph.D.Dr. M. K. Raina, Ph.D.Dr. A. V. Rama Rao, Ph.D. (Tech.), D.Sc.Prof. M. N. Saraf, M.Pharm., Ph.D.Dr. G. N. Singh, M.Pharm., Ph.D.Dr. Raman Mohan Singh, Ph.D.Dr. Ashok Vaidya, M.D., Ph.D., F.A.I.M.
Editorial BoardProf. K. G. Akamanchi, Ph.D. (Tech.)Dr. Evans Coutinho, Ph.D. (Tech.)Prof. Padma Devarajan, M.Pharm., Ph.D. (Tech.)Dr. Prashant M. Dikshit, Ph.D.Prof. A. K. Gadad, M.Pharm., Ph.D.Dr. K. N. Ganesh, Ph.D.Dr. (Mrs.) Gopa Ghosh, Ph.D.Dr. Parthajyoti Gogoi, Ph.D.Dr. Nirmala D. Grampurohit, Ph.D.Prof. Shreerang Joshi, Ph.D. Dr. (Mrs.) S. S. Mahajan, M.Sc. (Tech.), Ph.D. Dr. A. A. Natu, Ph.D.Dr. Laxman Parthiban, Ph.D.Prof. Bhushan Patwardhan, Ph.D.Dr. M. N. A. Rao, Ph.D.Dr. Ashwinikumar Raut, M.D.Dr. Sanjay Singh, M.Pharm., Ph. D.Dr. Saranjit Singh, M.Pharm., Ph.D.Dr. N. G. N. Swamy, Ph.D.Prof. N. Udupa, M.Pharm., Ph.D.
*Inga Laboratories P. Ltd., Inga House, Mahakali Road, Andheri (East), Mumbai 400 093, INDIA
INDIAN DRUGS 57 (05) MAY 2020 5
ED
ITOR
IAL
REPURPOSING OR SECONDARY USE OF KNOWN DRUGS
Dear Reader,
Repurposing of drugs was most often a strategic approach, though by “serendipity” in few instances.
Early practice of pharmacovigilance in USA, led to near ‘serendipitous’ addition to market value for many “first use” drugs. Glaxo-Welcome’s Bupropion brand “Wellbutrin” was first approved for treatment of depression. While “Wellbutrin” received an average response in the anti-depression segment, pharmacovigilance reports to FDA was perplexing. Patients gave conflicting response. While a few wanted to change to another drug, many others wanted to continue the use of the prescribed drug even after being advised to stop. The reason when investigated, was that the patients felt the urge to discontinue smoking, which was acceptable to some, while not to others. When this finding was received by the FDA from multiple specialists, Glaxo-Welcome was asked to conduct a full study on a priority basis. Consequently, FDA granted a fast track approval for use of Bupropion for smoking cessation. Antidepressant “Wellbutrin” opened up new pathway for smokers to “kick the Butts” through its second use “Avatar”, in form of “Zyban” for smoking cessation and become the first global breakthrough drug of choice for this second use. Pharmacovigilance helps in developing a road map for secondary use.
There are many more Repurposed drugs which have been strengthening the therapeutic armoury over the years. An outstanding example is Zidovudine (azidothymidine or AZT!) which was initially investigated in 1964 as an anticancer medication, but failed. Just like Zyban, AZT was reborn in its new incarnation globally for the treatment of HIV/AIDS. Another typical example for repurposing is Erlotinib (TARCEVA) which was originally approved for non-small-cell lung cancer, turned out as global blockbuster for the treatment of pancreatic cancer and other therapeutic indications.
Aspirin has been in use for hundreds of years to treat inflammation, in its earliest incarnation from the extract of willow bark. Synthetic Aspirin emerged as the most popular anti-inflammatory, analgesic and antipyretic of our times, once introduced by Bayer in 1990. After living its role for nearly 100 years, Aspirin got a new life with its revolutionary use in cardiovascular and cerebrovascular disease. The “Aspirin story” is now becoming the trend in the new millennium as “Repurposing of drugs” is attracting attention like never before, for multiple reasons. Aspirin’s successor Paracetamol has now emerged as a front-line treatment for near asymptomatic patients of COVID-19. Reportedly, ibuprofen is also being investigated. Closer home, HCQ (Hydroxychloroquine) emerged from the antimalarial family as a treatment for rheumatoid arthritis, lupus and other diseases in combination with methotrexate. HCQ has further proved effective in a combination with Nitazoxanide and even with Azithromycin in the latest repurposing for treatment of Sars-cov-2, popularly known as Covid-19.
Repurposing of the drugs have emerged as the most potent strategy to treat Covid-19. Task Force constituted by the Government’s Principal Scientific Advisor, Dr. K. Vijaya Raghvan for inter-disciplinary assessment of drug candidates (Repurposing) have identified nearly thirty molecules as potential candidates for the treatment of Covid-19. Remdesivir developed for treatment of Ebola is now emerging as a front-runner along with Tocilizumab and Favipiravir. Even well-known drugs like Famotidine, Lisinopril, Losartan, Ivermectin+Doxycycline, Dexamethasone, Interferon Beta, Lopinavir+ Ritonavir, Ribavirin, Darunavir and others are emerging useful against Covid-19, often in combinations. However, there is an emerging global conflict against cheaper options of
INDIAN DRUGS 57 (01) JANUARy 2020 5 4 INDIAN DRUGS 57 (01) JANUARy 2020 INDIAN DRUGS 57 (01) JANUARy 2020 5 4 INDIAN DRUGS 57 (01) JANUARy 2020
A CLARION CALL TO ALL INDIANS TO FACILITATE INTENSE RESEARCH ON NATURAL PRODUCTS
Dear Reader,
More than 100 years before India was recognised as the “Generic Capital” or “Pharmacy of the World, India was acclaimed for the “Materia Medica” and the extensive research on natural products by Sir Col. Dr. Ram Nath Chopra, the father of Indian Pharmacology. In fact, Dr. M.L. Shroff got inspired from Sir R.N. Chopra and started the first Pharmacy course at Banaras Hindu University in 1932. Every student (even veterans) of Pharmacy should know that Drugs Act, 1940 (later renamed as Drugs and Cosmetics Act) as well as formal courses in Ayurveda, Siddha and Unani were started under his mentorship. DTAB (Drugs Technical Advisory Committee) was set up under his stewardship. The first edition of the Indian Pharmacopoeia (evolved from Indian pharmacopoeial list 1946) 1955 was published with his contributions. “We are reaping the fruits of the plant grown by their sown seeds. We should make the same available to our future generations” said the biographer, Roja Rani. Prof.Harkishan Singh in J.Young Pharm, 2009 (Vol.1/No.3) had profiled Dr.R.N. Chopra (which is reproduced herein). Later Dr. K.M. Nadkarni came out with “Indian Materia Medica” in two volumes in 1908 with Foreword by Dr. R.N. Chopra. These are extremely valuable reference books for further research in Indian Natural Products.
There were many more books and publications on Indian Herbal resources and their medicinal applications. Among them, the following are of high interest to herbal researchers:
- British Herbal Pharmacopoeia – 1996
- Ayurvedic Pharmacopoeia of India – many volumes
- Ayurvedic Formulary of India – many volumes
- Indian Herbal Pharmacopoeia Vol. I & II compiled and published by Indian Drug Manufacturers’ Association jointly with Regional Research Laboratory, Jammu.
IDMA has been “naturally” active “herbally” in the early days till the nineties. Early interest in the Indian pharmaceutical industry as well as IDMA in herbal drugs was high with IDMA members such as Zandu, Inga, Charak, Amsar, Cipla and many more. While global giants such as Lever and P&G continue to be interested in herbal and natural products, of late both Indian companies such as Himalaya, Patanjali, Laila, Dabur and global cosmetic and nutraceutical leaders such as Colgate, Unilever, L’Oreal, Herbalife and others continue to develop herbal cosmetics, nutraceuticals and healthcare products. The Indian pharma industry’s interest in herbal research was eventually almost laid to rest with the hasty and untimely Biodiversity Act, 2002 and the Rules 2004 thereof, with extremely harsh and impractical enforcement efforts. The last nail on the coffin of Indian herbal research initiatives was the intrusion of National Green Tribunal through the Biodiversity Act to penalise the herbal researchers and users in India. The “benefit-sharing” objectives of the Nagoya Protocol and the International “Convention on Biodiversity (CBD)” was hijacked by vested NGOs and the protagonists of the distorted and anti-benefit generation activists, so much so that the Indian research on Natural resources got discouraged. Efforts were made to reduce and soften the impact of the Biodiversity Act by IDMA and other likeminded organisations. Consequently, the NBA (National Biodiversity Authority) has repeatedly amended the “guidelines” to make the Act and Rules more friendly for the users, researchers and herbal industry. However, the blatantly illegal demands from State Biodiversity Boards (SBB) and NGT continues even today. It is imperative and extremely necessary for these agencies to seize and desist from further damaging the research potential in herbal and natural resources.
ED
ITOR
IAL
6 INDIAN DRUGS 57 (05) MAY 2020
If you would like to comment on the editorial please write to us at [email protected]
About The EditorDr. Gopakumar G. Nair is a Ph.D in Organic Chemistry (1966) from National Chemical Laboratory, Pune (Pune University). He was a Post-Doctoral fellow at IIT Bombay, Powai (1967) before joining the Pharma Industry. He was Director of Bombay Drug House P. Ltd., later Chairman of BDH Industries Ltd. as well as CMD of Bombay Drugs & Pharma Ltd., which was merged with Strides Arcolab Ltd. in 2001. Dr. Nair served IDMA as office bearer for many years from 1972 onwards and was Chairman of various Committees for nearly 4 decades. He was the President of IDMA in 1999/2000. Currently, Dr. Nair is the Chairman of the IPR Committee in IDMA.
Having moved into the Intellectual Property field, he was the Dean of IIPS (Institute of Intellectual Property Studies) at Hyderabad in 2001/2002. Later, he set up his own boutique IP firm, Gopakumar Nair Associates, as well as Gnanlex Hermeneutics Pvt. Ltd., having done his L. L. B. from Mumbai University. He is also CEO of Patent Gurukul and President of Bharat Education Society, Kurla, Mumbai, managing many educational institutions in and around Mumbai.
well-known drugs used for years and proven to be safe in preference to costlier newly invented patented molecules. India needs to continue the repurposing exercise to exhaust all options in its armoury against treatment of Covid-19, at least till an effective vaccine option emerges. Even after vaccines enter the market, it is absolutely essential that continued research into new uses of known drugs must continue. Today, it may be for Covid-19, tomorrow for another emerging disease. The momentum gained through new initiative of repurposing must keep up beyond Covid-19. Many of the safe and effective drugs in the market could be evaluated for secondary indications as a need-based research strategy based on market needs. The roadmap for future research for the Indian pharma industry and academia should be in repurposing or second medical use. The options are enormous, the opportunity is there for grabbing. The need in the market place or treatment gaps are too strong to be ignored.
The current crisis has also created more awareness on the need for strong immunity building to prevent or avoid co-morbidities. Awareness on need for monitoring and maintaining general well-being has opened up opportunities of nutraceutical, herbal supplements, probiotics and food supplements. Herbal options are being clinically evaluated not only for immunity -building, but also for prophylactic and even therapeutic use. AQCH based on Cissampelos Pareira ( Patha, velvet leaf, Hirtusa Abuta) is being clinically evaluated for the treatment of Covid-19. It is time that Indian Pharma researcher redirects attention to repurposing or newer use of known drugs. The patentability or non-patentability considerations should not deter these valuable frontiers for need-based research. New use of known drugs (not patentable in India under Section 3 (d) of Patents Act, 1970) is patentable in all developed countries. Even in India, a novel and inventive formulation developed distinctively and distinguishingly over the prior primary use dosage form, could be patented when developed for the secondary use. Let us jump into this new “Kurukshetra” domain to fight against emerging threat to health and well being using the weapons we already possess in our armoury with effective use of observations and reports received through the sales-force or though pharmaco-vigilance.
INDIAN DRUGS 57 (01) JANUARy 2020 7 6 INDIAN DRUGS 57 (01) JANUARy 2020 INDIAN DRUGS 57 (01) JANUARy 2020 7 6 INDIAN DRUGS 57 (01) JANUARy 2020
Many life-saving medicines were isolated from herbal resources in the past. Commencing from Alkaloids such as Quinine, Reserpine, Ephedrine, Atropine and anti-cancer drugs such as Vincristine, Vinblastine (Catharanthus roseus), Taxol (Taxus), Camptothecin, Colchicine, Anti-malarials such as Artemisinin, Artemether, Arteether (Artemisia annua) and latest blockbusters like Oseltamivir (Tamiflu) provides ample examples of the opportunity to discover new drugs from the herbal kingdom. Potent drugs from Opium and Cannabis are once again receiving attention for medicinal uses for their low toxicity and high efficacy.
Countries with high density or broad range of Biodiversity have higher potential for bioprospecting. India and China are leading countries with high harnessing potential. However, India is likely to fall far behind China, since China’s Biodiversity Conservation Legislation of 2013 is by far research, economic utilization and benefit-sharing friendly compared to India’s Biodiversity Act, Rules, guidelines and procedures. Scope for biomolecules such as newer proteins, lipids, enzymes, nucleic acids, antigens and antibodies including newer antibiotics are high for Indian researchers. Let us hope to see revived interest in R&D for biomolecules and discovery of new molecular entities (NMEs) and newer nutritional ingredients in coming years. CSIR institutes such as IHBT (Institute of Himalayan Bioresource Technology, Palanpur, H.P.), IIIM (Indian Institute of Integrative Medicine, Jammu – former RRL) and others will hopefully succeed in their mission to develop products and processes from bioresources using cutting edge technology.
Happy Reading!
Dr. Gopakumar G. NairEditor,
Indian Drugs
If you would like to comment on the editorial please write to us at [email protected]
About the EditorDr. Gopakumar G. Nair is a Ph.D in Organic Chemistry (1966) from National Chemical Laboratory, Pune (Pune University). He was a Post-Doctoral fellow at IIT Bombay, Powai (1967) before joining the Pharma Industry. He was Director of Bombay Drug House P. Ltd., later Chairman of BDH Industries Ltd. as well as CMD of Bombay Drugs & Pharma Ltd., which was merged with Strides Arcolab Ltd. in 2001. Dr. Nair served IDMA as office bearer for many years from 1972 onwards and was Chairman of various Committees for nearly 4 decades. He was the President of IDMA in 1999/2000. Currently, Dr. Nair is the Chairman of the IPR Committee in IDMA.
Having moved into the Intellectual Property field, he was the Dean of IIPS (Institute of Intellectual Property Studies) at Hyderabad in 2001/2002. Later, he set up his own boutique IP firm, Gopakumar Nair Associates, as well as Gnanlex Hermeneutics Pvt. Ltd., having done his L. L. B. from Mumbai University. He is also CEO of Patent Gurukul and President of Bharat Education Society, Kurla, Mumbai, managing many educational institutions in and around Mumbai.
INDIAN DRUGS 57 (05) MAY 2020 7
REVIEW ARTICLES
BIODEGRADABLE NANOSPHERES - CURRENT STATUS
Yarraguntla Srinivasa Rao a* and Kamala Kumari P. V.a
(Received 1 December 2018) (Accepted 27 March 2019)
ABSTRACT
Nanospheres are polymeric matrix of spherical shape that ranges in size between 10- 200 nm in diameter. The drug is dissolved, entrapped, encapsulated or attached to the matrix of polymer. The nature of nanospheres can be amorphous or crystalline, and they potentiate to protect the drug from chemical and enzymatic degradation. In the matrix of this polymer, a drug will evenly distribute as well as physically and uniformly disperse and can enclose a variety of drugs, enzymes and, genes, providing a long circulation time. Nanospheres have the capability to convert poorly soluble, poorly absorbed and labile biologically active substance into promising deliverable drugs. This review focuses on the mechanism for synthesis of nano-based drug delivery systems, characterization, and application of biodegradable nanospheres and mainly on successful formulations based on biodegradable nanospheres.
INTRODUCTIONDrug delivery has become an integral part of drug
development, because it can significantly enhance the therapeutic efficacy of drugs1. Furthermore, newer drugs prepared by recombinant technology, though comparatively more potent and specific in their pharmacological action, require efficient drug delivery systems. These drugs are either unstable in the biological environments2or are unable to cross the biological barriers effectively3. Nanospheres are submicron size colloidal particles with a therapeutic agent either entrapped in the polymer matrix or bound on to the surface. The nanospheres formulated with polylactic polyglycolic acid co-polymer (PLGA), for example, were 100-150 nanometer in diameter with the drug entrapped into the nanosphere polymer matrix4,5.
Nanosphere can be divided into two categories: biodegradable nanospheres and non-biodegradable nanospheres. Biodegradable nanospheres include albumin nanospheres, poloxypropylene dextran nanospheres, gelatin nanospheres, modified starch nanospheres, and polylactic acid nanospheres such as poly-lactic acid (PLA), poly-D-L-glycolide (PLG), poly-D-L-lactide-co-glycolide (PLGA), and poly-cyanoacrylate (PCA)6. In the case of non-biodegradable nanospheres,
a Department of Pharmaceutics, Vignan Institute of Pharmaceutical Technology, Visakhapatnam - 530 049, Andhra Pradesh, India*For Correspondence: E-mail: [email protected]
polylactic acid is the only polymer that is approved to be used as a controlled release agent and used by the people. Moreover, immune nanospheres and magnetic nanospheres have also become very common in the recent years. Immune nanospheres possess the immune competence as a result of antibody and antigen coated or adsorbed on the polymer nanospheres. Magnetic nanospheres possess a distinctive magnetic feature, which is their reaction to a magnetic force and are generally coated with protective shells as magnetic polymer nanoparticles.
Nanospheres could provide many advantages. Since nanospheres are submicron in size, they could be taken up more efficiently by the cells than the larger size particles. Furthermore, nanospheres could cross the cell membrane barriers by transcytosis. Drug molecules such as proteins and peptides and also DNA, which have a larger hydrodynamic diameter because of the charge and the bound water, could be entrapped into the nanosphere polymer matrix. Thus, the cellular uptake and transport of such macromolecules across biological membranes could be significantly improved by condensing them into nanosphere1. Nanospheres could protect the entrapped agent(s) from enzymatic and hydrolytic degradation. This is important because many drugs such as oligonucleotides or DNA, and also proteins and peptides, are susceptible to degradation due to nucleases
8 INDIAN DRUGS 57 (05) MAY 2020
and other enzymes present in the body7. Furthermore, the sustained release characteristics of nanospheres could be useful for many therapeutic agents that require repeated administration for their pharmacologic effects or to cure the disease completely. Such drugs could be delivered using nanospheres as a single-dose therapy. Dependent efflux pump of the cell membrane substrates for the p-glycoprotein are anticancer agents. Because of the efflux pump, anticancerdrugs are either not taken up by the cells or are thrown out rapidly.Intracellular delivery of such drugs could be improved by encapsulating them into nanospheres.Thus, nanospheres could offer a solution to many problems related to the delivery of therapeutic agents.
Oral bioavailability of drugs whichare not very well absorbed by the gastrointestinal tract either due to their instability to the enzymes (cytochrome P) present in the intestinal tract or because of the efflux pump,P-glycoprotein, can be improved by formulation with nanospheres. In general, the uptake of drugs through the gastrointestinal tract could be via two different routes.
1. Paracellular route(intercellular)
2. Transcellular route (intracellular)
The paracellular route is probably less significant in the transport of nanospheres as compared to the transcellular route. The tight junctions in between the cells may not allow nanospheres to cross the intestinal epithelial barrier via paracellular route. The transcellular route could allow a restrict passage of the nanospheres across the intestinal mucosal layer because of their moderate size range. There are many issues with oral delivery of nanospheres, such as the efficiency of uptake, reproducibility, and the pathophysiologic condition of the patient that could affect the retention of orally administered nanospheres.
Mechanism for synthesis of nano-based drug delivery systems
There are a number of mechanisms that have been developed to synthesize nano-based drug delivery system as per the application and type of nanoparticles.
Thin film hydration methodIn order to prepare drug-loading nanoparticles, thin
film hydration method is a widely used method. For this
method, the stock solutions of drug and nanocarrier are prepared in appropriate organic solvents such as methanol and chloroform. After solubilization, both the solvents in required amounts are mixed in round-bottomed flasks. The ratio of drug and nanocarrier can be prepared as per the requirement. Thereafter, the solvent is evaporated by rotary evaporation, which leads to formation of thin polymeric film. In order to remove remaining solvent, the film is kept in vacuum overnight at room temperature. Thereafter, the film is rehydrated in triple distilled water or in phosphate buffered saline (PBS) (0.01M,pH 7.4) (pre-warmed at 370C) by sonication or vortexing. This results in formation of drug encapsulated micelles8.
Solvent evaporation methodThe most frequently used method for synthesis
of nanoparticles is the solvent evaporation method. In the previously used emulsification solvent evaporation method, the drug was dissolved or dispersed in polymer/solvent solution and then the next step was the emulsification of polymer solution into the aqueous phase. After emulsification, the polymer solvent was evaporated and polymer precipitated as nanospheres (Fig. 1). Thereafter, to remove the residue or free drug and to collect the nanoparticles, the solution was centrifuged and washed with distilled water. Then the nanoparticles were lyophilized for long- term storage9.
Emulsion-diffusion methodIn this method, the moderately water-miscible
solvents such as benzyl alcohol and propylene carbonate are used to dissolve encapsulating polymer. Two equally saturated phases are primed: water and solvent. The saturated water includes 8-10% of solvent and the saturated solvent includes 2-3% of water. The stabilizer is added and dissolved in saturated water at ambient temperature. Thereafter, the drug, oil and encapsulating polymer are saturated with aqueous medium. Then the
28
Fig.1: Solvent evaporation method
Fig.1: Solvent evaporation method
INDIAN DRUGS 57 (05) MAY 2020 9
29
Fig.2: Emulsion diffusion and solvent- evaporation method
Fig. 2: Emulsion diffusion and solvent- evaporation method
prepared organic solution is poured into saturated water in which stabilizer was added (Fig. 2). The resultant solution is stirred with a rotor-stator device in a cylindrical vessel. The emulsion oil/water (o/w) forms at room temperature. The dispersed droplets are converted into nanocapsules or nanospheres by addition of a large volume of water in order to induce the solvent diffusion. After the addition of water on the emulsion and mixing at 300 rpm, the nanocapsules are formed. The solvent and part of the water are removed by evaporation under reduced pressure to get a purified and concentrated suspension.
Double emulsion and evaporation method
The drawback of the emulsion and evaporation method is the poor encapsulation of hydrophilic drug. Therefore, to overcome this limitation, another technique has been employed called double-emulsion technique. In this method the aqueous drug solution is added to organic polymer solution with vigorous stirring to prepared water (w/o) emulsions. Thereafter w/o emulsion is added in second aqueous phase along with continuous stirring to prepare the water/oil/water (w/o/w) emulsion (Fig. 3). The solvent is evaporated from the emulsion and nanoparticle is centrifuged at higher speed to isolate and remove residual or free drug. At the final stage the nanoparticle is lyophilized for long-term storage 10,11.
30
Fig. 3: Double emulsion method
Fig. 3: Double emulsion method
Solvent displacement/precipitation method
In solvent displacement method, the polymer is precipitated from an organic solution and the organic solvent diffuses in the aqueous medium in the presence or absence of surfactant. Semipolar water miscible solvent (acetone, ethanol) is used to dissolve polymers, drug, and/or lipophilic surfactant. Thereafter under magnetic stirring conditions, the prepared solution is added into an aqueous solution containing stabilizer. By rapid solvent diffusion, nanoparticles are formed instantly. Under reduced pressure, the solvent is removed from the suspension. The particle size depends upon the rate of addition of organic phase in aqueous medium. The particle size and drug entrapment is decreased as the rate of
mixing of the two phases increases 12. Nanoprecipitation method is more appropriate for encapsulation of hydrophobic drugs. Depending on the preparation parameters, the size of the nanosphere, yield and drug release can be controlled 13.
10 INDIAN DRUGS 57 (05) MAY 2020
Salting out methodSalting out method is built on the basis of salting
out effect, which involves the separation of a water-miscible solvent from aqueous solution14. In this method, salting out agents are used such as electrolytes like magnesium chloride and calcium chloride, or non-electrolytes like sucrose. Primarily, the polymer and drug are dissolved in a solvent (acetone, methanol), which is consequently emulsified into an aqueous gel containing the salting out agent and a colloidal stabilizer such as hydroxyethylcellulose or polyvinylpyrrolidone. In order to augment the diffusion rate of solvent in aqueous phase, adequate volume of water or aqueous phase is used to dilute the o/w emulsion. This results in formation of nanospheres. Depending on the variation in manufacturing parameters such as internal/external phase ratio, type of electrolyte concentration, the type of salting out agent plays a key role in encapsulating efficiency of the drug. The salting out agent and solvent are eliminated by the process of cross-flow filtration. This method is useful in the case of heat-sensitive substances as it does not require an increase in temperature15. This technique is useful to synthesize and potentiate the efficiency of nanospheres such as ethylcellulose nanospheres, PLA and poly (methacrylic) acids16.
Emulsion polymerization techniqueEmulsion polymerization is the method for preparation
of nanoparticles that is one of the fastest and most readily scalable methods. This was one of the first methods for the production of nanoparticles. The emulsion poly-merization formulation contains water, monomer, water-soluble initiator, and surfactant. This method tends to be complex because growth, nucleation, and stabilization of the polymer particles are controlled by free radical polymerization mechanism in combination with various colloidal phenomena. Depending on the organic or aqueous continuous phase, this method can be classified into two categories. In continuous organic phase, the monomers disperse into inverse microemulsion or in non-solvent material 14. Polyacrylamide nanospheres have been synthesized by this method 17. In order to prevent the aggregation in the early stage of polymerization,protective soluble polymers or surfactants were used for production of nanoparticles. The drawback of this method is that it requires toxic organic solvent,monomers and surfactants. These toxic components are excluded from the final product. Polyacrylamide is the nonbiodegradable polymer and due to this being a difficult procedure, there needs to be an alternative approach. Later, poly(ethylcyanoacrylate)(PECA), poly(methylmethacrylate)(PMMA) and poly(butylcyanoacrylate) nanoparticles were produced via
surfactants by dispersion into solvents such as n-pentane (ICH, class 3), cyclohexane (ICH, class 2) and toluene (ICH, class 2) as the organic phase. In the aqueous continuous phase, there is no need for surfactants or emulsifiers and the monomer is dissolved in the continuous aqueous phase. At the initial phase of polymerization process, monomer molecules collide with waterborne free radicals or ions that act as initiator molecules. As an outcome, the hydrophobicity of oligomeric radical will increase. Alternatively, by high- energy radiation such as ultraviolet light, γ-radiation, or strong visible light, the monomer molecules can be transformed into an initiating radical. The oligomeric radicals become hydrophobic and attain strong tendency to enter inside the micelles and react with monomer molecules therein, once they achieve critical chain length and persist the propagation. As a result, monomer-swollen micelles are effectively transformed into particle nuclei. Before or after the termination of the polymerization reaction, the phase separation and synthesis of solid particles takes place18,19.
co-stabilizer, monomer mixture, initiator and surfactant. In this method the compounds used as co-stabilizers are of lower molecular mass;also, the high shear device such as ultrasound is used. In order to reach steady state, the mini-emulsion requires high shear and its interfacial tension is greater than zero. Moreover, they are critically stabilized 20-22.
Microemulsion polymerizationMicroemulsion polymerization is one of the
novel approaches for synthesis of nanoparticles. The significant difference between emulsion and microemulsion polymerization is in its kinetics. They both come out as similar approaches due to similarity in production of colloidal polymer of higher molecular mass, but they are kinetically different. The sizes of particles and average number of chains per particle are significantly reduced in microencapsulation polymerization. The thermodynamically stable aqueous phase of microemulsion contains swollen micelles in which a water-soluble initiator has been added. The polymerization initiates from this state and depends on elevated amounts of surfactant systems, which acquire and interfacial tension at the oil/water interface close to zero. This system utilizes higher amount of surfactant and therefore the particles are fully enclosed with surfactant. At initiation, in few droplets, the polymer chains will form because it is difficult to initiate the formation of polymer chains in all microdroplets. The particle size will increase
INDIAN DRUGS 57 (05) MAY 2020 11
due to elastic and osmotic influence of the chain, which destabilizes the microemulsion and leads to formation of micelles as well as secondary nucleation. Small latexes approximately in the range of 5-50 nm in size with micelles will be obtained as the final product. The critical factors that affect the kinetics of microencapsulation polymerization and properties of polymeric nanoparticle (PNP) are surfactant, types of initiator and concentration, reaction temperature and monomer9,23.
Interfacial polymerization In this method, two reactive monomers or agents,
which are dissolved in two respective phases, polymerize. This is known as step polymerization 24,25. The reaction between the two phases takes place at the interface of two liquids. As an outcome of an interfacial cross-linking reaction (polycondensation, polyaddition, or radical polymerization), hollow nanoparticle will be synthesized. 26, 27.The monomers are polymerized at the interface of oil/water and it leads to formation of nanocapsules containing oil28. In this method, water- miscible organic solvent acts as a vehicle for monomer. During emulsification, oil droplets are formed. On the surface of these oil droplets, the interfacial polymerization of the monomer tends to occur29.The acetone and acetonitrile solvents are recommended to synthesize nanocapsules while protic solvents such as isopropanol, ethanol, and n-butanol are used to induce the synthesis of nanocapsules as well as nanospheres. Alternatively, in water-in–oil microemulsions, interfacial polymerization occurs, which results in formation of water-containing nanocapsules. The nanocapsule’s shell formed by the precipitation of the polymer, is synthesized locally at thewater-oil interface30.
Controlled/living radical polymerizationOne of the major hurdles in radical polymerization is
the inability to control the molar mass and molar mass distribution, the macromolecular architecture, and the end functionalities. This is due to termination reaction between fast radicals. There is a need to modify the old polymerizing technique in a way to obtain controlled or living radical polymerization (C/LRP) processes31,32. This technique tends to provoke the application of hydrophilic polymer in medical and pharmaceutical fields along with superior environmental concerns. An aqueous dispersed system, specifically emulsion polymerization, is used for industrial radical polymerization. The salient feature of this technique is to hold the polymer characteristics, such as molar mass and molar mass distribution, the macromolecular architecture, and functions. The polymeric nanoparticle with narrow size distribution and precise particle size
will form at industrial level by implementation of (C/LRP) in aqueous dispersed system33. There is a range of controlled/living radical polymerization methods that are broadly studied: 1. Atom transfer radical polymerization34, 2. Nitrooxide- mediated polymerization35 and 3. Reversible addition and fragmentation transfer chain polymerization36.The key contribution in determination of particle size is monomer, the nature and concentration of the control agent, and initiator.
DialysisDialysis method facilitates the synthesis of polymeric
nanoparticles with narrow size distribution. The salient feature of this method is ease of preparation with an effective outcome12,37. Dialysis tube of appropriate molecular weight is used to pour organic solvent in which the polymer is dissolved. This technique is performed against a non-solvent miscible with the former miscible. The solvent present inside the membrane will be displaced with simultaneous aggregation of polymer. This aggregation of polymer is an outcome of homogenous suspension of nanoparticles. This method is closely related to the nanoprecipitation method. This technique is used to synthesize a number of polymers and copolymers38,39.The particle size distribution and morphology of the nanoparticle relies on the type of solvent used in the synthesis. The solution of the polymer poured in the dialysis membrane allows the passive transport of the solvents. This facilitates the slow and sustained mixing of non-solvent with polymer solution. This is an osmosis- based method and is used to synthesize synthetic as well as natural polymeric nanoparticles40.
Supercritical fluid technologyThis method has great importance as it is an
environmentally friendly method for the development of nanoparticles. In this method, environmentally friendly solvents that tend to produce highly pure nanoparticle without trace of organic solvents are used. Supercritical fluid extraction by using carbon dioxide has been widely known as a green technology41.
There are two principles for the production of nanoparticles by supercritical fluid technology:
1. Rapid expansion of supercritical solution (RESS)
2. Rapid expansion of supercritical solution into liquid solvent (RESOLV)
Rapid expansion of supercritical solution In rapid expansion of supercritical solution (RESS), the
supercritical fluid is used to dissolve the solute to form a
12 INDIAN DRUGS 57 (05) MAY 2020
solution. The rapid expansion of the solution occurs across a capillary nozzle or orifice into ambient air. Well-dispersed particles will form due to the homogenous nucleation, which is an outcome of the high degree of supersaturation due to the rapid reduction in pressure in the expansion. By using this method, poly (perfluoropolyetherdiamide) droplets were created from the rapid expansion of carbon dioxide solutions.
There are three major units in the RESS experimental apparatus
1. High-pressure stainless steel mixing cell.
2. Syringe pump
3. Pre-expansion unit.
The preparation of polymer solution in the CO2 is done at ambient temperature. The solution is passed through the pre-expansion unit by utilizing syringe pump and heated at the pre-expansion temperature. After this, the solution leaves the nozzle. At ambient pressure, via nozzle the supercritical solution is allowed to expand. For RESS, the particle size and morphology are greatly affected by the concentration and degree of saturation of the polymer42,43.
Rapid expansion of supercritical solution into liquid solvent
Rapid expansion of supercritical solution into liquid solvent (RESOLV) is a modification of rapid expansion of supercritical solution (RESS). In this method, the expansion of supercritical solution occurs into a liquid solvent in place of ambient air. This is a simple technique with considerable modification to RESS. Poly(heptadecafluorodecyl acrylate) nanoparticles were prepared with an average size of less than 50 nm. The prime product produced by RESS technique was obtained in microscale, which is the major demerit of RESS. This can be overcome by RESOLV technology. In this technique, inside the expansion jet, liquid solvent suppresses the particle growth, which leads to production of particles with nanosize44,45.
Characterization of NanoparticlesBased on the size, morphology and surface charge,
using such advanced microscopic techniques as atomic force microscopy (AFM), scanning electron microscopy
Chemical analysis of surface Static secondary ion mass spectrometrySorptometer
Drug stability Bioassay of drug extracted from NanoparticlesChemical analysis of drug
Nanoparticle dispersion stability Critical flocculation temperature (CFT)
Particle size and distribution Atomic force microscopy Laser defractometryPhoton correlation spectroscopy (PCS) Scanning electron microscopy Transmission electron microscopy
Release profile In vitro release characteristics under physiologic and sink conditions
Surface hydrophobicity Rose Bengal (dye) binding Water contact angle measurementX-ray photoelectron spectroscopy
Table I: Various characterization tools and methods for nanoparticles
INDIAN DRUGS 57 (05) MAY 2020 13
(SEM) and transmission electron microscopy (TEM), characterization of nanoparticles is carried out. Features like physical stability and re-dispersibility ofthe polymer dispersion as well as their in vivo performance are affected by the surfacecharge of the nanoparticles. Different characterization tools and methods fornanoparticles are mentioned in Table I.
Particle sizeCharacterization of nanoparticles isprimarily by particle
size distributionand morphology with the aid of electron microscopy. It is now possible toascertain the morphology of nanoparticles. Application ofnanoparticles in drug release and drug targeting can be conveniently determined byparticle size of nanoparticles46and hasprofound effect on the drug release, as shown in Table I.
Smaller size of nanoparticles results in faster drugrelease. Drug(loaded) when exposed to the particle surface area causes significantdrug release. In contrast, inside the nanoparticle, slow diffusion of largerparticles occurs. During storageand transportation of nanoparticle dispersions, there is a mutual compromisebetween maximum stability and small size of nanoparticles47. Particle size also affects degradation of the polymer, e.g., the extent ofpoly (lactic-co-glycolic acid) degradation was found to increase with increasingparticle size in vitro48.
Determination of nanoparticle size by various techniques:
Photon-Correlation Spectroscopy (PCS) or Dynamic LightScattering (DLS)
Photon-correlation spectroscopy(PCS) or dynamic light scattering (DLS), is most widely used to determine the size of nanoparticles in colloidal suspensions in the nano and submicron ranges.Spherical particles in Brownian motion cause aDoppler shift when they are exposed against shining laser monochromatic light. Such monochromatic light exposure hits the moving particle, which results in changing the wavelength of the incoming light.The extent of this change in wavelengthdetermines the size of the particle. This parameter assists in evaluation of the sizedistribution as well as the particle’s motion in the medium, which may further assist in measuringthe diffusion coefficient of the particle and using the autocorrelation function.Dynamic light scattering (DLS) offer the most frequently used techniquefor accurateestimation of the particle size and size distribution49.
Scanning Electron Microscopy (SEM)Scanning electron microscopy-based technique
determines the size, shape and surfacemorphology with direct visualization of the nanoparticles andoffers several advantages in morphological and sizing analysis.In SEM, solution of nanoparticlesshould be initially converted into a dry powder. This dry powder is then furthermounted on a sample holder followed by coating with a conductive metal (e.g. gold)using a sputter coater. Whole sample is then analyzed by scanning with a focused fine beam of electrons50. Surface characteristics of the sample can be determined by the surface electrons emitted from the sample.Average mean size evaluated by SEM is comparable with results obtained bydynamic light scattering. In addition, these techniques are time consuming, costlyand frequently need complementary information about size distribution51.
Transmission Electron Microscope (TEM)Experimental difficulties in studying nanostructures
start from their small size,which limits the use of traditional techniques for measuring their physical properties. Imaging, diffractionand spectroscopic information, either simultaneously or in a serial manner, of thespecimen with an atomic or a sub-nanometer spatial resolution is provided by TEM. Sample preparation is complex and time consuming because of its requirement tobe ultra-thin for electron transmittance. High-resolution TEM imaging, whencombined with nano-diffraction, atomic resolution electron energy-loss spectroscopyand nanometer resolution X-ray energy dispersive spectroscopy techniques, iscritical to the fundamental studies of importance to nanoscience and nanotechnology.
Nanonoparticle samples can also beexposed to liquid nitrogen temperatures after embedding in vitreous ice. When a beam of electrons is transmitted through an ultra-thin sample, it interacts with the sample as it passes through51. Benefits of TEM include:
• Specimen information is collected using a high angular annular dark field (HAADF) detector (in which the images registered have different levelsof contrast related to the chemical composition of the sample).
• It can be utilized for the analysis of biological samples due to its contrast for thickstained sections, since high angular annular dark field images have better contrast than those obtained by othertechniques.
• Combining HAADF-TEM imaging leads to imaging the atomistic structure andcomposition of nano-structures at a sub-angstrom resolution.
14 INDIAN DRUGS 57 (05) MAY 2020
Atomic Force Microscopy (AFM)This technique is also known as scanning force
microscopy (technique that formsimages of surfaces using a probe that scans the specimen), very high-resolution typeof scanning probe microscopy, with reported resolution on the order of fractions ofa nanometer, more than 100 times better than the optical diffraction limit. Atomic force microscopy is based on the physical scanning of samples at sub-micronlevel using a probe tip of atomic scale and offers ultra-high resolution in particlesize measurement52. One of the prime advantages of AFM isits ability to image non-conducting samples without any specific treatment. Thisfeature allows the imaging of delicate biological and polymeric nano- and microstructures53. Moreover, AFM (without any mathematical calculation) providesthe most accurate description of size, size distribution and realtime picture which helpsin understanding the effect of various biological conditions54.
Surface ChargeIt determines the interaction of nanoparticles with
thebiological environment as well as their electrostatic interaction with bioactive compounds.Stability of colloidal material is usually analyzed through zeta potential of nanoparticles. Zeta potential can beobtained by evaluating the potential difference between the outer Helmholtz planeand the surface of shear. Zeta potential values (high zeta potentialvalues, either positive or negative) are got in order to ensure stability and avoidaggregation of the particles. Zeta potential values can be utilized in evaluating surfacehydrophobicity and the nature of material encapsulated within the nanocapsulesor coated onto the surface55.
partitioning,adsorption of probes and contact angle measurements can be utilized for the determinationof surface hydrophobicity.Moderntechniques such as X-ray photon correlation spectroscopy not only determine surfacehydrophobicity but also permit the identification of specific chemical groups on thesurface of nanoparticles56.
Drug Release It is very essential to determine the extent of the
drug release.In order to obtain suchinformation, most release methods require that the drug and its delivery vehicle beseparated. Drug loading capacity of the nanoparticles isthe amount of drugbound per mass of polymer or in another term it is the moles of drug per mg polymeror it could also be given as percentage relative to the polymer.Different techniques such as UV
spectroscopy or highperformance liquid chromatography (HPLC) after ultracentrifugation, ultra filtration, gel filtration, or centrifugal ultrafiltration are used to determine this parameter. Methods that are employed fordrug release analysis are drug loading assay, which is more oftenassessed for a period of time to evaluate the drug release mechanism57,58.
APPLICATIONS OF NANOSPHERES
Targeted delivery
Targeted drug delivery could increase the specificity of the pharmacologic action of the drug and also could reduce the dose and the toxic effects of the drug with the help of nanospheres. Cell or tissue specific ligands could be coupled.
Oral delivery
The research in oral delivery of nanospheres is focused on vaccines to induce mucosal and systemic immunity59. Nanospheres could gain entry into the lymphoidal tissue of the gut through the Peyer’s patches, a group of specialized tissues in the gut consisting of the antigen presenting cells, the B cells and the T cells. The fate of the particles following their uptake by the lymphoidal tissue is mainly governed by their size.
Gene delivery
Gene therapy could be used to cure genetic diseases by adding a missing gene or correcting the defective gene60. However, one of the major limiting factors in gene therapy is the efficiency of gene transfer61.Non-viral methods of gene transfer are relatively less efficacious compared to viral methods of gene transfer but are considered safe.
Vaccine delivery
The goal of vaccine delivery system is to combine the primary and the booster doses in a single dose injection62.The advantage of a single dose vaccine system is that it could prevent the fall-out in the number of people receiving the complex immunization with the increase in the number of injections.
Cancer chemotherapy
The major limitation in cancer chemotherapy is the poor uptake of anticancer agents by the cancer cell. By using nanospheres as a carrier system, higher cellular uptake of drugs and greater therapeutic effects were observed. The proposed mechanism is the drug in nanospheres (entrapped or bound) could bypass the efflux
INDIAN DRUGS 57 (05) MAY 2020 15
Table II: Research work and successful formulations based on biodegradable nanospheres
action of the p-glycoprotein pump63.The other therapeutic approach to treat cancers is to block the expression of certain oncogenes such as C-raf and C-ras and kinases64. Nanospheres could facilitate the uptake of oligonucleotides and also stabilize them against nuclease attack.
Intramuscular delivery
The nanospheres could form a depot at the site of injection from which the drug could be released slowly in the local tissue and then absorbed for the systemic effect. The strategy is useful for drugs which require repeated administration such as growth factors or hormones65,66.
Localized drug delivery
The infused nanospheres could provide a localized sustained drug effect with minimal toxicity. Intravenous or oral delivery of drugs is usually ineffective because it
does not provide a therapeutic dose of the drug to the diseased artery for a sufficient period of time. Nanospheres mobilized into the arterial wall could provide sustained drug levels in the diseased artery. The localized delivery of drug using nanospheres could significantly reduce the total dose of the drug and also could prevent the toxic effects of the drug67.Research work and successful formulations based on biodegradable nanospheres are shown in Table II.
CONCLUSIONNanospheres could be used as an effective drug
delivery tool to improve the therapeutic efficacy of drugs in various pathophysiologic conditions. It is important to understand the pathobiology of the disease, so that one can determine the desired nanosphere formulation in terms of the release kinetics and the duration of drug release. At the same time, knowledge of the cell surface
16 INDIAN DRUGS 57 (05) MAY 2020
and receptors could help in developing a targeted drug delivery system using nanospheres. Gene delivery using nanospheres needs to be optimized to increase the transfection efficiency.
ACKNOWLEDGEMENTThe authors are thankful to Dr.L.Rathaiah, Chairman,
Vignan’s Group of Institutions for providing necessary information to carryout the above review work.
REFERENCES1. Davis S. S., Biomedical applications of nanotechnology—
implications for drug targeting and gene therapy,Trends Biotechnol., 1997, 15(6), 217-24.
2. Chavany C., Saison-Behmoaras T., Le Doan T., Puisieux F., Couvreur P., Hélène C., Adsorption of oligonucleotides onto polyisohexylcyanoacrylate nanoparticles protects them against nucleases and increases their cellular uptake,Pharm. Res, 1994,11(9), 1370-8.
3. Bender A.R., Von Briesen H., Kreuter J., Duncan I.B., Rübsamen-Waigmann H., Efficiency of nanoparticles as a carrier system for antiviral agents in human immunodeficiency virus-infected human monocytes/macrophages in vitro,Antimicrob.Agents Chemother., 1996,40(6),1467-71.
4. Labhasetwar V.D., Dorle A.K., Nanoparticles—a colloidal drug delivery system for primaquine and metronidazole, J. Control.Rel., 19901, 12(2),113-9.
5. Labhasetwar V., Nanoparticles for drug delivery, Pharmaceutical News,1997;4(6):28-31.
6. Labhasetwar V.D., Dorle A.K., Nanoparticles—a colloidal drug delivery system for primaquine and metronidazole, J. Control. Rel., 1990, 12(2):113-9.
8. Sahu A., Kasoju N., Goswami P., Bora U., Encapsulation of curcumin in Pluronic block copolymer micelles for drug delivery applications,J. Biomater.Appl., 2011, 25(6):619-39.
9. Song C.X., Labhasetwar V., Murphy H., Qu X., Humphrey W.R., Shebuski R.J., Levy R.J., Formulation and characterization of biodegradable nanoparticles for intravascular local drug delivery,J. Control. Rel., 1997, 43(2-3),197-212.
10. Vandervoort J., Ludwig A., Biocompatible stabilizers in the preparation of PLGA nanoparticles: a factorial design study,Int.J.Pharm.,2002, 238(1-2),77-92.
11. Ubrich N., Bouillot P., Pellerin C., Hoffman M., Maincent P., Preparation and characterization of propranolol hydrochloride nanoparticles: a comparative study,J. Control. Rel., 2004, 97(2), 291-300.
13. Chorny M., Fishbein I., Danenberg H.D., Golomb G., Lipophilic drug loaded nanospheres prepared by nanoprecipitation: effect of formulation variables on size, drug recovery and release kinetics,J.Control.Rel.,2002, 83(3),389-400.
14. Reis C.P., Neufeld R.J. Nanoencapsulation I., Methods for preparation of drug loaded polumeric nanoparticles. Nanomed.Nanotechnol. Biol. Med., 2006, 2(1), 8-21.
15. Lambert G., Fattal E., Couvreur P., Nanoparticulate systems for the delivery of antisense oligonucleotides.Adv.Drug Del.Rev., 2001, 47(1), 99-112.
16. Quintanar-Guerrero D., Allémann E., Fessi H., Doelker E., Preparation techniques and mechanisms of formation of biodegradable nanoparticles from preformed polymers. Drug Dev.Indl. Pharm., 1998, 24(12), 1113-28.
17. Ekman B., Sjöholm I., Improved stability of proteins immobilized in microparticles prepared by a modified emulsion polymerization technique, J.Pharm.Sci., 1978, 67(5),693-6.
18. Vauthier C., Dubernet C., Fattal E., Pinto-Alphandary H., Couvreur P., Poly (alkylcyanoacrylates) as biodegradable materials for biomedical applications,Adv.Drug Del.Rev., 2003, 55(4),519-48.
19. Kreuter J., On the mechanism of termination in heterogeneous polymerization, J. Polymer Sci.: Polymer Letters Edition. 1982, 20(10), 543-5.
20. Arias J.L., Gallardo V., Gomez-Lopera S.A., Plaza R.C., Delgado A.V., Synthesis and characterization of poly (ethyl-2-cyanoacrylate) nanoparticles with a magnetic core,J. Control. Rel., 2001,77(3), 309-21.
21. Ham H.T., Choi Y.S., Chee M.G., Chung I.J.,Singlewall carbon nanotubes covered with polystyrene nanoparticles by in-situ miniemulsion polymerization, J. Polymer Sci. Part A: Polymer Chemistry. 2006, 44(1):573-84.
22. Ziegler A., Landfester K., Musyanovych A., Synthesis of phosphonate-functionalized polystyrene and poly (methyl methacrylate) particles and their kinetic behavior in miniemulsion polymerization,Coll.Polymer Sci., 2009, 287 (11),1261.
23. Puig J. E., Microemulsion polymerization,Polymeric Mater.Encycloped., 1996, 6, 4333-41.
24. Nagavarma B.V., Yadav H.K., Ayaz A., Vasudha L.S., Shivakumar H.G., Different techniques for preparation of polymeric nanoparticles-a review, Asian J. Pharm. Clin. Res., 2012, 5(3), 16-23.
25. Wu M., Dellacherie E., Durand A., Marie E. Poly (n-butyl cyanoacrylate) nanoparticles via miniemulsion polymerization (1): Dextran-based surfactants. Colloids and surfaces B: Biointerfaces, 2009, 69(1),141-6.
26. Crespy D., Stark M., Hoffmann-Richter C., Ziener U., Landfester K., Polymeric nanoreactors for hydrophilic reagents synthesized by interfacial polycondensation on miniemulsion droplets,Macromolecules, 2007, 40(9), 3122-35.
INDIAN DRUGS 57 (05) MAY 2020 17
27. Sarkar D., El-Khoury J., Lopina S.T., Hu J., An effective method for preparing polymer nanocapsules with hydrophobic acrylic shell and hydrophilic interior by inverse emulsion radical polymerization, Macromolecules, 2005, 38(20), 8603-5.
28. Fallouh N.A., Roblot-Treupel L., Fessi H., Devissaguet J.P., Puisieux F., Development of a new process for the manufacture of polyisobutylcyanoacrylatenanocapsules, Int. J. Pharm., 1986, 28(2-3), 125-32.
29. Aboubakar M., Puisieux F., Couvreur P., Deyme M., Vauthier C., Study of the mechanism of insulin encapsulation in poly (isobutylcyanoacrylate) nanocapsules obtained by interfacial polymerization. J. Biomed. Mater. Res., 1999,47(4), 568-76.
30. Watnasirichaikul S., Davies N.M., Rades T., Tucker I.G., Preparation of biodegradable insulin nanocapsules from biocompatible microemulsions, Pharm.Res., 2000, 17(6):684-9.
31. Matyjaszewski K., Xia J., Atom transfer radical polymerization,Chem. Rev.,2001,101(9):2921-90.
32. Zetterlund P.B., Kagawa Y., Okubo M., Controlled/living radical polymerization in dispersed systems. Chem.Rev., 2008, 108(9), 3747-94.
33. Nicolas J., Charleux B., Guerret O., Magnet S., Nitroxide-mediated controlled free-radical emulsion polymerization using a difunctional water-soluble alkoxyamine initiator. Toward the control of particle size, particle size distribution, and the synthesis of triblock copolymers. Macromolecules, 2005, 38(24), 9963-73.
34. Min K., Gao H., Yoon J.A., Wu W., Kowalewski T., Matyjaszewski K., One-pot synthesis of hairy nanoparticles by emulsion ATRP,Macromolecules, 2009, 42(5),1597-603.
35. Farcet C., Lansalot M., Charleux B., Pirri R., Vairon J.P., Mechanistic aspects of nitroxide-mediated controlled radical polymerization of styrene in miniemulsion, using a water-soluble radical initiator,Macromolecules, 2000, 33(23), 8559-70.
36. Rieger J., Zhang W., Stoffelbach F., Charleux B., Surfactant-free RAFT emulsion polymerization using poly (N, N-dimethylacrylamide) trithiocarbonate macromolecular chain transfer agents, Macromolecules, 2010 ,43(15), 6302-10.
37. Jeong Y.I., Cho C.S., Kim S.H., Ko K.S., Kim S.I., Shim Y.H., Nah J.W., Preparation of poly (DL-lactide-co-glycolide) nanoparticles without surfactant,J. Appl. Polymer Sci., 2001, 80(12):2228-36.
38. Jung S.W., Jeong Y.L., Kim Y.H., Kim S.H., Self-assembled polymeric nanoparticles of poly (ethylene glycol) grafted pullulan acetate as a novel drug carrier, Arch.Pharm.Res., 2004, 27(5), 562.
39. He X., Ma J., Mercado A.E., Xu W., Jabbari E., Cytotoxicity of paclitaxel in biodegradable self-assembled core-shell poly (lactide-co-glycolide ethylene oxide fumarate) nanoparticles,Pharm. Res., 2008, 25(7), 1552-62.
40. Chronopoulou L., Fratoddi I., Palocci C., Venditti I., Russo M.V., Osmosis based method drives the self-assembly of
polymeric chains into micro-and nanostructures, Langmuir, 2009, 25(19), 11940-6.
41. Deshpande P.B., Kumar G.A., Kumar A.R, Shavi GV, Karthik A, Reddy MS, Udupa N., Supercritical fluid technology: concepts and pharmaceutical applications, PDA J. Pharm. Sci. Technol., 2011, 65(3), 333-44.
42. Chernyak Y., Henon F., Harris R.B., Gould R.D., Franklin R.K., Edwards J.R., DeSimone J.M., Carbonell R.G., Formation of perfluoropolyether coatings by the rapid expansion of supercritical solutions (RESS) process, Part 1: Experimental results,Ind.Eng.Chem.Res., 2001, 40(26), 6118-26.
43. Blasig A., Shi C., Enick R.M., Thies M.C., Effect of concentration and degree of saturation on RESS of a CO2-soluble fluoropolymer,Ind. Eng.Chem. Res., 2002, 41(20),4976-83.
44. Meziani M.J., Pathak P., Hurezeanu R., Thies M.C., Enick R.M., Sun Y.P., Supercritical-fluid processing technique for nanoscale polymer particles,Angew.Chemie Internat. Ed, 2004, 43(6), 704-7.
45. Meziani M.J, Pathak P., Wang W., Desai T., Patil A., Sun Y.P., Polymeric nanofibers from rapid expansion of supercritical solution. Ind. Eng. Chem.Res., 2005, 44(13), 4594-8.
46. Vyas S.P., Khar R.K.,Targeted Controlled Drug Delivery, CBS publishers and distributers.New Delhi. 2002, 1, 331–43.
47. Redhead H.M., Davis S.S., Illum L. J. Control. Rel.,2001,70,353.
49. DeAssis D.N., Mosqueira V.C., Vilela J.M., Andrade M.S., Cardoso V.N., Release profiles andmorphological characterization by atomic force microscopy and photon correlation spectroscopyof 99m Technetium—fluconazole nanocapsules, Int. J. Pharm., 2008,349,152–60.
50. Jores K., Mehnert W., Drecusler M., Bunyes H., Johan C., MAder K., Investigation on the stricterof solid lipid nanopartuicles and oil-loaded solid nanoparticles by photon correlation spectroscopy,field flow fractionasition and transmission electron microscopy,J. Control.Rel., 2004,17, 217–27.
51. Molpeceres J., Aberturas M.R., Guzman M., Biodegradable nanoparticles as a delivery systemfor cyclosporine: preparation and characterization,J Microencapsul.,2000, 17, 599–614.
52. Muhlen A.Z., Muhlen E.Z., Niehus H., Mehnert W., Atomic force microscopy studies of solid lipid nanoparticles. Pharm Res., 1996,13, 1411–6.
53. Shi H.G., Farber L., Michaels J.N., Dickey A., Thompson K.C., Shelukar S.D., Hurter P.N., ReynoldsS.D., Kaufman M.J., Characterization of crystalline drug nanoparticles using atomic forcemicroscopy and complementary techniques, Pharm Res., 2003, 20, 479–84.
54. Polakovic M., Gorner T., Gref R., Dellacherie E., Lidocaine loaded biodegradable nanospheres.II. Modelling of drug release, J. Control. Rel..,1999, 60, 169–77.
18 INDIAN DRUGS 57 (05) MAY 2020
55. Pangi Z., Beletsi A., Evangelatos K., PEG-ylated nanoparticles for biological and pharmaceuticalapplication. Adv. Drug Del. Rev., 2003, 24, 403–19.
56. Scholes P.D., Coombes A.G., Illum L., Davis S.S., Wats J.F., Ustariz C., Vert M., Davies M.C.,Detection and determination of surface levels of poloxamer and PVA surfactant on biodegradablenanospheres using SSIMS and XPS, J. Control. Rel., 1999, 59, 261–78.
57. Kreuter J., Physicochemical characterization of polyacrylic nanoparticles, Int. J. Pharm., 1983, 14, 43–58.
58. Magenhein B., Levy M.Y., Benita S., A new in vitro technique for the evaluation of drug release profile from colloidal carriers ultrafiltration technique at low pressure,Int. J. Pharm.,1993, 94,115–23.
59. O’Hagan D.T, Palin K.J, Davis S.S., Poly (butyl-2-cyanoacrylate) particles as adjuvants for oral immunization, Vaccine, 1989, 7(3), 213-6.
60. Dickler H.B., Collier E., Gene therapy in the treatment of disease, J. Allergy Clin.Immunol., 1994, 94(6), 942-51.
61. Afione S.A., Conrad C.K., Flotte T.R.,Gene therapy vectors as drug delivery systems, Clin. Pharmacokin. 1995, 28(3), 181-9.
62. Cleland J.L., Barrón L., Berman P.W., Daugherty A., Gregory T., Lim A., Vennari J., Wrin T., Powell M.F., Development of a single shot subunit vaccine for HIV1. 2. Defining optimal autoboost characteristics to maximize the humoral immune response, J.Pharm.Sci., 1996, 85(12), 1346-9.
63. Couvreur P., Roblot-Treupel L., Poupon M.F., Brasseur F., Puisieux F., Nanoparticles as microcarriers for anticancer drugs, Adv. Drug Del. Rev., 1990, 5(3), 209-30.
64. Monia B.P., Johnston J.F., Geiger T., Muller M., Fabbro D., Antitumor activity of a phosphorothioate antisense oligodeoxynucleotide targeted against C-raf kinase,Nature Medicine, 1996, 2(6), 668.
65. Hashimoto T., Wada T., Fukuda N., Nagaoka A., Effect of Thyrotropinreleasing Hormone on Pentobarbitoneinduced Sleep in Rats: Continuous Treatment with a Sustained Release Injectable Formulation,J. Pharm.Pharmacol., 1993, 45(2):94-7.
66. Ball B.A., Wilker C., Daels P.F., Burns P.J., Use of progesterone in microspheres for maintenance of
pregnancy in mares. Amer. J. Vet. Res., 1992, 53(8), 1294-7.
67. Fuster V., Falk E., Fallon J.T., Badimon L., Chesebro J.H., BadimonJ.J.,The three processes leading to post PTCA restenosis: dependence on the lesion substrate,ThrombosisHaemostasis, 1995, 73(01), 552-9.
68. Niwa T., Takeuchi H., Hino T., Kunou N., Kawashima Y., Preparations of biodegradable nanospheres of water-soluble and insoluble drugs with D, L-lactide/glycolide copolymer by a novel spontaneous emulsification solvent diffusion method, and the drug release behavior,J. Control. Rel., 1993, 25(1-2), 89-98.
69. Baimark Y.O., Preparation and Characterization of Biodegradable Nanospheres of Amphiphilic Methoxy poly (ethylene glycol)-b-poly (D, L-lactide-co-glycolide-co-γ-caprolactone) for Controlled Drug Delivery, Int. J. Drug Deliv.,2012, 3(4),661-8.
70. Doi N., Jo J.I., Tabata Y., Preparation of biodegradable gelatin nanospheres with a narrow size distribution for carrier of cellular internalization of plasmid DNA, J.Biomater. Sci., Polymer Edition. 2012, 23(8):991-1004.
71. Kim S.Y., Shin I.G., Lee Y.M., Preparation and characterization of biodegradable nanospheres composed of methoxy poly (ethylene glycol) and DL-lactide block copolymer as novel drug carriers, J. Control. Rel., 1998, 56(1-3), 197-208.
72. Kim S.Y., Shin I.G., Lee Y.M., Amphiphilic diblock copolymeric nanospheres composed of methoxy poly (ethylene glycol) and glycolide: properties, cytotoxicity and drug release behavior,Biomater., 1999, 20(11),1033-42.
73. Dalpiaz A., Leo E., Vitali F., Pavan B., Scatturin A., Bortolotti F., Manfredini S., Durini E., Forni F., Brina B., Vandelli M.A., Development and characterization of biodegradable nanospheres as delivery systems of anti-ischemic adenosine derivatives,Biomater., 2005, 26(11), 1299-306.
74. Görner T., Gref R., Michenot D., Sommer F., Tran M.N., Dellacherie E., Lidocaine-loaded biodegradable nanospheres. I. Optimization of the drug incorporation into the polymer matrix, J. Control. Rel.,1999, 57(3), 259-68.
INDIAN DRUGS 57 (05) MAY 2020 19
SYNTHESIS CHARACTERIZATION AND DOCKING STUDY OF HCV NS3/4A PROTEASE INHIBITOR MOLECULESKumar Satisha*, Santra P. K.b and Aryan R. C.b
(Received 21 January 2020) (Accepted 25 February 2020)
ABSTRACT
A sharp rise in the number of hepatitis C patients has been observed worldwide in the last decades. This has attracted the attention of researchers and medical professionals to provide cheap and effective treatment options for the eradication of hepatitis C. The present study describes the synthesis of a number of novel molecules which may be capable of inhibiting progression of the virus. The molecules were synthesized using the general techniques of the peptide bond preparation. Synthesized molecules were characterized by elemental analysis and spectral studies such as Mass, UV/Vis, IR and NMR. Purity of the molecules was determined by HPLC. Molecules with purity more than 98.5 % were selected for further docking studies to determine their interaction with binding site of NS3/4A protease on HCV RNA.
In the last few decades, a manifold increase in the number of hepatitis C patients has been observed. Currently, there are approximately 177.5 million people infected from hepatitis C and 71 million people have chronic hepatitis C1-3. In one of its reports, WHO estimated that about 4 lakh people died due to hepatitis C in 2016. In the Indian context, one survey revealed that there are approximately 6-12 million hepatitis C virus (HCV) infected people. Earlier, there were few options for the treatment of hepatitis C, but in recent years significant increase in number of options for the treatment of hepatitis C is observed4-5.These drugs targets various protease sites on HCV RNA (ribonucleic acid). In the structure of hepatitis C virus, there are two types of proteins in HCV RNA - structural and nonstructural proteins. Non-structural6 proteins are NS2, NS3, NS4A, NS4B, NS5A, and NS5B7. HCV drugs are categorized according to the mechanism they follow by inhibiting replication of RNA by blocking the particular protein site of RNA. These drugs block the particular protein site on HCV like nucleoside and nucleotide NS5B polymerase inhibitors, directed inhibitors (NS5A inhibitors), non-nucleoside
a Department Of Chemistry, Banasthali Vidyapith, Banasthali - 304 022, Rajasthan, Indiab API Research, Sun Pharmaceutical Industries Ltd, Sec-18, Gurgaon - 122 015, Haryana, India*For Correspondence: E-mail: [email protected]
NS5B polymerase inhibitors and NS3/4A8 inhibitors or serine protease inhibitor antiviral medications. NS3/4A protease inhibitors, NS5A polymerase inhibitors and NS5B polymerase inhibitors combined are known as direct acting antivirals (DAA)9,10, because they directly act on the target.
Both NS3 and NS4A protein are bound to ER membrane. NS4A acts as a cofactor to the NS3 protein. The NS3 protein have 185 amino acid chain, while NS4A has 54 amino acid chain. Nowadays, the most prominent drugs in the market are direct acting antivirals such as sofosbuvir11, ledipasvir, daclatasvir, valptasavir, grazoprevir, asunaprevir12, faldaprevir, paritaprevir, vaniprevir13, boceprevirand and telaprevir14-15. Although sofosbuvir is the most prominent drug for treatment of hepatitis C, it is not pan genotypic i.e. it cannot inhibit all the genotypes of HCV. Telaprevir is marketed as NS3/4A serine protease inhibitor or NS3/4A inhibitor. Other NS3/4A protease inhibitor drugs are asunaprevir, faldaprevir, paritaprevir, vaniprevir and telaprevir. Telaprevir16 is also effective against only genotype 1 of HCV out of total nine genotypes. There are many other drugs which have been introduced into the market in recent years. But none of these drugs can inhibit all the HCV genotypes. To counter this problem, combination drug therapy is introduced wherein two or more direct acting antiviral’s (DAA’s) are
20 INDIAN DRUGS 57 (05) MAY 2020
combined together to prepare a new formulation. These drugs targets more than one nonstructural protein site on HCV RNA, thus decreasing the virus load in patients sharply. But these drugs which are effective in combination are costly and cannot be generally afforded by people in underdeveloped countries. So, there is always a need to develop drugs which are effective against all the genotype of hepatitis C virus and are at the same time also affordable.
The present study describes the synthesis of affordable and pan genotype HCV drug molecules. Molecules were designed to be effective against HCV and targeting NS3/4A protease enzyme on HCV RNA. The α-keto amide molecules inhibit active site at NS3/4A, thus inhibiting the replication of RNA and Hepatitis C Virus spread is thereby controlled. The molecules are small peptidomimetic molecules which are synthesized using natural and synthetic amino acids. Synthesis was carried out by solution phase approach of peptide synthesis using different amidation reagents such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC.HCl), TBTU, HBTU and other reagents. The molecules were characterized by using elemental analysis and spectral studies such as Mass, UV/Vis, IR and NMR. The docking studies were conducted by using Schrödinger’s docking software called as Glide to measure the compatibility of molecules with the active site. More the resemblance of the active site with the molecule, more effective is the molecule against hepatitis C.
EXPERIMENTAL
Material and MethodsLR grade chemicals and solvents were used
throughout the study. Solvents were freshly distilled prior to use. Amino acids used for synthesis of the molecules were procured from G L Biochem (Shanghai) Ltd. and were purified further by acid base treatment. Progress of reaction was monitored by TLC (TLC Silica Gel F254 Aluminum sheets, Merck). Purification of compounds was done by using crystallization technique or in some cases by column chromatography using silica gel (JD Fine Chemical having mesh size of 100-200 or 60-120). Molecules were characterized by using instruments such as NMR (Bruker Avance III 400 MHz) and FT-IR (Perkin Elmer Spectrum One FT-IR). Melting points were determined by differential scanning calorimetry DSC 821e of Mettler Toledo. Purity of molecules was determined by using HPLC of Waters model E2695 having PDA Detector 2998. The moisture content of raw materials and molecules was determined by Karl Fischer µAquaCal50, manufactured by Analab Scientific Instruments Pvt. Ltd., wherever required.
The general synthesis procedure followed is as given below:
Procedure of coupling reaction/amide formation using EDC HCl:
1.0 mol of acid moiety was charged into round bottom flask having 10-20 volume equivalent of dichloromethane. To this solution was added 1.0 mol of 1-hydroxy-benzotriazole (HOBt) and 1.2 mol of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC.HCl) to obtain a clear solution. Amino acid moiety having free amine (1.0 mol) was added to the clear solution. Solution was stirred for 10 min and then 2.0 mol of diisopropyl ethyl amine was added slowly drop wise at ambient temperature. Stirring was continued for 4 to 24 h and the progress of the reaction was monitored by using TLC. After completion, reaction mixture was treated with water, sodium bicarbonate solution and hydrochloric acid solution. Organic layer was concentrated to obtain a solid residue, which was further purified by treating with hexane and ethyl acetate.
Procedure of coupling reaction/amide formation using TBTU/HBTU:
1.0 mol of amino acid moiety having free acid group was added to a round bottom flask having 10 volume equivalent of acetonitrile. To this solution, 1.2 mol of O-(benzotriazol-1-yl)-N,N,N,N-tetramethyluronium tetrafluoroborate (TBTU) or O-(benzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphate (HBTU) was added as the coupling reagent. After stirring of 10-20 min, 1.0 mol of amino acid moiety having free amino group was added to the clear solution. 1.0 mol of triethyl amine was added to the reaction mass. It was then stirred at ambient temperature for 10-16 h. Progress of the reaction was monitored by TLC. On completion of the reaction, 10-20 volume of dichloromethane was added to the reaction mass and the reaction mass was treated with water, sodium bicarbonate solution and hydrochloric acid solution. Organic layer was collected and concentrated to obtain a solid residue, which was further purified by treating with hexane and ethyl acetate.
Procedure for protection of amino acids with N-benzyloxycarbonyl (CBz) group
Solution of amino acid in 2-4 volumes of toluene was cooled to 3±2oC and 10-11% solution of lithium hydroxide in water (10-12 volumes) was added. 5 volumes of 40% benzyl chloroformate solution in toluene was added drop wise at 5-10oC. Reaction mixture was warmed and allowed to stir at 15-20oC for 10-15h. Progress of reaction
INDIAN DRUGS 57 (05) MAY 2020 21
was monitored by HPLC and TLC. After completion of reaction, the layers were separated. Aqueous layer was washed with 5 volumes of toluene. pH of the aqueous layer was adjusted to 1.5 using 2N hydrochloric acid. It was then extracted with 8-10 volumes of ethyl acetate. Ethyl acetate extract was then concentrated under vacuum to obtain solid residue as CBz-protected amino acid.
Procedure for deprotection of N-benzyloxycarbonyl (CBz) group
To the clear solution of CBz-protected compound in 10-15 volumes of methanol was added palladium on carbon (50 % wet with water) in an autoclave. Reaction was stirred at 30±20C for 2-3h under hydrogen pressure of 2 bar. Progress of reaction was monitored by TLC. After completion of the reaction, the reaction mass was filtered through hyflo bed and washed with methanol. Filtrate was collected and solvent was distilled out at 36±20C to give a solid residue. The solid residue further purified using hexane and ethyl acetate to give the desired compound.
1. Synthesis of (1S,3aR,6aS)-2-[(2S)-2-({(2S)-2-cyclohexyl-2-[(pyridin-2-ylcarbonyl)amino]acetyl}amino)-4-methylpentanoyl]-N-[(3S)-1-(cyclopropylamino)-1,2-dioxohexan-3-yl]octahydrocyclopenta[c]pyrrole-1-carboxamide (A-1)
2. Synthesis of (1S,3aR,6aS)-2-[(2S)-2-{[(2S)-2-cyclohexyl-2-{[(5-methylpyrazin-2-yl)carbonyl]amino}acetyl]amino}-4-methylpentanoyl]-N-[(3S)-1-(cyclopropylamino)-1,2-dioxohexan-3-yl]octahydrocyclopenta[c]pyrrole-1-carboxamide (A-3)
Off white powder, yield: 21%,MP by DSC: 155.1; MW:
4. Synthesis of (1S,3aR,6aS)-2-[(2S)-2-{[(2S)-2-cyclohexyl-2-{[(5-methylpyrazin-2-yl)carbonyl]amino}acetyl]amino}-3,3-dimethylbutanoyl]-N-[(3S)-1-(cyclopropylamino)-1,2-dioxohexan-3-yl]octahydrocyclopenta[c]pyrrole-1-carboxamide (A-6)
5. Synthes is o f (1S ,3aR,6aS) -N- [ (3S) -1 -(cyclopropylamino)-1,2-dioxohexan-3-yl]-2-[(2S)-3,3-dimethyl-2-({(2S)-3-phenyl-2-[(pyridin-2-ylcarbonyl)amino] propanoyl}amino) butanoyl]octahydrocyclopenta[c]pyrrole-1-carboxamide (A-7)
6. Synthes is o f (1S ,3aR,6aS) -N- [ (3S) -1 -(cyclopropylamino)-1,2-dioxohexan-3-yl]-2-[(2S)-3,3-dimethyl-2-{[(2S)-2-{[(5-methylpyrazin-2-yl)carbonyl]amino}-3-phenylpropanoyl]amino}butanoyl]octahydrocyclopenta[c]pyrrole-1-carboxamide (A-9)
13. Synthes is o f (1S ,3aR,6aS) -N- [ (2S) -1 -(cyclopropylamino)-1-oxopentan-2-yl]-2-[(2S)-3,3-dimethyl-2-({(2S)-3-phenyl-2-[(pyrazin-2-ylcarbonyl)amino]propanoyl} amino)butanoyl]octahydrocyclopenta[c]pyrrole-1-carboxamide (V-1)
14. Synthes is o f (1S ,3aR,6aS) -N- [ (2S) -1 -(cyclopropylamino)-1-oxopentan-2-yl]-2-[(2S)-4-methyl-2-({(2S)-3-phenyl-2-[(pyrazin-2-ylcarbonyl)amino]propanoyl} amino)pentanoyl]octahydrocyclopenta[c]pyrrole-1-carboxamide (V-3)
Table I: Structure of molecules with docking score
Sr. No. Molecule Code
Structure Docking Score
1 A-1
12
peaks of four –CH protons of amino acids, beside peptide bonds. The M+1 peak in mass spectra
confirms the molecular mass of molecules. The NMR and mass spectra match with the
molecules.
To determine the anti HCV activity of the molecules, docking was done using Glide program of
Schrödinger. The binding energy is obtained by interaction of molecule with active site of
NS3/4A enzyme on Hepatitis C Virus RNA. The docking scores of all the molecules are given in
Table I. Docking score suggests the binding energy between the molecules and binding site,
more –ve the docking score more is the resemblance between molecule and active site i.e.
molecule will fit better into the active site and block the active site, thus inhibiting the replication
of RNA and stopping the spread of hepatitis C. In our study, we compared the docking score of
molecules with docking score telaprevir (-5.110482), which is also a NS3/4A protease inhibitor.
The docking score of B-3 (-6.231025) at serial no. 9 in Table IX and V-3 (-5.400139)) at serial
no. 14 in Table I are more than that of telaprevir. The docking score of A-3, V-1 and V-17 at
serial no. 2, 13 and16, respectively, in Table I are nearby to that of telaprevir. Hence, these
molecule can have better chances of inhibiting HCV. To determine the activity of these
molecules, further in vitro studies are required.
Table I: Structure of molecules with docking score
Sr. No
.
Molecule Code
Structure Docking Score
1 A-1
N
NH
O
O
NHN
O
O
NH
CH3
O
O
NHCH3
CH3
Molecular Formula = C37H54N6O6 Formula Weight = 678.86126 (1S,3aR,6aS)-2-[(2S)-2-({(2S)-2-cyclohexyl-2-[(pyridin-2-ylcarbonyl)amino]acetyl}amino)-4-methylpentanoyl]-N-[(3S)-
Molecular Formula = C37H55N7O6 Formula Weight = 693.8759 (1S,3aR,6aS)-2-[(2S)-2-{[(2S)-2-cyclohexyl-2-{[(5-methylpyrazin-2-yl)carbonyl]amino}acetyl]amino}-4-methylpentanoyl]-N-[(3S)-1-(cyclopropylamino)-1,2-dioxohexan-3-yl]octahydrocyclopenta[c]pyrrole-1-carboxamide
-4.618015
3 A-4
N
NH
O
O
NHN
O
O
NH
CH3
O
O
NHCH3 CH3
CH3
Molecular Formula = C37H54N6O6 Formula Weight = 678.86126 (1S,3aR,6aS)-2-[(2S)-2-({(2S)-2-cyclohexyl-2-[(pyridin-2-ylcarbonyl)amino]acetyl}amino)-3,3-dimethylbutanoyl]-N-[(3S)-1-(cyclopropylamino)-1,2-dioxohexan-3-yl]octahydrocyclopenta[c]pyrrole-1-carboxamide
-4.390643
4 A-6
N
N
NH
O
O
NHN
O
O
NH
CH3
O
O
NHCH3 CH3 CH3
CH3
Molecular Formula = C37H55N7O6 Formula Weight = 693.8759 (1S,3aR,6aS)-2-[(2S)-2-{[(2S)-2-cyclohexyl-2-{[(5-methylpyrazin-2-yl)carbonyl]amino}acetyl]amino}-3,3-
Molecular Formula = C37H55N7O6 Formula Weight = 693.8759 (1S,3aR,6aS)-2-[(2S)-2-{[(2S)-2-cyclohexyl-2-{[(5-methylpyrazin-2-yl)carbonyl]amino}acetyl]amino}-4-methylpentanoyl]-N-[(3S)-1-(cyclopropylamino)-1,2-dioxohexan-3-yl]octahydrocyclopenta[c]pyrrole-1-carboxamide
-4.618015
3 A-4
N
NH
O
O
NHN
O
O
NH
CH3
O
O
NHCH3 CH3
CH3
Molecular Formula = C37H54N6O6 Formula Weight = 678.86126 (1S,3aR,6aS)-2-[(2S)-2-({(2S)-2-cyclohexyl-2-[(pyridin-2-ylcarbonyl)amino]acetyl}amino)-3,3-dimethylbutanoyl]-N-[(3S)-1-(cyclopropylamino)-1,2-dioxohexan-3-yl]octahydrocyclopenta[c]pyrrole-1-carboxamide
-4.390643
4 A-6
N
N
NH
O
O
NHN
O
O
NH
CH3
O
O
NHCH3 CH3 CH3
CH3
Molecular Formula = C37H55N7O6 Formula Weight = 693.8759 (1S,3aR,6aS)-2-[(2S)-2-{[(2S)-2-cyclohexyl-2-{[(5-methylpyrazin-2-yl)carbonyl]amino}acetyl]amino}-3,3-
Molecular Formula = C37H55N7O6 Formula Weight = 693.8759 (1S,3aR,6aS)-2-[(2S)-2-{[(2S)-2-cyclohexyl-2-{[(5-methylpyrazin-2-yl)carbonyl]amino}acetyl]amino}-4-methylpentanoyl]-N-[(3S)-1-(cyclopropylamino)-1,2-dioxohexan-3-yl]octahydrocyclopenta[c]pyrrole-1-carboxamide
-4.618015
3 A-4
N
NH
O
O
NHN
O
O
NH
CH3
O
O
NHCH3 CH3
CH3
Molecular Formula = C37H54N6O6 Formula Weight = 678.86126 (1S,3aR,6aS)-2-[(2S)-2-({(2S)-2-cyclohexyl-2-[(pyridin-2-ylcarbonyl)amino]acetyl}amino)-3,3-dimethylbutanoyl]-N-[(3S)-1-(cyclopropylamino)-1,2-dioxohexan-3-yl]octahydrocyclopenta[c]pyrrole-1-carboxamide
-4.390643
4 A-6
N
N
NH
O
O
NHN
O
O
NH
CH3
O
O
NHCH3 CH3 CH3
CH3
Molecular Formula = C37H55N7O6 Formula Weight = 693.8759 (1S,3aR,6aS)-2-[(2S)-2-{[(2S)-2-cyclohexyl-2-{[(5-methylpyrazin-2-yl)carbonyl]amino}acetyl]amino}-3,3-
Molecular Formula = C38H50N6O6 Formula Weight = 686.8402 (1S,3aR,6aS)-N-[(3S)-1-(cyclopropylamino)-1,2-dioxohexan-3-yl]-2-[(2S)-3,3-dimethyl-2-({(2S)-3-phenyl-2-[(pyridin-2-ylcarbonyl)amino]propanoyl}amino)butanoyl]octahydrocyclopenta[c]pyrrole-1-carboxamide
-3.505345
6 A-9
N
N
NH
O
O
NHN
O
O
NH
CH3
O
O
NHCH3 CH3
CH3
CH3
Molecular Formula = C38H51N7O6 Formula Weight = 701.85484 (1S,3aR,6aS)-N-[(3S)-1-(cyclopropylamino)-1,2-dioxohexan-3-yl]-2-[(2S)-3,3-dimethyl-2-{[(2S)-2-{[(5-methylpyrazin-2-yl)carbonyl]amino}-3-phenylpropanoyl]amino}butanoyl]octahydrocyclopenta[c]pyrrole-1-carboxamide
Molecular Formula = C38H50N6O6 Formula Weight = 686.8402 (1S,3aR,6aS)-N-[(3S)-1-(cyclopropylamino)-1,2-dioxohexan-3-yl]-2-[(2S)-3,3-dimethyl-2-({(2S)-3-phenyl-2-[(pyridin-2-ylcarbonyl)amino]propanoyl}amino)butanoyl]octahydrocyclopenta[c]pyrrole-1-carboxamide
-3.505345
6 A-9
N
N
NH
O
O
NHN
O
O
NH
CH3
O
O
NHCH3 CH3
CH3
CH3
Molecular Formula = C38H51N7O6 Formula Weight = 701.85484 (1S,3aR,6aS)-N-[(3S)-1-(cyclopropylamino)-1,2-dioxohexan-3-yl]-2-[(2S)-3,3-dimethyl-2-{[(2S)-2-{[(5-methylpyrazin-2-yl)carbonyl]amino}-3-phenylpropanoyl]amino}butanoyl]octahydrocyclopenta[c]pyrrole-1-carboxamide
Molecular Formula = C38H50N6O6 Formula Weight = 686.8402 (1S,3aR,6aS)-N-[(3S)-1-(cyclopropylamino)-1,2-dioxohexan-3-yl]-2-[(2S)-4-methyl-2-({(2S)-3-phenyl-2-[(pyridin-2-ylcarbonyl)amino]propanoyl}amino)pentanoyl]octahydrocyclopenta[c]pyrrole-1-carboxamide
-2.648008
8
B-1
NHO
NH
CH3
N
O
O
NH
NH
ON
N
O
CH3
CH3
CH3
Molecular Formula = C33H45N7O5 Formula Weight = 619.7543 N-(pyrazin-2-ylcarbonyl)-L-phenylalanyl-3-methyl-L-valyl-L-prolyl-N-cyclopropyl-L-norvalinamide
9
B-3
NHO
NH N
O
O
NH
NH
ON
N
O
CH3
CH3
CH3 Molecular Formula = C33H45N7O5 Formula Weight = 619.7543 N-(pyrazin-2-ylcarbonyl)-L-phenylalanyl-L-leucyl-L-prolyl-N-cyclopropyl-L-norvalinamide
Molecular Formula = C38H50N6O6 Formula Weight = 686.8402 (1S,3aR,6aS)-N-[(3S)-1-(cyclopropylamino)-1,2-dioxohexan-3-yl]-2-[(2S)-4-methyl-2-({(2S)-3-phenyl-2-[(pyridin-2-ylcarbonyl)amino]propanoyl}amino)pentanoyl]octahydrocyclopenta[c]pyrrole-1-carboxamide
-2.648008
8
B-1
NHO
NH
CH3
N
O
O
NH
NH
ON
N
O
CH3
CH3
CH3
Molecular Formula = C33H45N7O5 Formula Weight = 619.7543 N-(pyrazin-2-ylcarbonyl)-L-phenylalanyl-3-methyl-L-valyl-L-prolyl-N-cyclopropyl-L-norvalinamide
9
B-3
NHO
NH N
O
O
NH
NH
ON
N
O
CH3
CH3
CH3 Molecular Formula = C33H45N7O5 Formula Weight = 619.7543 N-(pyrazin-2-ylcarbonyl)-L-phenylalanyl-L-leucyl-L-prolyl-N-cyclopropyl-L-norvalinamide
Molecular Formula = C38H50N6O6 Formula Weight = 686.8402 (1S,3aR,6aS)-N-[(3S)-1-(cyclopropylamino)-1,2-dioxohexan-3-yl]-2-[(2S)-4-methyl-2-({(2S)-3-phenyl-2-[(pyridin-2-ylcarbonyl)amino]propanoyl}amino)pentanoyl]octahydrocyclopenta[c]pyrrole-1-carboxamide
-2.648008
8
B-1
NHO
NH
CH3
N
O
O
NH
NH
ON
N
O
CH3
CH3
CH3
Molecular Formula = C33H45N7O5 Formula Weight = 619.7543 N-(pyrazin-2-ylcarbonyl)-L-phenylalanyl-3-methyl-L-valyl-L-prolyl-N-cyclopropyl-L-norvalinamide
9
B-3
NHO
NH N
O
O
NH
NH
ON
N
O
CH3
CH3
CH3 Molecular Formula = C33H45N7O5 Formula Weight = 619.7543 N-(pyrazin-2-ylcarbonyl)-L-phenylalanyl-L-leucyl-L-prolyl-N-cyclopropyl-L-norvalinamide
Molecular Formula = C33H45N7O5 Formula Weight = 619.7543 N-(pyrazin-2-ylcarbonyl)-L-phenylalanyl-3-methyl-L-valyl-L-prolyl-N-cyclopropyl-L-valinamide
-4.229773
11
B-13
N
N
O
NHNH
O
O
N
O
NH
CH3
O
NH
CH3CH3CH3
Molecular Formula = C31H41N7O5 Formula Weight = 591.70114 N-(pyrazin-2-ylcarbonyl)-L-phenylalanyl-3-methyl-L-valyl-L-prolyl-N-cyclopropyl-L-alaninamide
-3.839055
12
B-17
N
N
O
NHNH
O
O
N
O
NH
CH3
O
NH
CH3CH3
Molecular Formula = C31H41N7O5 Formula Weight = 591.70114 N-(pyrazin-2-ylcarbonyl)-L-phenylalanyl-L-leucyl-L-prolyl-N-cyclopropyl-L-alaninamide
-3.749998
13
V-1
N
N
O
NHNH
O
O
N
O
NH O
NH
CH3CH3CH3
CH3 Molecular Formula = C36H49N7O5 Formula Weight = 659.81816 (1S,3aR,6aS)-N-[(2S)-1-(cyclopropylamino)-1-oxopentan-2-yl]-
Molecular Formula = C33H45N7O5 Formula Weight = 619.7543 N-(pyrazin-2-ylcarbonyl)-L-phenylalanyl-3-methyl-L-valyl-L-prolyl-N-cyclopropyl-L-valinamide
-4.229773
11
B-13
N
N
O
NHNH
O
O
N
O
NH
CH3
O
NH
CH3CH3CH3
Molecular Formula = C31H41N7O5 Formula Weight = 591.70114 N-(pyrazin-2-ylcarbonyl)-L-phenylalanyl-3-methyl-L-valyl-L-prolyl-N-cyclopropyl-L-alaninamide
-3.839055
12
B-17
N
N
O
NHNH
O
O
N
O
NH
CH3
O
NH
CH3CH3
Molecular Formula = C31H41N7O5 Formula Weight = 591.70114 N-(pyrazin-2-ylcarbonyl)-L-phenylalanyl-L-leucyl-L-prolyl-N-cyclopropyl-L-alaninamide
-3.749998
13
V-1
N
N
O
NHNH
O
O
N
O
NH O
NH
CH3CH3CH3
CH3 Molecular Formula = C36H49N7O5 Formula Weight = 659.81816 (1S,3aR,6aS)-N-[(2S)-1-(cyclopropylamino)-1-oxopentan-2-yl]-
Molecular Formula = C33H45N7O5 Formula Weight = 619.7543 N-(pyrazin-2-ylcarbonyl)-L-phenylalanyl-3-methyl-L-valyl-L-prolyl-N-cyclopropyl-L-valinamide
-4.229773
11
B-13
N
N
O
NHNH
O
O
N
O
NH
CH3
O
NH
CH3CH3CH3
Molecular Formula = C31H41N7O5 Formula Weight = 591.70114 N-(pyrazin-2-ylcarbonyl)-L-phenylalanyl-3-methyl-L-valyl-L-prolyl-N-cyclopropyl-L-alaninamide
-3.839055
12
B-17
N
N
O
NHNH
O
O
N
O
NH
CH3
O
NH
CH3CH3
Molecular Formula = C31H41N7O5 Formula Weight = 591.70114 N-(pyrazin-2-ylcarbonyl)-L-phenylalanyl-L-leucyl-L-prolyl-N-cyclopropyl-L-alaninamide
-3.749998
13
V-1
N
N
O
NHNH
O
O
N
O
NH O
NH
CH3CH3CH3
CH3 Molecular Formula = C36H49N7O5 Formula Weight = 659.81816 (1S,3aR,6aS)-N-[(2S)-1-(cyclopropylamino)-1-oxopentan-2-yl]-
Molecular Formula = C33H45N7O5 Formula Weight = 619.7543 N-(pyrazin-2-ylcarbonyl)-L-phenylalanyl-3-methyl-L-valyl-L-prolyl-N-cyclopropyl-L-valinamide
-4.229773
11
B-13
N
N
O
NHNH
O
O
N
O
NH
CH3
O
NH
CH3CH3CH3
Molecular Formula = C31H41N7O5 Formula Weight = 591.70114 N-(pyrazin-2-ylcarbonyl)-L-phenylalanyl-3-methyl-L-valyl-L-prolyl-N-cyclopropyl-L-alaninamide
-3.839055
12
B-17
N
N
O
NHNH
O
O
N
O
NH
CH3
O
NH
CH3CH3
Molecular Formula = C31H41N7O5 Formula Weight = 591.70114 N-(pyrazin-2-ylcarbonyl)-L-phenylalanyl-L-leucyl-L-prolyl-N-cyclopropyl-L-alaninamide
-3.749998
13
V-1
N
N
O
NHNH
O
O
N
O
NH O
NH
CH3CH3CH3
CH3 Molecular Formula = C36H49N7O5 Formula Weight = 659.81816 (1S,3aR,6aS)-N-[(2S)-1-(cyclopropylamino)-1-oxopentan-2-yl]-
Molecular Formula = C36H49N7O5 Formula Weight = 659.81816 (1S,3aR,6aS)-N-[(2S)-1-(cyclopropylamino)-1-oxopentan-2-yl]-2-[(2S)-4-methyl-2-({(2S)-3-phenyl-2-[(pyrazin-2-ylcarbonyl)amino]propanoyl}amino)pentanoyl]octahydrocyclopenta[c]pyrrole-1-carboxamide
-5.400139
15
V-13
N
N
O
NHNH
O
O
N
O
NH
CH3
O
NH
CH3CH3CH3
Molecular Formula = C34H45N7O5 Formula Weight = 631.765 (1S,3aR,6aS)-N-[(2S)-1-(cyclopropylamino)-1-oxopropan-2-yl]-2-[(2S)-3,3-dimethyl-2-({(2S)-3-phenyl-2-[(pyrazin-2-ylcarbonyl)amino]propanoyl}amino)butanoyl]octahydrocyclopenta[c]pyrrole-1-carboxamide
-4.143548
16
V-17
N
N
O
NHNH
O
O
N
O
NH
CH3
O
NH
CH3
CH3
Molecular Formula = C34H45N7O5 Formula Weight = 631.765 (1S,3aR,6aS)-N-[(2S)-1-(cyclopropylamino)-1-oxopropan-2-yl]-2-[(2S)-4-methyl-2-({(2S)-3-phenyl-2-[(pyrazin-2-ylcarbonyl)amino]propanoyl}amino)pentanoyl]octahydrocyclopenta[c]pyrrole-1-carboxamide
Molecular Formula = C36H49N7O5 Formula Weight = 659.81816 (1S,3aR,6aS)-N-[(2S)-1-(cyclopropylamino)-1-oxopentan-2-yl]-2-[(2S)-4-methyl-2-({(2S)-3-phenyl-2-[(pyrazin-2-ylcarbonyl)amino]propanoyl}amino)pentanoyl]octahydrocyclopenta[c]pyrrole-1-carboxamide
-5.400139
15
V-13
N
N
O
NHNH
O
O
N
O
NH
CH3
O
NH
CH3CH3CH3
Molecular Formula = C34H45N7O5 Formula Weight = 631.765 (1S,3aR,6aS)-N-[(2S)-1-(cyclopropylamino)-1-oxopropan-2-yl]-2-[(2S)-3,3-dimethyl-2-({(2S)-3-phenyl-2-[(pyrazin-2-ylcarbonyl)amino]propanoyl}amino)butanoyl]octahydrocyclopenta[c]pyrrole-1-carboxamide
-4.143548
16
V-17
N
N
O
NHNH
O
O
N
O
NH
CH3
O
NH
CH3
CH3
Molecular Formula = C34H45N7O5 Formula Weight = 631.765 (1S,3aR,6aS)-N-[(2S)-1-(cyclopropylamino)-1-oxopropan-2-yl]-2-[(2S)-4-methyl-2-({(2S)-3-phenyl-2-[(pyrazin-2-ylcarbonyl)amino]propanoyl}amino)pentanoyl]octahydrocyclopenta[c]pyrrole-1-carboxamide
Molecular Formula = C36H49N7O5 Formula Weight = 659.81816 (1S,3aR,6aS)-N-[(2S)-1-(cyclopropylamino)-1-oxopentan-2-yl]-2-[(2S)-4-methyl-2-({(2S)-3-phenyl-2-[(pyrazin-2-ylcarbonyl)amino]propanoyl}amino)pentanoyl]octahydrocyclopenta[c]pyrrole-1-carboxamide
-5.400139
15
V-13
N
N
O
NHNH
O
O
N
O
NH
CH3
O
NH
CH3CH3CH3
Molecular Formula = C34H45N7O5 Formula Weight = 631.765 (1S,3aR,6aS)-N-[(2S)-1-(cyclopropylamino)-1-oxopropan-2-yl]-2-[(2S)-3,3-dimethyl-2-({(2S)-3-phenyl-2-[(pyrazin-2-ylcarbonyl)amino]propanoyl}amino)butanoyl]octahydrocyclopenta[c]pyrrole-1-carboxamide
-4.143548
16
V-17
N
N
O
NHNH
O
O
N
O
NH
CH3
O
NH
CH3
CH3
Molecular Formula = C34H45N7O5 Formula Weight = 631.765 (1S,3aR,6aS)-N-[(2S)-1-(cyclopropylamino)-1-oxopropan-2-yl]-2-[(2S)-4-methyl-2-({(2S)-3-phenyl-2-[(pyrazin-2-ylcarbonyl)amino]propanoyl}amino)pentanoyl]octahydrocyclopenta[c]pyrrole-1-carboxamide
15. Synthes is o f (1S ,3aR,6aS) -N- [ (2S) -1 -(cyclopropylamino)-1-oxopropan-2-yl]-2-[(2S)-3,3-dimethyl-2-({(2S)-3-phenyl-2-[(pyrazin-2-ylcarbonyl)amino]propanoyl} amino)butanoyl]octahydrocyclopenta[c]pyrrole-1-carboxamide (V-13)
16. Synthes is o f (1S ,3aR,6aS) -N- [ (2S) -1 -(cyclopropylamino)-1-oxopropan-2-yl]-2-[(2S)-4-methyl-2-({(2S)-3-phenyl-2-[(pyrazin-2-ylcarbonyl)amino]propanoyl} amino)pentanoyl]octahydrocyclopenta[c] pyrrole-1-carboxamide(V-17)
Off White powder, yield: 21%,MP by DSC: 100.98; MW.: 631.76; Purity by HPLC : 95.83% ; IR (KBr, frequency in cm-1), 3391 (N-H),3304 (N-H),3064 (C-H),2957 (C-H),2870 (C-H),1657 (C=O),1632 (C=O),1525 (C=O),1453 (C-H),1368 (C-N),1154 (C-N),1020 (C-H); 1H NMR (400 MHz,DMSO-d6, value
The synthesis of all the molecules required 8-10 synthetic steps by condensation of amino acids. The peptide chain was end-capped with cyclic amine at acidic end and with Pyrazine-2-carboxlic acid, pyridine-2-carboxylic acid and 4-methyl-2-pyrazine carboxylic acid at the amine end. Various coupling reagents were used to couple N-CBz (carbobenzyloxy) protected amino acids such as EDC.HCl/HOBt, TBTU or HBTU in combination with bases such as triethyl amine and diisopropyl amine. The yield of coupling reaction varied from 65% to 90% for different steps involving different amino acids. The CBz- protection of N-terminal of amino acids was carried out using benzyl chloroformate and inorganic base such as lithium hydroxide or sodium hydroxide and de-protection of CBz-group to get free amine group was carried with the help of palladium on carbon (Pd/C) in methanol. The yield of de-protection step ranged from 80-95%. The overall yield ranged between 12-24%.
Melting points of the synthesized molecules were determined by differential scanning calorimetry (DSC).
30 INDIAN DRUGS 57 (05) MAY 2020
The synthesized molecules were characterized by mass, IR and NMR spectral studies. The purity of the molecules was determined by high performance liquid chromatography (HPLC). UV/VIS spectrophotometry was used to determine wavelength of maximum absorbance i.e. λmax of molecules. IR showed typical characteristic peaks in the regions with wave numbers 3392-1 (N-H), 3319-1 (N-H), 3030-1 shows NH stretching of molecules while 1657-1 (C=O), 1632-1 (C=O), 1525-1 (C=O), and a typical characteristic peak at 1730-1 (C=O) stretching of alpha keto-amide. 1338-1 and 1153-1 peaks shows typical CO-NH amide carbonyl group stretching.
The chemical shifts at δ 0.63 and 0.38 in 1HNMR spectra are the peaks of –CH2, confirming the presence of cyclopropyl amine. The peaks between δ 9.4 and 8.4 are characteristic chemical shifts of pyrazine or pyridine –CH. The four peaks of 1H proton each between δ 5.0-3.5 are the peaks of four –CH protons of amino acids, beside peptide bonds. The M+1 peak in mass spectra confirms the molecular mass of molecules. The NMR and mass spectra match with the molecules.
To determine the anti HCV activity of the molecules, docking was done using Glide program of Schrödinger. The binding energy is obtained by interaction of molecule with active site of NS3/4A enzyme on Hepatitis C Virus RNA. The docking scores of all the molecules are given in Table I. Docking score suggests the binding energy between the molecules and binding site, more –ve the docking score more is the resemblance between molecule and active site i.e. molecule will fit better into the active site and block the active site, thus inhibiting the replication of RNA and stopping the spread of hepatitis C. In our study, we compared the docking score of molecules with docking score telaprevir (-5.110482), which is also a NS3/4A protease inhibitor. The docking score of B-3 (-6.231025) at serial no. 9 in Table I and V-3 (-5.400139)) at serial no. 14 in Table I are more than that of telaprevir. The docking score of A-3, V-1 and V-17 at serial no. 2, 13 and16, respectively, in Table I are nearby to that of telaprevir. Hence, these molecule can have better chances of inhibiting HCV. To determine the activity of these molecules, further in vitro studies are required.
CONCLUSION
All the synthesized molecules were characterized using Physico-chemical techniques and supported the suggested structures. Activity of the molecules was inferred with the help of docking studies on the crystal structure of HCV RNA on NS3/4A protease enzyme. The docking study predicted that many molecules fit well on
the binding site, thus having properties of inhibiting the propagation of cell division by stopping replication of RNA. On further investigation, there is every possibility that these molecule may turn out to be cheaper and prospective anti HCV drugs.
REFERENCES1. Petruzziello A., Marigliano S., Loquercio G., Cozzolino A.,
and Cacciapuoti C.: Global epidemiology of hepatitis C virus infection: An up-date of the distribution and circulation of hepatitis C virus genotypes, World J. Gastroenterol., 2016, 22(34) 7824-7840.
2. Jefferies M., Rauff B., Rashid H., Lam T. and Rafiq S.: Update on global epidemiology of viral hepatitis and preventive strategies, World J.Clin.Cases., 2018, 6(13) 589-599.
3. WHO Website, Hepatitis Key Facts, https://www.who.int/news-room/fact-sheets/detail/hepatitis-c,viewed 13 Jan 2020.
4. Jennifer L., Silva H., and Vargas H. E.,: New Therapies for Hepatitis C Virus Infection, Gastroenterol. Hepatol., 2017, 13(1) 22-31
5. Xavier F. and Christoph S.: Treatment of chronic hepatitis C, J. Hepatol., 2018, 69(2) 544-546.
6. Usman A. A., Tariq J., Sidra R., Zafar N. and Sheikh R.: An overview of HCV molecular biology replication and immune responses, Virol. J.,2011, 8:161.
7. Dubuisson J. and Cosset F. L.: Virology and cell biology of the hepatitis C virus life cycle: an update, J. Hepatol., 2014, 61 (1) S3–S13.
8. Bukh J.: The history of hepatitis C virus (HCV): Basic research reveals unique features in phylogeny, evolution and the viral life cycle with new perspectives for epidemic control, J. Hepatol., 2016, 65(1) S2–S21.
9. Li D.K. and Chung R.T.: Overview of Direct-Acting Antiviral Drugs and Drug Resistance of Hepatitis C Virus, Methods Mol. Biol., 2019, 1911: 3-32.
10. Gupta V., Kumar A., Sharma P., and Arora A.: Newer direct-acting antivirals for hepatitis C virus infection: Perspectives for India, Indian J. Med. Res.,2017, 146(1): 23–33.
11. Bhatia H. K., Singh H., Grewal N., and Natt N. K.: Sofosbuvir: A novel treatment option for chronic hepatitis C infection, J. Pharmacol. Pharmacother., 2014, 5(4) 278–284.
12. Scola P.M., Sun L.Q., Wang A.X., Chen J., Sin N., Venables B.L., Sit S.Y., Chen Y., Cocuzza A., Bilder D.M., D’Andrea S.V., Zheng B., Hewawasam.P, Tu Y., Friborg J., Falk P., Hernandez D., Levine S., Chen C., Yu F., Sheaffer A.K., Zhai G., Barry D., Knipe J.O., Han Y.H., Schartman R., Donoso M., Mosure K., Sinz M.W., Zvyaga T., Good A.C., Rajamani R., Kish K., Tredup J., Klei H.E., Gao Q., Mueller L., Colonno R.J., Grasela D.M., Adams S.P., Loy J., Levesque P.C., Sun H., Shi H., Sun L., Warner W., Li D., Zhu J., Meanwell N.A. and McPhee F.: The discovery of asunaprevir (BMS-650032), an orally efficacious NS3 protease inhibitor for the treatment of Hepatitis c virus infection, J. Med. Chem., 2014, 57(5) 1730-52.
INDIAN DRUGS 57 (05) MAY 2020 31
13. McCauley J. A., McIntyre C. J., Rudd M. T., Nguyen K. T., Romano J. J.,Butcher J. W., Gilbert K.F., Bush K.J., Holloway M.K., Swestock J., Wan B.L., Carroll S.S., DiMuzio J. M., Graham D.J., Ludmerer S.W., Mao S.S., Stahlhut M.W., Fandozzi C.M., Trainor N., Olsen D.B.,. Vacca J.P., and Liverton N.J.: Discovery of vaniprevir (MK-7009), a Macrocyclic Hepatitis C Virus NS3/4a Protease Inhibitor, J. Med. Chem., 2010, 53(6) 2443-2463.
14. Pan Q., Peppelenbosch M. P., Janssen H. L. A. and Knegt R. J. D.: Telaprevir/Boceprevir era: From bench to bed and back, World J. Gastroenterol., 2012, 18(43) 6183-6188.
15. Rajani A.K., Ravindra B.K. and Dkhar S.A.: Telaprevir: Changing the standard of care of chronic hepatitis, J. Postgrad. Med.,2013, 59 (1) 42-47.
16. Cunningham M. and Foster G. R.: Efficacy and safety of Telaprevir in patients with genotype 1 hepatitis C infection, Ther. Adv. Gastroenter., 2012, 5(2) 139–151.
NOW AVAILABLE ! IDMA-APA GUIDELINES / TECHNICAL MONOGRAPHS
Copies are available at IDMA Office, Mumbai. We do not mail any publications against VPP payment. All payments to be made in advance as Cheque/DD/RTGS/NEFT in favour of
“INDIAN DRUG MANUFACTURERS’ ASSOCIATION” at Mumbai.
For more details please contact: PUBLICATIONS DEPARTMENT Tel.: 022 - 2494 4624 / 2497 4308 Fax: 022 - 2495 0723 E-mail: [email protected], Website: www.idma-assn.org/www.indiandrugsonline.org
TECHNICAL MONOGRAPH NO. 3 TECHNICAL MONOGRAPH NO. 4 INVESTIGATION OF OUT OF SPECIFICATION PHARMACEUTICAL PREFORMULATION (OOS) TEST RESULTS ANALYTICAL STUDIES
PHARMACOGNOSTICAL AND PHYSICO-CHEMICAL SCREENING OF EUPHORBIA TIRUCALLI STEM-BARK
Mali P. Y.a* and Goyal S.a
(Received 30 December 2019) (Accepted 27 January 2020)
ABSTRACT
Euphorbia tirucalli L. is a flowering shrub or tiny tree, indigenous to temperate regions. It is useful in the treatment of whooping cough, asthma, dyspepsia, colic, jaundice and some more ailments. Aim of the present investigation was to study pharmacognostical and physico-chemical screening of E. tirucalli stem-bark. Fresh and dried stem-bark with powder of E. tirucalli was evaluated using macroscopic and microscopic appraisal. Physico-chemical, preliminary phytochemical, fluorescence and quantitative evaluation along with primary HPTLC fingerprinting analysis were performed. Macroscopic appraisal confirmed that E. tirucalli has herbaceous odour with tongue sensitizing bitter taste. Dried stems are greenish brown and surface is longitudinally finely striated. Microscopic appraisal of stem-bark consists of epidermis, cortex and vascular cylinder. Foreign matter was found to be 0.26%. Total ash, acid insoluble ash and water soluble ashes were found to be 3.66%, 0.33% and 3.39%, respectively. Loss on drying was 6.58%. Swelling and foaming index were 6.92 mL and 200 mL, respectively. Quantitative evaluation suggested that the stem-bark has 0.38% and 4.60% alkaloid and saponin contents, respectively. HPTLC fingerprinting of ET6 extract fraction showed Rf values 0.04 (255 nm, 365 nm), 0.20 (400 nm), 0.21 (290 nm), 0.27 (400 nm) and others at various concentrations. Present investigation aids in developing the quality control parameters for standardization of E. tirucalli stem-bark.
Keywords: Euphorbia tirucalli; Physico-chemical; Standardization; Macroscopic; Microscopic; HPTLC
INTRODUCTION
Euphorbia tirucalli L. Sp. Pl. (452.1753) belongs to Euphorbiaceae family. It is a flowering shrub or tiny tree, indigenous to temperate regions. It has pencil like twigs from which it derives its vernacular name, pencil tree1. E. tirucalli is broadly distributed in hotter parts of India and planted as a hedge plant in gardens and along cultivated fields2. Latex of E. tirucalli is vesicant and rubefacient and is used in treating rheumatism, warts, cough, asthma, ear-ache, tooth-ache and neuralgia3,4. Milky juice is alexiteric, carminative and purgative. It is useful in treatment of whooping cough, gonorrhea, asthma, leprosy, dropsy, dyspepsia, enlargement of spleen, colic, jaundice and stone in bladder. The fresh milky juice is a good alternative in syphilis and a good application in neuralgia5. Bark is used in treatment of fractures3. Poultices prepared from the stem are useful to repair the broken bones. Boiled
a B. R. Nahata College of Pharmacy, Mandsaur University, Mandsaur - 458001, Madhya Pradesh, India *For Correspondence E-mail: [email protected]
root liquid acts as an emetic in cases of snake-bite and for infertility in women. The wood is used for rafters, toys and veneering purposes. It is also useful against leprosy and foot paralysis subsequent to childbirth6.
E. tirucalli is reported to have β-sitosterol, euphorbol hexacosonate, 12-deoxy-4β-hydroxyphorbol-13-phenyl acetate-20-acetate, 12, 20-dideoxyphorbol-13-isobutyrate, glut-5-en-3-β-ol, euphol, hentriacontene, 4-deoxyphorbol ester, hentriacontanol, β-sitosterotchouc, cyclotirucanenol, cycloeupordenol, corilagin, casuarin, euphorbins, euphorone, euphorcinol, euphol, gallic acids, ellagic acids, glucosides7,8, terpenic alcohol, isoeuphorol, taraxasterol, tirucallol9, stigmasterol, campesterol, palmitic acid and linoleic acid10 as active phytoconstituents. E. tirucalli possesses activity in human-lymphocytes11 and as analgesic12, anthelmintic13, antiarthritic14, antibacterial / antifungal / antimicrobial13,15-17, anti-HIV17, anti-inflammatory12,18, antioxidant19,20, antiviral21,22 and CNS depressant and to alleviate neuropathic pain18,23.
INDIAN DRUGS 57 (05) MAY 2020 33
It also exhibits cytotoxicity / anticancer9,22,24-26 and immunomodulatory27,28 pharmacological activities.
E. tirucalli is not mentioned in Ayurvedic Phar-macopeia of India and should be incorporated. This plant can also be incorporated into the syllabus of undergraduate and postgraduate botany, Ayurveda and pharmacy courses. There are very few patents on E. tirucalli reported so far29. This investigation updates and validates the pharmacognostical and physico-chemical information for classification and standardization of E. tirucalli stem-bark crude drug.
MATERIAL AND METHODS
Chemicals and instrumentsAnalytical grade chemicals like toluene, ethyl acetate,
n-butanol, formic acid (98-100%), acetone (Merck, Mumbai), chloroform, methanol and petroleum ether (40-60°C) (SD Fine-Chem Ltd, Mumbai) were used. Leica photographic microscope (Leica Microsystems, Mumbai, DM-3000), U. V. chamber (Dolphin, Mumbai) and Afcoset digital balance (Afcoset Balances, Mumbai, E-R-180A) were used for the study.
Authentication of E. tirucalli plant and its stem-barkFresh E. tirucalli plant and its stem-bark were procured
from the region of Jalgaon district, Maharashtra, India in August-September month of 2012 and authenticated
Macroscopic appraisalMacroscopic appraisal of fresh and dried E. tirucalli
stem-bark was performed with reference to the procedure of Brain and Turner30. The colour, odour, taste, condition, fracture, texture, size and shape features were considered for the appraisal.
Microscopic appraisalMicroscopic appraisal of fresh E. tirucalli stem-bark
was assessed based on the procedure mentioned in the Ayurvedic Pharmacopoeia of India31. The phloem and pith with vascular bundle, vascular bundle with xylem, proto and meta xylem, epidermis, cuticle with epidermis and chlorenchyma cells, spongy parenchyma, intercellular spaces in pith region, cortex region, cuticle zone with chlorenchyma and the starch grains were studied. The dried powder of stem-bark was cleaned with NaOH and mounted in glycerin medium after staining. Different staining reagents such as toluidine blue, safranin, fast green and iodine were used to examine stone cells, sclerenchyma fibres, xylem parenchyma, non-articulated laticiferous cells, calcium oxalate crystals, group of fibres, parenchyma cells with prismatic crystals, tracheids and laticiferous cells. The photographs of different magnifications were taken by Leica photographic microscope.
Extraction and fractionation of E. tirucalli stem-bark
Freshly collected E. tirucalli stem-bark was dried in shade. The fine powder (#44) of stem-bark (300g) was extracted successively with petroleum ether, toluene, chloroform (non-polar solvents), ethyl acetate, n-butanol and distilled water (polar solvents) using maceration process of extraction for the period of seven days with each solvent with occasional stirring. The macerated mixtures were filtered two times with muslin cloth separately and thereafter extracts obtained were concentrated, dried and designated as ET1 (petroleum ether), ET2 (toluene), ET3 (chloroform), ET4 (ethyl acetate), ET5 (n-butanol) and ET6 (aqueous). The percentage yields and consistencies of all the extracts were recorded.
Physico-chemical evaluationForeign matter32, total ash, acid insoluble ash, water
soluble ash31, loss on drying33, swelling31 and foaming index32 were evaluated.
Preliminary phytochemical studiesPreliminary phytochemical studies of extracts of E.
tirucalli stem-bark are helpful to recognize the presence
28
Fig. 1: Photograph and herbarium of E. tirucalli Linn. (Source: At Bilkhede Village, Amalner Tahsil,
Jalgaon District, Maharashtra, India; Aug. 2012)
28
Fig. 1: Photograph and herbarium of E. tirucalli Linn. (Source: At Bilkhede Village, Amalner Tahsil,
Jalgaon District, Maharashtra, India; Aug. 2012)
Fig. 1: Photograph and herbarium of E. tirucalli Linn. (Source: At Bilkhede Village, Amalner Tahsil, Jalgaon
District, Maharashtra, India; Aug. 2012)
by taxonomist, Department of Botany, Pratap College, Amalner-425401, Maharashtra, India. Voucher specimen (No. PCA/Bot-P1639) was deposited in the same. Photograph and herbarium of E. tirucalli are shown in Fig. 1.
34 INDIAN DRUGS 57 (05) MAY 2020
of phytoconstituents such as alkaloids, amino acids, carbohydrates, flavonoids, glycosides, proteins, saponin, steroids and tannins and phenols33,34.
Fluorescence examinationDried powder of E. tirucalli stem-bark was assessed for
fluorescence examination as such and also after treating alone with water, 1 N of HCl, HNO3, H2SO4, NaOH, KMnO4, KOH, alcoholic NaOH, KOH and ammonia using normal and U.V. light (254 nm). Colour reaction of petroleum ether, toluene, chloroform, ethyl acetate, n-butanol and distilled water extracts were also observed using normal and U.V. light (254 nm)35,36.
Quantitative evaluation
Alkaloid assessmentAlkaloid assessment was studied with reference to
procedure described by Harbone37.
Saponin assessmentSaponin assessment was studied with reference to
method described by Obdoni and Ochuko38.
Primary HPTLC fingerprinting studiesBased on the percentage yield and presence of
phyto-constitutents in the different extract fractions, ET6 extract fraction was studied using HPTLC technique for normal phase separation of components.
Preparation of solution of ET6 extract fractionAccurately weighed 500 mg of ET6 extract fraction
was dissolved in 5 mL of methanol in a volumetric flask. It was then sonicated for 20 min. The solution was kept aside for 30 min to settle down the aliquots. The aliquots of stock solution of ET6 extract fraction were
29
Fig. 2: Macroscopic photographs of E. tirucalli stem-bark
B A C
E D F
Fig. 2: Macroscopic photographs of E. tirucalli stem-bark
Fig. 3: Microscopic photographs of E. tirucalli stem-bark. A: Pith with vascular bundle; B: Epidermis; C: Vascular bundle with xylem, phloem and pith; D: Cuticle with epidermis and chlorenchyma cells; E: Spongy parenchyma; F: Intercellular spaces in pith region; G: Cortex region; H: Cuticle zone with chlorenchyma; I: Proto xylem; J: Meta xylem; K: Latex release; L: Starch grains
29
Fig. 2: Macroscopic photographs of E. tirucalli stem-bark
B A C
E D F
30
Fig. 3: Microscopic photographs of E. tirucalli stem-bark. A: Pith with vascular bundle; B:
Epidermis; C: Vascular bundle with xylem, phloem and pith; D: Cuticle with epidermis and
region; H: Cuticle zone with chlorenchyma; I: Proto xylem; J: Meta xylem; K: Latex release; L:
Starch grains
H G I
L K J
transferred to 10 mL volumetric flask and volume was adjusted with methanol to get the final concentration of 100 µg/µL of extract fraction. Further, this solution was used for identification of extracted phytocompounds.
Mobile phase used for primary HPTLC studiesToluene:chloroform:ethanol (4:4:2 V/V/V).
INDIAN DRUGS 57 (05) MAY 2020 35
Chromatography and HPTLC fingerprinting evaluation
A chromatographic study was performed on prewashed and preactivated 20.0 x 10.0 cm aluminum Lichrosphere HPTLC plates pre-coated with silica gel 60 F254 of 0.2 mm thickness layer (Merck KGaA, Darmstadt, Germany). Spots of extract fraction ET6 was applied at 2, 4, 6, 8, 10 and 15 µL at application position 8.00 mm with band length 8.00 mm by a CAMAG Linomat-V automatic TLC sample spotter (CAMAG Muttenz, Switzerland) equipped with a 100 µL syringe (Hamilton) under a continuous drying stream of nitrogen gas at a constant application speed of 150 nL/s. The linear ascending development with the above mobile phase was in a 20.0 x 10.0 cm twin trough glass chamber (CAMAG) previously saturated with mobile phase for 15 min at room temperature (25± 2 °C) and relative humidity 40%. The development distance was 80.00 mm (development time 20 min with filter paper) and 20 mL mobile phase was used. After development, the plate was dried with a stream of hot air and densitometric scanning was performed at 254 nm in absorption-reflectance mode and 366 nm in absorption-fluorescence mode by using a CAMAG TLC scanner 3 and CAMAG visualizer with automatic digital camera linked to winCATS software (Version 1.4.6). The slit dimension of scanner was set at 6.00 x 0.45 mm (Micro) with 100 µm / step data resolution and 20 mm/s scanning speed. Later, the plate was dipped in anisaldehyde sulfuric acid derivatising reagent and plate was dried with a stream
31
c b a
f e d
32
Fig. 4: Microscopic photographs of powder of E. tirucalli stem-bark. a: Stone cells; b:
Table II: Preliminary phytochemical analysis of extracts of E. tirucalli stem-bark
Test ET1 ET2 ET3 ET4 ET5 ET6Alkaloids
Dragendorff’s test
Wagner’s test
+
-
-
+
+
+
-
+
+
-
+
+Amino acids
Ninhydrin test
Test for cysteine
-
-
-
-
-
-
-
+
-
+
+
+Carbohydrates
Molisch’s test
Fehling’s test
Iodine test
-
+
-
-
-
+
+
-
-
+
-
-
-
+
-
+
+
-Flavonoids
Test with conc. H2SO4 & Mg (A)
Test with aq. NaOH (B)
Test with conc. H2SO4 (C)
Shinoda test
Pew’s test
-
-
+
-
-
-
-
-
+
-
-
+
-
-
-
+
+
-
+
-
-
-
-
+
+
+
-
+
-
+Glycosides
Legal’s test
Killer –Killani test
+
+
+
-
-
+
-
-
+
-
+
+Proteins
Biuret test
Millon’s test
Precipitation test
-
-
-
-
-
-
-
-
-
-
+
-
+
-
+
+
+
+Saponin
Foam test
Molisch’s test
+
-
+
-
+
-
+
-
+
+
+
+Steroids
Salkowski test
Liebermann-Burchard test
+
+
+
+
+
+
+
-
+
-
+
-Tannins & phenols
Phenol test
Test with 5% of FeCl3 (A)
Test with lead acetate (B)
Test with acetic acid (C)
Test with dilute iodine (D)
Test with dilute KMnO4 (E)
-
+
-
-
-
+
+
-
+
-
+
-
-
-
-
+
+
-
-
-
+
-
+
-
-
+
-
+
-
-
+
-
+
+
-
+
where, ET1- Petroleum ether; ET2-Toluene; ET3-Chloroform; ET4-Ethyl acetate; ET5-n-butanol; ET6-Aqueous; “+” = Presence of constituents; “-” = Absence of constituents
of hot air and scanned at 540 nm in absorption-reflectance mode with the same software system. The volume of sample applied, color of the resolved band, peak number, Rf value, peak height and peak area were noted. The UV-visible spectra were also scanned at 254 nm under D2 lamp with 200-400 nm start and end wavelengths.
RESULTS
Macroscopic appraisal of E. tirucalli stem-bark
Macroscopic appraisal confirmed that E. tirucalli is an herbaceous plant. Fresh stem is green and from it milky exudates oozes out after breaking. It has herbaceous odour with tongue sensitizing bitter taste, rough hard texture and round rod-like structure with slight curvatures. The stems are cylindrical in shape with diameter in 0.9-2.5 cm. Dried stems are greenish brown and surface longitudinally finely striated. Fractures are short and fibrous. Taste is acrid and odour is not characteristic. Macroscopic photographs of E. tirucalli stem-bark are shown in Fig. 2.
Microscopic appraisal of E. tirucalli stem-bark
The primary structure of stem-bark consists of epidermis, cortex and vascular cylinder. The epidermis sinks to cortex where stomata are formed. No much intercellular space exists in cortex and pith. Its structural characters prevent water losing from plant body, which enhance the function of gas exchange between environment, plant and adaptation to the arid environment. Different sizes and shapes of vascular bundles exist in the stem. Different distances exist between vascular bundles. Lots of laticifers are distributed in the primary structure stem cortex. The secondary transverse section structure of stem
INDIAN DRUGS 57 (05) MAY 2020 37
Fig. 5: Extraction and fractionation scheme for E. tirucalli stem-bark
Table IV: Fluorescence analysis of powder of E. tirucalli stem-bark in normal and U.V. light
Powder + Solvent ObservationNormal / Day
LightU.V. Light (254 nm)
Dry powder Brown cream Dark cream
Powder + water Dark brown Reddish brown
Powder + HCl Dark brown Sandal brown
Powder + HNO3 Light brown Grayish brown
Powder + H2SO4 Light brown Dark gray
Powder + NaOH Reddish brown Grayish brown
Powder + KMnO4 Dark brown Grayish brown
Powder + KOH Gray brown Dark brown
Powder + Alc. NaOH Gray brown Grayish brown
Powder + Alc. KOH Gray brown Grayish brown
Powder + Ammonia Reddish dark brown
Dark brownish green
33
Fig. 5: Extraction and fractionation scheme for E. tirucalli stem-bark
34
Fig. 6: Scanned HPTLC developed plates. A: Scanned HPTLC normal white plate; B: Scanned
HPTLC plate at 254nm; C: Scanned HPTLC plate at 366 nm
B A
C
34
Fig. 6: Scanned HPTLC developed plates. A: Scanned HPTLC normal white plate; B: Scanned
HPTLC plate at 254nm; C: Scanned HPTLC plate at 366 nm
B A
C
Fig. 6: Scanned HPTLC developed plates. A: Scanned HPTLC normal white plate; B: Scanned HPTLC plate at 254nm; C: Scanned HPTLC plate at 366 nm
(A) (B)
(C)
38 INDIAN DRUGS 57 (05) MAY 2020
Table V: HPTLC fingerprinting and spectrum scan of ET6 extract fraction at 254 nm
Volume applied Color of band Peak No. Rf value Peak height Peak area Spectrum scan (200-400 nm)2 µL Maroon 1 0.05 62.8 954.6
--2 0.32 16.6 359.4
3 0.45 76.8 1605.9
4 0.55 70.2 1489.8
4 µL Dark pink 1 0.05 91.6 1505.1
--2 0.32 25.4 632.9
3 0.45 123.2 2849.6
4 0.55 118.7 2626.0
5 0.73 14.4 390.9
6 µL Pink 1 0.05 104.8 1897.9
--2 0.32 35.7 835.1
3 0.45 155.3 4007.9
4 0.55 151.1 3528.6
5 0.73 18.9 587.7
8 µL Light pink 1 0.04 105.2 2123.0
--2 0.32 45.9 1161.4
3 0.46 181.1 5239.2
4 0.56 174.0 4239.1
5 0.73 21.3 648.4
10 µL Purple 1 0.04 101.4 2230.1 255
2 0.21 14.0 436.4 290
3 0.32 53.7 1315.0 269
4 0.46 197.7 6828.1 255
5 0.56 190.8 4667.6 255
6 0.73 28.5 866.1 283
15 µL Dark blue 1 0.04 86.2 1972.2 365
2 0.20 19.9 675.4 400
3 0.27 22.9 496.8 400
4 0.32 73.0 1866.2 368
5 0.35 42.9 811.1 384
6 0.46 227.7 7786.0 254
7 0.56 211.0 5418.8 255
8 0.63 12.4 250.0 383
9 0.73 34.9 1110.5 386
consists of periderm, cortex and vascular cylinder. Phellogen and cambium produce the secondary structure together. Phellogen comes from parenchyma cells close to epidermis. The periderm forms at a later time. So the epidermis, especially the cortex of stem can exist for very long time. But laticifers are mostly distributed in the stem cortex. Many typical laticifers can be observed in longitudinal section structures of stem. The innermost layers of the cortex in the stems of mat vascular plants
may contain abundant starch and this layer is known as starch sheath. The cortex is often parenchymatous and consists of thin walled, oval or rounded cells with a large number of intercellular spaces. The innermost layers of cortex are called endodermis, which consists of cells with casparian strip thickenings. Cortex is chlorenchymatous in E. tirucalli, while stems are green and take up the function of photosynthesis in the absence of leaves. Pericycle is a thick cylinder of tissue which separates the cortex. Pith is
INDIAN DRUGS 57 (05) MAY 2020 39
Table VI: HPTLC fingerprinting of ET6 extract fraction at 366 nm and 540 nm
an almost cylindrical body of tissue in the centre of the axis, enclosed by the vascular tissue. It is made up of chiefly paranchymatous cells which are arranged rather loosely with pronounced intercellular spaces. Outer few layers of cortical cells are assimilatory in nature due to presence of chloroplasts marked in phylloclade. Microscopic photographs of E. tirucalli stem-bark are shown in Fig. 3. The microscopic examination of powder of E. tirucalli stem-bark showed presence of stone cells, sclerenchyma fibres, xylem parenchyma, non-articulated laticiferous cells, calcium oxalate crystals, group of fibres, parenchyma cells with prismatic crystals, tracheids, laticiferous cells and more. The microscopic photographs of powder of E. tirucalli stem-bark are shown in Fig. 4.
Extraction and fractionation The scheme for extraction and fractionation of E.
tirucalli stem-bark is shown in Fig. 5 and percentage yields and consistencies of all the extracts are shown in Table I.
Physico-chemical evaluationForeign matter was found to be 0.26%. Total ash,
acid insoluble ash and water-soluble ashes were found to be 3.66%, 0.33% and 3.39%, respectively. Loss on drying was found 6.58%. Swelling and foaming index were found 6.92 mL and 200 mL, respectively.
Preliminary phytochemical studiesThe results of preliminary phytochemical studies
of extracts of E. tirucalli stem-bark are summarized in Table II.
Fluorescence examination of extracts and powder of E. tirucalli stem-bark
The results of fluorescence examination of extracts of E. tirucalli stem-bark are tabulated in Table III and of the powder in Table IV.
Quantitative evaluationThe result of quantitative evaluation of phytoconstitu-
ents indicates that E. tirucalli stem-bark has alkaloid and saponin contents of 0.38% and 4.60%, respectively.
Primary HPTLC fingerprinting studiesHPTLC fingerprinting of ET6 extract fraction showed
the Rf values at 254 nm with their respective UV-visible spectrum wavelengths scanned in between 200-400 nm. They are 0.04 (255 nm, 365 nm), 0.20 (400 nm), 0.21 (290
nm), 0.27 (400 nm), 0.32 (269 nm, 368 nm), 0.35 (384 nm), 0.46 (254 nm, 255 nm), 0.56 (255 nm), 0.63 (383 nm), 0.73 (283 nm, 386 nm) at various concentrations of applied sample. HPTLC plate was also scanned at 366 nm and densitogram showed the different peaks with their respective Rf values being 0.04, 0.05, 0.20, 0.32, 0.33, 0.34, 0.45, 0.46, 0.55, 0.56, 0.63 and 0.71. At 540 nm, the peaks having Rf values of 0.01, 0.02, 0.06, 0.12, 0.17, 0.34, 0.35, 0.37, 0.47, 0.48, 0.55, 0.56, 0.63, 0.64 and 0.73 were found. The scanned HPTLC white normal plate at 254 and 366 nm are shown in Fig. 6 and the volume of sample applied, color of the resolved band, peak number, Rf value, peak height and peak area at 254 nm are shown in Table V, and at 366 nm and 540 nm are shown in Table VI.
DISCUSSION AND CONCLUSION
In spite of ease of understanding of important hyphenated techniques, recognition and assessment of plant drugs using pharmacognostical and physico-chemical consideration is still more reliable, specific and efficient. According to WHO, macroscopic and microscopic appraisal of plant is the first step towards establishing its identity and purity. It must be carried out earlier to any other tests being performed33. Bearing this in mind, the authors have evaluated macroscopic and microscopic studies of fresh and dried stem-bark using their powder of E. tirucalli. Results of macroscopic appraisal will be beneficial for identifying it from its substitutes or adulterants. Microscopic appraisal results of drug will help to classify the ordered cellular structure of drug material by their identified histological appearance such as phloem and pith with vascular bundle, vascular bundle with xylem, proto- and meta-xylem, epidermis, cuticle with epidermis and chlorenchyma cells, spongy parenchyma, intercellular spaces in pith region, cortex region, cuticle zone with chlorenchyma and starch grains. The application of a variety of reagents or stains helps to distinguish cellular organization depending on their chemical make-up. Results of E. tirucalli stem-bark powder suggest that more extractives yields are to be found in aqueous and ethyl acetate solvents followed by toluene, n-butanol, petroleum ether and chloroform.
Physico-chemical evaluation helps in judging the purity and superiority of drug. Foreign matter was present in trace amounts due to the first hand collection of plant material from the non-polluted region. Ash values were used to identify the presence of any siliceous materials and water-soluble salts. These values are essential quantitative standards as it is valuable in shaping genuineness and purity of drugs38. The result of total ash,
INDIAN DRUGS 57 (05) MAY 2020 41
acid insoluble ash and water-soluble ash were found to be 3.66%, 0.33% and 3.39%, respectively. Low level of total ash is indication of little amounts of carbonates, phosphates and silica in the selected stem-bark. There is a chance of presence of non-physiological substances in the crude drugs. Hence, total ash value is not always reliable. Therefore, the authentication of acid insoluble ash was also studied. Loss on drying was found to be 6.58%, which signifies substantial amount of moisture or mucilage in the stem-bark. Percent active chemical constituent is usually expressed on air-dried basis in crude drugs. Moisture content of drug was studied for making the solutions of exact strength. It should be minimized for preventing the decay of crude drug because of microbial infectivity or chemical alteration. Swelling and foaming index was found in the range of 6.92 mL and 200 mL due to presence of mucilage in the stem-bark.
Results of preliminary phytochemical analysis showed the presence of various phytoconstituents in the stem-bark which are known to have different biological potencies in treating diseases. ET6 extract has shown the presence of amino acids, alkaloids, flavonoids, carbohydrates, proteins, glycosides, saponin, tannins, steroids and phenols. Previous research reports suggested that saponins, flavonoids, tannins, alkaloids and phenols have anti-inflammatory activities whereas flavonoids, glycosides, alkaloids and tannins have hypoglycemic effects39,40. The results of fluorescence examination of extracts of E. tirucalli stem-bark showed its characteristic fluorescent colours in tested inorganic and organic chemicals. The fluorescent phenomenon of drug powder determines the purity and quality of plant material. Fluorescence features exhibited by many chemical constituents which indicate fluorescence is visible in day light. U.V light shows fluorescence in various herbal drugs. As per literature reports, alkaloid such as berberine is not visible in day light. It is due to decomposition of drug or the derivative is not fluorescent after treating with different chemical reagents41. The result of quantitative evaluation of phytoconstituents indicates that the E. tirucalli stem-bark has higher percentage of saponin content (4.60%) than alkaloid (0.38%). Hence, quantitative and fluorescence standard of powder provides valuable information to substantiate and authenticate the phytomedicine. Primary HPTLC fingerprinting studies showed that there is a presence of certain phytocompounds with respect to their Rf values and UV-visible spectra’s (200-400 nm) scanned at 254 nm, 366 nm and after derivatization at 540 nm. These phytocompounds may comprise steroidal saponins, terpenes, glycosides, flavonoids, tannins, phenolic acids, alkaloids, etc. with promising pharmacological actions which can be utilized to expand prospective drugs. Based
on the results, there is a recommendation to co-relate these phytocompounds present in the ET6 extract with reference to recognition, quantification and validation of HPTLC protocols by different standard marker entities. This investigation is an additional standardized research and supports previous reports and will be helpful for qualitative and quantitative standardization of herbal formulation containing E. tirucalli, as E. tirucalli stem-bark might be utilized as a potential source of functional therapeutics. Further research is in pipe-line on ET6 extract of stem-bark with reference to recognition, quantification and validation of HPTLC protocols by different standard marker entities. The biological potentials are also under investigation for justification of its ethno-pharmacological uses.
ACKNOWLEDGEMENTS
Authors are grateful to Scientist, Botany, Vindhya Herbal Testing and Research Laboratory, MFP-PARC, Barkhdea Pathani, Bhopal and Anchrom Test Lab Pvt. Ltd., Mumbai for providing necessary facilities to perform this research work.
REFERENCES1. Julius M., Damme P.V.: Euphorbia tirucalli L. (Euphorbiaceae)-
The miracle tree: Current status of available knowledge. Sci. Res. Essays. 2011, 6(23), 4905-4914.
2. Gupta A. K., Tandon N., Sharma M.: Quality standards of Indian medicinal plants. Indian Council of Medical Research, New Delhi, Vol. 2, 2005.
3. Nadkarni K.M., Nadkarni A. K.: Indian materia medica. Popular Prakashan, Bombay, 3rd ed., Vol. I, 2007.
4. Anonymous.: The Wealth of India, A Dictionary of Indian Raw Materials and Industrial Products (Raw Materials). Central Institute of Medicinal and Aromatic Plants (Reprinted), New Delhi, Vol. III (D-E), 2003. pp. 226-228.
5. Kirtikar K. R., Basu B. D.: Indian Medicinal Plants. Lalit Mohan Basu, Allahabad, 2nd ed., Vol. III., 2006, pp. 2201-2204.
6. Duke J. A.: Handbook of Energy Crops. Purdue University Centre for new crops and plant products. 1983.
7. Baslas R. K., Gupta N. C.: Constituents with potential effective agents from the latex of some Euphorbia species. Herba Hung. 1984, 2, 67-69.
8. Khan A. Q., Malik A.: A new macrocyclic diterpene ester from the latex of Euphorbia tirucalli. J Nat Prod. 1990, 53, 728-731.
9. Cataluna P., Taxa S.M.K.: The traditional use of the latex from Euphorbia tirucalli Linnaeus (Euphorbiaceae) in the treatment of cancer in South Brazil. Acta Horticulture. 1999, 501, 289-296.
10. Yoshikazu Y., Ryuzo M., Yasuyuki Y.: Chemical constituents of cultured cells of Euphorbia tirucalli and Euphorbia millii. Plant Cell Reports. 1981, 1, 29-30.
11. Doris S. L.C., Laura Y.G.D., Leidy P.S.Q., Lady J.P., Fernando
42 INDIAN DRUGS 57 (05) MAY 2020
T., Fernando E., et al.: New promising Euphorbiaceae extracts with activity in human lymphocytes from primary cell cultures. Immunopharmacol. Immunotoxicol. 2010, 1-12.
12. Prabha M. N., Ramesh C.K., Kuppasta I.J., Mankani K.L.: Studies on anti-inflammatory and analgesic activities of Euphorbia tirucalli L. latex. Int. J. Chem. Sci. 2008, 6(4), 1781-1787.
13. Asha S.K., Ramesh C.K., Paramesha M., Srikanth A.V.: Evaluation of anthelmintic and antimicrobial activities of Euphorbia tirucalli L. latex. Nat. Prod. 2009, 5(2), 45-49.
14. Sarang B., Anpurna K., Beenish K., Vijay Kumar G., Naresh Kumar S., Krishan Avtar S., Ghulam Nabi Q.: Anti-arthritic activity of a biopolymeric fraction from Euphorbia tirucalli. J. Ethnopharmacol. 2007, 110, 92–98.
15. Rathi S.G., Patel K.R., Bhaskar V.H., Isolation of herbal plants: Antifungal and antibacterial activities. J. Pharm. Sci. Biosci. Res. 2012, 2(1), 25-29.
16. Swapna N.L., Prasad M.: Efficacy of Euphorbia tirucalli (L) towards microbicidal activity against human pathogens. Int. J. Pharm. Bio. Sci. 2011, 2,12-18.
17. Bhuvaneshwar U., Singh K.P., Kumar A.: Ethno-medicinal, phytochemical and antimicrobial studies of Euphorbia tirucalli L. J. Phytol. 2010, 2, 65-77.
18. Rafael C.D., Kathryn Ana B.S.S., Allisson F.B., Rodrigo M., Ana F.P., Flávia C.M., Luiz F.P., João B.C.: Euphol, a tetracyclic triterpene produces antinociceptive effects in inflammatory and neuropathic pain: The involvement of cannabinoid system. Neuropharmacol. 2012, 63, 593-605.
19. Chanda S.V., Baravalia Y.: Screening of some plant extracts against some skin diseases caused by oxidative stress and microorganisms. African J. Biotech. 2010, 9(21), 3210-3217.
20. Jyothi T.M., Shankariah M.M., Prabhu K., Lakshminarasu S., Srinivasa J.M., Ramachandra S.S.: Hepatoprotective and antioxidant activity of Euphorbia tirucalli. Iranian J. Pharmacol. Therap. 2008, 7(1), 25-30.
21. Ramesh C.K., Prabha M.N., Deepak S.A., Madhusudhan K.N.: Screening of antiviral property against tobamoviruses in latex of Euphorbia tirucalli L. Biotech. 2009, 3(1). Available at http://tsijournals.com/btaij/Vol03_01/ABS01.htm.
22. Betancur-Galvis L.A., Morales G.E., Forero J.E., Roldan J.: Cytotoxic and antiviral activities of Colombian medicinal plant extracts of the Euphorbi genus. Memorias do Instituto Oswaldo Cruz. 2002, 97(4), 541–546.
23. Prabha M.N., Ramesh C.K., Kuppast I.J., Mankani K.L.: CNS depressant activity of Euphorbia tirucalli L. latex. Nat. Prod.-An Indian J. 2008, 4(2), 117-119.
24. Xiao-yang W., Li-ping L., Rui-xin Z., Ting-guo K., Ling-jiang T., Hua X., Hong-bing W.: Cytotoxic activities of some selected medicinal plants of the genus Euphorbia. J. Med. Plants Res. 2011, 5(31), 6766-6769.
25. Abul-fetouh A., Aisha I.M., Heba A.M., Fleur F. A., Ibrahim S.: Cytotoxic and apoptotic enhancing activities of the crude juice of an ornamental plant Euphorbia tirucalli on human cancer cell lines. Tanta Med. Sci. J. 2006, 1(10), 78-92.
26. Mainen J.M., Zakaria H.M., Ramadhani S.O.N., Pax J.M., Appolinary K., Modest C.K., Pascal T., Marco R.: Evaluation of ethnomedical claims and brine shrimp toxicity of some plants used in Tanzania as traditional medicines. Afr. J. Trad. Complem. Altern. Med. 2006, 3(3), 48-58.
27. Chanda S., Baravalia Y.: Novel leads from herbal drugs for infectious skin diseases. In: Current Research, Technology and Education Topics in Applied Microbiology and microbial biotechnology. A. Mendez Vilas (Eds). Formatex. 2012.
28. Bethania A. A., Felipe J.N.L., Renato S.A., Mathias W., Elaine M.S., Mirian T.P.L., Olindo A.M., Gustavo E.A.B.: The crude latex of E. tirucalli modulates the cytokine response of leukocytes, especially CD4+ T lymphocytes. Brazilian J. Pharmacog. 2011,21(4), 662-667.
29. Mali P.Y., Panchal S.S.: Euphorbia tirucalli L.: Review on morphology, medicinal uses, phytochemistry and pharmacological activities. Asian Pacific J. Trop. Biomed. 2017, 7(7), 603-613.
30. Brain K.R, Turner T.D.: The practical evaluation of phytopharmaceuticals. Wright-Scientechnica, Bristol, 1975.
31. The Ayurvedic Pharmacopoeia of India, Controller of Publications, Ministry of Health and Family Welfare, Department of Indian Systems of Medicine and Homoeopathy, Government of India. National Institute of Science Communication (CSIR), New Delhi, Part-I, Vol. I, 1st Ed., 2001.
32. World Health Organization. Quality control methods for medicinal plant material. Geneva, 1998.
35. Chase C. R., Pratt R., Fluorescence of powdered vegetable drugs with particular reference to development of a system of identification. J. Pharm. Sci. 1949, 38(6), 324-331.
36. Kokoski C,J, Kokoski R. J., Slama F.J.: Fluorescence of powdered vegetable drugs under ultraviolet radiation. J. Pharm. Sci. 1958, 47(10), 715–717.
37. Harbone J.B.: Phytochemical Methods- A Guide to Modern Techniques of Plant Analysis, Chapman and Hall, London, 3rd ed., 1998.
38. Obdoni B.O., Ochuko P.O.: Phytochemical studies and comparative efficacy of the crude extracts of some homeostatic plants in Edo and Delta states of Nigeria. Global J. Pure Appl. Sci. 2001, 8b, 203-208.
39. Orhan I., Kupeli E., Sener B., Yesilada E.: Appraisal of anti-inflammatory potential of the clubmoss, Lycopodium cuvatum L. J. Ethnopharmacol. 2007, 109, 146-150.
40. Sharma B., Balomajumder C., Roy P.: Hypoglycemic and hypolipidemic effects of flavonoid rich extract from Eugenia jambolana seeds on streptozotocin induced diabetic rats. Food Chem. Toxicol. 2008, 46(7), 2376-2383.
41. Mukherjee P.K.: Quality control of herbal drugs: An approach to evaluation of botanicals. Business Horizons, New Delhi, 2010.
INDIAN DRUGS 57 (05) MAY 2020 43
ASSESSMENT OF PHYTOCONSTITUENTS OF MEDICINAL IMPORTANCE FROM MILLETTIA PEGUENSIS ALI (SYN. MILLETTIA OVALIFOLIA KURZ)
Kaur Arshpreet*a and Sidhu M. C.a
(Received 03 August 2019) (Accepted 23 April 2020)
ABSTRACT
The present study was aimed to investigate the biologically active compounds present in the seed and leaf extracts of Millettia peguensis. Screening was done through preliminary qualitative analysis of phytochemicals and Gas Chromatography-Mass Spectroscopy (GC-MS) method. The study revealed the presence of alkaloids, carbohydrates, flavonoids, glycosides, gum and mucilage, phenolics, quinones, steroids and terpenoids in both aqueous and ethanol extracts of leaves and seeds. The GC-MS analysis of ethanol extracts of seeds and leaves have yielded 29 and 23 phytoconstituents, respectively. These observations were further validated by Fourier transform infrared spectroscopy (FT-IR) of the leaves and seeds powder. FT-IR has provided detailed information of the various functional groups associated with the compounds present in the samples. A single compound was isolated through Column Chromatography and Thin Layer Chromatography whose characterization was done through mass spectroscopy and IR spectroscopy.
Keywords: FT-IR; GC-MS; Phytochemicals; Plant parts; Medicinal value
INTRODUCTION
The medicinal importance of plants is known to the human beings since long times. According to the World Health Organization (WHO), about 80% of the people throughout the World still rely on traditional medicines such as medicinal plants species for healthcare requirements1. Several secondary metabolites that are known to exhibit a wide spectrum of biological activities are reported in plants2. Though the synthetic drugs have their own place in the medical world, in recent years focus has shifted towards the exploration of natural plant products in the pharmacological sector. Genus Millettia, a member of family Fabaceae, has around 150 species occurring throughout the world. Different species of this genus contain many important phytochemicals, including flavonoids3. The leaves of Millettia griffonianus are employed for fumigation after mixing with some other materials. The flowers can be used as a soap-substituent in the soap making industry4. Millettia dura has traditionally been used in Africa for the treatment of diarrhoea, hernia and menstrual problems5. It has been reported that leaf extract of Millettia auriculata possesses antimicrobial activity against Bacillus subtilis, Escherichia coli, Salmonella typhi and Staphylococcus
a Department of Botany, Panjab University, Chandigarh - 160 014, India * For Correspondence E-mail: [email protected]
aureus6. The leaves and stem extracts of Millettia auriculata contain steroids, triterpenes, phenolics and flavonoids7. Millettia conraui has shown insecticidal and molluscicidal activities8. Similarly, Millepurone present in Millettia atropurpurea possesses anti-tumor activity9. Millettia pachycarpa extracts can be employed as a natural pesticide10. Furthermore, the inhibitory action of Millettia pachycarpa towards murine retroviral reverse transcriptase and human DNA polymerases has also been reported11. Millettia ovalifolia has a noteworthy, anti-inflammation activity12. The phytochemical study of the bark of this species has revealed the presence of flavonoid characterized as 3,7-dihydroxy-2-phenyl-4H-chromen-4-one, cinnamic acids characterized as (E)-ethyl 13-(3,4-dimethoxyphenyl) acrylate, (E)-methyl 3-(3,4-dimethoxyphenyl) acrylate and N-Ethylacetamide13. This genus in general and Millettia peguensis in particular has a significant role to play in healthcare practices. Keeping this in view, the present study was undertaken to study the phytoconstituents of M. peguensis.
MATERIALS AND METHODS
Collection of MaterialThe leaves and seeds of M. peguensis were collected
from Panjab University Campus, Sector-14, Chandigarh, India. The identification of the specimens was done by
44 INDIAN DRUGS 57 (05) MAY 2020
using different flora, manuals and by consulting the herbarium, Department of Botany, Panjab University, Chandigarh. Fresh leaves were washed thoroughly 4-5 times with water to get rid of the dust. After being dried properly, a fine powder of leaves was made using an electric grinder. In a similar manner, the seeds were also powdered. Both these powders were then packed into properly labeled airtight bags.
Preparation of Aqueous Extract20 g each of seed and leaf powder was mixed well
in 100 mL of distilled water, taken in two separate conical flasks, and kept on a rotary shaker for about 24 hours. The extracts were then filtered through muslin cloth and then through Whatman filter paper. The final extract obtained was filled in air-tight vials and stored in the refrigerator till future use.
Preparation of Ethanol ExtractThe seed and leaf extracts in ethanol were prepared
using Soxhlet apparatus. Powdered material (10 g) was placed in the Soxhlet Extractor and extraction was done using ethanol (150 mL) as the solvent. When the color of the solvent in siphon tube of the extractor became colorless, the completion of the extraction was indicated. The extracts obtained by this method were then evaporated by incubating them overnight and then stored in vials and kept in the refrigerator till further use.
Phytochemical analysisA standard procedure was applied for screening
of different phytochemicals in aqueous and ethanol seed and leaf extracts14-17. Various phytochemicals like alkaloids, amino acids, betaxanthin, carbohydrates,
17
⁕ Pubchem (https://pubchem.ncbi.nlm.nih.gov/)
# Dr. Duke’s Phytochemical and Ethnobotanical Database (https://phytochem.nal.usda.gov/)
Fig. 1 (a-b): a- The branches of M. peguensis bearing inflorescence; b- flowers borne in
a raceme inflorescence
Fig. 2 (a-b): a-Compound leaves bearing leaflets of M. peguensis; b- Seeds of M.
peguensis
a
b
b
a Fig. 1 (a-b): a-The branches of M. peguensis bearing inflorescence;
b-flowers borne in a raceme inflorescence
Fig. 2 (a-b): a-Compound leaves bearing leaflets of M. peguensis; b- Seeds of M. peguensis17
⁕ Pubchem (https://pubchem.ncbi.nlm.nih.gov/)
# Dr. Duke’s Phytochemical and Ethnobotanical Database (https://phytochem.nal.usda.gov/)
Fig. 1 (a-b): a- The branches of M. peguensis bearing inflorescence; b- flowers borne in
a raceme inflorescence
Fig. 2 (a-b): a-Compound leaves bearing leaflets of M. peguensis; b- Seeds of M.
peguensis
a
b
b
a
coumarins, flavonoids, glycosides, gum and mucilage, oxalate, phenolics, phlobatannins, quinones, steroids, tannins and terpenoids were screened.
Fourier transform-Infrared (FT-IR) Spectroscopy
FT-IR was carried out to understand the various functional groups associated with the metabolites present in the seeds and leaves samples18.The test was performed using Perkin Elmer Spectrum 400 FT-IR/ FT-FIR spectrometer in the range 400-4000 cm-1 at Central Instrumentation Laboratory (CIL), Panjab University, Chandigarh.
Gas Chromatography-Mass Spectroscopy (GC-MS)
It was performed to identify the specific compounds present in the ethanol extract using a Thermo Trace 1300GC coupled with Thermo TSQ 8000 Triple Quadruple MS (for GC-Thermo Trace 1300 GC; for MS-Thermo TSQ 8000) at Central Instrumentation Laboratory (CIL), Panjab University, Chandigarh. The chromatogram indicated the presence of compounds with separate retention times.
Thin Layer Chromatography (TLC)The ethanol extract (seeds) was
concentrated and dried using a rotary evaporator. Silica gel of pore size 60-120 mesh and dichloromethane were used in
INDIAN DRUGS 57 (05) MAY 2020 45
order to prepare the slurry. After this, column was packed with silica gel activated at 110ºC for 1 hour for column chromatography. The slurry was allowed to flow down the vertical column. The extraction was performed by eluting the column with petroleum ether/ethyl acetate at different concentrations starting from 2 percent. Various fractions obtained at different concentrations of the solvent were first concentrated and then monitored through Thin Layer Chromatography. The solvent system used was 20% petroleum ether/80% ethyl acetate (V/V). Each TLC plate consisted of three spots: crude seed extract spot, co-spot (seed extract + product), spot for the fraction to be analyzed. TLC plates were observed in the UV chamber for results.
RESULTS AND DISCUSSION
Morphological StudyM. peguensis is a deciduous tree. The trunk usually
bears a greyish-brown bark. The plant blooms in the months of March –April in city belts of Chandigarh (UT). The raceme inflorescence has blush colored flowers (Fig 1 a-b). The leaves are imparipinnately compound with an uneven number of leaflets and a terminal leaflet at the top. The individual leaflet is long, elliptical, acute to acuminate (Fig2 a). The glabrous fruits (pods) on an average are 6-7 cm long and 1-2 cm broad. Each pod carries spherical and smooth 5-7 seeds (Fig 2 b).
Phytochemical CharacterizationDifferent phytochemicals reported during the present
study are listed in Table I. The ethanol extract of seeds contain more phytoconstituents as compared to ethanol extract of leaves. Betaxanthin, coumarins, flavonoids, glycosides, phenolics, quinones and terpenoids were present in all (four) extracts, but in variable amounts, whereas amino acids were absent in both extracts (ethanol and aqueous extracts of leaves and seeds). Oxalates in traces were present only in the ethanol extract of leaves. Similarly, phlobatannins were present only in the leaf extract. Carbohydrates were present in good quantity in the seed extract but were absent or present traces in aqueous and ethanol extracts of leaves, respectively. Alkaloids, gum and mucilage were present in different extracts except ethanol extract of seeds. Similarly, steroids were exclusively present in seed extracts. In addition to this, phenolics and quinones were found in the aqueous seeds extract. Based on the results obtained, it can be inferred that the extraction of phytochemicals from the plant samples depends on the solvent used. A good amount of carbohydrates has been reported in the seed oil of M. griffonianus4. According to the available literature, not
Table I: Phytochemical Details of Millettia peguensis
The symbols -, +, ++, +++ represent absent, present, moderately present and presence in abundance respectively.
much work has been done concerning the phytochemical account of M. peguensis.
FT-IR SpectroscopyFT-IR spectra of the leaves and seeds powder were
studied separately (Fig 3, 4). Various functional groups were reported at different absorption peaks in the spectra. The FT-IR spectra at wave number 3264.27 cm-1 pertains to O-H stretch in alcohols. The peak at 2916.01 cm-1 can be assigned to C-H (stretch) in alkanes. The peaks at 2848.97 and 1732.22 cm-1 are due to C-H aldehyde and C=O aldehyde, respectively. The peak at 1600.89 cm-1 can indicate C=C (stretch) whereas the peak at 1241.89 cm-1 indicates C-O stretching vibrations. On comparing the FT-IR spectra of leaves and seeds, one peak was found to be dissimilar at 1377.33 cm-1, which can be assigned to C-H (bending). The functional groups presented by the FT-IR spectroscopy of the leaf and the seed were associated with the phytochemicals reported during the preliminary analysis. It has further corroborated the number and nature of the screened phytochemicals.
GC-MS AnalysisGas Chromatography - Mass Spectroscopy (GC-MS)
analysis revealed the presence of 23 and 29 compounds
46 INDIAN DRUGS 57 (05) MAY 2020
18
Fig 3. FT-IR spectrum of M. peguensis Leaf
Fig 4. FT-IR spectum of M. peguensis Seed
Fig 5. GC-MS Chromatogram of M. peguensis leaf
Fig. 3: FT-IR spectrum of M. peguensis Leaf Fig. 4: FT-IR spectum of M. peguensis Seed
18
Fig 3. FT-IR spectrum of M. peguensis Leaf
Fig 4. FT-IR spectum of M. peguensis Seed
Fig 5. GC-MS Chromatogram of M. peguensis leaf
Table II: GC-MS analysis of ethanol extract of leaves
Single spot obtained at 10% Petroleum Ether/ Ethyl acetate (White coloured compound isolated)
Crude Seed extract
Co-spot (Product + Crude)
19
Fig 6. GC-MS Chromatogram of M. peguensis seed
Fig 7. TLC observed under Short UV light
Single spot obtained at 10% Petroleum Ether/ Ethyl acetate (White coloured compound isolated)
Crude Seed extract
Co-spot (Product + Crude)
Fig 5: GC-MS Chromatogram of M. peguensis leaf
Fig 6: GC-MS Chromatogram of M. peguensis seed
in the ethanol extracts of leaf and seed, respectively (Tables II and III).The height of peaks in chromatogram is suggestive of its relative concentrations in the sample. The maximum peak height in leaf and seed extracts was at RT value 5.47. It has covered 94.19% and 90.67% peak area in the leaf and seed extract, respectively (Fig 5, 6). Bicyclo (2.2.1) heptan-2-one, 1, 7, 7-trimethyl-, (1S); camphor; (+)-2-bornanone were the three compounds recorded at this retention time. The activity of all these compounds was recorded from Dr. Duke’s Phytochemical and Ethnobotanical Databases and PubChem as shown in Table IV19-20. Many compounds with anti-inflammatory, anti-cancer, antidote, free radical scavenging and antitumor activity have been reported. Camphor can be utilized as an antidote. 3-Deoxy-d-mannitol has a significant role as Central Nervous System depressant, decalcifier and as an anticancer agent against duodenal cancer. (1, 3)-Dioxolane-4, 5-dicarboxylic acid, diethyl ester, besides, is an RNA stimulant and also effective against rabies and rectum cancer.
L-Proline,1-[O-(1-oxohexyl)-N-[N-[N6-(1-oxohexyl)-N2-[N-(1-oxohexyl)-L-valyl]-L-lysyl]-L-valyl]-L-tyrosy l]-, methyl ester is useful in liver, lung, breast and prostate cancer. Earlier, GC-MS analysis of petroleum ether extract of leaves of M. peguensis yielded 10 bioactive compounds (3 major and 7 minor) on the basis of their peak percent area. Pentadecane, tetradecane and octadecane were among the major compounds whereas eicosane, undecane, 5-methyl-, 9-methyl-hepatdecane,
Table IV: GC-MS analysis of M. peguensis (leaves and seeds)
Name of Compound Source Importance[1,3]Dioxolane-4,5-dicarboxylic acid, diethyl ester
Benzofuran, octahydro-6-methyl-3-methylene- Leaves and Seeds Catechol-O-methyl-transferase-Inhibitor#
* Pubchem (https://pubchem.ncbi.nlm.nih.gov/)
# Dr. Duke’s Phytochemical and Ethnobotanical Database (https://phytochem.nal.usda.gov/)
sulfurous acid, dodecyl hexyl ester, heptadecane, 2, 6, 10, 15-tetramethyl-, 2 bromododecane and heneicosane were the minor compounds21. Another study on the ethanol bark extract of M. ovalifolia revealed the presence of
INDIAN DRUGS 57 (05) MAY 2020 49
Fig 7: TLC observed under Short UV light
Fig 8: IR Spectrum of isolated compound
20
Fig 8. IR Spectrum of isolated compound
Fig 9. Mass spectrum of isolated compound
Fig 9: Mass spectrum of isolated compound
21
7-(4-methoxyphenyl)-9H-furo (2, 3-f) chromen-9-one, 3, 7-dihydroxy-2-phenyl-4H-chromen-4-one, (E)-ethyl13 (3,4-dimethoxyphenyl) acrylate, (E)-methyl-3-(3, 4-dimethoxyphenyl) acrylate and N-ethylacetamide. The flavonoid 7-(4-methoxyphenyl)-9H-furo (2, 3-f) chromen-9-one has significant medicinal importance in the treatment of cystic fibrosis, epilepsy and leukemia3. The compound undecane has also been reported in the present study.
Thus, isolation of the compounds and testing of their biological activity may provide some new, best or alternative raw material for the preparation of cost-effective, highly efficient medicines to take care of the human healthcare issues and enhance the pharmaceutical potential of M. peguensis.
Identification of CompoundDifferent fractions were monitored under UV chamber
and all have shown multiple bands except the fraction eluted with 10% petroleum ether/ethyl acetate in which a single band was observed (Fig 7). This indicated a single pure compound in the form of a white precipitate. This compound was further characterized through IR spectroscopy and mass spectroscopy. The IR spectrum has shown different functional groups (Fig 8). The band at 3006.49 cm-1 indicates C-H (stretching) in alkanes. The bands at 2989.49, 2924.96 and 2855.22 cm-1 represent CH2 and CH3 groups. The bands at 2351.83, 2318.63 and 2302.00 cm-1 show the presence of CγC bonds. CH2 bending is denoted by 1461.22 cm-1, while C-O stretching is indicated by a band at 1276.00 cm-1. The presence of an aldehyde group is shown by the band at 1737.26 cm-1. Mass spectrum of the compound has shown different fragmentation peaks (Fig 9). Further, to elucidate the complete structure of the compound, Nuclear Magnetic Resonance (NMR) spectroscopy analysis is required.
CONCLUSIONS
The ethanol extract of seeds and leaves of M. peguensis contain more phytoconstituents as compared to the aqueous extract. It can be inferred that the extraction of phytochemicals also depends upon the solvent used. The FT-IR spectroscopy of the leaf and the seed powder has supported the findings of preliminary phytochemical analysis by providing the detail of the associated functional groups. Further, the GC-MS analysis has revealed the presence of 29 and 23 compounds in the seed and the leaf extract, respectively. Some of the compounds are of medicinal importance for example for anti-cancer, antitumor, antidote and anti-inflammatory activities. A single white colored compound was isolated from the seed extract through chromatography. The isolation and testing of biological activity of this compound may be a milestone and offer interesting pharmacological results. The characterization of this compound is still in progress.
ACKNOWLEDGEMENTS
Authors are grateful to the Chairperson, Department of Botany, Panjab University, Chandigarh for providing the necessary facilities during the present investigation.
Conflict of Interest Statement We declare that we have no conflict of interest.
Fig 7. TLC observed under Short UV light
Single spot obtained at 10% Petroleum Ether/ Ethyl acetate (White coloured compound isolated)
Crude Seed extract
Co-spot (Product + Crude)
50 INDIAN DRUGS 57 (05) MAY 2020
REFERENCES1. Peter H., Canter H.T. and Edzard E.: Bringing medicinal
plants into cultivation: opportunities and challenges for biotechnology, Trends Biotechnol., 2005, 23 180-185.
2. Kabila B., Sidhu M.C. and Ahluwalia A.S.: Phytochemical Profiling of Different Cassia Species A: Review, Int. J. Pharm. Biol. Archiv., 2017, 8 12-20.
3. Rahman T.U., Khattak K.F., Liaqat W., Zaman K. and Mussharaf S.G.: Characterization of one Novel Flavone and four New Source Compounds from the Bark of Millettia ovalifolia and In Vitro Inhibition of Carbonic Anhydrase-II by the Novel Flavonoid, Records Nat. Prod., 2015, 9 553-560.
4. Adewuyi A., Oderinde R.A., Rao B.V.S.K., Prasad R.B.N. and Nalla M.: Proximate analysis of the seeds and chemical composition of the oils of Albizia saman, Millettia griffonianus and Tamarindus indica from Nigeria, Ann. Food Sci. Technol., 2011, 12 123-129.
5. Banzouzi J.T., Prost A., Rajemiarimiraho M. and Ongoka P.: Traditional uses of the African Millettia species (Fabaceae), Int. J. Bot., 2008, 4 406-420.
6. Ganorkar R. P., Satpute S.V. and Deshmukh R.S.: Antimicrobial Efficiency of Leaves Extracts of Millettia auriculata, Plant Int. J. Pharm. Pharm. Res., 2016, 6 195-200.
7. Das S. and Ganapaty S.: In vitro anthelmintic activity of Millettia auriculata leaves and stems, Asian Pac. J. Trop. Dis., 2014, 4 870-873.
8. Fuendjiep V.: Conrauinones C and D, two isoflavones from stem bark of Millettia conraui, Phytochem., 1998, 47 113-115.
9. Ito C.: Anti-tumor-promoting effects of isoflavonoids on Epstein–Barr virus activation and two-stage mouse skin carcinogenesis, Cancer Lett., 2000, 152 187-192.
10. Ningonbum A., Ahluwalia A., Srivastava C. and Walia S.: Antifeedent activity and phytochemical investigation of Millettia pachycarpa extracts against Tobacco leaf eating caterpillar, Spodoptera litura (Fabricus) (Lepidoptera: Noctiudae), J. Asia-Pac. Entomol., 2017, 20 381-385.
11. Ono K., Nakane H., Meng Z.M., Ose Y., Sakai Y. and Mizuno M.: Differential inhibitory effects of various herb
extracts on the activities of reverse transcriptase and various deoxyribonucleic acid (DNA) polymerases, Chem. Pharm. Bull., 1989, 37 1810-1812.
12. Rehman T.U., Zeb M.A., Liaqat W. and Xiao W.L.: Molecular Docking of 3, 7-Dihydroxy-2-phenyl-4Hchromen-4-one as a LOX Inhibitory Compound, PSM Biol. Res., 2018, 3 120-124.
13. Rahman T.U., Zeb M.A., Hussain S. and Liaqat W.: Physiochemical extraction, spectroanalytical identification, antibacterial and docking studies of four new source phytochemicals from the bark of Millettia ovalifolia, Int. J. Biosci., 2017, 11 222-231.
14. Kokate C.K., Practical Pharmacognosy, 4th (Ed.), VallabhPrakashan, New Delhi, India 1994, pp.115-117.
15. Harborne J.B.,Textbook of Phytochemical Methods: A guide to modern techniques of plant analysis, 5th (Ed.), Chapman & Hall Ltd., London 1998, pp.21-72.
16. Jyoti S. and Rajeshwari S.: Evaluation of phytochemical constituents in conventional and non-conventional species of Curcuma, Int. Res. J. Pharm., 2012, 3 203-204.
17. Sidhu M.C. and Thakur S.: Phytochemical and Elemental Exploration of Nothoscordum gracile (Aiton) Stearn for Its Medicinal Potential, J. Chem. Pharm. Sci., 2016, 9 2627-2631.
18. Puri S., Sidhu M.C., Tewari R. and Sharma A.: Study of Phytochemicals, Trace Elements and Antibacterial Activity of Silybum marianum (L.) Gaertn., J. Plant Sci. Res., 2015, 2 122.
19. Dr. Duke’s Phytochemical and Ethnobotanical Databases, U.S. Department of Agriculture, Agricultural Research Service. Available online: https://phytochem.nal.usda.gov/
20. PubChem, U.S. National Library of Medicine, National Centre for Biotechnological Information. Available online: https://pubchem.ncbi.nlm.nih.gov/
21. Manikandan G., Vimala Rani A., Divya C. and Ramasubbu, R.: GC -MS analysis of phytochemical constituents in the petroleum ether leaf extracts of Millettia peguensis, Int. Res. J. Pharm., 2017, 8 144-150.
Have you renewed your subscription for
INDIAN DRUGSIf not, please do so: Kindly Contact IDMA Secretariat at:
A NOVEL VALIDATED LC-MS/MS ANALYTICAL METHOD FOR THE ESTIMATION OF MIDODRINE HYDROCHLORIDE IN PHARMACEUTICAL FORMULATION
Narenderan S. T.a, Meyyanathan S. N.a*, Babu B.a and Karthik Y.a
(Received 18 March 2017) (Accepted 01 April 2019)
ABSTRACT
A novel simple, precise, accurate and validated, liquid chromatography-tandem mass spectroscopy method has been developed for the quantitative determination of midodrine hydrochloride from a commercially available formulation. Caffeine was used as the internal standard. Isocratic separation was achieved using Jones C18 column (4.6mm x 150mm, 3µm) as the stationary phase. The mobile phase consists of 10mM ammonium formate (pH 4.0):methanol (30:70 V/V) with a flow rate of 0.5 mL/min. Detection was carried out in positive ionization with a mass transitions of 255→237.1 and 195→138.1 to monitor midodrine hydrochloride and caffeine. The method was found to be linear over the concentration range of 0.30 – 110 ng/mL with a regression analysis of 0.9991. The percentage recovery of the present method was found to be 99.5 ± 1.20. The LC-MS/MS method was validated as per ICH guidelines.
Midodr ine (MD) , 2 -amino-N - [ (E ) -2 - (2 ,5-dimethoxyphenyl) ethenyl] acetamide, is a hypotensive agent. It is a prodrug (Fig. 1), forming an active metabolite, desglymidodrine, which is an α1-receptor agonist. It exerts its actions via activation of the alpha-adrenergic receptors of the arteriolar and venous vasculature, producing an increase in vascular tone and elevation of blood pressure1,2.
a Department of Pharmaceutical Analysis, JSS College of Pharmacy, (JSS Academy of Higher Education & Research, Mysuru), Udhagamandalam, Tamil Nadu - 643 001, India
Literature survey reveals that midodrine hydrochloride has been estimated by HPLC3-5, spectrometric6, HPTLC7 and tandem mass spectroscopy in plasma of ascetic CHECK? patients8. There is no reported method for the estimation of midodrine (MD) in bulk drug by LC-MS/MS method and hence, a validated analytical method for the estimation of MD in bulk drug and in commercial formulation9 as per ICH guidelines10 was undertaken.
MATERIALS AND METHODS
Chemicals and reagentsWorking standards of midodrine hydrochloride
was provided as a gift sample by PAR Formulations, Chennai. Internal standard caffeine was purchased from Sigma Aldrich; methanol of LC-MS/MS grade from Sigma Aldrich, ammonium formate and formic acid from Qualigen Fine Chemicals, ammonium acetate from Rankem Fine Chemical Limited and water of LC-MS/MS grade from Milli-Q RO system (Millipore, Bedford, USA) were used.
Equipment and chromatographic conditionsLC system coupled with tandem quadrupole mass
spectrometry (Shimadzu 8030, Tokyo, Japan) equipped with electrospray ionization (ESI) interface, LC-20AD pump, SPD-M20 PDA detector, CTO-20AC column oven, CBM-20 Alite controller and SIL-20AC autosampler.
2
recovery of the present method was found to be 99.5 ± 1.20. The LC-MS/MS method was validated as
Midodrine (MD), 2-amino-N-[(E)-2-(2,5-dimethoxyphenyl)ethenyl]acetamide, is a hypotensive agent.
It is a prodrug (Fig. 1), forming an active metabolite, desglymidodrine, which is an α1-receptor agonist.
It exerts its actions via activation of the alpha-adrenergic receptors of the arteriolar and venous
vasculature, producing an increase in vascular tone and elevation of blood pressure1,2.
Fig. 1: Structure of Midodrine
Literature survey reveals that midodrine hydrochloride has been estimated by HPLC3-5,
spectrometric6, HPTLC7 and tandem mass spectroscopy in plasma of ascetic CHECK? patients8.
There is no reported method for the estimation of midodrine (MD) in bulk drug by LC-MS/MS method
and hence, a validated analytical method for the estimation of MD in bulk drug and in commercial
formulation9 as per ICH guidelines10 was undertaken.
MATERIALS AND METHODS
Fig. 1: Structure of Midodrine
52 INDIAN DRUGS 57 (05) MAY 2020
The data were recorded using Lab Solution data station software. Isocratic separation was achieved using Jones C18 column (4.6 x 150 mm, 3µm) using 10mM ammonium formate (pH adjusted to 4.0 using formic acid): methanol (30:70 V/V) as a mobile phase with 0.5 mL/min flow and an injection volume of 10 µL was employed.
Selection of mass rangeA sample containing 1000 ng/mL of midodrine
hydrochloride and caffeine was directly infused into the mass spectrometer and the operation conditions were optimized. Transitions 255→237.1 and 195→138.1 m/z were used to monitor MD and caffeine (IS) (Fig. 2). Fig. 2: Mass spectra of (A) midodrine hydrochloride, (B) caffeine (IS) and corresponding product ion (A1 & B1) in positive mode (LC-MS/MS)
Preparation of working standard solution Midodrine hydrochloride solution was prepared by
dissolving 10 mg in 10 mL of water; this solution was refrigerated at 2-8°C. Working solutions were obtained by diluting the stock solution with diluent methanol: water (50:50, V/V).
Fig. 2: Mass spectra of (A) midodrine hydrochloride, (B) caffeine (IS) and corresponding product ion (A1 & B1) in positive mode (LC-MS/MS)
Preparation of working caffeine solution (IS) Caffeine solution internal standard was prepared by
dissolving 10 mg of caffeine in 10 mL of water and this solution was refrigerated at 2-8°C. Working solutions were obtained by diluting the stock solution with diluent methanol: water (50:50, V/V).
Preparation of sample solution The weight equivalent to 0.13 g of midodrine
hydrochloride was weighed and transferred into a 100 mL volumetric flask, the content dissolved with water and the volume made up with water to obtain a concentration of 25 µg/mL of MD. The above solution was further diluted with the diluent methanol: water (50:50, V/V) to obtain concentrations of 0.5, 33 and 110 ng/mL (LQC, MQC and HQC).
Method ValidationThe method was validated for specificity, linearity,
accuracy, precision, range, quantitation limit, detection limit, robustness and system suitability as per the ICH guidelines11.
Fig no clear
INDIAN DRUGS 57 (05) MAY 2020 53
8
where Y is the peak area ratio of the analyte to the IS (Caffeine) and X is the concentration of
an analyte in ng/mL (Fig. 4).
Fig. 4: Calibration curve of midodrine hydrochloride
Accuracy
The accuracy of the method was carried for three quality control (LQC, MQC and HQC)
samples by standard addition method, and the accuracy was found to be 99 ± 0.12 % (Table I). The
developed method was applied for the estimation of MD in the commercial formulation (Table II).
Fig. 4: Calibration curve of midodrine hydrochloride
7
RESULTS AND DISCUSSION
Specificity
The specificity test results demonstrate that the used excipients did not interfere with the peak
of the main compound. No peaks were eluted along with the retention time of MD (Fig. 3). Hence, the
results showed that the developed method was selective for determination of MD in the formulation.
Fig. 3: MRM standard chromatogram of midodrine hydrochloride and caffeine (IS)
The linearity of the method was evaluated by six determinations at twelve concentration levels
with a range of 0.3 – 110 ng/mL for MD. The calibration curve was found to be linear with a mean
regression of equation
Y=0.0389 X + 0.0124, r2 0.999, SD= 1.28
Fig. 3: MRM standard chromatogram of midodrine hydrochloride and caffeine (IS)
SpecificitySpecificity is the ability of the method to measure
the analyte response in the presence of other drugs, excipients and their potential impurities.
LinearityLinearity was evaluated using the average of six
determinations at twelve concentration levels covering the range of 0.30 – 110 ng/mL for MD. The linearity of the proposed method was evaluated by using calibration curve to calculate the coefficient correlation, slope and intercept values.
ACCURACY
The accuracy of the method was determined by recovery studies by standard addition method according to ICH guidelines. The pre-analyzed samples were spiked with standard drug MD.
PRECISION
The precision of the method was evaluated by inter-day and intra-day precision studies. Samples of three concentration levels at six replicates were prepared as low (LQC), medium (MQC) and high (MQC) quality controls, corresponding to 0.50, 33.0 and 110 ng/mL, respectively. The relative standard deviation (%RSD) of the regressed concentration was used to report precision.
Limit of detection and limit of quantification
The limit of detection was determined by the signal-to-noise ratio of 3:1. The limit of quantification was also determined by the signal-to-noise ratio, where the drug can be quantified with minimum peak area in the ratio of 10:1.
Robustness
The robustness of the method was studied by changing the experimental conditions (operator, the source of reagents and column of similar type) and the optimized conditions (pH, mobile phase ratio and flow rate).
System suitability
System suitability test is an integral part of method development. Retention time (RT), number of theoretical plates (N) and Tailing factor (T) were evaluated for three replicates injections of the sample solution.
54 INDIAN DRUGS 57 (05) MAY 2020
Table I: Recovery studies for MD
QC Levels Conc. of Drug Added
Mean Conc. of MD Recovered (ng/mL) in Mobile Phase
Relative Recovery (%)
% RSD
Level I 0.5 0.48 ± 0.02 99.65 1.37
Level II 33 31.9 ± 0.19 99.63 0.13
Level III 110 107.6 ± 0.11 99.98 0.26
Table II: Recovery studies for formulation
Sr. No Formulation Label claim Amount taken for the assay (ng/mL)
SpecificityThe specificity test results demonstrate that the used
excipients did not interfere with the peak of the main
compound. No peaks were eluted along with the retention time of MD (Fig. 3). Hence, the results showed that the developed method was selective for determination of MD in the formulation.
LinearityThe linearity of the method was evaluated by six
determinations at twelve concentration levels with a range of 0.3 – 110 ng/mL for MD. The calibration curve was found to be linear with a mean regression of equation
Y=0.0389 X + 0.0124, r2 0.999, SD= 1.28
where Y is the peak area ratio of the analyte to the IS (Caffeine) and X is the concentration of an analyte in ng/mL (Fig. 4).
AccuracyThe accuracy of the method was carried for three
quality control (LQC, MQC and HQC) samples by
INDIAN DRUGS 57 (05) MAY 2020 55
standard addition method, and the accuracy was found to be 99 ± 0.12% (Table I). The developed method was applied for the estimation of MD in the commercial formulation (Table II).
Precision
The precision of the method was calculated by intra-day and inter-day precision studies at three different concentrations. It found to be within the limits (Table III).
Limit of detection and limit of quantification
The limit of detection for this method and the lowest limit detected for the drug MD, were found to be 0.1 ng/mL, based on the signal-to-noise ratio 3:1. Due to the increase in the sensitivity of the method, quantification was done at 0.5 ng/mL and 0.30 ng/mL for midodrine. This method was found to have a high percentage of recovery at low concentration at the acceptable limit (Table IV).
System suitability
The results of system suitability were within acceptable limits (Table IV), which is an integral part of the developed analytical method. A system suitability test can be defined as a test to ensure that the method can generate results of acceptable, accuracy and precision. The requirement for system suitability is usually designed after method development. Besides, the method has been validated as per ICH guidelines.
Robustness
No significant changes in the chromatographic parameters were observed when the experimental conditions were changed, proving that the developed method was robust.
CONCLUSION
A novel simple, precise and accurate liquid chromatography-tandem mass spectroscopy method has been developed and validated The developed method was found be successfully applied for the estimation of MD in commercial formulation and in bulk drug.
ACKNOWLEDGEMENTS
The authors thank the Tamil Nadu Pharmaceutical Sciences Welfare Trust for providing financial assistance to carry out this research work and PAR formulation Chennai for providing the standard midodrine hydrochloride as a gift sample.
REFERENCES1. Gilden. J.L., Midodrine, Adrenergic Agonist and Antagonist,
in: Primer on the Autonomic nervous system, 3rd (Ed.), Elsevier 2012, 621-625.
2. Midodrine, Available at https://www.drugbank.ca/drugs/DB00211 and accessed on Nov 2016.
3. Yoshida H., Ohno Y., Yoshikuni K., Tododroki K., Nohta H., Yamaguchi M.: Determination of Midodrine in human plasma by High performance liquid chromatography with fluorescence detection, Anal. Sci., 2003, 19 317-19.
4. Barth T., Aleu J., Pupo M.T., Bonato P.S., Collado I.G.: HPLC analysis of Midodrine and Desglymidodrine in culture medium: Evaluation of static and shaken condition on the biotransformation by fungi, J. Chromatogr. Sci. 2013, 51 460-67.
5. Sattar O., Rezk M., Bandawy A., Khattab O.: Selective determination of Midodrine hydrochloride in the presence of its acid degradation product, Anal. Chem. 2013, 12(5) 182-187.
6. Jain H.K., Gujar K.N., Radhe V.A.: First order derivative spectrometric method development and validation for Midodrine hydrochloride in bulk and tablets. World J. Pharm. Sci. 2016, 5(7) 1760-67.
7. Damle M.C. and Salunke S.R.: Stability indicating HPTLC for the determination of Midodrine hydrochloride. Eur. J. Pharm. Med. Res. 2016, 3(9) 202-207.
8. Ali A.A., Al-Ghobashy M.A., Farid S.F., Kassem M.A.: Development and validation of LC-MS/MS method for the determination of the prodrug Midodrine and its active metabolite Desglymidodrine in plasma if ascetic patients: Application to individualized therapy and comparative pharmacokinetics. J. Chromatogr. B. 2015, 991 34-40.
9. GUTRON, Available at http://www.medsafe.govt.nz/profs/Datasheet/g/Gutrontab.pdf and accessed on Dec 2016.
10. Available at ICH, Q3B validation of analytical procedures: methodology, International Conference on Harmonization, November 1996, accessed on Dec 2016.
56 INDIAN DRUGS 57 (05) MAY 2020
STABILITY INDICATING UPLC METHOD FOR ESTIMATION OF METFORMIN HYDROCHLORIDE AND NATEGLINIDE SIMULTANEOUSLY
A novel Ultra Performance Liquid Chromatography (UPLC) method was developed and validated for the simultaneous determination of antidiabetic drugs metformin hydrochloride and nateglinide. The method was developed using a Waters ACQUITY UPLC SB C18 (100 × 2.1 mm, 1.8 µm) column. The mobile phase consisting of 0.01 % potassium dihydrogen phosphate buffer (pH 5.8): acetonitrile (50: 50 V/V) was used throughout the analysis. The flow rate was 0.3 mL/min, the injection volume was 1.0 µL, column temperature was 30 0C, run time 3 min and detection was carried at 238 nm using a TUV detector. The retention times of metformin hydrochloride and nateglinide were found to be 1.28 and 1.71 min, respectively. Metformin hydrochloride and nateglinide were found to be linear over the concentration range of 125-750 and 15-90 µg/mL. The limit of detection and the limit of quantification for metformin hydrochloride were found to be 0.22 and 0.68 µg/mL, respectively, and, for nateglinide, 0.02 and 0.6 µg/mL, respectively. Developed method was validated as per ICH guidelines. The specificity of the method was confirmed by forced degradation study. The suggested method is suitable for determination of metformin hydrochloride and nateglinide in bulk and pharmaceutical dosage forms.
Many oral antidiabetic drugs with different mechanisms of action have been developed to lower blood sugar and delay the occurrence of serious complications in patients with type II diabetes. For glycemic control in such cases, monotherapy with an oral antidiabetic agent is not adequate to achieve satisfactory blood glucose control. Thus, combination regimens which include drugs with different and complementary mechanisms of action are recommended. The combinational therapy for type II diabetes is frequently prescribed when mono therapy fails. The combination of metformin hydrochloride and nateglinide is approved by FDA for treatment of type II diabetes.
Metformin hydrochloride (Fig. 1) is chemically known as 3-(diaminomethylidene)-1,1-dimethylguanidine. It lowers blood glucose level in type II diabetes patients by suppressing hepatic glucose output and enhances peripheral glucose uptake and insulin sensitivity1-4.
a University College of Pharmaceutical Sceinces Acharya Nagjuna University, Nagarjuna Nagar Guntur, Andhra Pradesh - 522 510, India*For Correspondence E-mail: [email protected]
Nateglinide (Fig. 2) is chemically known as (2R)-2-({hydroxy[(1r,4r)-4-(propan-2-yl)cyclohexyl]methylidene}amino)-3-phenylpropanoic acid. It is a
3
quantification of nateglinide and metformin hydrochloride in combined dosage form14,15. UPLC
method has the advantage of rapid separation with increased sensitivity and resolution.
The aim of the present work was to develop and validate16 a new simple, rapid, selective,
cost effective and stability indicating RP-UPLC method for simultaneous determination of
metformin hydrochloride and nateglinide in pure and tablet dosage form.
Fig. 1: Metformin hydrochloride
Fig. 2: Nateglinide
MATERIALS AND METHODS
Instrumentation
The Waters ACQUITY UPLC system equipped with TUV detector and auto sampler
integrated with empower software was used to perform development and validation of the
3
quantification of nateglinide and metformin hydrochloride in combined dosage form14,15. UPLC
method has the advantage of rapid separation with increased sensitivity and resolution.
The aim of the present work was to develop and validate16 a new simple, rapid, selective,
cost effective and stability indicating RP-UPLC method for simultaneous determination of
metformin hydrochloride and nateglinide in pure and tablet dosage form.
Fig. 1: Metformin hydrochloride
Fig. 2: Nateglinide
MATERIALS AND METHODS
Instrumentation
The Waters ACQUITY UPLC system equipped with TUV detector and auto sampler
integrated with empower software was used to perform development and validation of the
Fig. 1: Metformin hydrochloride
Fig. 2: Nateglinide
INDIAN DRUGS 57 (05) MAY 2020 57
derivative of the amino acid D-phenylalanine, which acts directly on the pancreas β-cells to stimulate insulin secretion5,6.
Literature survey revealed several analytical methods being reported for the estimation of metformin hydrochloride and nateglinide in single and in combination with other antidiabetic agents7-13. There are very few analytical methods reported that permit the simultaneous quantification of nateglinide and metformin hydrochloride in combined dosage form14,15. UPLC method has the advantage of rapid separation with increased sensitivity and resolution.
The aim of the present work was to develop and validate16 a new simple, rapid, selective, cost effective and stability indicating RP-UPLC method for simultaneous determination of metformin hydrochloride and nateglinide in pure and tablet dosage form.
MATERIALS AND METHODS
InstrumentationThe Waters ACQUITY UPLC system equipped with
TUV detector and auto sampler integrated with empower software was used to perform development and validation
of the method. An ACQUITY HSS SB C18, 100 x 2.1 mm column with particle size of 1.8 µm was used as the stationary phase for chromatographic separation and determination of metformin hydrochloride and nateglinide. Sartorius analytical balance was used for all weighing, Metsar pH meter was used for the pH measurement of buffer solution, Labman sonicator was used to dissolve the standard and samples.
Reagents and materialsMetformin hydrochloride was obtained from
Aurobindo Pharmaceuticals Ltd. (Hyderabad, India). The pharmaceutical grade pure sample of nateglinide was procured from Hetero Drugs Ltd. Dosage form Glinate MF tabletswere purchased from a local pharmacy. HPLC-grade acetonitrile, methanol, ortho-phosphoric acid (OPA) and potassium dihydrogen phosphate were purchased from Merck Ltd, Mumbai, India and used in the study.
Preparation of solutions
Preparation of standard stock solutions Accurately weighed 50 mg of metformin hydrochloride
and 6 mg of nateglinide were transferred to 10 mL of volumetric flasks separately. 8 mL of diluent was added to both of these flasks and sonicated for 10 min. Flasks were
Table XI: Assay in marketed tablets by the proposed method
Drug Label claim Quantity found % Assay Metformin hydrochloride 500 mg 497.7 mg 99.54
Nateglinide 60 mg 59.99 mg 99.99 *Average of six determinations
Fig. 3: Representative chromatogram of standard preparation
Fig. 4: Representative chromatogram of sample preparation
Fig. 3: Representative chromatogram of standard preparation
Fig. 4: Representative chromatogram of sample preparation
made up with diluents and labeled as standard stock solution 1and 2 (5000 µg/mL of metformin hydrochloride and 600 µg/mL of nateglinide).
Preparation of standard working solutions (100% solution)
1 mL of each stock solution was pipetted out and taken into a 10 mL volumetric flask and made up with diluents (500 µg/mL of metformin hydrochloride and 60 µg/mL of nateglinide).
Preparation of marketing sample stock solution
Ten tablets were weighed and the average weight of each tablet was calculated. The weight equivalent to one tablet was transferred into a 10 mL volumetric flask, 8 mL of diluent was added and sonicated for 25 min, further the volume was made up with diluent and filtered by UPLC filter
58 INDIAN DRUGS 57 (05) MAY 2020
(5000 µg/mL of metformin hydrochloride and 600 µg/mL of nateglinide). From the filtered solution, 1 mL was pipetted out into a 10 mL volumetric flask and made up to 10 mL with diluents (500 µg/mL of metformin hydrochloride and 60 µg/mL of nateglinide).
1.36 g of potassium dihydrogen phosphate was taken into a 1000 mL of volumetric flask and about 100 mL of Milli-Q water added and dissolved. Final volume was made up to 1000 mL with Milli-Q water pH adjusted to 5.8 using 0.1% OPA.
Diluent preparationBased upon the solubility of the drugs, diluent was
selected, acetonitrile and water were taken in the ratio of 50:50.
ChromatographyChromatographic analysis was performed on
Waters ACQUITY UPLC SB C18 (100 × 2.1 mm, 1.8 µm) column. The mobile phase, consisting of 0.01% potassium dihydrogen phosphate buffer (pH 5.8): acetonitrile (50:50 V/V) was used throughout the analysis. The flow rate was 0.3 mL/min, the injection volume was 1.0 µL, column temperature was 30ºC, run time was 3 min and detection was carried at 238 nm using a TUV detector.
Calibration curve of metformin hydrochlorideAliquots of working standard solution (500 µg/mL) of
metformin hydrochloride (0.25, 0.5, 0.75, 1, 1.25 and 1.5 mL) were transferred into a series of 10 mL volumetric flasks and the volume was adjusted to the mark with diluents to get concentrations 125, 250, 375, 500, 625 and 750 µg/mL. Solutions were injected into the system with optimized chromatographic conditions. The graph of area of peak obtained versus respective concentration was plotted.
Calibration curve of nateglinideAliquots of working standard solution (60 µg/mL) of
nateglinide (0.25, 0.5, 0.75, 1, 1.25 and 1.5 mL) was transferred into a series of 10 mL volumetric flasks and volume was adjusted to the mark with diluent to get concentrations 15, 30, 45, 60, 75 and 90 µg/mL. Solutions were injected into the system with optimized chromatographic conditions. The graph of area of peak obtained versus respective concentration was plotted.
METHOD VALIDATION
System suitability parametersThe system suitability parameters were determined
by preparing standard solutions of metformin hydrochloride (500 µg/mL) and nateglinide (60 µg/mL) and the solutions were injected six times and parameters like peak tailing, resolution and USP plate count were determined.
LinearityLinearity was demonstrated from 25% to 150% of
standard concentration using minimum six calibration levels (25%, 50%, 75%, 100%, 125% and 150%) for both the title drugs. The responses against concentration were checked for all levels. By plotting a graph of peak area versus concentration (on X-axis concentration and on Y-axis Peak area), the correlation coefficient was determined.
PrecisionThe precision of an analytical method gives information
on the random error. The precision of the method was performed as repeatability precision, method precision and intermediate precision.
Repeatability precisionTo study the repeatability precision, six replicates
mixed standard solutions of metformin hydrochloride and nateglinide were injected.
Method precisionThe method precision study was carried out on six
preparations from the same tablet samples of metformin hydrochloride and nateglinide and percent amount of both were calculated.
Intermediate precisionThe intermediate precision study was carried out on
different days (interday and intraday) from the same tablet of metformin hydrochloride and nateglinide.
AccuracyThe accuracy of an analytical method expresses the
nearness between the reference value and found value. To find out the accuracy, a known amount of standard drug was added to the fixed amount of pre-analyzed sample solution at three different concentration levels (i.e. 50%, 100%, and 150%) in triplicate. Percent recovery of the drug was calculated by comparing the area before and after the addition of the standard drug.
INDIAN DRUGS 57 (05) MAY 2020 59
Robustness Robustness of the method was determined to ensure
its capacity to remain unaffected by small deliberate variation in the method parameters such as a mobile phase ratio, temperature of the column and flow rate of the mobile phase.
LOD & LOQIncreasingly dilute solution of each drug was injected
into the chromatograph and signal to noise (S/N) ratio was calculated at each concentration. The limit of detection (LOD) & limit of quantitation (LOQ) were calculated on the basis of signal to noise ratio of 3:1 and 10:1, respectively. The LOD is the smallest concentration of the analyte that gives a measurable response. The LOQ is the smallest concentration of the analyte which gives a response that can be accurately quantified.
Forced Degradation StudyA thorough verification of method selectivity was
carried out by forcing degradation studies, also known as stress testing. They are performed to determine possible degradation products, and confirm the ability of the developed method to detect and separate impurities, which can possibly arise during the lifetime of an API or drug product. Stress tests are conducted in conditions exceeding those used in accelerated stability testing. Stress studies were performed under conditions of dry heat (thermal studies), hydrolysis (in the presence of acidic, alkaline and neutral media), oxidation, and photolysis. A minimum of four samples was generated for every stress condition, the blank solution stored under normal conditions. 100% sample solution was subjected to stress treatment. Hydrolytic decomposition of metformin hydrochloride and nateglinide was conducted at 30ºC for 24 h in 2 N HCl, water and 2 N NaOH. For oxidative stress studies, sample was dissolved in 20% H2O2 and kept for one day at room temperature. For photolytic study, drug solution was exposed to UV light for one day.
RESULTS
System suitability parametersThe retention time of metformin hydrochloride
and nateglinide were found to be 1.28 and 1.71 min, respectively. The peaks obtained for metformin hydrochloride and nateglinide were sharp and had clear baseline separation. It was observed from the results that the system suitability parameters met the requirement of method validation. System suitability results are summarized in Table I.
LINEARITY
The response of the drug was found to be linear over the concentration range of 125-750 µg/mL for metformin hydrochloride and 15-90 µg/mL for nateglinide. The method of linear regression was used for data evaluation. The correlation coefficient values of metformin hydrochloride and nateglinide are 0.999 and 0.999 (Table II, Figs. 5 and 6).
Table II: Linearity data for metformin hydrochloride and nateglinide
% level (Approx)
Metformin hydrochloride Nateglinide
Conc (μg/mL)
Peak area
Conc (μg/mL) Peak area
25 125 256844 15 87099
50 250 497927 30 166939
75 375 794011 45 253992
100 500 1027206 60 335317
125 625 1301683 75 410218
150 750 1516489 90 507771
Slope 2048 5566.5Intercept 2388.4 1127.1
R2 0.9993 0.9994
Table I: System suitability parameters for metformin hydrochloride and nateglinide
PrecisionThe percent relative standard deviation (% RSD) was
calculated and it was found to be 1.2 and 0.7 for metformin hydrochloride and nateglinide, respectively, which were well within the acceptable criteria, i.e. % RSD should not more than 2.0. Results of repeatability precision studies are shown in Table III.
Table III: System precision data for metformin hydrochloride and nateglinide
Injection Peak area metformin hydrochloride
Peak area nateglinide
1 1018129 352822
2 1025218 352621
3 1024177 357678
4 1021200 350463
5 1016850 352042
6 1049714 350689
Mean 1025881 352719SD 12123.7 2618.4
% RSD 1.2 0.7
The % RSD of the assay result of six preparations in method precision study was found to be 0.75 and 0.8 for metformin hydrochloride and nateglinide, respectively, which were well within the acceptable criteria of not
more than 2.0. The results obtained are presented in Table IV.
Table IV: Method precision data for metformin hydrochloride and nateglinide
The % RSD of the six preparations for the intra day intermediate precision study was 0.7 and 0.8, inter day intermediate precision were 0.8 and 0.8 for metformin hydrochloride and nateglinide, respectively, which were well within the acceptable criteria of not more than 2.0. The results of intermediate precision study are reported in Table V.
Table V: Results of intermediate precision of metformin hydrochloride and nateglinide
AccuracyPercent recovery of the drugs was calculated
by comparing the area before and after the addition of the standard drug. Percent recovery of metformin hydrochloride ranged from 98.21% to 100.70% and, for nateglinide, 99.52% to 101.24%, showing better accuracy
17
Fig. 5: Linearity graph of metformin hydrochloride
Fig. 6: Linearity graph of nateglinide
Fig. 7: Acid degradation chromatogram of metformin hydrochloride and nateglinide
CONCLUSION 17
Fig. 5: Linearity graph of metformin hydrochloride
Fig. 6: Linearity graph of nateglinide
Fig. 7: Acid degradation chromatogram of metformin hydrochloride and nateglinide
CONCLUSION
Fig. 5: Linearity graph of metformin hydrochloride
Fig. 6: Linearity graph of nateglinide
INDIAN DRUGS 57 (05) MAY 2020 61
Table VI: Accuracy data for metformin hydrochloride
% Level Amount spiked (μg/mL)
Added STD drug (μg/mL)
Peak area
Total amount found (μg/mL)
Amount recovered (μg/mL)
% recovery
50 %
250 500 1540618 751.0889 251.08887 100.44
250 500 1539603 750.5933 250.59326 100.24
250 500 1536762 749.2061 249.20605 99.68
100 %
500 500 2052139 1000.855 500.85498 100.17
500 500 2056026 1002.753 502.75293 100.55
500 500 2040337 995.0923 495.09229 99.02
150 %
750 500 2566349 1251.934 751.93408 100.26
750 500 2573108 1255.234 755.23438 100.70
750 500 2534862 1236.56 736.55957 98.21
Mean % recovery
99.92
SD 0.82% RSD 0.82
Table VII: Accuracy data for nateglinide
% Level Amount spiked (μg/mL)
Added STD drug (μg/mL)
Peak area
Total amount found (μg/mL)
Amount recovered (μg/mL)
% recovery
50 % 30 60 502049 90.19925 30.199245 100.66
30 60 500136 89.85555 29.855552 99.52
30 60 500642 89.94646 29.946461 99.82
100 % 60 60 670479 120.4598 60.459756 100.77
60 60 671676 120.6748 60.674811 101.12
60 60 669059 120.2046 60.204635 100.34
150 % 90 60 838140 150.5821 90.582106 100.65
90 60 841112 151.1161 91.116062 101.24
90 60 836173 150.2287 90.22871 100.25
Mean % recovery
100.49
SD 0.57
% RSD 0.56
of the method. The results obtained are presented in Table VI and VII.
RobustnessNo significant effect was observed on system suitability
parameters such as theoretical plates, purity angle and purity threshold, when small but deliberate changes were made for chromatography conditions such as change in flow rate, organic content and temperature. The results are summarized in Table VIII.
LOD and LOQ The lower limit of detection for metformin hydrochloride
and nateglinide was found to be 0.22 and 0.02 µg/mL, respectively. The lowest limit of quantitation for metformin hydrochloride and nateglinide was found to be 0.68 and 0.06 µg/mL respectively (Table IX).
Forced degradation studyThe peak purity angle is smaller than that of peak
threshold angle, indicating that there was no interface
62 INDIAN DRUGS 57 (05) MAY 2020
with the analyte peak from degradation products. Major degradation occurred for metformin hydrochloride and nateglinide under acid hydrolysis condition up to 4.90% and 4.86 % respectively (Fig. 7). The degradation products
Table VIII: Robustness data for metformin hydrochloride and nateglinide
Condition Metformin hydrochloride NateglinideRT (min) Peak area % RSD RT (min) Peak area % RSD
Mobile phase composition 45B:55A 1.28 1058467 0.3 1.66 348194 0.5
Mobile phase composition 55B:45A 1.29 950060 0.8 1.78 305986 0.8
Temperature at 25 oC 1.28 1041995 0.6 1.66 339973 0.6
Temperature at 35 oC 1028 1029536 0.5 1.71 340803 0.4
B-Buffer A- Acetonitrile
Table IX: LOD and LOQ of metformin hydrochloride and nateglinide
Drug name LOD LOQMetformin hydrochloride 0.22 µg/mL 0.68 µg/mL
Nateglinide 0.02 µg/mL 0.6 µg/mL
17
Fig. 5: Linearity graph of metformin hydrochloride
Fig. 6: Linearity graph of nateglinide
Fig. 7: Acid degradation chromatogram of metformin hydrochloride and nateglinide
CONCLUSION
Table X: Degradation data of metformin and nateglinide
Type of degradation
Metformin hydrochloride Nateglinide Peak area % Recovered % Degraded Peak area % Recovered % Degraded
Acid 976580 95.10 4.90 335927 95.14 4.86
Base 999551 97.34 2.66 342572 97.03 2.97
Peroxide 1008203 98.18 1.82 346112 98.03 1.97
Thermal 1021730 99.50 0.50 350462 99.26 0.74
UV 1022187 99.54 0.46 350948 99.40 0.60
Water 1017022 99.54 0.46 350984 99.41 0.59
Fig. 7: Acid degradation chromatogram of metformin hydrochloride and nateglinide
obtained were well resolved from the pure drugs with significantly different Rf values. The results obtained are presented in Table X.
Assay of Marketed Formulation The proposed method was applied to the determination
of metformin hydrochloride and nateglinide in their combined tablet dosage form. The results of the assays (n = 6) undertaken yielded 99.54% and 99.99% of label claim for metformin hydrochloride and nateglinide,
INDIAN DRUGS 57 (05) MAY 2020 63
respectively. The results of this assay (Table XI) indicate that the developed method is selective for the analysis of both metformin hydrochloride and nateglinide without interference from the excipients.
Table XI: Assay in marketed tablets by the proposed method
Drug Label claim
Quantity found
% Assay
Metformin hydrochloride
500 mg 497.7 mg 99.54
Nateglinide 60 mg 59.99 mg 99.99
*Average of six determinations
DISCUSSION
The development of RP-UPLC methods for the determination of drugs has received more attention recently because of their speed, resolution, sensitivity, less solvent consumption, cost effective and more productivity, which is important in the quality control of drugs and drug products. This work was intended to develop a rapid, precise and reliable method in reverse-phase UPLC separation combined with TUV detection for simultaneous estimation of metformin hydrochloride and nateglinide.
In this work we developed a UPLC method for the estimation of metformin hydrochloride and nateglinide in pure and tablet dosage form. The basic chromatographic condition was designed to be simple and easy to use and reproduce and were selected after testing the different condition that affect UPLC analysis, for example column, aqueous and organic components of mobile phase, proportion of mobile phase components, detection wavelength, diluents and concentration of analyte. A Waters ACQUITY UPLC SB C18 (100 × 2.1 mm, 1.8 µm) column was used because of its advantages of high resolving capacity, better reproducibility and low tailing. Better peak shape was obtained when the mobile phase consisted of 0.01 % potassium dihydrogen phosphate buffer (pH 5.8): acetonitrile (50: 50 V/V).
After development of the analytical method, it was validated in accordance with ICH guidelines. System suitability data were found to be satisfactory. The typical retention times of metformin hydrochloride and nateglinide are 1.28 and 1.71 min, respectively with a total chromatographic run time of 3 min. The proposed method was also evaluated by studying the precision as % RSD. The % RSD was found to be less than 2 for all the drugs, which indicates that the method is precise. The results of accuracy study specify that the method
enables highly accurate simultaneous determination of both drugs.
The results of robustness were found to be within the acceptable limits, which indicate that the method is highly robust. LOD and LOQ values indicate that the sensitivity of the method is adequate. The method has proven specificity as the peaks of degraded products are well separated from the peaks of metformin hydrochloride and nateglinide. Both the drugs were found to be most susceptible to degradation under acidic condition more than any stress conditions.
CONCLUSION
This paper reports for the first time a novel method for the simultaneous estimation of metformin hydrochloride and nateglinide in pure and dosage forms by RP-UPLC. The analytical method was validated according to the ICH guidelines and the results revealed that the method is selective, precise and accurate. Developed method is rapid and direct, and can be used for the routine analysis in the finished dosage form.
The results of forced degradation studies reveal that it was a stability indicating method. The proposed RP-UPLC method was economical and rapid and can be used for routine analysis in quality control laboratories.
REFERENCES1. Ministry of Health and Family Welfare., Indian Pharma-
copoeia, The Indian Pharmacopoeia Commission, Ghaziabad, India., 2014, 2, pp. 2186-2187.
2. Sean C. Sweetman, Martindale., The Complete Drug Reference, Pharmaceutical Press, London, England, U.K., 2011, A, pp. 491-492.
3. S. Budawari., The Merck Index, Merck & Co. Inc, Whitehouse Station, NJ, 2006, pp. 1025.
4. U.S. Pharmacopeial convention., The United States of Pharmacopeia, United Book Press, Inc., Batimore, MD. 2016, 3, pp. 4764-4765.
5. Sean C. Sweetman, Martindale., The Complete Drug Reference, Pharmaceutical Press, London, England, U.K., 2011, A, pp. 493.
6. U.S. Pharmacopeial convention., The United States of Pharmacopeia, United Book Press, Inc., Batimore, MD., 2016, 3, pp. 5004-5007.
7. Sakhare RS, Pekamwar SS and Mohkare DP.: Development and Validation of Stability Indicating HPTLC method for the determination of Metformin Hydrochloride and Benfotiamine in Bulk and Combined Dosage Form, Indian J. Pharm. Educ. Res, 2017, 51(2S) S8-S16.
8. K. Neelima and Y. Rajendra Prasad.: Analytical method development and validation of metformin, voglibose, glimepiride in bulk and combined tablet dosage form
64 INDIAN DRUGS 57 (05) MAY 2020
by gradient RP-HPLC, Pharm Methods, 2014, 5(1) 27-33.
9. Himal Paudel Chhetri, Panna Thapa and Ann Van Schepdael.: Simple HPLC-UV method for the quantification of metformin in human plasma with one step protein precipitation, Saudi Pharm. J., 2014, 22, 483-487.
10. Kanij Fatema, Md. Zakiur Rahman, Tasnuva Haque and Mohammad Abul Kalam Azad., Development and validation of a simple method for simultaneous estimation of metformin hydrochloride and gliclazide in tablets by using reversed phase high performance liquid chromatography, Dhaka Univ. J. Pharm. Sci, 2010, 9(2) 83-89.
11. Nihar RanjanPani, LilakantNath, AkhileshVikramSingh, and Santosh KumarMahapatra.: Development and validation of analytical method for the estimation of nateglinide in rabbit plasma. J. Pharm. Anal, 2012, 2(6) 492–498.
12. Cijo Madathil Xavier, Kanakapura Basavaiah and Pavagada Jaganathamurthi Ramesh.: RP-UPLC method development and validation for the determination of nateglinide in bulk
drug and pharmaceutical formulations: A quality by design approach, Malays. J. Pharm. Sci, 2012, 10(1) 23–44.
13. Ashutosh KS, Manidipa D, Seshagiri RJVLN and Gowri SD.: New Validated Stability Indicating RP-HPLC Method for Simultaneous Estimation of Metformin and Alogliptin in Human Plasma, J. Chromatogr. Sep. Tech, 2015, 6(6) 1-6.
14. Asha Byju Thomas, Shrikrushna Digambar Patil and Rabindra Kumar Nanda.: Stability-indicating HPTLC method for simultaneous determination of nateglinide and metformin hydrochloride in pharmaceutical dosage form. Saudi Pharm. J, 2011, 19, 221–231.
15. Asha B Thomas and Shrikrushna D Patil., Simultaneous spectrophotometric estimations of nateglinide and metformin hydrochloride in pharmaceutical formulation. Der. Pharma. Chemica, 2011, 3(3) 271-276.
16. ICH, Q8 (R2): Harmonized tripartite guideline, Pharmaceutical development, IFPMA. In proceedings of the International Conference on Harmonization, Geneva, 2009, 1-28.
INDIAN DRUGS ONLINEPUBLISHED ON 28th OF EVERY MONTH
ADVERTISEMENT BANNER RATES FOR INDIAN DRUGS WEBSITE (Rates in Rupees per insertion)
Position Size RATE VALIDITY
Right Side Banner 180 X 150 Pixel 25,000 3 MONTHS
Left Side Banner 180 X 150 Pixel 25,000 3 MONTHS
Terms and Conditionsl All payments by DD in advance only to be made in favour of Indian Drug Manufacturers’ Association, payable
at Mumbai
l 25% discount applicable only for IDMA members
l 15% discount is applicable on Annual Contract for Non IDMA Members
l Please provide Banner Artwork as per the size for advertisements before the deadline
l Advertisement material must reach us 10 days before the date of release
For more details please contact: Publications Department Indian Drug Manufacturers’ Association
102-B, Poonam Chambers, Dr A B Road Worli, Mumbai 400 018. Tel: 24944624/24974308 Fax: 24950723 Email: [email protected]/ [email protected] / Website: www.idma-assn.org / www.indiandrugsonline.org
INDIAN DRUGS 57 (05) MAY 2020 65
HEPATOPROTECTIVE ACTIVITY OF WHOLE FLOWER OF HIBISCUS ROSA-SINENSIS LINN EXTRACTS IN WISTAR RATS
Agrawal Krishna Kumar a*
(Received 20 February 2020) (Accepted 5 May 2020)
ABSTRACT
The aim of the present study was to evaluate the hepatoprotective effect of ethanolic and aqueous extracts of Hibiscus rosa-sinensis Linn flowers..Carbon tetrachloride-induced hepatic injury in rats and pentobarbitone induced hypnosis methods were used to evaluate the hepatoprotective activity. Alteration in biochemical parameters of hepatic damage, such as alanine transminase (ALT), aspartate transminase (AST), alkaline phosphatase (ALP), triglycerides (TG), cholesterol, total bilirubin, direct bilirubin and pentobarbitone induced sleeping time were tested in different groups of study. Carbon tetrachloride (1mL/kg i.p.) enhances the level of biochemical markers of hepatic damage and increases the sleeping time in mice. Treatment with ethanolic and aqueous extracts of H. rosa-sinensis flowers (200mg/kg and 400mg/kg) has significantly brought back the altered levels of biochemical markers to near normal levels in a dose dependent manner, as compared to silyamarin (100mg/kg) and control group (1% w/V CMC).The result indicaes that the hepatoprotective activity shown may be due to the presence of flavonoids, mucilage, tannins or alkaloids, which justify its folkloric use.
Hibiscus is a large genus that contains herbs, shrubs and trees widely distributed in the tropical and sub-tropical region of the world. Hibiscus rosa-sinensis Linn (Malvaceae) is commonly known as jasut in Hindi and China rose in English. It is a native of China and is grown throughout in India as an ornamental plant. Plant can be propagated by cutting from mature wood of current growth. It is an evergreen, woody, glabrous, showy shrub of 5-8 ft in height. Leaves are bright green, ovate, coarsely toothed above; flower are solitary, axillary, bell shaped, large 4-6 inch in diameter with pistil and stamens projecting from the centre1.
The dark red petals in the form of mucilaginous infusion are used in ardor-urinae, strangury, cystitis and other irritable conditions of the genito-urinary tract2. The flower of H. rosa-sinensis were reported to possess various activity such as analgesic3, anticonvulsant4, anti-diabetic5, anti-pyretic6, wound healing7, anti-bacterial8, immunomodulatory9, anti-estrous10, anti-oxidant11 and hair growth12.
a Faculty of Pharmacy, Raja Balwant Singh Engineering Technical Campus Bichpuri, Agra - 283 105, Uttar Pradesh, India* For Correspondenc E-mail: [email protected]
MATERIAL AND METHODS
Plant materialThe flowers of H. rosa-sinensis Linn were collected
in the month of August from the gardens of Mathura (27.60560N, 77.59300E) district, Uttar Pradesh and authenticated by Birbal Sahni Institute of Palaeobotany, Lucknow, Uttar Pradesh, India (Ref. No. 13374).
Preparation of extractsThe flowers of H. rosa-sinensis were collected from
gardens in Mathura (Uttar Pradesh) and dried in shade and coarsely powdered. It was then passed through sieve no. 20. A weighed quantity (360g) of the powder drug was extracted with petroleum ether (60-800C) using Soxhlet extractor. Defatted drug was subjected to ethanolic extraction and extract was dried by distilling off the solvent and then dried in desiccator. The marc collected after ethanolic extraction was subjected to aqueous extraction by maceration process for seven days consecutively. The extract was then dried by evaporating the water and stored for further activity.
AnimalThe experimental protocol described in the present
66 INDIAN DRUGS 57 (05) MAY 2020
study was approved by the Institutional animal ethical committee (Approval no. GLAIPR/IAEC/2011-12/134) of Institute of Pharmaceutical Research, GLA University, Uttar Pradesh, India, with the permission from committee for the purpose of control and supervision of experiments on animals (CPCSEA). Healthy Wistar rats (200-300g) and mice (25-30g) were used for the study. Animals were housed in small cages in environmentally controlled (25 ± 20C, 12h light and dark cycle, with free access to food and water ad libitum). Animals were fed with the standard laboratory chow diet during the period of study.
Drugs and chemicalsPetroleum ether (60-800C) (CDH), absolute ethanol
(ChangshuYangyun Chemical China), silymarin (Medens Healthcare as a gift sample), carboxy methyl cellulose (CDH), carbon tetra-chloride (Qualikem), pentobarbitone sodium (Rhone-Poulenc, France) and olive oil (Figaro) were used in various parts of the experiment.
Phytochemical screeningThe preliminary phytochemical screenings were
conducted for the petroleum ether, ethanol and aqueous extracts of H. rosa-sinensis flower to find out the presence of various phytochemical constituents such as alkaloids, sterol, protein, amino acid, glycosides, tannins, flavonoids, mucilage, reducing sugar and saponin13.
CCl4 Induced hepatotoxicity in rats14
A total of 48 rats were divided into eight groups of six rats each and experiment was done by the method described by Vudaet al. with minor modification.
Group I served as a normal control for both prophylactic and curative studies and received 1.0% CMC for14days orally and on the 14th day olive oil (1.0 mL/kg, i.p.). Group II served as a toxic control for both prophylactic and curative studies and received 1.0% CMC for 14 days orally and on the 14th day CCl4 (1.0 mL/kg i.p.) in1:1dilution with olive oil. Group III act as pre-treatment group (prophylactic) and received ethanolic extract at a dose of 400mg/kg, orally for 14days, and on the 14thday received CCl4 (1.0 mL/kg i.p.) in1:1dilution with olive oil, 2h after administration of the last dose of extract. Group IV served as pre-treatment group (prophylactic) and received aqueous extract at a dose of 400mg/kg, orally for 14 days, and on the 14th day received CCl4 (1.0 mL/kg, i.p.) in 1:1 dilution with olive oil, 2h after administration of the last dose of extract. Group V served as pre-treatment group for standard drug silymarin (prophylactic). Rats received silymarin (100mg/kg, orally) for 14 days and on the 14th day received CCl4 (1.0 mL/kg i.p.) in1:1 dilution with olive
oil, 2h after administration of the last dose of silymarin. Group VI served as post-treatment group (curative). They received 1.0% CMC orally for 14 days and on the 14th day they received CCl4 (1.0 mL/kg i.p.) in a 1:1dilution with olive oil, followed by an ethanolic extract of H. rosa-sinensis at a dose of 400 mg/kg orally at 2, 6, 24, and 48h after CCl4 intoxication. Group VII served as post-treatment group (curative) and received 1.0 % CMC orally for 14 days and on the 14th day they received CCl4 (1.0 mL/kg i.p.) in 1:1 dilution with olive oil, followed by an aqueous extract of H. rosa-sinensis at a dose of 400mg/kg orally at 2, 6, 24 and 48h after CCl4 intoxication. Group VIII served as a post-treatment group for standard drug silymarin. The rats received 1.0 % CMC orally for 14 days, and on the 14thday received CCl4 (1.0 mL/kg i.p.) in a 1:1 dilution with olive oil, followed by silymarin (100 mg/kg, orally) at 2, 6, 24, and 48 h after CCl4 administration
All rats were sacrificed by high dose of diethyl ether 50 h after CCl4 administration and blood was collected from heart puncture. Serum was separated by centrifugation at 2500rpm for 15 min and used for biochemical estimations. The activities of serum aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase(ALP) and total bilirubin were determined with the help of bioanalyzer.
Determination of pentobarbitone-induced hypnosis time in mice15
A total of 36 mice were divided into six groups of six mice in each group and experiment was done by the method described by Rani et al. with minor modification.
Group I served as control group and received 1.0 % w/v CMC orally for 14 days and on 14th day the sleeping time of all mice in this group was determined by the injection of pentobarbitone sodium at dose of 45mg/kg b.w. On the next day, all the mice received CCl4 in olive oil in 1:1 dilution at dose of 1.0 mL/kg i.p., 2h after the CCl4 intoxication, the sleeping time of mice was determined by the injection of pentobarbitone sodium at the same dose. Group II and III served as treatment groups (extract) and received ethanolic extract of H. rosa-sinensis flower at dose of 200mg/kg and 400mg/kg, respectively, for 14 days and on the 14th day, the sleeping time of all mice was determined by injection of pentobarbitone sodium. On the next day, all mice received CCl4 in olive oil in 1:1 dilution at dose of 1.0 mL/kg i.p., 2h after the CCl4 intoxication, the sleeping time of mice was determined by the injection of pentobarbitone sodium (45mg/kg). Group IV and V was served as treatment groups (extract) and received aqueous extract at dose of 200mg/kg and
INDIAN DRUGS 57 (05) MAY 2020 67
400mg/kg, respectively, for 14 days and on the 14th day, the sleeping time of all mice was determined by injection of pentobarbitone sodium (45mg/kg). On the next day, all mice received CCl4 in olive oil in 1:1 dilution at dose of 1.0 mL/kg i.p., 2h after the CCl4 intoxication, the sleeping time of mice was determined by the injection of pentobarbitone sodium. Group VI served as standard group and received silymarin orally at the dose of 100mg/kg b.w. for 14 days and on the 14th day, the sleeping time was determined by pentobarbitone injection (45mg/kg). On the next day, all mice received CCl4 in olive oil in 1:1 dilution at dose of 1.0 mL/kg i.p., 2h after the CCl4 intoxication the sleeping time of mice was determined by the injection of pentobarbitone sodium (45 mg/kg)16.
RESULTS
Results of the preliminary phytochemical screening of different extracts of H. rosa-sinensis are shown in Table I. The qualitative screening indicates that the extracts contain alkaloids, tannins, flavonoids, sterols, glycosides and mucilage.
Table II: Effect of ethanolic and aqueous extracts on serum biochemical parameters in CCl4-induced rats
Group Treatment ALT(IU/L) AST(IU/L) ALP(IU/L) TB(mg/dl)Group I Control 69.63±1.63 134.74±2.41 139.4±4.38 0.16±0.03
Group II Control+CCl4 453.93±38.38** 370.75±30.02** 395.58±3.98** 0.94±0.61**
Group III HEE+CCl4 168.14±8.30** 156.1±3.97 172.22±4.85* 0.28±0.05
Group IV HAE+CCl4 226.17±14.51** 216.02±28.85* 190.39±10.9** 0.64±0.12**
Group V Silymarin+CCl4 140.78±6.56* 176.7±6.28 161.88±6.25 0.24±0.03
Group VI CCl4+HEE 141.84±3.17* 141.45±3.75 154.57±3.47 0.26±0.31
Group VII CCl4+HAE 194.16±8.10** 183.21±3.89 169.02±10.69* 0.39±0.01*
Group VIII CCl4+Silymarin 118.17±2.68 139.42±9.86 139.33±4.45 0.22±0.12
All values are Mean±SEM for N=6, Statistical comparison was performed by Graph pad prism software using ANOVA followed by Dunnett’s test, **P<0.01, *P<0.05 when all compared with the normal control group.
Treatment groups
Leve
l of b
ioch
emic
al p
aram
eter
s
Control 4
Control+CCl 4
HEE+CCl 4
HAE+CCl 4
Silymari
n+CCl+HEE4
CCl +HAE4
CCl+Sily
marin
4CCl
0
200
400
600
ALT(IU/L)AST(IU/L)ALP(IU/L)
Figure 1. Evaluation of liver biochemical parameters of extracts
Treatment groups
Leve
l of T
otal
bil
irub
in
Control 4
Control+CCl 4
HEE+CCl 4
HAE+CCl 4
Silymari
n+CCl
+HEE
4CCl +H
AE
4CCl
+Sily
marin
4CCl
0.0
0.5
1.0
1.5
2.0
TB (mg/dl)
Figure 2. Evaluation of total bilirubin after treatment of extracts
Fig. 1: Evaluation of liver biochemical parameters of extracts
The hepatoprotective, curative effects of H. rosa-sinensis aqueous and ethanolic extract on serum biochemical parameters in CCl4-intoxicated rats are shown in Table II. Rats treated with CCl4 (Group II) showed significant increase in serum AST, ALT, ALP, and total bilirubin levels compared to control animals (Group I). Pretreatment with H. rosa-sinensis aqueous and ethanolic extract at 400mg/kg for 14 days (Group III and IV) and post treatment with H. rosa-sinensis aqueous and ethanolic extract 400mg/kg at 2, 6, 24 and 48 h after CCl4 intoxication (Group VI and VII) showed significant hepatic protection in serum AST, ALT, ALP and total bilirubin levels (Fig. 2), compared to the toxic control group (Group II). However, post treatment with ethanolic extract (Group VI) at 400mg/kg dose showed better activity as compared to post treatment with aqueous extract (Group VII). Pre-and post-treatment with silymarin (Group V and VIII) also reduced all measured serum biochemical activities toward normalization (Fig.1).
Table I: Phytochemical screening of different extracts
Sr. No.
Chemical constituents
Pet. Ether extract
Ethanolic extract
Aqueous extract
1. Alkaloids - + +
2. Sterols + - -
3. Proteins - - -
4. Tannins - + +
5. Amino acid - - -
6. Glycosides - + +
7. Mucilage - + +
8. Flavonoids - + +
9. Reducing sugar
- + +
10. Saponin - - -
68 INDIAN DRUGS 57 (05) MAY 2020
Table III. Effect of aqueous and ethanolic extracts on CCl4-induced change in liver weight and volume
in rats
Group Treatment Dose (mg/kg)
Weight g/ 100g± SEM
Volume mL/100g
±SEMGroup
I Control 1.0 % w/v CMC
3.92±0.29 4.92±0.50
Group II
Control+ CCl4
1.0 % w/v CMC
4.08±0.32** 6.22±0.49**
Group III HEE+CCl4 400 2.90±0.10* 3.63±0.56**
Group IV HAE+CCl4 400 3.55±0.05 4.01±0.44**
Group V
Silymarin+ CCl4
100 2.86±0.25* 2.64±0.43**
Group VI CCl4+HEE 400 3.27±0.25 4.70±0.32
Group VII CCl4+HAE 400 3.50±0.22 3.39±0.19**
Group VIII
CCl4+ Silymarin 100 3.45±0.22 3.67±0.22**
All values are Mean±SEM for N=6, Statistical comparison was performed by Graph pad prism software using ANOVA followed by Dunnett’s test, **P<0.01, *P<0.05 when all compared with the control group.
Fig. 3: Evaluation of extracts on rat liver weight & volume
Treatment groups
Liv
er W
eigh
t & v
olum
e
Control 4
Control+CCl 4
HEE+CCl 4
HAE+CCl 4
Silymari
n+CCl
+HEE
4CCl +H
AE
4CCl
+Sily
marin
4CCl
0
2
4
6
8
Liver Weight (g/100g)Liver volume (ml/100g)
Figure 3. Evaluation of extracts on rat liver weight & volume
Treatment groups
Slee
ping
tim
e(m
in)
Control
HEE 200
HEE 400
HAE 200
HAE 400
Silymarin0
50
100
150
200
250Before CCl4After CCl4
Figure 4. Evaluation of hypnosis time (min) of Hibiscus rosa-sinensis extracts
Table IV. Effect of aqueous and ethanolic extract on pentobarbital sodium induced hypnosis time in
CCl4 treated mice
Group Treatment Dose (mg/kg)
Sleeping time (min) ± SEM
Before CCl4
After CCl4
Group I
Control 1.0 % CMC Suspension
158.00± 4.93
207.33± 1.45
Group II
HEE 200 110.00± 4.62**
129.00± 4.93**
Group III
HEE 400 95.00± 2.89**
106.67± 3.33**
Group IV
HAE 200 144.67± 1.45
170.00± 2.89**
Group V
HAE 400 134.67± 1.45**
152.33± 6.23**
Group VI
Silymarin 100 65.00± 5.00**
89.33± 2.91**
All values are Mean±SEM for N=6 rats, Statistical comparison was performed by Graph pad prism software using ANOVA followed by Dunnett’s test, **P<0.01 when all compared with the control group.
The change in liver weight and volume was measured after the carbon tetra-chloride administration and results are shown in Table III. Rats treated with CCl4 (Group II) showed significant increase in liver weight and volume compared to control group (Group I). Pre-and post-treatment (Groups III, IV, VI and VII) with aqueous and ethanolic extract at dose of 400 mg/kg showed significant
Treatment groups
Leve
l of b
ioch
emic
al p
aram
eter
s
Control 4
Control+CCl 4
HEE+CCl 4
HAE+CCl 4
Silymari
n+CCl+HEE4
CCl +HAE4
CCl+Sily
marin
4CCl
0
200
400
600
ALT(IU/L)AST(IU/L)ALP(IU/L)
Figure 1. Evaluation of liver biochemical parameters of extracts
Treatment groups
Leve
l of T
otal
bil
irub
in
Control 4
Control+CCl 4
HEE+CCl 4
HAE+CCl 4
Silymari
n+CCl
+HEE
4CCl +H
AE
4CCl
+Sily
marin
4CCl
0.0
0.5
1.0
1.5
2.0
TB (mg/dl)
Figure 2. Evaluation of total bilirubin after treatment of extracts
Fig. 2: Evaluation of total bilirubin after treatment of extracts
(P<0.01, P<0.05) reduction in liver weight and volume. Pre- and post-treatment (Groups V and VIII) with silymarin also reduced liver weight and volume (Fig. 3).
As shown in Table IV, before the liver injury, the sleeping time of extracts was compared with control and standard. The data showed that both the extracts have dose-dependent effect on sleeping time when compared to control group. Sleeping time of extracts after carbon tetra-
INDIAN DRUGS 57 (05) MAY 2020 69
chloride was measured and the results showed that both the extracts have significant (P<0.01) hepatoprotective activity (Fig. 4).
DISCUSSION
The liver is one of the vital organs in our body, responsible for detoxification of toxic chemicals and drugs. Thus, it is the target organ for all toxic chemicals. CCl4 is known to cause hepatotoxicity. The tri-chloromethyl free radical (CCl3•), an active metabolite of CCl3, is mainly associated with CCl4-inducedhepaticdamage. These radicals are suggested to react with sulfhydryl groups of glutathione and protein thiols. The covalent binding of these radicals to sulfhydryl-containing proteins in cells will initiate chain of events leading to membrane lipid peroxidation and cell necrosis17. When there is hepatocyte necrosis or membrane damage, these enzymes will be released into the circulation, as indicated by elevated serum enzyme levels. This increase in the serum AST, ALT and ALP enzyme levels in CCl4- treated animals indicates hepatic cell damage. In the present study, the elevated levels of all these marker enzymes observed in CCl4-treated rats (GroupII) indicate liver damage induced by toxins18. Bilirubin is the breakdown product of heme in red blood cells and hyper-bilirubinemia reflects the patho-physiology of liver. It is a most useful clinical indicator of the severities of necrosis, and its accumulation is a measure of the binding, conjugation and excretory capacity of liver cells. Pre- and post-treatment with H. rosa-sinensis aqueous and ethanolic extract reduced the increase in serum AST, ALT, ALP and total bilirubin, indicating its hepato-protective and curative activities. The restoration of serum enzyme levels to normal levels in CCl4-treated rats after pre -and post-treatment with aqueous and ethanolic extract of H. rosa-sinensis indicates prevention of the leakage of intra-cellular enzymes by stabilizing the hepatic cell membrane. Restoration of increased hepatic serum enzyme levels to normal levels reflects protection against the hepatic damage caused by liver toxins.
Fig. 4: Evaluation of hypnosis time (min) of Hibiscus rosa-sinensis extracts
Treatment groups
Liv
er W
eigh
t & v
olum
e
Control 4
Control+CCl 4
HEE+CCl 4
HAE+CCl 4
Silymari
n+CCl
+HEE
4CCl +H
AE
4CCl
+Sily
marin
4CCl
0
2
4
6
8
Liver Weight (g/100g)Liver volume (ml/100g)
Figure 3. Evaluation of extracts on rat liver weight & volume
Treatment groups
Slee
ping
tim
e(m
in)
Control
HEE 200
HEE 400
HAE 200
HAE 400
Silymarin0
50
100
150
200
250Before CCl4After CCl4
Figure 4. Evaluation of hypnosis time (min) of Hibiscus rosa-sinensis extracts
The results conclude that hepatoprotective activity may be due to the presence of flavonoids, mucilage, tannins or alkaloids, which justify its folkloric uses in curing various ailments.
ACKNOWLEDGEMENTS
Author is thankful to Director, Institute of Pharma-ceutical Research, GLA University for providing the necessary facilities and support to carry out this work.
REFERENCES1. Hibiscus rosa-sinensis In. Sastri BN (chief editor) The
Wealth of India.(vol.5) New Delhi National Institute of Science Communication CSIR, p.91
2. Asolkar LV, Kakkar KK, Chakre OJ. Glossary of Indian medicinal plants with active principle part 1New Delhi National Institute of Science communication & Information Resource CSIR 2005, p.353
3. Analgesic Activity of Hibiscus rosa-sinensis Linn in Rat, Sawarkar A, Jangde CR, Thakre PD, Kadoo R and Shelu S, Veterinary World (2009), Vol.2(9):353-354
4. Evaluation of anticonvulsant activity of Hibiscus rosa-sinensisflower extracts, Birari RB, Singh A, Giri IC, Saxena N, Shaikh MI and Singh A, IJPSR (2010), Vol. 1, Issue 5, 83-88
5. Anti-diabetic activity of flowers of Hibiscus rosa-sinensis, Venkatesh S, Thilagavathi J, Shyamsundar D, Fitoterapia 79 (2008) 79–81.
6. An evaluation of antipyretic and analgesic potentials of aqueous root extract of Hibiscus rosa-sinesisLinn. (malvacae), Soni D, Gupta A, Int. J. Res. Phytochem. Pharmacol., 1(3), 2011, 184-186.
7. Effects of Hibiscus rosa-sinensis L (Malvaceae) on wound healing activity: a preclinical study in a Sprague Dawley rat, Nayak B S, Raju SS, Orette FA, Chalapathi Rao AV, Int J Low Extrem Wounds. 2007 Jun;6(2):76-81.
8. Antibacterial Potentiality of Hibiscus rosa-sinensisSolvent Extract and Aqueous Extracts Against Some Pathogenic Bacteria, Hena J V, Herbal Tech Ind. , November 2010, 21-23
9. Comparative Screening of Immunomodulatory Activity of Hydro-alcoholic Extract of Hibiscus rosa-sinensisLinn. and Ethanolic Extract of Cleome gynandraLinn, Gaur K, Kori M L and Nema RK, Global J Pharmacol, 3 (2): 85-89, 2009.
10. Antiestrogenic activity of Hibiscus rosa-sinensis Linn. flowers, Kholkute SD, Udupa KN, Indian J Exp Biol. 1976 Mar;14(2):175-6.
11. Phytochemical screening and in vitro antioxidant activities of the ethanolic extract of Hibiscus rosasinensisL, Bhaskar A A, Nithya V and Vidhya VG, Ann. Biol. Res., 2011, 2 (5) :653-661
12. In vivo and In Vitro evaluation of hair growth potential of Hibiscus rosasinensisLinn. Adhirajan N, Kumar RT, Shanmugasundaram N, Babu M,J. Ethnopharmacol. 88 (2003) 235–239.
70 INDIAN DRUGS 57 (05) MAY 2020
INDIAN DRUG MANUFACTURERS’ ASSOCIATION102-B, “A”-Wing, Poonam Chambers, Dr A B Road, Worli, Mumbai 400 018.Tel: 2494 4624 / 2497 4308 Fax: 022- 2495 0723 E-mail: [email protected]/[email protected], Website: www.idma-assn.org / www.indiandrugsonline.org
KINDLY NOTE:
• Mailing of IDMA Bulletin and Indian Drugs by Post will commence prospectively only after receipt of payment.• All payments may be made in advance by Cheque / DD / RTGS / NEFT only in favour of: “Indian Drug Manufacturers’ Association”. • For RTGS / NEFT: Account Name: Indian Drug Manufacturers’ Association, Bank: CITIBANK N.A, Bank Account Number:0036274115,
IFSC Code: CITI0100000, Branch: Fort Branch, Mumbai – 400 001.• Courier charges for Publications under Serial Nos. 4 to 7 will be extra as applicable. • Please intimate us details through email immediately after making the remittance through RTGS/NEFT, so as to enable us to do the
needful promptly. • GST will be charged extra, as applicable.
Sr. No. Name of Publications Cost in `1. IDMA BULLETIN (Annual Subscription – 48 Issues) (Published on 7th, 14th, 21st and 30th of every month)
• Members 1000/- p.a.
• Government Research / Educational Institutions 2000/- p.a.
• Non-Members 4000/- p.a.
2. INDIAN DRUGS (Annual Subscription – 12 Issues) (Published on 28th of every month)
• Members 1000/- p.a
• Students 1000/- p.a.
• Government Research / Educational Institutions 2000/- p.a.
• Non-Members 4000/- p.a.3. IDMA APA Forum
• Annual Membership 500/-
• Life Membership 5000/-4. TECHNICAL MONOGRAPHS
NO. 1: STABILITY TESTING OF EXISTING DRUG SUBSTANCES AND PRODUCTS 400/-
NO. 2: PRIMARY & SECONDARY CHEMICAL REFERENCE SUBSTANCES 400/-
NO. 3: INVESTIGATION OF OUT OF SPECIFICATION (OOS) TEST RESULTS 400/-
14. Vuda, M., D’souza, R., Upadhya, S., Kumar, V., Rao, N., Kumar, V., Boillat, C. and Mungli, P., 2011. Hepatoprotective and antioxidant activity of aqueous extract of Hybanthusenneaspermus against CCl4-induced liver injury in rats. Exper. Toxicol. Pathol., article in press.
15. Rani, A., Nema, R.K. and Gupta, J.K., 2007. A preliminary pharmacological screening of Leucascephalotes (Labiatae)
for its Hepatoprotective activity. Planta Indica, Vol. 3, Issue 2, pp.11-12.
17. Slater, T.F. and Sawyer, B.C., 1971. The Stimulatory Effects of Carbon Tetrachloride and other Halogenoalkanes on Peroxidative Reactions in Rat Liver Fractions in-vitro, Biochem. J. Vol. 123, pp.805-814.
18. Kamboj P., Sardana S., Kaur G., Goyal R., Hepatotoxicity: mechanism and assessment, Pharm Aspire, 1, 2009, 105-108.
INDIAN DRUGS 57 (05) MAY 2020 71
SIMULTANEOUS DETERMINATION OF DOMPERIDONE AND SOME PRESERVATIVES IN ORAL FORMULATION: RP-HPLC METHOD
ABSTRACT
A novel RP-HPLC method was developed and validated for the determination of compounds in an oral solution. The method describes the determination of domperidone along with sodium methylparaben, sodium propylparaben and sodium benzoate in liquid oral formulation. Chromatographic separations were performed using BDS Hypersil 5 µm C18 column and gradient elution (solvent A: phosphate buffer, pH 3.5 and solvent B: methanol), keeping a flow rate of 1.5 mL/min. Detection was done at dual wavelength (232 nm for domperidone and sodium benzoate, and 257 nm for sodium methylparaben and sodium propylparaben). Analysis time was <17 min. The retention times for domperidone, sodium benzoate, sodium methylparaben and sodium propylparaben were found to be 10.0, 6.5, 8.0, and 13.5 min., respectively. The calibration curves for domperidone, sodium benzoate, sodium methylparaben and sodium propylparaben were found to be linear in the range of 250-750, 50-150, 50-150, and 5-15 µg/mL.
Domperidone (DP) is an antidopaminergic drug, which stimulates gastro-intestinal motility and is used as an antiemetic. Combinations of preservatives are used in pharmaceutical products to prevent their degradation or chemical alteration. Sodium propylparaben (SPP), sodium methylparaben (SMP), sodium benzoate (SB) and sorbic acid are the preservatives that are commonly used preservatives in pharmaceutical preparations. Various analytical methods are available for estimation of DP alone or in combination with other drugs1,9. A number of analytical procedures have been also reported for the determination of MP and PP preservatives separately or in combination with other drugs by HPLC or other techniques2,5,6,8-10. However, there is no single method available for the simultaneous determination of DP, SB, SMP, and SPP in a single chromatographic run. The present research describes the analysis of DP, SB, SMP, and SPP in pharmaceutical oral solutions using an RP-HPLC method.
EXPERIMENTAL
Chromatographic SystemChromatographic measurements were made on
HPLC (WATERS model e2695), five sample carousels, a UV–Visible and a PDA detector.
Chemicals and Reagents
Domeperidone was obtained asa gift sample from Cadila Pharmaceuticals Limited, Ahmadabad. HPLC grade methanol was obtained from Sigma-Aldrich. Hydrochloric acid, orthophosphoric acid and potassium dihydrogen orthophosphate were obtained from Merck. Deionized water was purified by a Milli-Q system.
Chromatographic conditions
The mobile phase for the proposed method consisted of phosphate buffer, pH 3.5: methanol. The final selected optimized conditions were as follows: Injection volume: 10 µL, flow-rate: 1.5 mL/min; detection wavelength: 232 nm for DP and SB, and 257 nm for SMP and SPP. Fig.1 shows a chromatogram of standard solutions and standard solutions of DP, SB, SMP, and SPP.
Preparation of diluents
Diluents was prepared by mixing equal volumes of 0.1 M NaOH and methanol (50:50 V/V).
Sample Preparation
About 6 g of sample (containing 50 mg of API) was accurately weighed and transferred to a 100 mL volumetric flask and dissolved by adding 70 mL of diluents. The solution was mixed and sonicated for 20 min. Final volume was completed with diluents, to obtain a solution containing DP (500 µg/mL), SB (100 µg/mL), SMP (90 µg/mL) and SPP (10 µg/mL), then filtered through 0.45 micron filter and 10 µL of it was injected.
72 INDIAN DRUGS 57 (05) MAY 2020
RESULTS AND DISCUSSION
Method ValidationThe proposed method was validated as per ICH
guidelines (Q2 R1) (ICH, 1996) (Table I).
LinearitySample solutions were prepared by diluting
the stock solutions of DP, SB, SMP, and SPP. The regression coefficients obtained demonstrate excellent
relationship between peak area and concentration of analyte.
PrecisionMethod prec is ion was
established by assaying six sample preparations, each injected in replicates under same conditions. The corresponding peak areas were measured and % RSD was calculated.
AccuracyThe accuracy of the analytical
method for assay of drug was established at three levels in triplicate, viz. 50 %, 100 % and 150 % of the test concentration. The results of the recovery studies and its statistical validation data are given in Table I and indicate high accuracy of the proposed method.
RobustnessRobustness of the proposed
Table I: Results of Validation parameters
Parameter Domperidone Sodium Benzoate
Sodium Methylparaben
Sodium Propyl paraben
Linearity range (µg/mL) 250-750 50-150 50- 150 5-15
Mean % Recovery (accuracy) 98.6±0.2 98.6±0.2 99.4±0.2 100±0.2
Precision (RSD) 0.9 0.3 0.3 0.8
Robustness Robust Robust Robust Robust
Fig. 1. Chromatogram of standard solutions and standard solutions of DP, SB, SMP, and SPP
method was performed by changing the following conditions:
i. Decreasing the flow rate of mobile phase from 1.5 mL/min to 1.3 mL/min.
ii. Decreasing the column temperature from 40 to 38 0C.
Standard solutions were injected six times for each change and system suitability parameters were recorded and were found within the acceptable limits. Six test
INDIAN DRUGS 57 (05) MAY 2020 73
samples were analyzed in duplicate for each change. Relative standard deviations and recoveries were calculated for each component and found to be <2.0% and 98.0-102%, respectively.
SpecificityThe chromatograms of diluent, standard, sample
and placebo and their interference were observed. As no interference was found, the method can be termed as highly specific.
System SuitabilityThe standard solutions of DP, SB, SMP, and SPP
were injected five times. The peak area, theoretical plates and tailing factor were measured and showed that the parameters evaluated were within acceptable range (RSD < 2.0 %, tailing factor < 2, theoretical plates > 2000) indicating that the system was suitable for the analysis.
Stability of SolutionThe stability of sample solutions was observed by
keeping the samples in a tightly capped volumetric flask at 100C for 24 hours.
CONCLUSION
The newly developed RP-HPLC method is simple, economical, precise, accurate and robust. There was no interference from placebo and diluents, hence the method is specific. Therefore, it can be applied for the simultaneous determination of DP, SB, SMP, and SPP in pharmaceutical formulations.
ACKNOWLEDGEMENTS
Authors are thankful to Principal of R. C. Patel Institute of Pharmaceutical Education and Research, Shirpur, Dist: Dhule (MS) for providing necessary laboratory facility.
a Department of Pharmaceutical Analysis, H. R. Patel Institute Patel Aasha.a, Firke Sandip D.b*, Patil Ravindra R.b, of Pharmaceutical Education and Research, Kalaskar Mohan G.b, Bari Sanjay B.a and Surana Sanjay J.b, Shirpur, Dist. Dhule - 425 405, Maharashtra, India b Department of Pharmaceutical Analysis, R. C. Patel Institute of Pharmaceutical Education and Research, Shirpur, Dist. Dhule - 425 405, Maharashtra, India For Correspondence E-mail: [email protected] (Received 6 January 2018) (Accepted 5 August 2018)
REFERENCES1. Al-Khamis K.I., Hagga M.E. and Al-Khamees H.A.:
Spectrophotometric Determination of Domperidone Using δA Method, Anal. Lett., 1990, 23(3) 451–460.
2. Austin K.L. and Mather L.E.: Simultaneous quantitation of morphine and paraben preservatives in morphine injectables, J. Pharm. Sci., 1978, 67(11) 1510–1511.
3. British Pharmacopoeia, The Stationary Office, Medicines and Healthcare products regulatory agency, British Pharmacopoeia Commission Office, London 2009, pp. 2091-2095
4. Denyer S.P. and Baird R.M.: Guide to Microbiological Control in Pharmaceuticals, England 1990, pp. 314–340.
5. Dvorak J., Hajkova R., Matysova L., Novakova L., Koupparis M.A., and Solich P.: Simultaneous HPLC determination of ketoprofen and its degradation products in the presence of preservatives in pharmaceuticals, J. Pharm. Biomed. Anal., 2004, 36(3), 625–629.
6. Hajkova R., Solich P., Dvorak J., and Sicha J.: Simultaneous determination of methylparaben, propylparaben, hydrocortisone acetate and its degradation products in a topical cream by RP-HPLC, J. Pharm. Biomed. Anal., 2003, 32(4-5), 921–927.
7. ICH Q2 (R1) Validation of analytical procedure methodology. International Conference on Harmonization, 1996:1-18
8. Kang S., and Kim H.: Simultaneous determination of methylparaben, propylparaben and thimerosal by high-performance liquid chromatography and electrochemical detection, J. Pharm. Biomed. Anal., 1997, 15(9-10), 1359–1364.
9. Kobylinska M., and Kobylinska K.: High-performance liquid chromatographic analysis for the determination of domperidone in human plasma, J. Chromatogr. B Biomed. Sci. Appl., 2000, 744(1), 207–212.
10. Kollmorgen D. and Kraut B.: Determination of methylparaben, propylparaben and chlorpromazine in chlorpromazine hydrochloride oral solution by high-performance liquid chromatography, J. Chromatogr. B Biomed. Sci. Appl., 1998, 707(1-2), 181–187.
74 INDIAN DRUGS 57 (05) MAY 2020
DETERMINATION OF BIOACTIVE CONTENTS AND IN VITRO ANTIOXIDANT ACTIVITY OF POLY HERBAL FORMULATIONS
ABSTRACT
Free radicals are reactive molecules involved in many physiological processes and have been associated with many diseases, such as ageing, cancer, arthritis and liver injury and cardiac complications. In polyherbal formulations HAF-I and HAF-II, described below, the total phenolics content were found to be 34.4±0.10 and 27.6±1.20 mg gallic acid equivalent (GAE)/g and total flavonoids contents, total tannin contents were 24.7±0.25 and 18.1±1.20 RE/g and 12.31±0.25 and 9.48±1.85 GAE/g, respectively. Free radical scavenging activity was determined according to the elimination of DPPH radicals. Total phenol content was determined by the Folin–Ciocalteu reaction. The relative antioxidant ability of the polyherbal formulations were investigated through two in vitro models, namely, antioxidant capacity by radical scavenging activity using γ, γ-diphenyl-γ-picrylhydrazyl (DPPH) and nitric oxide (NO) methods. The extracts were used at 20, 40, 60, 80 and 100 µg/mL concentrations and radical scavenging activity was determined in terms of inhibition percentage. The IC50 (concentration required for 50% inhibition) were calculated for each radical. The present study was designed to evaluate the free radical scavenging activity of hydro-alcoholic extracts of polyherbal formulations (HAF-I & HAF-II) various in-vitro models using ascorbic acid and rutin as references. The in vitro free radical DPPH activities were found to be 74.17±0.18 & 75.30±0.18 and NO antioxidant activity were found to be 75.3±1.10 & 76.17±1.24 at maximum concentration of 100 µg/mL. The in-vitro anti-oxidant activity of these polyherbal formulations may be due to the presence of polyphenols.
Climatic changes are giving rise to a variety of free radicals, and plants have to deal with them in order to survive. Many reactive oxygen species, such as singlet oxygen, superoxide ion, hydroxyl ion and hydrogen peroxide, are highly reactive, toxic molecules, which are generated normally in cells during metabolism. They cause severe oxidative damage to proteins, lipids, enzymes and DNA by covalent binding and lipid peroxidation, with subsequent tissue injury1,2. Reactive oxygen and nitrogen species (RONS) or radical and oxygen derived, non radical species, referred to as pro-oxidants, are involved in deleterious/beneficial biological process such as mutation, ageing, carcinogenesis, inflammation, neurological disorders and liver toxicity. The RONS is a predictable product of aerobic metabolic pathways that encompass membrane-bound reduced nicotinamide adenine dinucleotide phosphate (NADPH)-dependent oxidase, lip-oxygenase, cytochrome P-450, and xanthine oxidase activities. Numerous reports have shown that oxidative stress injuries are metabolic outcomes of noxious chemical agents or impaired metabolic events3-5.
2,2-Diphenyl-1-picrylhydrazyl (DPPH) is a stable free radical used for ascertaining the capacity of tissue extracts
to act as free radical scavengers and to measure their antioxidant activity in vitro. The reaction of DPPH with antioxidant of tissue extracts produces a corresponding reduced compound (hydrazine DPPH2), which can be monitored by color change from purple to yellow with maximum absorptivity (γmax) within the range of 515-528 nm6,7. Nitric oxide (NO) and reactive nitrogen species (RNS) are free radicals that are derived from the interaction of NO with oxygen or reactive oxygen species. Nitric oxide (NO) is generated from amino acid L-arginine by the enzymes in the vascular endothelial cells, certain neuronal cells, and phagocytes. NO is a diffusible free radical that plays many roles as an effector molecule in diverse biological systems including neuronal messenger, vasodilatation, and antimicrobial and antitumor activities8,9.
The present study sought to investigate the capacity of single and polyherbal formulations of individual herbs and blends of Bergenia ciliata, Pedalium murex, Tribulus terrestris, Tinospora cordifolia, Sphaeranthus indicus, Saccharum officinarum, Saccharum spontaneum, Saccharum munja, Desmostachya bipinnata, Imperata cylindrica and Piper longum to act as RONS and nRRS antagonists using in vitro antioxidant models. Phenolic, flavonoids compound and other polyphenolics are widespread in plant kingdom where they act as antioxidant and free radical scavengers. The objective of this study was to focus on determination of total phenolics and flavonoids,
INDIAN DRUGS 57 (05) MAY 2020 75
total tannin contents and in-vitro antioxidant activity of hydro-alcoholic extracts of polyherbal formulations (HAF-I & HAF-II)9-18.
MATERIALS AND METHODS
Collection and preparation and extraction of herbal samples
All the medicinal plant materials were collected from different geographical areas of districts Fatehpur, Deoria and Agra, Uttar Pradesh, India. All the medicinal plants were authenticated from National Institute of Science Communication and Information Research
(NISCAIR), New Delhi, India under supervision of scientist Dr. Sunita Garg with different authentication numbers. (Table I)
Plant extractionThe plants materials of different parts of the plants
used in formulations were then dried for two weeks under shade, then at room temperature, subsequently subjected to size reduction with a crusher and then passed through sieve no.40 to get uniform powder. Around 250 g of powdered plant material were subjected to extraction with solvent such as petroleum ether (for the purpose of defatting) or alcohol (60%). The hydro-alcoholic (40:60)
Table I: Composition of Polyherbal Formulations
Sr. N. Name of Drug Authentication No. Part used Quantity(mg) Formulation-I (HAF-I)
Fig1: Determination of bioactive contents (a) Total Phenolic, Total Flavonoid & Total Tannin Content of Formulation-I, (b) Total Phenolic, Total Flavonoid & Total Tannin Content of Formulation-II (c) Data comparison of Formulations HAF-I & HAF-II, calculated in Mean ± SEM.
Fig. 1: Determination of bioactive contents (a) Total Phenolic, Total Flavonoid & Total Tannin Content of Formulation-I, (b) Total Phenolic, Total Flavonoid & Total Tannin Content of Formulation-II (c) Data comparison of
Formulations HAF-I & HAF-II, calculated in Mean ± SEM.
76 INDIAN DRUGS 57 (05) MAY 2020
Fig 3: (a) Nitric oxide (NO) free radical scavenging activity of polyherbal formulations (b) Graphical calculations of samples and standards
Fig2: In vitro DPPH Model (a) DPPH Free radical scavenging activity of polyherbal formulations HAF-I &
HAF-II (b) Graphical calculations of samples and standards
Fig. 2: In vitro DPPH Model (a) DPPH Free radical scavenging activity of polyherbal formulations HAF-I & HAF-II (b) Graphical calculations of samples and standards
INDIAN DRUGS 57 (05) MAY 2020 77
extracts were subjected for maceration process of cold extraction. Each extract was then distilled to dryness under reduced pressure using rotatory evaporator to yield the respective dried extracts19-21.
Herbal formulationsThe amount in individual extracts of composition
of polyherbal formulations (powder extracts) contained the ethanolic (60%) extracts were used for polyherbal formulations mentioned in Table I 21-23.
Bioactive content determinations
Determination of total phenolic content flavonoid contents and tannin contents
The total phenolic content of the extract was determined by the Folin-Ciocalteu method. Quantitative analysis/total phenolic contents of individual plants and its polyherbal formulations (HAF-1 & HAF-2) were performed by spectrophotometric method using gallic acid as a standard24-30.
Measurement of radical scavenging capacities of polyherbal formulations
The hydro-alcoholic extracts medicinal plants and its blends in to polyherbal formulations had strong antioxidant activity against all the free radicals investigated44-47.
In vitro anti-oxidant activity (DPPH Test)The ability of individual plant extracts to scavenge the
DPPH• radicals were assessed by using spectrophotometric method. The hydro-alcoholic extracts of individual plant extracts and polyherbal formulations were performed and absorbances were recorded at 517 nm using UV/Vis spectrophotometer. The measurements were taken thrice, and scavenging effect was calculated based on the percentage of DPPH scavenged.
Percentage radical scavenging activity was calculated by the following formula:
% Radical Scavenging Power = ×100
[Ac–(As–Ao)]
Ac
where Ac = Absorbance of control (DPPH); As = Absorbance of sample/ standard+DPPH,
Ao = Absorbance of sample / standard without DPPH interaction32-37.
Nitric oxide scavenging activity Nitric oxide radical generation was performed and
absorbances were recorded at 546nm with or without the polyherbal formulations (HAF-I & HAF-II) at different concentrations. The formula of % inhibition of polyherbal formulations is given below
% inhibition = ×100
[(A0-A1)
A0]
Where: A0-absorbance of blank, A1- absorbance of extract38-43.
The DPPH assayThe DPPH methods revealed that the scavenging
activity of polyherbal formulations were found to be 30±0.14, 40.78 ±0.15, 65.34 ±0.12, 68.34±0.12, 74.17 ±0.18 and 26.55±0.176, 35.65±0.156, 56.30±0.12, 65.32±0.14, 75.30±0.18 at 20, 40, 60, 80 and 100 µg/mL, respectively. DPPH scavenging activities were maximum at 74.17±0.18 and 75.30±0.18 at a concentration of 100 µg/mL while that of the ascorbic acid and rutin were found to be 89.17±0.18 and 91.46±2.64 % .(Fig 3). In the DPPH assay, the IC50 of HAF-I, HAF-II, ascorbic acid and rutin were found to be 50.17, 57.21, 45.18 and 19.90 shown in Fig. 2.
Nitric oxide radical quenching activity In the present study, the nitric oxide radical quenching
activity of the polyphenolics extract was detected and compared with the standards rutin and ascorbic acid. The NO methods revealed that the scavenging of the free radicals was found to be 32.55±1.24, 36.65±1.12, 50.3±1.20, 64.32±1.25, 75.3±1.10 and 34.12±0.25, 43.78±0.54, 52.34±0.25, 65.45±1.20, 76.17±1.24 at 20, 40, 60, 80 and 100 µg/mL, respectively, for polyherbal formulation-I and polyherbal formulation-II. In the NO assay, the IC50 of HAF-I, HAF-II, ascorbic acid and Rutin were found be 56.87, 72.20, 45.64 and 20.63 respectively and displayed in Figure 3.
RESULTS AND DISCUSSION
Total phenolic, flavonoids and tannin contentsAs this is the first report on the anti-oxidant activity
of polyherbal formulations (HAF-I & HAF-II), thorough phytochemical analyses were done to identify the active phenolics, flavonoids and tannin components. The total phenolics contents were found to be 34.4±0.10 and 27.6±1.20 mg gallic acid equivalent (GAE)/g and total
78 INDIAN DRUGS 57 (05) MAY 2020
flavonoids contents, total tannin contents were 24.7±0.25 & 18.1±1.20 RE/g and 12.31±0.25 & 9.48±1.85 GAE/g, respectively, of polyherbal formulations (HAF-I & HAF-II). Data are shown in Fig. 1.
Anti-oxidant activity CHECK MANUSCRIPT
Statistical analysisThe experimental results were expressed as
mean ± SEM of three replicates (Triplicate). The IC50
values were calculated by the formula
Y = 100*A1/(X + A1),
where A1 = IC50, Y = response (Y = 100% when X = 0), X = inhibitory concentration. The IC50 values were compared by paired t tests. p < 0.05 was considered significant.
CONCLUSION
The results indicate a direct correlation between the antioxidant activity and the polyphenolics contents of the polyherbal formulations, which may the foremost contributors to the antioxidant activity of these polyherbal formulations. The present study confirmed that the hydro-alcoholic polyherbal formulations are potential sourceS of natural antioxidants.
AKS: Performed the study. Design, analysis and acquisition of data.
VKL: Supervised the study design and drafted the manuscript.
DK: Supervised the study design and drafted the manuscript.
scavenging activities, and neuroprotection of five medicinal plant extracts. Evidence-based. Comp. Alt.Med.; 2012, 1-8.
2. Ames BN, et al. Oxidants and the degenerative diseases of ageing. Proc Nati Acad Sci. 1993. 90: 7915-22.
3. Masoko P., Eloff JN., Screening of twenty-four South African Combretum and six Terminalia species for antioxidant activites. Afr J Trad, Compl Alter Med.; 2007, 4(2): 231–239.
5. Hodzic Z., et.al. (The influence of total phenols content on antioxidant capacity in the whole grain extracts. Europ J Sci Res. 2009, 28(3): 471–477.
6. Sies H. Strategies of antioxidant defense. Euro J Biochem, 1993, 215:213-219.
7. Molyneux P. The use of the stable free radical diphenylpicrylhydrazyl (DPPH) for estimating antioxidant activity. Songklanakarin J Sci Technol.; 2004, 26: 211-219.
8. Gunalan G. et alIn vitro antioxidant analysis of selected coffee bean varieties. J Chem Pharma.Res; 2012, 4(4): 2126–2132.
9. Yokozawa T.et al Direct scavenging of nitric oxide by traditional crude drugs. Phytomed. 2000, 6:453-463.
10. Kaneria M et al. Assessment of effect of hydroalcoholic and decoction methods on extraction of antioxidants from selected Indian medicinal plants. Asian Pac J Trop Biomed; 2012, 2(3):195–202.
11. Kähkönen MP, et alAntioxidant activity of plant extracts containing phenolic compounds. J agric food chem.; . 1999, 47(10): 3954-3962.
12. Vinson JA et al. Plant flavonoids, especially tea flavonols, are powerful antioxidants using an in vitro oxidation model for heart disease. J agric food chem.,1995, 43(11):2800-2802.
13. Singh R et.al. Total phenolic, flavonoids and tannin contents in different extracts of Artemisia absinthium. J Intercult Ethnopharmacol., 2012, 1 (2): 101-104.
14. AfifyAel-M., et al. (2012). Biochemical changes in phenols, flavonoids, tannins, vitamin E, β-carotene and antioxidant activity during soaking of three white sorghum varieties. Asian Pac J Trop Biomed, 2012, 2(3): 203-9.
15. Muthu kumaran P. et al. Assessment of total phenolic, flavonoid, tannin content and phytochemical screening of leaf and flower extracts from Peltophorum pterocarpum (DC.) Backer ex K.Heyne: a comparative study. Pharmacog J.; 2016, 8(2):25-32.
16. Saeed et al. Antioxidant activity, total phenolic and total flavonoid contents of whole plant extracts Torilis leptophylla L. BMC Compl Alter Med, 2012, 12:221.
17. Hossain D.M. et al. Investigation of total phenolic content and antioxidant activities of Azadirachta indica roots, Avicenna J Phytomed, 2014, 4(2): 97–102.
18. Tseng TH, et al. Protective effects of dried flower extracts of Hibiscus sabdariffa L. against oxidative stress in rat primary hepatocytes. Food Chem Toxicol; 1997, 35(12):1159-1164.
19. Salwe KJ, et al. Evaluation of antidiabetic, hypolipedimic and antioxidant activity of hydroalcoholic extract of leaves and fruit peel of Punica granatum in male Wistar albino rats. J Nat Sci BioMed, 2015, 6(1):56-62.
20. Bhalodia NR. et al. (2013). In-vitro antioxidant activity of hydro alcoholic extract from the fruit pulp of Cassia fistula Linn. Ayu.; 34(2):209-314.
21. Naik G.H. et al. Evaluating the antioxidant activity of different plant extracts and herbal formulation. Res Chemica Intermed, 2005, 31(1-3) :145–151
INDIAN DRUGS 57 (05) MAY 2020 79
22. Bose S. et al.Comparative study of antioxidant activity of herbal drugs and their formulations using Asparagus racemosus and Centella asiatica. Ame. J Pharm. Tech Res; 2012, 2(2):391-398.
23. Balaraman R, et al.Antioxidant activity of DHC-1--a herbal formulation. J Ethnopharmacol.; 2004, 94(1):135-41
24. Singh R et.al.Total phenolic, flavonoids and tannin contents in different extracts of Artemisia absinthium. J Intercult Ethnopharmaco, 2012, 1 (2): 101-104
25. Muthu kumaran P. et.al Assessment of total phenolic, flavonoid, tannin content and phytochemical screening of leaf and flower extracts from Peltophorum pterocarpum (DC.) Backer ex K.Heyne: a comparative study. Pharmacog. J ; 2016, 8(2):25-32.
26. Sreejayan N, Rao MN. Nitric oxide scavenging by curcuminoids. J Pharm Pharmacol , 1997, .49; 105-7.
27. Saeed et al. Antioxidant activity, total phenolic and total flavonoid contents of whole plant extracts Torilis leptophylla L. BMC Compl Alter Med, 2012, 12:221.
28. Augusto TR. et.al. Phenolic compounds and antioxidant activity of hydroalcoholic extracts of wild and cultivated murtilla (Ugni molinae Turcz.) Food Sci. Technol, Campinas., 2014, 34(4): 667-673.
29. Gil MI. Effect of postharvest storage and processing on the antioxidant constituents (flavonoids and vitamin C) of fresh-cut spinach. J Agri Food Chem, 1999, 47(6):2213-2217.
30. Vinson JA, et al. Plant flavonoids, especially tea flavonols, are powerful antioxidants using an in vitro oxidation model for heart disease. J Agri Food Chem, 1995, 43(11):2800-2802.
31. Marcocci L., et al. Antioxidant action of Ginkgo biloba extract EGB 761. Methods Enzymol. 1994, 234: 462–475.
32. Chiang LY., et al. Free radical scavenging activity of water-soluble fullerenols. J Chem Soc Chem Commun.; 1995, 1283–1285
33. Blois MS. (1958). Antioxidant determinations by the use of a stable free radical, Nature.; 1958, 181,:1199–1200.
34. Bondet V, et al. Kinetics and mechanisms of antioxidant activity using the DPPH free radical method. Lebensmitt Wissenschaft Technologie Food Sci Technol.;1997,30: 609–615.
35. Molyneux P. The use of the stable free radical diphenylpicrylhydrazyl (DPPH) for estimating antioxidant activity. Songklanakarin J. Sci. Technol., 2004, 26:211-219.
36. Rafat A., et al. Antioxidant potential and phenolic content of ethanolic extract of selected Malaysian plants. Res. J. Biotechnol.; 2000, 5:16-19.
37. Soares JR., et al. (1997). Antioxidant activity of some extracts of Thymus zygis, Free Radic. Res, 1997, 26: 469-478.
38. Balavoine GG, et al. Peroxynitrite scavenging by different antioxidants. Part 1: convenient study. Nitric oxide.1999, 3(1):40-54.
39. Tsai PJ et al. Evaluation of NO-suppressing activity of several Mediterranean culinary spices. Food Chem Toxicol, 2007, 45(3): 440-447.
40. Amaeze OU. et al. Evaluation of antioxidant activity of Tetracarpidium conophorum (Müll. Arg) Hutch & Dalziel leaves. Oxidative Med Cell Long, 2011, 7.
41. Duh PD, et al. Antioxidant activity of water extracts of Harng Jyur (Chrysenthemum morifolium Ramat) Lebnes wiss Technol.; 1999, 32: 269–277.
42. Jagetia GC, et al. The Evaluation of Nitric Oxide Scavenging Activity of Certain Herbal Formulations in vitro: A Preliminary Study. Phytother. Res, 2004, 18: 561–565,
43. Vriesman MF et al. A method for measuring nitric oxide radical scavenging activity. Scavenging properties of sulfur-containing compounds. Pharm World Sci, 1997, 19(6): 283-286.
44. Chaulya NC et al. In vitro free radical scavenging activity of methanol extract of rhizome of Cyperus tegetum Roxb.(Cyperaceae). Int J Curr Pharm Res, 2010, 2(3):39-43.
45. Soare JR, et al.Antioxidant activities of some extracts of Thymus zygis. Free Rad. Res, 1997, 26(5):469-478.
46. Prachayasittikul S. et al. Antimicrobial and anti-oxidative activities of bioactive constituents from Hydnophytum formicarum Jack. Molecules, 2008, 13(4):904-921.
47. Nogueira, JCR, et al. (2008). In vitro antimicrobial activity of plants in Acute Otitis Externa. Revista Brasileira de Otorrinolaringologia, 2008, 74(1): 118-124.
aAnand College of Pharmacy, Keetham, Agra - 282 007, Uttar Pradesh, India. Srivastava A. Ka*, Kaushik D.b and Lal V. K. bAgra Public Pharmacy College, Agra - 282 007, Uttar Pradesh, India. For correspondence Email: [email protected] (Received 24 March 2018) (Accepted 18 April 2018)
80 INDIAN DRUGS 57 (05) MAY 2020
OBITUARY
Dr. V. K. Parameswaran was an exemplary Pharmaceutical Technologist, who believed in teaching and sharing his knowledge unconditionally - whether in a professional or teaching capacity. He left behind a legacy full of achievements, grateful colleagues and students, and an enriched Pharma community.
Born in a small village in Kerala, Dr. Parameswaran did his schooling at a local government school, post which his brother brought him to Mumbai (then Bombay) in 1955 for further studies. He completed B.Sc. with Chemistry from Khalsa College in Bombay University in 1959, and that evolved in him a life defining love of chemistry. His educational record is as below:
• B.Sc. with Chemistry from Khalsa College / Bombay University
• B.Sc. Tech. (Pharmaceuticals) - UDCT
• M.Sc. Tech. (Pharmaceuticals) - UDCT
• Guided by late Professor M. L. Khurana, and awarded Industrial Research Fellowship from Pfizer Ltd
• He developed a new procedure for manufacture of Hydrazine Hydrate and Isonicotinic Acid.
• He holds 3 Patents in his name for his work on Hydrazine Hydrate and oxidation of Picolines.
• Ph.D. Tech in Medicinal Plant Chemistry
Dr. Parameswaran spent 21 years in UDCT, and along with acquiring his degrees, was involved with the following activities:
• 1968 to 1972 : Lecturer for Pharmaceutical Chemistry. Taught B.Pharm , M.Pharm , BSc (Tech) and MSc (Tech) classes
• 1972 to 1980 : Reader (Assistant Professor) in Pharmaceutical Chemistry
• 1968 to 1980 : Recognized as Guide by Bombay University for Master’s Degree in Tech and Ph.D. Tech.
• Guided a number of students for M–Pharm, MSc Tech and Ph.D. Tech degrees.
• He published many research papers, including an International Paper on Biological Correlations: Hansch Analysis on Phenothiazines, which he presented at a Pharmaceutical Conference in Basel, Switzerland in the year 1979.
• He was also a consultant through UDCT to Themis Pharmaceuticals Pvt. Ltd.
His students held Dr. V. K. Parameswaran in very high regard, and described him as highly dedicated, very intelligent, sincere, and an excellent teacher. No student wanted to miss his lectures, and they remember what he taught them to date - mainly due to his ability to simplify a complex topic, so that even a novice would clearly understand. His students recount his simple yet innovative and interactive style of teaching, which involved conducting weekly tests where each student would get a different set of questions – evidently painstakingly prepared and assessed with utmost sincerity. He helped many of his friends and students to develop and establish pharmaceutical manufacturing businesses.
In 1980, Dr. Parameswaran joined Roche Products as Manager - Research & Development at their Fine Chemical Plant, Balkum Village, Thane. He retired in March 1997 from Piramal Healthcare Ltd. (Roche was taken over by Piramal Group in 1993) and was looking after Research & Development and Quality Assurance. Besides, he was also overseeing Environmental studies. Post retirement, the company retained him as a full time consultant till 1999. His colleagues in Roche remember him as a perfect technologist - meticulous, very thorough with the subject, humble, and a man of few words and more action.
Dr V. K. Parameswaran remained associated with Bombay University as an Examiner for BSc Tech, B-Pharm, MSc. Tech, M-Pharm theory classes through his work career and after retirement. He played a vital role as an expert for framing the four-year, semester-based B. Pharm. syllabus.
Dr. Parameswaran was married to Mrs. S. P. Warrier, who was earlier in Glaxo Laboratories and later on moved to UDCT as a faculty. Both were simultaneously teaching faculty members in the same Department for around ten years and Mrs. Warrier retired many years later from the Department. They were blessed with a son and a daughter. Dr. V. K. Parameswaran enjoyed a happy retired life with his family and friends. He continued to touch the hearts of everyone he came into contact with. He was particularly proud of his granddaughters and would not stop talking about them. He breathed his last on 06th May this year, leaving behind a legacy of humility and knowledge which will continue to live on through his family, friends, students, and colleagues.
(1937 – 2020)
INDIAN DRUGS 57 (05) MAY 2020 81
*Sr. No.
Application No. Title Publication Date
Abstract
23. 201918049164 FORMULATION FOR ANTI-A4ß7
ANTIBODY AND METHODS
THEREOF
17/01/2020 A stable liquid pharmaceutical formulation comprising a mixture of an anti-γ4γ7 antibody at a concentration of 60 mg/ml to 170 mg/ml, citrate, and at least one amino acid, wherein the formulation is in liquid form, and wherein the anti-γ4γ7 antibody comprises a heavy chain variable region comprising amino acids 20-140 of SEQ ID NO: 2 and a light chain variable region comprising amino acids 20-131 of SEQ ID NO: 4 or amino acids 21-132 of SEQ ID NO: 5.
24. 201911049029 A NOVEL FORMULATION OF 5-FLUOROURACIL
FOR TREATING DIABETIC
RETINOPATHY
20/12/2019 The present invention discloses a novel formulation of 5-fluorouracil for treatingdiabetic retinopathy. The formulation is applied topically by non-invasive methodby patient itself. The formulation consists of 5-fluorouracil loaded in the nanoliquid carrier.
25. 201911049022 A NOVEL GEL FORMULATION AND PROCESS
FOR ANTI-ARTHRITIC ACTIVITY
20/12/2019 The present invention discloses a novel gel formulation for anti-arthritic activity.The gel formulation consists of curcumin, serratiopeptidase and prednisolonecyclodextrincomplex. The curcumin and serratiopeptidase are loaded intransethosomes. The formulation has synergistic action, shows better andprolonged drug diffusion through skin and better skin retention of drug.
26. 201911049027 A NOVEL PROTEIN RICH DIETARY FORMULATION AND PROCESS
THEREOF
20/12/2019 The present invention discloses a novel protein enriched dietary formulation andprocess thereof. The process of formulation is simple and cost effective. No extraprotein source is used in the preparation of the formulation.
27. 201911048915 A NOVEL FORMULATION FOR TREATING
BREAST CANCER
13/12/2019 A novel formulation of Cannabis sativus for cancer treatment. The formulation is known to be more effective in breast cancer compared to other therapeutic treatments like chemotherapy and other radiation therapies.
28. 201917048299
WO/2018/205022
OCULAR DRUG DELIVERY
FORMULATION
24/01/2020 There is provided an ocular drug delivery formulation comprising a delivery carrier comprising a cellulosic polymer and an anionic polysaccharide and nanoparticles comprising an amphiphilic non-ionizable block copolymer and a cannabinoid. The formulation has a gel point of about 30°C to about 37°C.
Recent Publication From Indian Patent Office Journal With PCT/Wipo Number
(*Sr. Nos. 1 to 5 and 6 to 22 were already published in Indian Drugs Issues Vol. 57 (03) March 2020 and Vol. 57 (04) April 2020, respectively)
82 INDIAN DRUGS 57 (05) MAY 2020
29. 201911047946 A NOVEL PHARMACEUTICAL
FORMULATION FOR ANTI-ARTHRITIC ACTIVITY
06/12/2019 The present invention discloses s novel pharmaceutical formulation for anti-arithmetic activity. The said formulation is liposomes and consist of a prodrug of sulfapyrdine. The said formulation has increases antiarithritic activity.
30. 201911047531 A NOVEL ORAL GOLD
NANOPARTICLE FORMULATION CONTAINING
INSULIN
06/12/2019 The present invention discloses a novel oral gold nanoparticle formulation of insulin. The said formulation is prepared by reduction of chloroauric acid with modified apple polysaccharide and the conjugation with insulin. The said formulation decreases blood glucose level and improves body weight, lipid profile, urea, creatinine and liver parameters.
31. 201941046289 FORMULATION AND CHARACTERI-ZATION OF DOU-
BLE EMULSION CA-PABLE OF CARRY-ING THREE DRUG
COMPONENTS
29/11/2019 Emulsions are metastable systems. Due to their kinetic stability, emulsions are used as potent drug carriers. Multiple emulsions are capable of carrying multiple drugs with different solubility. Double emulsion, a multiple emulsion system are reported as the carriers of two drugs. Aim of this study was to formulate a double emulsion, which can carry 3 drugs (double tertiary emulsion system). With the methods adopted, the formulation resulted in a model that can carry two hydrophobic and one hydrophilic drug. This system, to the best of our knowledge has not been reported so far and moreover, the protocol adopted for synthesis was a very simple one. Combination of surfactants with different Hlb values was used to bring two immiscible phases together, to get stable emulsions. Formulated emulsions were analysed by Dynamic Light Scattering (DLS) to measure their hydrodynamic diameter followed by HR-TEM to confirm the final formulation. All the individual components used for formulation and 1 °, 2”and 3°emulsions were given for Fourier Transform Infrared Spectroscopy (FTIR) and results were analysed. In addition, inimitably, this work also attempted to characterize the formulation by image processing captured by bright field compound microscope, using defined algorithms.
(Compiled and contributed by Ms. Shweta Patankar Vichare & Ms. Kavya Pillai of Gopakumar Nair Associates.)
INDIAN DRUGS 57 (05) MAY 2020 83
INDIAN DRUGSADVERTISEMENT TARIFF (Effective from 01.11.2017)
PUBLISHED ON 28th DAY OF EVERY MONTH Magazine size: 21.5 cm x 27.5 cm/Print Area 18.5 cm x 23.5 cm
(Rates in Rupees per insertion)
Position (Print Area Dimensions) B/W Colour
Full Page (18.5 cm x 23.5 cm) 6,500 10,000
Half Page(18.5 cm x 11.5 cm) (Horizontal) 4,000 7,500
Half Page (9 cm x 23.5 cm) (Vertical) 4,000 7,500
Quarter Page (9 cm x 11.5 cm) 2,000 5,500
Strips Advt (4.5 cm x 11.5 cm) 2,000 -
Inside Front Cover - 12,500
Inside Back Cover - 12,500
Back Cover - 18,000
TERMS AND CONDITIONS:
• All payments by Cheque/ Demand Draft/RTGS in advance only to be made in favour of “Indian Drug Manufacturers’ Association”, Payable at Mumbai
Please see the NEFT/RTGS details: ACCOUNT NAME : Indian Drug Manufacturers’ Association RTGS to BANK: CITIBANK N.A., BANK Account number : 0036274115 IFSC Code: CIT10100000, BRANCH: FORT BRANCH, MUMBAI — 400 001.
• Please provide Positives/Artwork/Typeset matter for advertisements.
• Advertisement material must reach us 10 days before the date of publication.
• Positioning of the Advt other than Cover Positions will be at our discretion.
• Only Colour Advts will be entertained on Cover Positions.
• GST will be charged extra, as applicable.
For further details, please contact Mr T R Gopalakrishnan — Cell: +9819035076, (Email: [email protected])
![57 2020 2020410 F] 27](https://static.documents.pub/doc/80x56/61d7b9e2c1432779c70f9be5/57-2020-2020410-f-27.jpg)
