Formulation and Evaluation of Polyherbal Formulations for...

Formulation and Evaluation of Polyherbal

Formulations for Canker Sore (Mouth Ulcer)

A

Dissertation

Submitted for the Degree of

MASTER OF PHARMACY

IN

THE FACULTY OF PHARMACY

(PHARMACOGNOSY)

TO

GANPAT UNIVERSITY, KHERVA

2008- 2009

Research Guide: Submitted by:

Mrs. Nikunjana Patel Karuna J. Patel

M. Pharm. B. Pharm.

S. K. PATEL COLLEGE OF PHARMACEUTICAL EDUCATION AND RESEARCHGANPAT UNIVERSITY, KHERVA-382711.

DIST – MEHSANA (GUJARAT), INDIA.

CERTIFICATECERTIFICATEI hereby certify that Miss Karuna J. Patel has completed her thesis for Master of

Pharmacy on the topic “FORMULATION AND EVALUATION OF

POLYHERBAL FORMULATIONS FOR CANKER SORE (MOUTH

ULCER)”. I further certify that the work done by her is of her own and original and

tends to the general advancement of knowledge. The work was carried out under my

supervision and guidance at Department of Pharmacognosy, S. K. Patel College of

Pharmaceutical Education and Research, Ganpat Vidyanagar, during the

academic year 2008-2009. This work is up to my satisfaction.

Research Guide:

Mrs. Nikunjana PatelM. Pharm.Assistant Professor, Department of Pharmacognosy.

Forwarded Through:

Dr. N. J. Patel

M. Pharm., Ph.D.,Principal,

S.K.Patel College of Pharmaceutical Education and Research,Ganpat University, Kherva-382711.

Date:

Place: Ganpat University

Head of Department:

Dr. R. K. PatelM. Pharm., Ph.D.Head & Associate Professor,Department of Pharmacognosy.

DECLARATIONDECLARATION

I, Karuna J. Patel hereby declare that the topic entitled “FORMULATION

AND EVALUATION OF POLYHERBAL FORMULATIONS FOR CANKER

SORE (MOUTH ULCER)” which is submitted to the Ganpat University, Kherva, in

partial fulfilment for the award for Degree of Master of Pharmacy in

Pharmacognosy. The result of the work done by me in Department of Pharmacognosy

under the guidance of Mrs. Nikunjana Patel, Assistant Professor S. K. College of

Pharmaceutical Education and Research, Ganpat University, Kherva. I further declare

that the results of this work have not been previously submitted for any degree of

fellowship.

Place: Ganpat University. Karuna J. Patel

Date: - (B. Pharm.) Department of Pharmacognosy, S.K.P.C.P.E.R. Ganpat University

ACKNOWLEDGEMENTACKNOWLEDGEMENTIt is great pleasure for me to acknowledge all those who have contributed towards the

conception, origin and nurturing of this project. This work was carried out at the

Department of Pharmaceutics and Pharmaceutical Technology, S. K. Patel College

of Pharmaceutical Education and Research, Kherva during the years 2008-2009

which affiliated with Ganpat University, Kherva.

With a deep sense of gratitude and the respect, I thank my esteemed guide and

preceptor Mrs. Nikunjana Patel, for her inestimable guidance, valuable suggestions

and constant encouragement during the course of this study. It is with affection and

reverence that I acknowledge my indebtness to her and her outstanding dedication,

often far beyond the call of duty. Apart from guiding me, her unwiring moral support

and advice.

I take this opportunity to place on record my indebtness to Dr. Rakesh K.

Patel ,Head & Associate Professor ; I take this opportunity to say many thanks to Mr.

Hardik Patel. Mr. Kapil Khambholja, Lecturers, Department of Pharmacognosy for

their valuable suggestions, directions and selfless support throughout the

investigation.

I also expressed my profound gratitude to Dr. N. J. Patel, Principal & H.O.D,

Department of Pharmacology and toxicology, S. K. Patel College of Pharmaceutical

Education and Research, Kherva who have been a constant source of inspiration to

steer me forward through the two years of study and also for allowing me to use

facility during my project work..

I special mention thanks to Dr. S .S. Pancholi, H.O.D, Department of quality

assurance, and other faculty members of quality assurance, for their valuable

suggestions, directions and selfless support throughout the investigation.

I am also thankful to Mr. H. R Patel, Lecturer, Department of Pharmaceutics, for his

valuable help in my project work.

I sincerely thank to Mr. P. I. Patel, Librarian and assistant Mahadevbhai and

Mukeshbhai for providing me library facilities and their untiring and really

appreciable help in my literature survey.

I would like to express my thanks to non-teaching staff especially, Rakeshbhai (Lab.

Assistant), Jaimin bhai and Madhuben for their untiring help and support

throughout my M. Pharm. course.

I also thankful to Mr. Manishbhai Patel, Mrs. Kiranben Patel, (Lab. Assistant.

pharmaceutics department), Mr. Dineshbhai Patel (Store in charge), Mr. Sushilbhai

Patel (Lab. Assistant. quality assurance department), Mr. Nrupeshbhai Patel and

Mr. Rajubhai Raval (office staff) for their support throughout my M. Pharm. Course.

I sincerely acknowledge the help rendered to me by colleagues of M. pharm, Megha,

Sangita, Abhilasha, Monika, Pathik, Kaushik, Asif, Piyush and Umang for being co-

operative. Without their constant support and ideas this work would have been

difficult for me.

I equally thankful to my junior colleagues Tejas, Jigar, Nirav, Nevil, Rahul, Priya,

Kajal, Urvashi , Riddhi and Sejal for kind co-operation and timely help in the

fulfillment of my course work.

Memories play an important part in keeping special people close at heart. I can not

forget the sweat memories of the time that I have spent with my friends Manish,

Nisha, Anita and Ripal, who contributed in one or another way knowing and

unknowingly to reach out giving an encouragement and constant support when I am

need.

I hearty expressed my deep gratitude to my parents Mr. Jayantibhai Patel and Smt.

Sushilaben Patel, my brother Devendra, and other family members for their moral

support, constant encouragement and patience absolutely needed to complete my

entire study. They never opposed me and allowed me to what I want to do. They gave

me all facility and support in every situation of life. Taught me key of successes and

how to become a good person in life. Without their support I was not able to reach at

this level. It was the blessing of them that gave me courage to face the challenges and

made my path easier.

Thank you papa and mom.

Thank you very much to every body.

Karuna J. Patel

Dedicated

to

my beloved

family

Chapter.1Introduction

Chapter.2Review

ofLiterature

Chapter.3Aim and Plan of the

work

Chapter.4Evaluation of Raw

materials

Chapter.5Formulation and

Evaluation ofGel

Chapter.6Formulation and

Evaluation ofPatch

Chapter.7Antimicrobial activity

ofselected formulations

Chapter.8Summary

andConclusion

List ofTables and Figures

Index

List of Tables

List of Tables

Table

No.Title Page No.

1.1 Physical and Chemical Properties of Carbopol 28

1.2 Viscosity range of different Carbopol Polymers 28

4.1 Physicochemical parameters of G. glabra powder 70

4.2Observation table for calibration curve of std. 18-β- G.A.

(Using HPTLC method)72

4.3 Physicochemical parameters of A. catechu powder 74

4.4Observation table for calibration curve of std. catechin


4.5 Results of performed physical parameters of clove oil 77

4.6Observation table for calibration curve of std. eugenol


5.1 Preparation of gel formulations 83

5.2 Evaluation Parameters of Gels 87

5.3 In vitro Release profile of gel formulations 90

6.1 Preparation of patch formulations 94

6.2 Evaluation Parameters of Patches 98

6.3 In vitro Release profile of patch formulations 101

7.1 Zone of Inhibition (cm) of gel formulations 106

7.2 Zone of Inhibition (mm) of patch formulation 107

M. Pharm. (Pharmacognosy) I

List of Figures

List of Figures

Figure

No.Title

Page

No.1.1 Glycyrrhiza glabra Plant and Root 11.2 Acacia catechu Plant and Extract 61.3 Syzygium aromaticum plant , flower buds and oil 111.4 Types of Intraoral dosage forms 171.5 Variation in buccal patches 231.6 General Structure of Carbopol Polymers 261.7 Schematic drawing of a molecular segment of a cross-

linked polyacrylic acid polymer

27

1.8 Schematic representation of Hypromellose 341.9 Mouth Ulcer 404.1 Morphology of Glycyrrhiza glabra 684.2 Microscopic characters of G. glabra powder sample 684.3 TLC profile of Glycyrrhiza glabra powder 694.4 HPTLC profile of G. glabra powder 714.5 HPTLC chromatogram of standard 18-β-G.A. 714.6 HPTLC chromatogram of G. glabra extract 724.7 Calibration curve of 18-β- G.A. by HPTLC method 724.8 Morphology of A. catechu 734.9 TLC profile of A. catechu powder 744.10 HPTLC profile of A. catechu powder 754.11 HPTLC chromatogram of standard catechin 754.12 HPTLC chromatogram of standard A. catechu extract 764.13 Calibration curve of catechin by HPTLC method 764.14 TLC profile of clove oil 784.15 HPTLC profile of clove oil 784.16 HPTLC chromatogram of standard Eugenol 794.17 HPTLC chromatogram of clove oil 794.18 Calibration curve of Eugenol by HPTLC method 805.1 Prepared Gel Formulation 865.2 Calibration curve of 18-β-G.A. by HPTLC method 865.3 Calibration curve of Catechin by HPTLC method 875.4 HPTLC profile of Gel 885.5 HPTLC chromatogram of gel 885.6 HPTLC chromatogram of standard 18-β-G.A. 895.7 HPTLC chromatogram of standard catechin 895.8 HPTLC chromatogram of standard Eugenol 895.9 In vitro release profile of 18-β-G.A. in gel in Phosphate

buffer pH 6.8 at 254 nm

90

5.10 Calibration curve of Catechin by HPTLC method 916.1 Prepared Patch Formulation 976.2 Calibration curve of 18-β-G.A.by HPTLC method 976.3 Calibration curve of Catechin by HPTLC method 986.4 HPTLC profile of Patch 99

M. Pharm. (Pharmacognosy) II

List of Figures

6.5 HPTLC chromatogram of Patch 996.6 HPTLC chromatogram of standard 18-β-G.A. 1006.7 HPTLC chromatogram of standard Catechin 1006.8 HPTLC chromatogram of standard Eugenol 100

6.9In vitro release profile of 18-β-G.A. in patch in phosphate


101

6.10In vitro release profile of catechin in patch in phosphate


102

7.1 Antimicrobial Activity of gel formulation 1057.2 Antimicrobial Activity of patch formulation 106

M. Pharm. (Pharmacognosy) III

Index

Index

Chapter

NoTitle

Page

No.

List of Tales

List of Figures

I

II

1 Introduction 1-421.1

1.2

1.3

1.4

Introduction of Plants

Introduction of Buccoadhesive Drug Delivery

Introduction of Polymers

Introduction of Mouth Ulcer

References2 Review of Literature 43-58

2.1

2.2

2.3

2.4

2.5

Review Literature on Glycyrrhiza glabra and

Glycyrrhizin

Review Literature on Acaia Catechu and Catechin

Review Literature on Clove and Eugenol

Review Literature on Buccoadhesive Drug Delivery

Review Literature on Mouth Ulcer

References

3 Aim and Plan of the work 594 Evaluation of Raw materials 60-81

4.1

4.1.1

4.1.2

4.1.3

4.1.4

4.1.5

4.1.6

4.2

4.3

Experimental

Procurement of Raw Materials

Evaluation of G. glabra Whole Material and Powder

Evaluation of A. catechu Whole Material and Powder

Evaluation of Clove oil

Preparation of Extracts

Standardization of Extracts by HPTLC

Results and Discussion

Conclusion

References

5 Formulation and Evaluation of Gel82-92

M.Pharm. (Pharmacognosy)

Index

5.1

5.1.1

5.1.2

5.1.3

5.1.4

5.1.5

5.2

5.3

Experimental

Materials

Instruments

Preparation of Gel Formulations

Preparation of Standard Calibration Curves

Evaluation of Gel Formulations


Conclusion

References

6 Formulation and Evaluation of Patch93-103

5.1

5.1.1

5.1.2

5.1.3

5.1.4

5.1.5

5.2

5.3

Experimental

Materials

Instruments

Preparation of Patch Formulations

Preparation of Standard Calibration Curves

Evaluation of Patch formulations


Conclusion

References

7 Antimicrobial Activity of Selected Formulations 104-107

7.1

7.2

7.3

7.4

Introduction

Material and Method


Conclusion

References8 Summary and Conclusion 108

M.Pharm. (Pharmacognosy)

Chapter: 1.1 Introduction of Plants

A. Glycyrrhiza glabra

Source 1

Drug consists of the dried roots of Glycyrrhiza gtabra Linn. (syn. Liquiritae

officinalis Moench. ), Fam. Fabaceae. The plant is cultivated in Punjab and sub-

Himalayan tracts.

Figure: 1.1 Glycyrrhiza glabra plant and root

Classical Names 2

Yashtimadhu, Yashti. Yashtimadhuka, Madhuyashtika, Madhuka, Kleetaka,

Yashtyahva.

Vernacular Names 2

Eng.- Liquorice

Hindi- Mulhatii, Jethimadh, Jethimadhu, Mulelhi, Muleti, Mulathi

Beng.- Jashtimadhu. Jai-shbomodhu

Guj.- Jethimadha, Jethimard

Kan.- Yashti madhuka, Atimadhura, Jesthamadhu, Madhuka

Mal.-Yashtimadhukam. Atimadhuram, Irattimadhuram

Mar.- Jeshthamadh

Punj.- Mulelhi. Jethimadh

Tam.- Atimadhuram

Tel.- Yashtli-madhukam, Atimadhuramu

Arab.- Aslussus

Assam- Jesthimadhu, Yeshtmadhu

Burm.-Noe-khiyu, Noe-khiyu-asui

Kash.- Multhi

Urdu- Mulelhi, Asl-us-sus.

M. Pharm. (Pharmacognosy) 1


Botanical Description 2

It is a hardy herb or undershrub attaining a height up to 2 m.; leaves multifoliolate,

imparipinnate; flowers in axillary spikes, papilionaceous, lavender to violet in colour;

pods compressed, containing reniform seeds.

Ayurvedic Properties 2

Rasa - Madhura

Guna - Guru, Snigdha

Veerya - Sheeta

Vipaka – Madhura

Doshaghnata - Vatapittashamaka

Rogaghnata - Vranashotha, Visha, Khalitya, Palitya, Shastrabhighataja vrana,

Vatavikara, Vatarakta, Amavata, Shiroroga. Vamana, Trishna,

Vibandha,Udarashoola, Amlapitta, Paittika apasmara. Hikka, Raktavikara,

Raktalpata, Raktapitta, Shwasa, Kasa, Swarabheda, Yakshma, Urogata vrana,

Urahkshata. Parshwashoola, Mootrakrichchhra, Pooyameha. Paittika

prameha,Shukrameha, Varnavikara, Kandu, Charma roga, Jeerna jwara,

Samanya daurbalya, Netra roga

Karma – Dahashamaka, Keshya, vedanasthapana, Shothahara, Nadibalya,

Medhya, Chhardinigrahuna, Trishnanigrahana, Vatanulomana, Mridurechana,

Shonitasthapana, Kaphanissaraka, Kanthya, Mootrala, Mootruvirajaneeya,

Shukravardhaka,Varnya.,Kandughna,Jwarashamaka,Jeevaneeya,Sandhaneea,

Rasayna. Balya, Chakshushya

Parts Used 3

Root

Chemical Constituents 1

Major: Triterpenoid saponin glycyrrhizin (2-9 %), a mixture of potassium and

calcium salts of glycyrrhizinic acid

Minor: Include other triterpenoid saponons viz.,glabranin A&B, Glycerrhetol,

glabrolide, iso- glabrolide;viz., formononetin, glabrone, neoliquiritin, hispaglabridin

A&B; coumarins viz., herniarin, umbelliferone; triterpene sterols viz., onocerin, β-

amyrin, stigmasterol



Glycyrrhizin

Quantitative Standards 1

Ash values

Total ash - Not more than 10.0 %

Acid insoluble ash - Not more than 2.5 %

Extractive values

Alcohol soluble extractive -Not less than 10.0 %

Water soluble extractive - Not less than 20.0 %.

Adulratants / Substitues 4

The root of Abrus precatorious Linn. are known in the trade of Indian liquorice. Root

and Rhizome of Glycyrrhiza uralensis Fisch. ex DC. and some related plant species

are used as substitutes and adulterants of liquorice. G. uralensis contains only a small

percentage of sugar and gives a rather pungent extract.

Pharmacology

The drug possesses potent demulcent, expectorant, and anti-inflammatory properties

and these are attributed to the presence of glycyrrhizin. The latter is 50 times sweeter

than sucrose 5. Glycyrrhizin is also credited with antihepatotoxic 5,antiviral and

antibacterial 6 activities.

The drug is also use in the treatment of peptic ulcer and deglycyrrhizinated liquorice

while being substantially free from mineralocorticoid side effects of liquorice root is

clinically effective for gastric and deuodenal ulcers. It also indicates that in addition to

glycyrrhizinic acid, other unidentified constituents of the drug contribute to the

antiulcer activity 7, 8.



Clinical Studies 2

During a controlled clinical trial conducted on 92 cases of post-operative traumatic

inflammation following tonsillectomy, 28 cases were given Yashtimadhu powder in a

dose of 3 gm t.d.s. In another series of 24 cases, oxyphenbutazone 2 tablets t.d.s. were

given. On sequential analysis, the anti-inflammatory response of Yashtimadhu was

found to be equivalent to that of oxyphenbutazone. It appeared to possess a more

potent anti-pyretic and anti-exudative activity as compared to oxyphenbutazone.

Contra Indication 10

The German Commission E lists cholestatic liver disorders, liver

cirrhosishypertension, hypokalaemia, severe kidney insufficiency and pregnancy as

contraindications. It is also Contraindicated in oedema and congestive heart failure.

Potassium loss due to other drugs, like thiazide diuretics, can be increased. Thus they

should not be taken together with glycyrrhizin.

Therapeutic Category 7

Anti-inflammatory, Antiulcer

Safety Profile 1

The drug when used within the recommended dosage the treatment period is devoid

of any adverse reactions. However, if taken in excessive amounts it can cause

matabolic disturbances known as pseudoaldosteronism ( due to the mineralocortecoid

effect of glycyrrhizin) leading to oedema, hypertension and weight gain.

Toxicology 2

LD50 of glycyrrhizin and glycyrrhizin- thiamine HC1 in rats is reported to be 1.94 and

0.764 g/kg s. c. respectively. Liquiritoside a root flavonoside is a low toxic substance.

Consumption of liquorice 10-45 g. / day is reported to cause raised blood pressure,

together with a block of aldosterone/ renin axis and electrocardiogram changes.

Dose 3, 9

Powdered drug: 2-4 gm

Liquorice compound powder: 4-8 ml

Liquorice liquid extract: 2-4 ml



Formulations and Preparations 2

Yashtyadi churna, Yashtimadhvadya taila, Kalyanavaleha, Angamardaprashamana

kashaya, Brihat ashwagandha ghrita, Brihachchhagaladya ghrita, Shatavaryadi

ghritla, Nasika churna, Guduchyadi taila, Pippalyadi taila, Vyaghri taila,

Kubjaprasarani tlaila, Vridhihara lepa.

References:

1. Anonymous, “Indian Herbal Pharmacopoeia”, Indian Drug Manufacture’s

Association, Mumbai 2002, 89-97.

2. Sharma P.C., Yelne M.B, Dennis T.J., Assisted by Joshi Aruna, Prabhune Y.S.,

Khade Kundan, Rawat M.S., Database of medicinal plants used in Ayurveda,

vol-III , 561-566.

3. Anonymous, “The Ayurvedic Pharmacopoeia of India”; Ministry of health &

family welfare, Department of Health, Govt. of India, Part I,1st ed.,vol-1,

1989,127.

4. Anonymous, “The wealth of India (Raw material)”, CSIR, New Delhi, 1956,

vol-IV, 152.

5. Chandler R.E., Can. Pharm. J., 1985,118,420.

6. Kiso Y., Johkin M., Hikino H., Hattori M., Sakamoto T., and Namba T., Planta

med., 1984,50, 298.

7. Kassir Z.A., Irish Med J., 1985,78,153.

8. Longman M.J.S., Drugs , 1977,14,105.

9. Anonymous, “Pharmacopoeia of India”, Ministry of Health, Gov. of India, 2nd

Edi. 1966,406.

10. Blumenthal M (editor), 1998. Complete German Commission E monographs:

Therapeutic Guide to Herbal Medicines. American Botanical Council,

Integrative Medicine Communications, Boston, Massachusetts.



B. Acacia catechu

Source 1

Drug consists of either dried bark or dried aqueous extract known as Black catechu or

cutch, prepared from the heartwood of Acacia catechu Willd. (Syn. A. catechuoides

Wall.) : Fam. Fabaceae. Sub. fam. - Mimosaceae. The plant is distributed throghout

india mostly in dry types of forests. Mainly eastern slopes of Wastern ghats,

Andhrapradesh, Bihar, Punjab and Himalaya up to an elevation of 1500 m.

Figure: 1.2 Acacia catechu plant and extract

Classical Names 2

Khadira, Balpatra, Bahushalya, Dantadhawana, Raktasara, Yadniya, Gayatri.

Vernacular Names 2

Eng. - Cutch tree

Hindi – Khair

Beng. – Khayar

Guj. - Kherio baval

Khair, Kathe,Kher

Kan.- Kalu, Kachu, Kaggali, Kanti, Kaggal.nara, Kachinamara, Koggigida

Mal. - Karingali, Khadiram

Mar.- Kaderi, Khair

Punj.-Khair

Tam.- Karunkali, Kadiram, Karngalli

Tel.- Podalimanu,kaviri, Kachu, Kadiramu, Sandra

Assam- Kharira, Khara, Khayar, Khoria

Kash.-Kath

Urdu - Chanbe kaath




A moderate sized, deciduous tree, up to 3 m high. Leaves pinnate, with a pair of

recurved prickles at the base of the rachis. Flowers pale yellow, in cylindrical spikes.

Pods glabrous, oblong.


Rasa - Tikta, Kashaya

Guna - Loghu, Ruksha

Veerya - Sheeta

Vipaka - Katu

Prabhava - Kushthaghna

Doshaghnata - Kaphapittashamaka

Rogaghnata - Aruchi, Atisara, Kaphaja kasa, Prameha, Kushtha, Twakaroga,

Jeernajwara, Raktapitta, Krimi.

Karma-Ruchivardhaka, Stambhana, Shonitasthapana. Mutrasangrahana,

Kushlhaghna, Kandughna, Vrana ropaka.

Parts Used 2

Bark, heartwood, catechu (kattha)

Chemical Constituents 3

Major: Tannins-condensed tannins : catechutannic acid(25-30%), catechin (2-12%),l-

epicaetchin, catechu red, gum (20-30%)

Minor: flavanoids flavanols- quercitrin, quercitin

Catechin



Quantitative standards 1

Heartwood

Foreign matter: Not more than 2 %,

Ash values:

Total ash - Not more than 2 %


Extractive values

Alcohol soluble extractive -Not less than 1 %

Water soluble extractive - Not less than 3 %.

Catechu

Ash values:

Total ash- Not more than 8 %

Acid insoluble ash- Not more than 0.5 %

Extractive values

Alcohol insoluble residue- Not more than 30 %

Water insoluble residue-Net more than 25 %

Adulratants / Substitutes 4

Catechu i.e. heart wood extract can be adultrated with mineral matter viz. Clay or

starch, etc. This can be easily detected by the higher percentage of total and acid

insoluble ash. Similar extract is also obtained from leaves and young shoots of

Uncaria Gambier Rox, Family Rubiaceae. It is known as pale catechu, which has a

pale colour, and lighter in weight. The Gambier fluorescin test can be applied to

distinguish the two. Black catechu does not respond to this test.

Pharmacology

It is useful in Passive diarrhea either alone or in combination with cinnamon or

opium.

The concentrated aqueous extract, known as khayer gum or cutch is astringent 5,

cooling and digestive 6, beneficial in cough and diarrhoea6, applied externally to

ulcers6, boils and eruptions of the skin6, and is used extensively in Ayurvedic

formulations.



The extract known as catechu or cutch is used medicinally as an astringent in fevers

and other maladies 6.

The bark, in combination with other drugs, is prescribed for snake bite 5.

Hypoglycemic activity of Acacia catechu (seeda) was observed when administered to

normal albino rats7.

A. catechu shows hypotensive action 8.

Agglutinins from seeds are administered to leukaemia patients 9.

A small piece of catechu with cinnamom and nutmeg is held in toothache, loss of

voice etc, also in cases of mercurial salivation, hoarseness, relaxed sore throat,

bleeding, ulcerations and sponginess of gums. It is used for bed sores 2.

Therapeutic category 11

Antiseptic, Astringent, Antidysentric

Safety profile 1

Usage of the drug in Indian system for centuries proves it’s safety. Both in acute and

sub- acute studies on the drug have found it safe. In higher doses, hemolytic action

may be observed.

Dose 10

Crude: 3-10 gm

Dried extract: 100-340 mg

Tincture (1:5): 2.5-5 ml

Formulations and Preparations2

Khadirashtaka churna, Kadhiradikvalha, Khadiradi vati, Arimedadi taila,

Khadirarishta



References:

1. Anonymous, “Indian Herbal Pharmacopoeia”, Indian Drug Manufacture’s

Association, Mumbai 2002, Page no.1-11.


Khade Kundan, Rawat M.S., Database of medicinal plants used in Ayurveda, Vol-

I , 216-218.

3. Anonymous , “The wealth of India (Raw material)”, CSIR, New Delhi, 1985,vol-

I: A,25

4. Shah C.S and Quadry J.S., A Textbook of Pharmacognosy, B.S. Shah Prakashan,

Ahmedabad,7th edition,1990,154,157,158.

5. Anonymous, “British Pharmacopoeia”, Department of Health, British

Pharmacopoeia commission, London 1999.

6. Kirtikar K.R., Basu B.D., “Indian medicinal plants”.2nd edi., L. M. Basu,

Ahmadabad, Vol. II,926,927.

7. Singh K.N., Mittal R.K., and Barthwal K.C., et al.; Indian J Med Res, 1976,

64,754.

8. Sham K. N., Chiu K. W., Hypotensive Action Of Acacia Catechu, Planta Med,

1984, 50,177.

9. Agrawal S. and Agrawal S.S. et al; Indian J Med Res, 1990, 92: 38-42.

10. Chaudhari R.D., Herbal Drug Industry, Eastern publishers New Delhi, 1996,321.

11. Nadkarni K.M., Indian Materia Medica, Popular Prakashan, Bombay, Vol-I,

1976,11-13.



C. Syzygium aromaticum

Source 1

Drug consists of flower buds of Syzygium aromaticum (Linn.) Merrill & Perry [Syn.

Eugenia aromatica Baill; Eugenia caryophyllata Thumb ; Caryophyllus aromaticus

Linn.] Family. Myrtaceae, A tree indigenous to the Molucca islands, grown in India

in certain parts of Tamilnadu (The Nilgiris and Kanniyakumari) and Kerala

(Kottarakara and Chengannur).

Figure: 1.3 Syzygium aromaticum plant , flower buds and Oil

Classical Names 2

Lavanga, Devakusuma, Shriprasuna, Chandanapushpaka, Varija.

Vernacular Names 2

Eng. - Clove tree. Cloves

Hindi- Lavanga, Laung, Lavamg, Laumg

Beng.-Lavang, Langa

Guj.- lavang, Laving

Kan.- lavanga

Mal.- Karampu, Karayampoovu, Grampu, Chanki, Karayampu,Krambu

Mar.- Lvang, Lawangta

Punj.- Laung, Long, Karanfal

Tam.- Kiramhu, Lavangam, Kiramber, Ilavangap-pu, Karuvat-pui crambu

Tel.- lavangalu, Lavangamu, devekusumamu

Assam- lavang, Lan. Long

Kash.- Rung, Raung




A pyramidal or conical evergreen tree up to 12 m high. Leaves simple, lanceolate, in

pairs, 7-13 cm in length and 2-4 cm in breadth, gland dotted, fragrant. Flower buds

greenish to pink, aromatic, clustered at the ends of branches. Fruits fleshy, dark pink

drupes. Seeds grooved on one side, oblong.


Rasa- Katui, 'T'ikta

Guna –laghu, teekshna

Veerya - Sheeta

Vipaka - Katu

Doshagnata - Kaphapittanhamaka

Rogaghnata- Shirahshoola, Pratishyaya, Mukharoga, Charmaroga, Amavata,

Katishoola, gridharasi, Dantashoola, Dhwajabhanga, Aruchi, Agnimandya, Ajeerna,

Adhmana, udarashoola. Arnlapilta. Chhardi, Trishna. Grahani,

Visuchika,Yakridvikara, Hridduaurbalya, Raktavikara, Kasa, Shwasa, Hikka,

Kshaya,Mootraikrichchhra, Jwara, Samanya daurbalya

Karma - Raktotkleshaka, Uttejaka, Krimighna, Deepana, Pachana, Ruchya,

Mukhadurgandhanashan, Vaishadyakar, Vatanulomana, Shoolaprashamana,

Shleshmahara, Shleshmaputihara, Shwasahara, Vajikarana. Stanyajanana,

Stanyashodhana, Mootrajanana, Amapachana, Jwaraghna, katupaushtika,

Chakshushya, Vranaropana, Vranashodhana

Parts Used 3

Flower buds, oil

Chemical constituents 1

Major: volatile oil (15-20 %) containing chiefly a phenol eugenol (55-85%) and β-

caryophyllene (10-20%)

Minor: Eugenol acetate and derivative of β-caryophyllene, α-humulene and its

epoxide, acetophenone, benzyl salicylate, α-cardinol, γ-decalactone, fenchone,

hexanal, 2-hexanone, methyl palmitate, γ-muurolene, palustrol, propylbenzoate and

α-thujene.



Eugenol

Quantitative standards 1

Foreign matter: Not more than 4.0%

Ash values

Total ash - Not more than 7.0 %


Extractive values

Alcohol soluble extractive -Not less than 15.0 %

Water soluble extractive - Not less than 14.0 %

Volatile oil: Not less than 15.0%

Adulratants/substitues4

Cloves are sometimes adulterated with mother-of-cloves, clove stems, exhausted

cloves, withered cloves, clove dust containing broken stamens and flowers,

farinaceous products, cereal starches, ground fruit-stones and unripe fruits of

Cinnamomum verum J.S. Presl.

Pharmacology

Clove has a positive effect on the healing of stomach ulers5.

The sesquiterpenes of the drug are known to posses activity on inducing the

detoxifying enzyme glutathione S-transferase in mouse liver and small intestine and

the ability of natural anticarcinogens to induce such detoxifying enzymes correlates

well with their ability to inhibit chemical carcinogenesis4, 6.

The oil is antiseptic, local anaesthetic, rubefacient and useful in catarrh, cough,

bronchitis, flatulence, colic, skin diseases, dyspepsia, vomiting, odontalgia, dental

caries and cephalalgia2.

Clinical studies2

An Ayurvedic toothpaste prepared from Zingiher officinale, Terminalia chebulu.

Acacia catechu, Piper nigrum, Syzygium aromaticum, Cimiamomum zeylanicum and



camphor etc. has been investigated in 50 patients suffering from dental diseases. The

paste was highly effective in controlling the diseases.

Therapeutic category1

Carminative, stomachic

Safety profile7

Clove buds are devoid of any major side effects/toxicity. However the clove oil

should be used with caution orally and should not be used on the skin.

Toxicology 2

Essential oil from flower buds showed typical toxicity to cowpea weevil. With 1%

dose, complete mortality was recorded within 24 hours. However some mortality was

reported in 4 days at 0.75% dose.

Dose 7

Clove: 120-300 mg

Clove oil: 0.05-0.2 ml

Formulations and Preparations 2

Dashanasanskwachurna, Lavangadi churna, Brihat chandrodayamakaradhwaja,

Ashwagandhadi churna, Kumariasava, Jeerakarishta Shrikhandasava,

Saubhagyashunthi, Lavangadi vati, Lavanga chatuhsama, lavangodaka, Avipattikara

churna, Devakusumadi rasa.



References:

1. Anonymous , “Indian Herbal Pharmacopoeia”, Indian drug manufacture’s

Association, Mumbai 2002,Page no.89-97


Khade Kundan, Rawat M.S., Database of medicinal plants used in Ayurveda, Vol-

IV , 358-362.

3. Anonymous, “The Ayurvedic Pharmacopoeia of India”; Ministry of Health &

Family Welfare, Department of Health, Govt. of India, Part I, 1st ed., Part- I, vol.

I, 1989, 80-81

4. Bisset N.G. Herbal Drugs and Phytopharmaceuticals, Medpharm, Stuttgart,

1994,130

5. Zaidi S.H., Singh G.B., and Khanna N.M., Indian J Med Res, 1958, 46,732.

6. Zheng G.Q., Kenney p.m., AND Lam L.K.T., J. Nat.Prod. 1992,55,999.

7. Newall C.A., Anderson L.A., and Phillipson J.D., Herbal Medicines, The

Pharmaceutical Press, London, 1996, 79.




Chapter: 1.2 Introduction of Buccoadhesive Drug Delivery System

1.2 Introduction of Buccoadhesive Drug Delivery

The oral route is the most preferred route of drug delivery as it is convenient,

inexpensive, and versatile. However, drug delivery by this route has certain

disadvantages such as first-pass metabolism by the liver and gastrointestinal

enzymatic degradation of the drug. Therefore, other transmucosal routes such as

nasal, rectal, vaginal, ocular, and oral mucosa are being considered as alternatives to

conventional oral dosage forms for drug delivery to avoid the above disadvantages

associated with conventional oral delivery (i.e., tablets, capsules, syrups, etc.). Of

these routes of delivery, the buccal oral mucosa has emerged as one of the target sites

for administration of drugs in a wide variety of dosage forms, particularly for those

drugs targeted for local delivery in the oral cavity and systemic absorption.

The buccal route of drug delivery provides the opportunity for drug absorption

through the buccal epithelial lining of the oral cavity (mucosa of the cheek) for it to

exhibit its action locally or systemically. The noninvasive nature of administration,

ease and convenience of administration, precise localization, and increased

permeability of the buccal mucosa compared to other transepithelial routes makes this

a promising route of delivery. Also, the rich supply of blood vessels and lymphatics in

the buccal mucosa results in rapid onset of drug action for those that have the

requisite physicochemical profile 1-3.

Drugs absorbed from the buccal mucosa may directly enter the systemic

circulation by way of the jugular vein, minimizing the first-pass liver metabolism and

gastric acid- or enzyme-mediated degradation (salivary fluid has lower enzymatic

activity than gut). The presence of food or variations in the gastric emptying rate has

little or no influence on drug delivery by the buccal route. The continuous exposure of

the oral mucosal tissues to a multitude of substances and its high cellular turnover rate

makes the buccal tissue robust and less prone to local toxicity or irritation from drugs,

their dosage forms, and formulation excipients. The absence of Langerhans cells in

the oral mucosal tissues imparts tolerance to potential allergens 4. Therefore,

prolonged administration of drugs (chronic use) to the oral cavity is less prone to

induction of local tissue sensitization and allergic reactions. When compared to other

mucosal delivery routes, buccal drug delivery offers a higher degree of control and

reproducibility; it also allows the removal of the systemic dosage form (i.e., chewing

gums, lozenges, patches, etc.) to terminate drug absorption if necessary.



Figure 1.4: Types of Intraoral dosage forms

1.2.1 Advantages of Oral Mucosal Drug Delivery 5, 6

The rationale and key advantages of drug delivery to the oral cavity for local and

Systemic absorption includes:

1. Alternative to injection

2. Improved patient compliance, “patient-friendly”

3. Convenience

4. Targeted delivery to treat local diseases of the oral cavity

5. Potential route for delivery of proteins/peptides/vaccines

6. Opportunity for product line extension of quick-dissolving dosage forms

7. Dosing or administration anywhere at anytime without water

8. Increase drug bioavailability in some cases

1.2.2 Disadvantages of Oral Mucosal Drug Delivery 5, 6, 7

There are, however, a number of technical challenges to overcome in order to

effectively deliver drugs to the oral cavity for local oral mucosal absorption and

systemic delivery. The absorption of drugs from the oral mucosal tissues has the

following disadvantages.

1. Drug transport is by passive diffusion, drug absorption is low, which results in a

low bioavailability.



2. Buccal mucosa, like the small intestine, offers a lipoidal barrier and this route is

usually practical for small lipophilic molecules.

3. Only a few hydrophilic drugs or compounds such as certain amino acids and

monosaccharides have been reported to be transported via a carrier-mediated process.

4. Taste masking may be necessary for drugs that are bitter or irritable.

5. The total area for drug absorption is small (100 to 170 cm2) when compared to the

total area of gastrointestinal absorption.

6. The dosage form must be kept in place for effective absorption because salivary

flow may wash away the dissolved drug and the dosage form may be swallowed prior

to drug dissolution.

1.2.3 Intraoral Drug Delivery

For transdermal drug delivery, the rate-limiting barrier for a compound to be

absorbed into the systemic circulation is the stratum corneum, which is the keratinized

layer of the skin. Because the lining mucosa of the oral cavity is not keratinized, is

thinner (100 to 500 μm), and more permeable than skin, the degree of resistance for a

drug to penetrate the lining mucosal membrane is much less than that of skin. Similar

to the epithelium of the GIT and the skin, the epithelial lining mucosa is composed of

a constantly renewing cell population, which is produced by cell division in the basal

region.

Intraoral drug delivery overcomes hepatic first-pass metabolism and promotes

rapid systemic delivery with improved bioavailability with selected drugs having the

required physiochemical and biopharmaceutical characteristics. The oral mucosa

provides accessibility to allow for the precise localization of the dosage form for

targeted drug delivery. It also provides the opportunity to directly modify tissue

permeability, inhibit protease activity, or decrease immunogenic responses to drugs.

Therefore, the oral mucosa has emerged as one of the target sites for administration of

drugs in a wide variety of dosage forms, particularly for those drugs targeted for local

delivery in the oral cavity and those that required systemic absorption as well.1-3

Due to the relatively short cellular turnover time of the buccal mucosa, a

properly designed buccal adhesive delivery system may remain in place, under the

best circumstances, for 1 to 24 hours.

Historically, lozenges and troches, which are sucked on by the patient, and

sublingual tablets were the traditional means for delivering drugs to the oral cavity.



More recently, quick-dissolve dosage forms have become more popular, and

bioadhesive tablets and gels have been used to provide a longer contact time with the

absorbing tissue and thus minimize drug loss through clearance by salivary outflow.

Drug delivery via the membranes of the oral cavity can be subdivided as follows:

A. Sublingual delivery, which is the administration of the drug via the sublingual

mucosa (the membrane of the ventral surface of the tongue and the floor of the

mouth) to the systemic circulation.

B. Buccal delivery, which is the administration of the drug via the buccal mucosa

(the lining of the cheek and area between the gums and upper and lower lips) to the

systemic circulation.

C. Periodontal, gingival, and odontal delivery, for the local treatment of conditions

of the oral cavity, principally aphthous ulcers, bacterial and fungal infections, and

periodontal disease. These oral mucosal sites differ greatly from one another in terms

of anatomy, permeability to an applied drug, and their ability to retain the delivery

system for the desired length of time.

1.2.7 Classification of Intraoral Dosage Forms

Dosage forms targeted for delivery to the intraoral cavity can be classified in terms of

their dissolution or disintegration kinetics as either quick-dissolving (QD), slow

dissolving (SD), or nondissolving (ND), which release the drug over a period of

seconds up to 1 minute, 1 to 10 minutes, and >10 minutes to hours, respectively.

These IODs may be intended to present the drug for local release in the mouth (i.e.,

drug dissolved in saliva) or to the GIT (i.e., drug released as microparticles but not

dissolved in saliva) for subsequent systemic absorption. The various IODs may

further be targeted for release and local topical absorption by the tissues in the oral

cavity for treatment of local diseases or for systemic absorption for treatment of

diseases remote from the site of application. The IODs may be further categorized as

noninvasive (i.e., QD formulations), semi-invasive (i.e., patches, periodontal

filaments, microparticles, needleless injectors), or invasive (i.e., injection via needles,

as used in dental anesthesia, or other drug delivery devices) depending upon their

interaction with mucosal tissues and the degree to which they physically breach the

membrane barrier to absorption.



A. Quick-Dissolving Delivery Systems

QD delivery systems undergo disintegration or dissolution in the saliva, generally

within a few seconds to a minute, releasing the drug and inactive ingredients into the

oral cavity the major fraction of the drug will eventually be swallowed with the saliva

and transported along the GIT where the drug is subsequently absorbed. The technical

advantages of these dosage forms include: ease of swallowing, administration without

water anywhere and anytime, quick onset of action with some drugs, supervised

administration, buccal or sublingual absorption, and local therapy of the oral mucosa.

Therapeutic benefits of the mouth-dissolving dosage forms for patients may include:

enhanced efficacy, improved convenience, and improved compliance. Pharmaceutical

companies may benefit from these dosage forms due to product differentiation, life-

cycle management, reduction of development costs, and outsourcing. The following

therapeutic categories have been reported to have market opportunities for QD

delivery systems: nonopioid analgesics, opioid analgesics, migraine headache, cough

and cold, allergy, gastrointestinal, cardiovascular, central nervous system, urology,

and other categories.

B. Slow-Dissolving Delivery Systems

SD intraoral delivery systems are generally dissolved in the oral cavity within 1 to 10

minutes and the following products are included in this category: chewable tablets,

sublingual tablets, “lollipops,” mucoadhesive tablets, and buccal tablets. Although

there are many commercial products on the markets in the first three categories, only

a few mucoadhesive or buccal tablets have recently been introduced (i.e., Striant ®).

However, there have been numerous reports of mucoadhesive tablets investigated in

the scientific and patent literature, and some products are in the R&D pipeline.

Numerous U.S. and foreign patents as well as patent applications have been published

on buccal drug delivery with the first appearing in the late 1960s involving various

mucoadhesive polymers. These polymers include but are not limited to polyacrylic

acid, hydroxypropylmethyl cellulose, polyvinylpyrrolidone, hydroxypropyl cellulose,

vinylpyrrolidone and its copolymers, polyethylene oxide, and sodium carboxymethyl

cellulose.

C. Nondissolving Delivery Systems

ND intraoral dosage forms do not dissolve entirely when placed in the mouth and can

provide for controlled drug delivery from 10 minutes to several hours, and up to a day

or longer under the best circumstances. Examples of ND intraoral delivery systems



include the following dosage forms: chewing gums, buccal and gingival patches,

periodontal fibers, and drug delivery devices. Medicated chewing gum technology

provides a new competitive advantage for product life-cycle management for mature

and novel pharmaceuticals. This delivery system technology adds a new benefit in

efficacy, convenience, and quality of life. Chewing gum formulations offer optimal

patient controlled dose titration, an appealing mouth feel and texture, controlled

release of active substances, and can be manufactured under cGMPs. The advantages

of drug delivery in a gum formulation include fast onset of action, doses in the mg

range, few side effects, drug release for up to 60 minutes, and reduced first-pass liver

metabolism compared to oral dosage forms. A medicated chewing gum delivery

system may be ideal for drugs having limited oral absorption in conventional tablets

(i.e., nicotine) and indications where a quick onset of action is desirable, such as

treatment of migraine, pain, allergy, angina, motion sickness, and smoking cessation

may benefit.

Technologies similar to transdermal patches have been developed and designed to

adhere to either the gingival or buccal mucosa. Mucoadhesive patches (Cydot ®) have

been developed and gingival local anesthetic patches have been commercialized

(Dentipatch ®) that can provide for drug delivery over a period of from 30 minutes to

24 hours depending upon the patch design and therapeutic indication. Other controlled

drug delivery systems have been developed for the treatment of periodontal disease in

the form of thin filaments and microparticles that are compacted into the periodontal

pocket to provide for prolonged delivery of antibiotics such as tetracycline (Actisite ®

for up to 10 days).

1.2.8 Oral Mucosal Drug Delivery Systems

Several bioadhesive dosage forms for oral mucosal drug delivery have been

developed and some are already commercially available. They are mostly in the form

of patches and tablets, and some are in the form of ointments, films, and powders. The

newest approach in this area has been the use of multifunctional polymers (e.g.,

polymers possessing both bioadhesive and phase change characteristics) and/or

polymers that are bioadhesive but also capable of being penetration enhancers, or

peptide stabilizers. The following section focuses on bioadhesive drug delivery

systems such as patches and tablets, as well as the potential application of phase

change polymers.



A. Bioadhesive Tablets

Bioadhesive tablets for oral mucosal drug delivery are usually based on an erodible

tablet 18. An example of this uses crosslinked hydroxypropyl cellulose (SynchronR) as

both a bioadhesive and a drug release regulator 19. The drug (nitroglycerin) is mixed

with the bioadhesive and then compressed into tablets. Due to adhesion of the

gradually eroding polymer to the buccal mucosa, this dosage form may remain in

place for three hours and the drug release profile showed zero-order release kinetics.

They claim that the patient may talk, drink, and even eat with the tablet in place.

A two-layered tablet with hydroxypropyl cellulose and carbopol 934 as the

bioadhesive layer and a lactose nonadhesive as the backing layer was designed20. The

role of the upper (lactose) layer is to prevent drug diffusing away from the absorption

site and to allow easy placement of the tablet in the mouth. The lower layer, which

constitutes an erodible bioadhesive, together with triamcinolone acetonide as an

active ingredient provides sustained release of the drug for the treatment of local

aphtous stomatitis.

A delivery system to deliver local anesthesia for toothache also proposed 21, 22. This

three-layer tablet consists of a core containing the drug and bioadhesives

(hydroxypropyl cellulose and Carbopol 934 as before).

B. Bioadhesive Patches

Although bioadhesive patches pose a relatively new technology to pharmacy,

they have developed very quickly with the recent development of bioadhesive

technology. Generally speaking, four different types of adhesive patches have been

studied for oral mucosal drug delivery, as shown in Figure 1.5.

In these cases, the adhesive polymer serves either as a drug carrier itself, or an

adhesive layer link between a drug-loaded layer and the mucosa, or a shield to cover a

drug-containing disc. The design of these patches provides either unidirectional or

bidirectional release of the drug. The size of such systems typically varies from 1 to

16 cm 2, depending on the specific purpose of the application. Usually, 1 to 3 cm2

patches are commonly used because of convenience and comfort. However, the

administration site is also a factor. Large-size patches can be administered at the

central position of the buccal mucosa, (i.e., center of the cheek), whereas the

sublingual and gingival sites require a rather small-sized patch.



Figure 1.5: Variation in buccal patches: (a) bidirectional release from adhesive

patch by dissolution or diffusion; (b) unidirectional release from patch

embedded in an adhesive shield: (c) bidirectional release from a laminated

patch; (d) unidirectional release from a laminated patch. M: mucosa; P: polymer

with peptide; D: drug depot; S: adhesive shield; A: adhesive layer; B:

impermeable backing layer 25

A variety of polymers can be used for oral mucosal patches. This includes

water-soluble and insoluble polymers of both ionic and nonionic types 18. With

soluble polymer systems, drug release is accompanied by dissolution of the polymer;

therefore the overall drug release rate and duration are determined by both polymer

dissolution and drug diffusion, whereas in a nonsoluble hydrogel system, drug release

follows fickian or nonfickian diffusion kinetics, depending on design. The duration of

mucosal adhesion of different bioadhesive patches varies from minutes to days

depending on the type of polymer used, its amount per patch, and additional factors

such as the drying technique used to prepare the patches. Adhesive patches consisting

of two ply laminates with an impermeable backing layer and a hydrocolloid polymer

layer containing the drug has evaluated 23. The polymers they investigated were

hydroxyethylcellulose, hydroxypropylcellulose, poly(vinylpyrrolidone) and

poly(vinylalcohol). Their in vivo results indicated the adhesion time for all the above

polymers on human buccal tissue is within the range of several minutes. A patch

consisting of a mucoadhesive basement membrane, a ratelimiting center membrane,

and an impermeable facing membrane has developed. The mucoadhesive

polycarbophil permits tight attachment to the buccal mucosal and allows the patch to

remain in place for approximately 17 hours in dogs and humans regardless of eating

and drinking. A bilayer mucoadhesive polymer system (Carbopol 934 and PVP). It

consists of a fast-release layer and a sustained-release layer to achieve sustained

release of a peptide drug has reported24. This system is claimed to adhere to

gingival/alveolar mucosa for over 24 hours.



References:

1. De Vries, M.E., Bodde, H.E., Verhoef, J.C., and Junginger, H.E. (1991).

Developments in buccal drug delivery. Crit. Rev. Ther. Drug Carrier Syst., 8(3),

271-303.

2. Shojaei, A. (1998). Buccal mucosa as a route for systemic drug delivery: A

review. J.Pharm. Pharmaceut. Sci., 1, 15-30.

3. Squier, C.A. (1991). The permeability of oral mucosa. Crit. Rev. Oral Biol. Med.,

2(1),13-32.

4. Bodde, H.E., de Vries, M.E., and Junginger, E.H. (1990). Mucoadhesive polymers

for the buccal delivery of peptides, structure-adhesiveness relationships. J.

Control. Release, 13, 225-231.

5. Washington, N., Washington, C., and Wilson, C. (2001a). Physiological

Pharmaceutics-Barriers to Drug Absorption (2d ed.), New York: Taylor and

Francis.

6. Washington, N., Washington, C., and Wilson, G.C. (2001b). Drug delivery to the

oral cavity or mouth. Biological Pharmaceutics: Barriers to Drug Absorption (pp.

37-58). New York: Taylor and Francis.

7. Shojaei, A.H., Berner, B., and Li, X. (1998). Transbuccal delivery of acyclovir (I):

In vitro determination of routes of buccal transport. Pharm. Res., 15(8), 1182-

1188.

8. Zhou, X.H. Overcoming enzymatic and absorption barriers to non-parenterally

administered protein and peptide drug, J. Control Rel. 29:239-252, 1994.

9. Gutniak, M.K., Larsson, H., Sanders, S.W., Juneskans, O., Holst, J.J., and Ahren,

B. GLP-1 Tablet in type 2 diabetes in fasting and post-prandial conditions,

Diabetes Care, 20:1874-1879, 1997.

10. Merkle HP, Anders R, and Wermerskirchen A. Mucoadhesive buccal patches for

peptide delivery. In: Lenaerts V, Gurny R, eds. Bioadhesive Drug Delivery

Systems. Boca Raton, FL: CRC, 1990:105-136.

11. Schor JM, Davis SS, Nigalaye A, and Bolton S. Susadrin transmucosal tablets.

Drug Dev Ind Pharm 1983; 9:1359-1377.

12. Nagai T, Machida Y, Suzuki Y, and Ikura H. Method and preparation for

administration to the mucosa of the oral or nasal cavity. U.S. Patent 4226848,

1980.



13. Nagai T and Machida Y. Mucoadhesive dosage forms. Pharm Int 1985; 6:196-

200.

14. Ishida M, Nambu N, and Nagai T. Mucosal dosage form of lidocaine for

toothache using hydroxypropyl cellulose and carbopol. Chem Pharm Bull 1982;

30:980-984.

15. Anders R and Merkle HP. Evaluation of laminated muco-adhesive patches for

buccal drug delivery. Int J Pharm 1989; 49:231−240.

16. Lee Y and Chien YW. Oral mucosa controlled delivery of LHRH by bilayer

mucoadhesive polymer systems. J Control Rel 1995; 37:251-261.

17. Merkle HP, Anders R, Sandow J, and Schurr W. Drug delivery of peptides: the

buccal route. In: Davis SS, Illum L, Tomlinson E, eds. Delivery Systems for

Peptide Drugs. New York: Plenum, 1986:159-176.


Chapter: 1.3 Introduction of Polymers

1.3.1 Carbopol

Carbopol polymers are polymers of acrylic acid cross-linked with polyalkenyl ethers

or divinyl glycol. They are produced from primary polymer particles of about 0.2 to

6.0 micron average diameter. The flocculated agglomerates cannot be broken into the

ultimate particles when produced. Each particle can be viewed as a network structure

of polymer chains interconnected via cross-linking 1.

Carbomers were first prepared and patented in 1957 2. Since then, a number of

extended release tablet formulations, which involve carbomer matrices, have been

patented3.

Carbomers readily absorb water, get hydrated and swell. In addition to its hydrophilic

nature, its cross-linked structure and its essentially insolubility in water makes

Carbopol a potential candidate for use in controlled release drug delivery system 4, 5.

1. Nonproprietary Names

BP: Carbomers

PhEur: Carbomera

USPNF: Carbomer

2. Description

Carbopol polymers are offered as fluffy, white, dry powders (100% effective).

The carboxyl groups provided by the acrylic acid backbone of the polymer are

responsible for many of the product benefits. Carbopol polymers have an average

equivalent weight of 76 per carboxyl group 6. The general structure

can be illustrated with fig. No.1.6.

Figure 1.6: General Structure of Carbopol Polymers

M Pharm. (Pharmacognosy) 26


Figure 1.7: Schematic drawing of a molecular segment of a cross-

linked polyacrylic acid polymer

Carbopol polymers are manufactured by cross-linking process. Depending upon the

degree of cross-linking and manufacturing conditions, various grades of Carbopol are

available. Each grade is having its significance for its usefulness in pharmaceutical

dosage forms7.

Carbopol 934 P is cross-linked with allyl sucrose and is polymerized in solvent

benzene. Carbopol 71G, 971 P, 974 P are cross-linked with allyl penta erythritol and

polymerized in ethyl acetate. Polycarbophil is cross-linked polymer in divinyl glycol

and polymerized in solvent benzene. All the polymers fabricated in ethyl acetate are

neutralized by 1-3% potassium hydroxide. Though Carbopol 971 P and Carbopol 974

P are manufactured by same process under similar conditions, the difference in them

is that Carbopol 971 P has slightly lower level of cross-linking agent than Carbopol

974 P. Carbopol 71 G is the granular form Carbopol grade8.

3. Physical Properties 9

The three dimensional nature of these polymers confers some unique characteristics,

such as biological inertness, not found in similar linear polymers. The Carbopol resins

are hydrophilic substances that are not soluble in water. Rather, these polymers swell

when dispersed in water forming a colloidal, mucilage-like dispersion.

Carbopol polymers are bearing very good water sorption property. They swell in

water up to 1000 times their original volume and 10 times their original diameter to

form a gel when exposed to a pH environment above 4.0 to 6.0. Because the pKa of



these polymers is 6.0 to 0.5, the carboxylate moiety on the polymer backbone ionizes,

resulting in repulsion between the native charges, which adds to the swelling of the

polymer. The glass transition temperature of Carbopol polymers is 105°C (221°F) in

powder form.

Table 1.1: Physical and Chemical Properties of Carbopol 10

Appearance Fluffy, white, mildly acidic polymerBulk Density Approximately 208 kg/m3 (13 lbs.ft3) *Specific gravity 1.41Moisture content 2.0% maximumEquilibrium moisture

content

8-10% (at 50% relative humidity)

PKa 6.0 ± 0.5pH of 1.0% water

dispersion

2.5 - 3.0

pH of 0.5% water

dispersion

2.7 - 3.5

Equivalent weight 76 ± 4Ash content 0.009 ppm (average) **Glass transition temperature 100-105C (212-221F)

4. Rheological properties

While the relationships between structure and properties have been of interest both

academically and in industry. Different grades of Carbopol polymers exhibit different

rheological properties, a reflection of the particle size, molecular weight between

crosslinks (Mc), distributions of the Mc, and the fraction of the total units, which

occur as terminal, i.e. free chain ends 11-18.

Table 1.2: Viscosity range of different Carbopol Polymers 21, 22

Polymer Viscosity*Carbopol 934 NF 30500 – 39400

Carbopol 934 P NF 29400 – 39400Carbopol 71 G NF 4000 – 11000

5. Applications of Carbopol polymers

The readily water-swellable Carbopol polymers are used in a diverse range of

pharmaceutical applications to provide:

Controlled release in tablets 1, 23-25.

Bioadhesion in buccal 26, ophthalmic 27, 28, intestinal 29, nasal 30, vaginal 31 and

rectal 32 applications.



Thickening at very low concentrations to produce a wide range of viscosities

and flow properties in topical, lotions, creams and gels, oral suspensions and

transdermal gel reservoirs 33.

Permanent suspensions of insoluble ingredients in oral suspensions and

topicals 34.

Emulsifying topical oil-in-water systems permanently, even at elevated

temperatures, with essentially no need for irritating surfactants.

6. Bioadhesive Applications 35

Bioadhesion is a surface phenomena in which a material may be of natural or

synthetic origin, adheres or stick to biological surface, usually mucus membrane. The

concept of bioadhesion is emerging as a potential application in drug delivery due to

its applicability for bioavailability enhancement, prolongation of residence time for

drug in GIT and better contact between drug and absorbing surface.

Many hydrophilic polymers adhere to mucosal surfaces as they attract water from the

mucus gel layer adherent to the epithelial surface. This is the simplest mechanism of

adhesion and has been defined as “adhesion by hydration” Various kinds of adhesive

force, e.g. hydrogen bonding between the adherent polymer and the substrate, i.e.

mucus, are involved in mucoadhesion at the molecular level. Carbopol polymers have

been demonstrated to create a tenacious bond with the mucus membrane resulting in

strong bioadhesion.

Many commercial oral and topical products available today and under investigation

have been formulated with Carbopol polymers, as they provide numerous benefits in

bioadhesive formulations.

Benefits in Bioadhesive Applications

Improve bioavailability of certain drugs.

Enhance patient compliance (fewer doses are needed per day)

Lower concentrations of the active ingredients can be used.

Provide excellent adhesion forces.

7. Oral Care Applications 36,37

Carbopol polymers impart several desirable characteristics to toothpaste formulations

like Viscosity, Yield Value, Low thixotropy and Clarity.

Imparting viscosity at very low concentrations to thicken a system is a primary

function of the polymers. Suspending abrasives and solid actives is accomplished

through the build of yield value at low polymer concentrations. The combination of



Carbopol polymers’ ability to build yield value with low thixotropy provides for a

clean, non-stringing ribbon of toothpaste. From aesthetic and practical perspectives

this means that Carbopol toothpaste formulations are pumpable, leave minimal solids

residue on the tube rim, stand up well on the brush, and can be used in clear

formulations.

Benefits in Oral care Applications:

Efficient co-binders at low usage levels.

Suspending agents for non-soluble actives or excipients.

Thicken peroxide gel systems while maintaining product stability.

Compatible with commonly used formulation ingredients.

References:

1. At Florence, Pu, Jani: “Novel Oral-Drug Formulations-Their Potential in

Modulating Adverse-Effects” Drug Saf., 1994., 410(3), 233-266.

2. H P Brown, US Patent No. 2798053 (1957)

3. B F Goodrich Bulletin: “Sustained Release Patents using Carbopol Resin” ,B F

Goodrich Company, Cleveeland, OH, 1987.



4. Carnali J O, Naser M S: “The Use of Dilute Solution Viscosity to Characterize

the Network Properties of Carbopo® Microgels,” Colloid& Polymer Science,

1992,270(2),183-193.

5. Garcia-Gonzalez N, Kellaway, I W, Blanco, Fuente H, Anguiano, Igea S,

Delgado, Charro B,Otero, Espinar F J, Mendez J: “Influence of

Glycerol Concentration and Carbopol Molecular Weight on Swelling and Drug

Release Characteristics of Metoclopramide Hydrogels” Int. J.Pharm.,1994,

104,107-113.

6. B F Goodrich Company Technical Literature: Carbopol Resin Handbook, 1991

7. Alexander P, Organic Rheological Additives, Mfg. Chem., 1986,57(10),81-84.

8. Noveon Company Technical Literature, 2005.

9. Martindale – The Complete Drug Reference, 33rd Edition 2002 (1499).

10. Handbook of Pharmaceutical Excipients, Washington DC, American

Pharmaceutical Association / Pharmaceutical Society of Great Britain, 1986,41-

42.

11. Lochhead R Y, Davidson J A, Thomas G M: “Polymers in Aqueous Media”,

1989,13-147.

12. Meyer R J, Cohen L, “The Rheology of Natural and Synthetic Hydrophilic

Polymer Solutions as Related to Suspending Ability,” J. Soc. Cosmetic Chemists,

presented November 1958, New York City, 1958.

13. Flory, Paul J., Principles of Polymer Chemistry, Cornell University Press.

14. Reloar L R G: “The Physics of Rubber Elasticity, Oxford University Press”, New

York, 1958.

15. Advances in Polymer Science, Vol. 109 & 110, Responsive Gels: I & II,

Springer-Verlag.

16. Peppas N A: “Characterization of Cross-linked Structure of Hydrogels,”

Hydrogels Med.Pharm. , 1986,1, 27-56.

17. Brannon-Peppas L, “Preparation and Characterization of Cross-linked Hydrophilic

Networks,” Sud. Polym. Sci. , 1990, 8, 45-66.

18. Hooper H H et al: “Swelling Equilibrium for Positively Ionized Poly(acrylamide)

Hydrogels,” Macromolecules, 23(4) , 1990, 1096-1104.

19. Taylor, Bagley: “Tailoring Closely Packed Gel-Particles Systems for Use as

Thickening Agents,” J. Appl. Polym. Sci. , 1977, 21, 113-122.



20. Taylor, Bagley: “Rheology of Dispersions of Swollen Gel Particles,”J. Polym.

Sci.: Polymer Physics Edition, 1975, 13, 1133-1144.

21. Nae H N, Reichert W W: “Rheological Properties of Lightly Cross-linked

Carboxy Copolymers in Aqueous Solutions,” Rheologica Acta,31, 1992 ,(4),

351-360.

22. Ishikawa S et al: “Evaluation of the Rheological Properties of Various Kinds of

Carboxyvinyl polymer Gels,” Chem. Pharm. Bull, 1988, 36(6),2118-2127.

23. Choulis N H, Papadopoulos H, Choulis M: “Long Acting Methadone,”

Pharmazie, 1976, 31, H 7.

24. Durrani, Manzer J, Whitaker, Roy, Benner, Samuel C: “A Comparative Study of

Controlled Release Agents- I: Effects of Compression Force and Polymer

Concentration,” Amer. Assoc Pharm. Sci., (Nov.), 1992.

25. Perez-Marcos B, Gutierrez C, Gomez-Amoza J L, Martinez-Pacheco R, Souto C,

Concheiro A: “Usefulness of Certain Varieties of Carbomer in the Formulation of

Hydrophilic Furosemide Matrices,” Int. J. Pharm. , 1991, 67(2), 113-121.

26. Guo J H: “Investigating the Bioadhesive Properties of Polymer Patches for Buccal

Drug Delivery (Carbopol934P),” J. Control. Release. ,1994, 28(1), 272-273.

27. Davies N M, Farr S J, Hadgraft J, Kellaway I W: “Evaluation of Mucoadhesive

in Polymers Ocular Drug Delivery-II. Polymer-Coated Vesicles ” Pharm. Res. ,

1992,9(9), 1137-44.

28. Akiyama Y, Nagahara N, Hirai S, Toguchi H: “ In Vitro and In Vivo Evaluation

of Mucoadhesive Microspheres Prepared for the Gastrointestinal Tract Using

Polyglycerol Esters of Fatty Acids and a Poly(Acrylic Acid) Derivative,”

Pharm. Res. , 1995,12(3), 397-405.

29. ElHady S S A, Mortada N D, Awad G A S, Zaki NM, Taha RA, “Development of

insitu gelling and mucoadhesive Mebeverine Hydrochloride solution for rectal

administration” Saudi Pharm. J. , 2003,11(4), 59-169.

30. Al-Khamis K I, Davis S S, Hadcraft, J: “Microviscosity and Drug Release from

Topical Gel Formulations,” Parma. Res. , 1986, 3(4), 214-217.

31. Briede R H: “ Application of Carbomer water Gel 1%”, Pharm. Week,1983,

118(9), 170-174.

32. Berney B M, Deasy P B, “ Evaluation of Carbopol 934P as a suspending agent

for Sulfademidine suspensions” Int. J. Pharm. , 1979,3(2-3), 73-80.



33. Anlar S, Capan Y, Hincal A., A: “Physico-Chemical and Bioadhesive Properties

of Polyacrylic Acid Polymers,” Pharmazie, 1993, 48(4), 285-287.

34. Chang H S, Park H, Kelly P, Robinson J R: “ Bioadhesive Polymers as Platforms

for Oral Controlled Release Drug Delivery-II: Synthesis and Evaluation of Some

Swelling, Water- insoluble Bioadhesive Polymers,” J. Pharmacol. Sci. 1985,

74, 229.

35. Smart J D: “Some Formulation Factors Influencing the Rate of Drug Release from

Bioadhesive Matrices,” Drug Devel. Ind. Pharm. , 1992, 18(2), 223-232.

36. Lehr C M, Bouwstra J A, Tukker J J, Verhoef A C, de Boer A G, Junginger, H E,

Breimer D D: “Oral Bioadhesive Drug Delivery Systems - Effects on G.I. Transit

and Peptide Absorption,” Pharm. Res., 7(9), Sep 1990 (Suppl.) PDD 7226, 1990.

37. Longer M A, Chang H S, Robinson J R: “Bioadhesive Polymers as Platforms for

Oral Controlled Drug Delivery- III: Oral Delivery of Chlorothiazide Using a

Bioadhesive Polymer,” J. Pharm. Sci. , 1985, 74(4), 406-411.

1.3.2 Hypromellose

1. Nonproprietary Names

BP: Hypromellose

JP: Hydroxypropylmethylcellulose

PhEur: Hypromellosum

USP: Hypromellose



2. Synonyms

Benecel MHPC; E464; hydroxypropyl methylcellulose; HPMC; Methocel;

methylcellulose propylene glycol ether; methyl hydroxypropylcellulose; Metolose;

Tylopur.

3. Chemical Name and CAS Registry Number

Cellulose hydroxypropyl methyl ether [9004-65-3]

4. Empirical Formula and Molecular Weight

The PhEur 2005 describes hypromellose as a partly O-methylated and O-(2-

hydroxypropylated) cellulose. It is available in several grades that vary in viscosity

and extent of substitution. Grades may be distinguished by appending a number

indicative of the apparent viscosity, in mPa s, of a 2% w/w aqueous solution at 20°C.

Hypromellose defined in the USP 28 specifies the substitution type by appending a

four-digit number to the nonproprietary name: e.g., hypromellose 1828. The first two

digits refer to the approximate percentage content of the methoxy group (OCH3). The

second two digits refer to the approximate percentage content of the hydroxypropoxy

group (OCH2CH(OH)CH3), calculated on a dried basis. Molecular weight is

approximately 10 000–1 500 000.

5. Structural Formula

where R is H, CH3, or CH3CH(OH)CH2

Figure 1.8: Schematic representation of Hypromellose

6. Functional Category

Coating agent; film-former; rate-controlling polymer for sustained release; stabilizing

agent; suspending agent; tablet binder; viscosity-increasing agent.

7. Applications in Pharmaceutical Formulation or Technology



Hypromellose is widely used in oral, ophthalmic and topical pharmaceutical

formulations.

In oral products, hypromellose is primarily used as a tablet binder, 1 in film-coating, 2–7

and as a matrix for use in extended-release tablet formulations 8–12. Concentrations

between 2% and 5% w/w may be used as a binder in either wet- or dry-granulation

processes. High viscosity grades may be used to retard the release of drugs from a

matrix at levels of 10–80% w/w in tablets and capsules.

Depending upon the viscosity grade, concentrations of 2–20% w/w are used for film-

forming

solutions to film-coat tablets. Lower-viscosity grades are used in aqueous film-coating

solutions, while higher-viscosity grades are used with organic solvents. Examples of

filmcoating materials that are commercially available include AnyCoat C, Spectracel,

and Pharmacoat.

Hypromellose is also used as a suspending and thickening agent in topical

formulations. Compared with methylcellulose, hypromellose produces aqueous

solutions of greater clarity, with fewer undispersed fibers present, and is therefore

preferred in formulations for ophthalmic use. Hypromellose at concentrations between

0.45–1.0% w/w may be added as a thickening agent to vehicles for eye drops and

artificial tear solutions.

Hypromellose is also used as an emulsifier, suspending agent, and stabilizing agent in

topical gels and ointments. As a protective colloid, it can prevent droplets and

particles from coalescing or agglomerating, thus inhibiting the formation of

sediments.

In addition, hypromellose is used in the manufacture of capsules, as an adhesive in

plastic bandages, and as a wetting agent for hard contact lenses. It is also widely used

in cosmetics and food products.

8. Description

Hypromellose is an odorless and tasteless, white or creamy-white fibrous or granular

powder.

10. Typical Properties



Acidity/alkalinity: pH = 5.5–8.0 for a 1% w/w aqueous solution.

Ash: 1.5–3.0%, depending upon the grade and viscosity.

Auto ignition temperature: 360°C

Density (bulk): 0.341 g/cm3

Density (tapped): 0.557 g/cm3

Density (true): 1.326 g/cm3

Melting point: browns at 190–200°C; chars at 225–230°C. Glass transition

temperature is 170–180°C.

Moisture content: Hypromellose absorbs moisture from the atmosphere; the amount

of water absorbed depends upon the initial moisture content and the temperature and

relative humidity of the surrounding air.

Solubility: Soluble in cold water, forming a viscous colloidal solution; practically

insoluble in chloroform, ethanol (95%), and ether, but soluble in mixtures of ethanol

and dichloromethane, mixtures of methanol and dichloromethane, and mixtures of

water and alcohol. Certain grades of hypromellose are soluble in aqueous acetone

solutions, mixtures of dichloromethane and propan-2-ol, and other organic solvents.

Specific gravity: 1.26

Viscosity (dynamic): A wide range of viscosity types are commercially available.

Aqueous solutions are most commonly prepared, although hypromellose may also be

dissolved in aqueous alcohols such as ethanol and propan-2-ol provided the alcohol

content is less than 50% w/w. Dichloromethane and ethanol mixtures may also be

used to prepare viscous hypromellose solutions. Solutions prepared using organic

solvents tend to be more viscous; increasing concentration also produces more

viscous solutions.

To prepare an aqueous solution, it is recommended that hypromellose is dispersed and

thoroughly hydrated in about 20–30% of the required amount of water. The water

should be vigorously stirred and heated to 80–90°C, then the remaining hypromellose

should be added. Sufficient cold water should then be added to produce the required

volume.

When a water-miscible organic solvent such as ethanol (95%), glycol, or mixtures of

ethanol and dichloromethane are used, the hypromellose should first be dispersed into

the organic solvent, at a ratio of 5–8 parts of solvent to 1 part of hypromellose. Cold

water is then added

to produce the required volume.



11. Stability and Storage Conditions

Hypromellose powder is a stable material, although it is hygroscopic after drying.

Solutions are stable at pH 3–11. Increasing temperature reduces the viscosity of

solutions. Hypromellose undergoes a reversible sol–gel transformation upon heating

and cooling, respectively. The gel point is 50–90°C, depending upon the grade and

concentration of material.

Aqueous solutions are comparatively enzyme-resistant, providing good viscosity

stability during long-term storage 13. The aqueous solutions are liable to microbial

spoilage and should be preserved with an antimicrobial preservative.

Hypromellose powder should be stored in a well-closed container, in a cool, dry

place.

12. Incompatibilities

Hypromellose is incompatible with some oxidizing agents. Since it is nonionic,

hypromellose will not complex with metallic salts or ionic organics to form insoluble

precipitates.

13. Method of Manufacture

A purified form of cellulose, obtained from cotton linters or wood pulp, is reacted

with sodium hydroxide solution to produce a swollen alkali cellulose that is

chemically more reactive than untreated cellulose. The alkali cellulose is then treated

with chloromethane and propylene oxide to produce methyl hydroxypropyl ethers of

cellulose. The fibrous reaction product is then purified and ground to a fine, uniform

powder or granules.

14. Safety

Hypromellose is widely used as an excipient in oral and topical pharmaceutical

formulations. It is also used extensively in cosmetics and food products.

Hypromellose is generally regarded as a nontoxic and nonirritant material, although

excessive oral consumption may have a laxative effect 14. The WHO has not specified

an acceptable daily intake for hypromellose since the levels consumed were not

considered to represent a hazard to health 15-16.



15. Handling Precautions

Observe normal precautions appropriate to the circumstances and quantity of material

handled. Hypromellose dust may be irritant to the eyes and eye protection is

recommended. Excessive dust generation should be avoided to minimize the risks of

explosion. Hypromellose is combustible.

References:

1. Chowhan ZT. Role of binders in moisture-induced hardness increase in compressed

tablets and its effect on in vitro disintegration and dissolution. J Pharm Sci 1980;

69: 1–4.

2. Rowe RC. The adhesion of film coatings to tablet surfaces – the effect of some

direct compression excipients and lubricants. J Pharm Pharmacol 1977; 29: 723–

726.

3. Rowe RC. The molecular weight and molecular weight distribution of

hydroxypropyl methylcellulose used in the film coating of tablets. J Pharm

Pharmacol 1980; 32: 116–119.

4. Banker G, Peck G, Jan S, Pirakitikulr P. Evaluation of hydroxypropyl cellulose and

hydroxypropyl methyl cellulose as aqueous based film coatings. Drug Dev Ind

Pharm 1981; 7: 693–716.

5. Okhamafe AO, York P. Moisture permeation mechanism of some aqueous-based

film coats. J Pharm Pharmacol 1982; 34 (Suppl.): 53P.

6. Alderman DA, Schulz GJ. Method of making a granular, cold water dispersible

coating composition for tablets. United States Patent No. 4,816,298; 1989.

7. Patell MK. Taste masking pharmaceutical agents. United States Patent No.

4,916,161; 1990.

8. Hardy JG, Kennerley JW, Taylor MJ, et al. Release rates from sustained release

buccal tablets in man. J Pharm Pharmacol 1982; 34 (Suppl.): 91P.

9. Hogan JE. Hydroxypropylmethylcellulose sustained release technology. Drug Dev

Ind Pharm 1989; 15: 975–999.

10. Shah AC, Britten NJ, Olanoff LS, Badalamenti JN. Gel-matrix systems exhibiting

bimodal controlled release for oral delivery. J Control Release 1989; 9: 169–175.

11. Wilson HC, Cuff GW. Sustained release of isomazole from matrix tablets

administered to dogs. J Pharm Sci 1989; 78: 582–584.



12. Dahl TC, Calderwood T, Bormeth A, et al. Influence of physicochemical

properties of hydroxypropyl methylcellulose on naproxen release from sustained

release matrix tablets. J Control Release 1990; 14: 1–10.

13. Banker G, Peck G, Williams E, et al. Microbiological considerations of polymer

solutions used in aqueous film coating. Drug Dev Ind Pharm 1982; 8: 41–51.

14. Anonymous. Final report on the safety assessment of hydroxyethylcellulose,

hydroxypropylcellulose, methylcellulose, hydroxypropyl methylcellulose and

cellulose gum. J Am Coll Toxicol 1986; 5(3): 1–60.

15. FAO/WHO. Evaluation of certain food additives and contaminants. Thirty-fifth

report of the joint FAO/WHO expert committee on food additives. World Health

Organ Tech Rep Ser 1990; No. 789.

16. Lewis RJ, ed. Sax’s Dangerous Properties of Industrial Materials, 11th edn. New

York: Wiley, 2004: 2054.


Chapter: 1.4 Introduction of Mouth Ulcer

Mouth ulcers are open sores, which appear in the mouth. They are white or yellow in color, and are normally accompanied with a sharp pain, which is felt most when the person is eating. The pain is quite sharp when salty or spicy food passes over the ulcer. Mouth ulcers can occur wherever in the mouth - on the inner surface of the cheeks, lips, tongue, palate and at the base of the gums 1.

Figure 1.9 Mouth Ulcer

Symptoms 2

The symptoms preceding the ulcer may vary according to the cause of the ulcerative

process.

Some oral ulcers may begin with a sharp stinging or burning sensation at the site of

the future mouth ulcer. In a few days, they often progress to form a red spot or bump,

followed by an open ulcer. Sometimes this takes a little bit longer, depending on the

cause of the ulcer.

The oral ulcer appears as a white or yellow oval with an inflamed red border.

Sometimes a white circle or halo around the lesion can be observed. The grey, white,

or yellow coloured area within the red boundary is due to the formation of layers of

fibrin, a protein involved in the clotting of blood. The ulcer, which itself is often

extremely painful, especially when agitated, may be accompanied by a painful

swelling of the lymph nodes below the jaw, which can be mistaken for toothache.

Causes of mouth ulcers1

1. Incorrect diet

2. Digestive problems like constipation

3. Hormonal imbalances, particularly during puberty in girls

4. Anemia

5. Constant stress

6. Genetic factors

7. Illness of the herpes simplex virus

M.Pharm. (Pharmacognosy) 40


8. Irritation by a few chemicals, tobacco and alcohol

In some cases there are infectious agents that are both bacterial and viral in nature that

are considered as causes of mouth ulcers. The various chemical compounds that are

found within the infectious agents are perhaps one of the reasons for mouth ulcers

forming 3.

Possible Complications 3

Cellulitis of the mouth, from secondary bacterial infection of ulcers

Dental infections (tooth abscesses)

Oral cancer

Spread of contagious disorders to other people

Treatment of mouth ulcers 2, 4

Treatments based on antibiotics and steroids are reserved for severe cases, and should

be used only under medical supervision.

Some doctors may also prescribe local anaesthetic, such as lidocaine, for cases of

multiple or severe oral ulcers.

Some people benefit from using the over-the-counter topical gel Bonjela, which

contains choline salicylate -- choline salicylate is a local analgesic that helps to reduce

the pain and inflammation associated with oral ulcers.

No single factor is solely responsible for the initiation of Aphthous lesion. In addition,

nutrient deficiencies need to be corrected and anti-inflammatory nutrients prescribed.

Herbs which are useful in the Treatment of Mouth Ulcers1

1. Licorice ( Glycyrrhiza glabra)

Licorice is used for a number of oral and dental problems. It is a piece of several

toothpaste brands. Its stem and leaves wash the mouth effectively. Apart from

controlling the sores, it can refresh the mouth and wash the teeth.

2. Kattha (Acacia catechu)

Kattha is an extract of the Acacia catechu plant, known normally in the west as the

catechu plant. This has caustic properties. It has a extraordinary place in Ayurvedic

medicine in oral treatment. It is used in many states or forms for the treatment of

ulcers.


http://adam.about.com/encyclopedia/infectiousdiseases/Tooth-abscess.htm

http://adam.about.com/encyclopedia/infectiousdiseases/Cellulitis.htm


3.Banyan(Ficusreligiosa)

A decoction of the bark of the banyan tree decreases the pain caused by mouth ulcers.

4. Chebulic myrobalan (Tarminalia chebula)

The chebulic myrobalan is a component of the Triphala choorna, of which the amalaki

is also a necessary component. Its bark helps in decreasing the pain of the ulcers. It

also helps in correcting the constipation troubles, which causes ulcers.

5. Fenugreek (Trigonella foenum graecum)

Fenugreek leaves assists in the treatment of mouth ulcers. A mixture of these leaves is

used for gargling. Fenugreek is a strong mediator on the ulcers. Hence it is used as a

medicine for the recurrent ulcers.

6. Henna (Lawsonium alba)

Henna is a cooling herb and this herb can provide a soothing effect on the ulcers. It is

used with water for gargling to prevent mouth ulcers.

7. Indian Gooseberry (Emblica officinalis)

The Indian gooseberry or amalaki has a dual impact on ulcers. Used as a gargling

solution, it can loosen up the pain of the ulcers. Secondly it can deal with

constipation, which is usually one of the important factors causing ulcers in the

mouth.

8. Turmeric (Curcuma longa)

Turmeric is also a cooling agent as this helps in relieving mouth ulcers. It is mixed in

water and the suspension can be used for gargling for mouth ulcers treatment.

References :

1) http://www.himalayahomeremedies.com/homeremedies_mouthulcers.htm

2) http://www.medic8.com/healthguide/articles/mouthulcers.html

3) http://ulcertreatmentinfo4u.com/Causes-Of-Mouth-Ulcers.php

4) Joseph E.P., Michale T.M., Text Book of Natural Medicine, Vol-II, II nd edi.

1085.


http://ulcertreatmentinfo4u.com/Causes-Of-Mouth-Ulcers.php

http://www.medic8.com/healthguide/articles/mouthulcers.html

http://www.himalayahomeremedies.com/homeremedies_mouthulcers.htm

Chapter: 2.1 Review of Literature on Glycyrrhiza glabra and Glycyrrhizin

Jeff burgess et al., reviewed Over-the-counter Treatments for Aphthous Ulceration

and Results from Use of a Dissolving Oral Patch Containing Glycyrrhiza Complex

Herbal Extract. The aim of this article was to present a review of over-the-counter

(OTC) treatment strategies used for aphthous ulcerations and to provide results from

the use of an herbal extract containing glycyrrhiza.

Haley, Jeffrey T have patented Licorice root extract oral patch for treating canker

sores. A method for treating mouth ulcers (canker sores/aphthous ulcers) with licorice

extracts oral patches to speed healing and relieve pain. If licorice extract is applied to

a mouth ulcer using an adhesive oral patch that delivers the medication for at least 30

minutes and the patches are used for at least two or more hours per day, the method

reduces the healing times from typically 10–14 days to typically 2 days. The licorice

extract patches also quickly reduces canker sore pain and, if used before commencing

a meal, reduces pain during the meal.

Deb. soumitra and mandal, Kumar S. have developed an estimation method of 18

B-glycyrrhetinic acid obtained from glycyrrhizin in Glycyrrhiza glabra (yastimadhu)

by Thin Layer Chromatography- Densitometric method. Beer’s law is obeyed in the

concentration range of 0.48 to 2.4 g/l. the recovery is 89-107%. The method is

simple and is applicable both for crude drug and polyherbal formulations containing

yastimadhu.

Chauhan S.K. et al., have developed and described a reverse phase high pressure

liquid chromatography method to determine glycyrrhizin in G. glabra and its extract.

The method involved separation of compound using the mobile phase Acetonitrile:

Water: Phosphoric acid (32:67:1) and detection of chromatogram at 250 nm using

photodiode array detector. The sensitivity of the method was observed to be 2.0 g

and the linearity was observed in the range of 2.0 g-16.0 g/l. The propose method

being precise, sensitive and reproducible, can be used for det5ection, monitoring and

quantification of glycyrrhizin from Glycyrrhiza glabra and its extract.

Chauhan S.K. et al., have developed and described a simple reproducible HPTLC

method for the determination of glycyrrhizin from Glycyrrhiza glabra and its

extract .The sensitivity was found to be linear in the range of 0.2 to 1.0 g. the

proposed method being precise, sensitive and reproducible can be used for detection,

monitoring and quantification of glycyrrhizin from Glycyrrhiza glabra and its

extract.



Racková et. Al., studied the Mechanism of anti-inflammatory action of liquorice

extract and glycyrrhizin. The antiradical activity, protective effect against lipid

peroxidation of liposomal membrane, and inhibitory effect on whole blood reactive

oxygen species (ROS) liberation of Glycyrrhiza glabra crude extract and glycyrrhizin,

its major compound, were assessed. The liquorice extract showed significant activity

in all the three assay systems used in a dose dependent manner. It displayed

remarkable reactivity with free stable 1,1'-diphenyl-2-picrylhydrazyl (DPPH) radical,

inhibitory efficacy in peroxidatively damaged unilamellar dioleoyl

phosphatidylcholine (DOPC) liposomes, and inhibition of ROS chemiluminescence,

generated by whole blood, induced by both receptor-bypassing stimuli (PMA) and

receptor operating stimuli (Opz) in the ranking order of stimuli PMA> Opz. These

activities may be attributed to phenolic antioxidants involving isoflavan derivatives,

coumarins and chalcones. Nonetheless, triterpene saponin glycyrrhizin exhibited no

efficacy in the system of DPPH reaction and peroxidation of liposomal membrane,

and negligible inhibition of chemiluminescence generated by inflammatory cells.

These results indicate that the mechanism of anti-inflammatory effect of glycyrrhizin

most probably does not involve ROS and this major constituent is not responsible for

the inhibition effects of liquorice extract on neutrophil functions.

Gupta V.K. et. al., investigated the antimicrobial potential of Glycyrrhiza glabra

roots. Antimycobacterial activity of Glycyrrhiza glabra was found at 500 μg/ml

concentration. Bioactivity guided phytochemical analysis identified glabridin as

potentially active against both Mycobacterium tuberculosis H37Ra and H37Rv strains at

29.16 μg/ml concentration. It exhibited antimicrobial activity against both Gram-

positive and Gram-negative bacteria. Our results indicate potential use of licorice as

antitubercular agent through systemic experiments and sophisticated anti-TB assay.



References:

1. Jeff burgess, Peter van der ven, Michael Martin, Jeffrey Sherman, The Journal of

contemporary dental practice, March 2008, 9(3).

2. Haley, Jeffrey T., Licorice root extract oral patch for treating canker sores , United

States Patent 7201930

3. Deb. Soumitra and Mandal, Kumar S., An estimation method of 18 B-

glycyrrhetinic acid obtained from glycyrrhizin in Glycyrrhiza glabra (yastimadhu)

by Thin Layer Chromatography- Densitometric method, Indian Drugs, November

1999, 36(11), 687-688.

4. Chauhan S.K. et.al., Estimation of glycyrrhizin from Glycyrrhiza glabra and its

extract by High pressure liquid chromatography, Indian drugs, Aug 1999,36(8),

521-524.

5. Chauhan S.K. et. al., Determination of glycyrrhizin from Glycyrrhiza glabra and

its extract by HPTLC, Indian journal of pharmaceutical science, July-Aug

1998,60(4),251-254.

6. Rackova, Lucia et al., Mechanism of anti-inflammatory action of liquorice extract

and glycyrrhizin, Natural Product Research, December 2007, 21(14), 1234-1241.

7. Gupta V. K. et al., Antimicrobial potential of Glycyrrhiza glabra roots, Journal of

Ethnopharmacology, March 2008, 116 (2), Pages 377-380.


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Chapter: 2.2 Review of Literature on Acaia Catechu and Catechin

Singh K.N. and Lal B., noted Traditional Uses of Khair (Acacia catechu Willd.) by

Inhabitants of Shivalik Range in Western Himalaya. Katha or decoction of heartwood

is applied in mouth and on tongue to cure mouth ulcer. It is also applied externally on

ulcers, boils, skin eruptions and on gums as disinfectant.

Desai D.S. and Laddha K. S. have developed a stability indicating high performance

thin layer chromatographic (HPTLC) method for quantification of catechin. The

mobile phase was Chloroform: Ethyl acetate: Methanol: Toluene: Formic acid

(12:8:4:2:2). The calibration curve of catechin in methanol was linear in the

concentaration range of 800-2800 ng. The mean values of correlation coefficient,

slope and intercept were 0.9986 + 0.046, 477.363 + 3.961,266329 + 7.492. The limit

of detection of catechin was 25ng and limit of quantification was 85ng.no interference

was found from decomposition products. The percent recovery of catechin using

described procedure was 102.91 + 0.0123 from green tea and 98.805 +1.438 from

pale catechu. Concentration of catechin from green tea, black tea pale catechu and

black catechu was estimated.

Stuart (1979) has referred the use of the boiled and strained aqueous extract of the

heartwood as an astringent for inflamed conditions of the throat, gums, and mouth and

also externally for boils and chronic ulcers.

Sane R.T. et al., studied Spectrophotometric method for the determination of

cyanidalol (+ catechin) from pharmaceutical preparations. The methods use the

formation of coloured species of the drug with reagent like para-amino-phenol,

resorcinol, phosphomolybdic acid, folin and ciocalteau’s phenol and

parapenylediamine dihydrochloride (PPDA) in basic medium. The absorbance values

of the coloured species are measured at the wavelength of maximum absorption. The

methods are statistically validated and are found to be precise and accurate.

Sawant S.S. et al., studied the method of estimation of catechin from Acacia catechu

by HPTLC. This method could be used for routine analysis of catechin from acacia

catechu since it is relatively simple, sensitive and accurate.

Veluri R. et al., studied Phytotoxic and Antimicrobial Activities of Catechin

Derivatives. (±) - Catechin is a potent phytotoxin, with the phytotoxicity due entirely

to the (−)-catechin enantiomer. (+)-Catechin, but not the (−)-enantiomer, has

antibacterial and antifungal activities. Tetramethoxy, pentaacetoxy, and cyclic

derivatives of (±)-catechin retained phytotoxicity. The results indicate that antioxidant

properties of catechins are not a determining factor for phytotoxicity. A similar


Chapter: 2.2 Review of Literature on Acaia Catechu and Catechin

conclusion was reached for the antimicrobial properties. Centaurea maculosa (spotted

knapweed) exudes (±)-catechin from its roots, but the flavanol is not re-absorbed and

hence the weed is not affected. The much less polar tetramethoxy derivative may,

however, be absorbed and hence be able to cause toxicity. Because of the combination

of phytotoxicity and antimicrobial activity, (±)-catechin could be a useful natural

herbicide and antimicrobial.

Li ZhongXing et al., studied in-vitro study for anti-bacterial activity of Acacia

catechu (L.) Willd in 308 strains of clinical isolates 100 g of A. catechu was decocted

to make 200 ml of 50% catechu solution. Then different catechu concentrations were

made and used to study their antibacterial activity. Bacteria were isolated from

patients' blood, urine, abscesses and phlegm. These were Staphylococcus aureus (112

strains), Staphylococcus epidermidis (112 strains), Enterobacter aerogenes (28

strains), Klebsiella pneumoniae (28 strains) and Escherichia coli (28 strains). It was

found that the minimum inhibitory concentration (MIC) 50 values for catechu were

0.59, 1.19 and 1.19 mg/ml for S. aureus, S. epidermidis and E. aerogenes,

respectively, while the MIC 90 values were 1.19, 2.38 and 1.19 mg/ml. The MIC 90

value was 1.19 mg/ml for both K. pneumoniae and E. coli. It was concluded that

catechu has strong inhibitory activity on Gram +ve cocci and Gram -ve bacilli.

References:

1. Singh K.N. Singh and Lal B., Traditional Uses of Khair (Acacia catechu Willd.)

by Inhabitants of Shivalik Range in Western Himalaya, Ethnobotanical Leaflets

10, 2006, 109-112.

2. Desai D.S. and Laddha K. S., Stability indicating HPTLC determination and ph

stability profile of catechin, Indian Drugs, February 2002, 39(2), 91-95.

3. Stuart M., Reference section. In: Stuart M (Ed.), The Encyclopedia of Herbs and

Herbalism. London: Orbis Publishing, 1979 .141-283.

4. Sane R. T. et. al., Spectrophotometric methods for the determination of Cynidanol

(+ Catechin) from Pharmaceutical Preparations, Indian Drugs, 1984, 22(1), 20-24.

5. Sawant S. S. et. al., Estimation of Catechin from Acacia catechu by HPTLC,

Indian Drugs, 1995, 32(9), 461-463.

6. Veluri R. et al., Phytotoxic and antimicrobial activities of Catechin derivatives, J.

Agric. Food Chem., 2004, 52 (5), 1077–1082

7. Li ZhongXing, et al., Chinese Journal of Information on Traditional Chinese Med.


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Chater:2.3 Review of Literature on Clove and Eugenol

Ayoola G. A. et al., studied Chemical analysis and antimicrobial activity of the

essential oil of Syzigium aromaticum (clove). The antimicrobial sensitivity of the

volatile oil against some Gram-negative bacteria (Escherichia coli ATCC 35218,

Escherichia coli, Klebsiella pneumoniae, Salmonella paratyphi, Citrobacter spp. and

Enterobacter cloacae), a Gram-positive bacterium (Staphylococcus aureus ATCC

25923), and a fungus (Candida albicans) showed a broad spectrum of activity.

Antioxidant screening of clove oil with 2,2-diphenylpicryl-hydrazyl radical (DPPH)

was positive, indicating the presence of free radical scavenging molecules which can

be attributed to the presence of eugenol, a phenolic compound.

Dr. Chetan has reviewed that the clove oil has antiseptic, antifungal, and anti-

bacterial properties and has been widely used to prevent oral diseases such as plaque

and mouth ulcers.

Dighe V.V et al. Have developed a simple , precise and quanititative HPTLC method

for determination of eugenol in Cinnamomum zeylanicum leaf powder. The

methanolic extract of cinnamomum zeylanicum were applied on TLC pre –coated

plate (merck) and was developed using Toluene: Ethyl acetate: Formic acid (9:1:0.1)

v/v/v as the mobile phase. Detection was carried out densitometrically using an UV

detector at 280 nm. The HPTLC proposed method is precise, accurate and rapid for

determination of eugenol.

Anandjiwala S. et al., have quantified 4 marker compounds, viz., eugenol, luteolin,

ursolic acid, and oleanolic acid, from the leaf of green and black varieties of O.

sanctum using high-performance thin-layer chromatography (HPTLC) with

densitometry. The methods were found to be precise, with relative standard

deviation (RSD) values for intraday analyses in the range of 0.52 to 0.91%, 0.77

to 1.29%, 0.11 to 0.16%, and 0.34 to 0.42% and for interday analyses in the

range of 0.73 to 0.96%, 1.02 to 2.08%, 0.11 to 0.12%, and 0.39 to 0.64% for

different concentrations of eugenol, luteolin, ursolic acid, and oleanolic acid,

respectively. Instrumental RSD values were 0.24, 0.39, 0.21, and 0.18% for

eugenol, luteolin, ursolic acid, and oleanolic acid, respectively. Accuracy of the

methods was checked by conducting a recovery study at 3 different levels for

the 4 compounds, and the average recoveries were found to be 99.73, 99.3,

100.58, and 100.57%, respectively. Eugenol content ranged from 0.175 to

0.362% (w/w) and luteolin from 0.019 to 0.046% (w/w) in the samples


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analyzed. The HPTLC-densitometry methods for the quantification of the 4

markers in O. sanctum leaf will have the applicability in quality control.

Dighe V. V. et al., developed A simple, rapid and precise reverse-phase high

performance liquid chromatographic method for the quantitative determination of

eugenol from the extract of dried powder of Cinnamomum tamala leaves and its

polyherbal formulation. Chromatographic analysis was carried out on Zorbax C18

column (150 mm · 4.6 mm, 5 lm) with a mobile phase of mixture of Water,

Acetonitrile and Methanol in the volume ratio of 50:40:10, at a flow rate of 1.0 ml/

min. Quantitation was performed using a UV-visible detector at 210 nm. Good

linearity was obtained over the ranges of 0.20–3.0l g /ml. for eugenol.

Pathak S. B. et al., have reported a simple TLC densitometric method for the

quantification of eugenol and gallic acid in clove. The method was validated for

precision, repeatability and accuracy. The contents of eugenol and gallic acid in

different samples of clove, as estimated by the proposed method, were found to be in

the range of 12.9–14.6% and 0.31–0.61% respectively. The proposed HPTLC method

for the estimation gallic acid and eugenol was found to be simple, precise, specific,

sensitive and accurate and can be used for routine quality control of clove.

Jyoti BB has reviewed Phytotherapeutics in conservative dentistry & endodontics. In

Conservative Dentistry & Endodontics, variety of dental materials & medicaments are

being used originated from plants. Identification & isolation of "Digoxin" from

Digitalis lanata, "Reserpine"from Rauwalfa serpentina, "Vincrystin "and "Vinblastin

"from Catharanthus rosea," Gutta percha "from Palaquiam species. "Eugenol "from

Svgygium aromaticum are some of the examples.

Lining Cai and Christine D. Wu studied the effect of crude MeOH extract of

Syzygium aromaticum (clove) exhibited preferential growth-inhibitory activity

against Gram-negative anaerobic periodontal oral pathogens, including

Porphyromonas gingivalis and Prevotella intermedia. By means of bioassay-directed

chromatographic fractionation, eight active compounds were isolated from this

extract and were identified as 5,7-dihydroxy-2-methylchromone 8-C-β-D-

glucopyranoside, biflorin, kaempferol, rhamnocitrin, myricetin, gallic acid, ellagic

acid, and oleanolic acid, based on spectroscopic evidence. The antibacterial activity

of these pure compounds was determined against Streptococcus mutans, Actinomyces

viscosus, P. gingivalis, and P. intermedia. The flavones, kaempferol and myricetin,



demonstrated potent growth-inhibitory activity against the periodontal pathogens P.

gingivalis and P. intermedia.

Ogata M. et al., studied Antioxidant activity of Eugenol and related monomeric and

dimeric cmpounds. Since the inhibitory effect of eugenol (a), which was isolated as

an antioxidative component from plant, Caryopylli flos, on lipid peroxidation was

less than that of alpha-tocopherol, we synthesized the eugenol-related compounds

dieugenol (b), tetrahydrodieugenol (c), and dihydroeugenol (d), to find new strong

antioxidants and assessed them for their inhibitory effect on lipid peroxidation and

scavenging ability for superoxide and hydroxyl radicals. The antioxidative activities

were in the order: (b)>(c)> (d)> (a) for the thiobarbituric acid reactive substance

(TBARS) formation. These results suggest that the dimerized compounds have higher

antioxidant activities than that of the monomers. Electron spin resonance (ESR) spin

trapping experiments revealed that eugenol and its dimer, having allyl groups in the

structure, scavenged superoxide, and that only eugenol trapped hydroxyl radicals

under the conditions used. These finding suggest that eugenol and dieugenol have a

different mechanism of antioxidation, i.e. eugenol may inhibit lipid peroxidation at

the level of initiation, however , the related dimeric compounds may inhibit lipid

peroxidation at the level of propagation of free radical chain reaction like alpha-

tocopherol.


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References:

1. Ayoola G.A., Chemical analysis and antimicrobial activity of the essential oil of

Syzigium aromaticum (clove), African Journal of Microbiology Research., July

2008,2 , 162-166,

2. Dr. Chetan, Dentistry and Dental Information, 5 December, 2007.

3. Dighe V.V et al., Quantification of Eugenol from Cinnamomum zeylanicum

blume. leaf powder using High Performance Thin Layer Chromatography, Indian

drugs,43(6),June 2006,493-496.

4. Anandjiwala S . et al., Quantification of Eugenol, Luteolin, Ursolic acid, and

Oleanolic acid in black and green varieties of Ocimum sanctum Linn. Using High-

Performance Thin-Layer Chromatography, Journal of AOAC Int., Nov-Dec 2006,

89(6):1467-74.

5. Dighe V.V et al., Quantitative Determination of Eugenol from Cinnamomum

tamala Nees and Eberm. Leaf Powder and Polyherbal Formulation Using Reverse

Phase Liquid Chromatography, Chromatographia, May 2005, 61(9-10), 443-446.

6. Pathak S. B. et al., TLC Densitometric Method for the Quantification of Eugenol

and Gallic Acid in Clove, Chromatographia, 2004,60, 241-244.

7. Jyoti BB , Phytotherapeutics in conservative dentistry & endodontics -a review

(2005) Medknow Publications, United States

8. Lining Cai and Christine D. Wu, Compounds from Syzygium aromaticum

Possessing Inhibitory Activity against Oral Pathogens, J. Nat. Prod., 1996, 59

(10), 987-990.

9. Ogata M ., et al., Chem Pharm Bull (Tokyo). Oct 2000, 48(10), 1467-1469.


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Chapter:2.4 Review of Literature on Buccoadhesive Drug Delivery

Mahdi A. B. et al., evaluated the efficacy of bioadhesive hydrogel patches, made of

a pharmaceutical grade cellulose derivative, in the control of pain and as an aid to

healing of aphthous ulceration. A significant reduction in stimulated pain was

recorded following application of the patches to the ulcers (P<0.01). The patches

were found to adhere longer to large ulcers in the early stages of ulceration, when they

achieved their maximum protective and pain-attenuating effects. The ulcer size was

recorded daily by the patient and patients claimed a reduction in healing time

following patch therapy.

Sang-Chul Shin et al., studied Mucoadhesive and Physicochemical Characterization

of Carbopol-Poloxamer Gels Containing Triamcinolone Acetonide. The viscosity

and bioadhesive property of Carbopol-Poloxamer gels containing triamcinolone

acetonide to mucosa were tested according to various concentrations of Carbopol

gels of various pH. The increase in Carbopol concentration caused increased

viscosity and bioadhesiveness. The neutralization of pH in various concentrations

of Carbopol gels showed the increased viscosity, showing the highest viscosity

and highest bioadhesiveness when neutralized to pH 6. According to FTIR and

XRD studies, the drug did not show any evidence of an interaction with the

polymers used and was present in an unchanged state.

Mizrahi B. et al., Studied the Mucoadhesive Polymers for Delivery of Drugs to the

Oral Cavity. Local therapy of the oral cavity is used to treat conditions such as

gingivitis, oral candidosis, oral lesions, dental caries, xerostoma and oral carcinomas.

Delivery systems used include mouthwashes, aerosol sprays, chewing gums,

bioadhesive tablets, films, gels and pastes. Prolonged contact time of a drug with body

tissue can significantly improve the clinical performance of many agents used for

treating oral disorders. These improvements range from better treatment of local

pathologies to improved drug bioavailability and controlled release to enhanced

patient compliance. There are abundant examples in the literature over the past 15

years of these improvements using bioadhesive polymers.

Chien, Yie, W.; NAIR, Mona; have patented the mucosal adhesive device for long-

acting delivery of pharmaceutical combinations in oral cavity. Mucosal

adhesive devices are provided for use in the oral cavity for therapy against

infections. The devices are dosage units which comprise a combination of

antimicrobial agents such as antifungal agents and anti-inflammatory agents,



optionally also a local anesthetic. The dosage units yield a gradual and

relatively constant release of the pharmaceuticals over at least a 12-hour period.

Salamat-Miller Nazila et al., have studied The use of mucoadhesive polymers in

buccal drug delivery. Buccal delivery of the desired drug using mucoadhesive

polymers has been the subject of interest since the early 1980s. Advantages associated

with buccal drug delivery have rendered this route of administration useful for a

variety of drugs. This review highlights the use of mucoadhesive polymers in buccal

drug delivery. Additionally, they focus on the new generation of mucoadhesive

polymers such as thiolated polymers, followed by the recent mucoadhesive

formulations for buccal drug delivery.

Ramana MV et.al., studied the Design and evaluation of mucoadhesive buccal drug

delivery systems containing Metoprolol tartrate. Mucoadhesive buccal tablets of

metoprolol tartarate were fabricated with objective of avoiding first pass metabolism

and prolonging duration of action. The mucoadhesive polymers used in formulation

were Carbopol-934, hydroxypropylmethylcellulose, hydroxyethylcellulose and

sodium carboxymethylcellulose. The formulations were characterized for

physiochemical parameters, in vitro release studies and in vivo placebo studies. The

best mucoadhesive performance and in vitro drug release profile were exhibited by

the tablets containing hydroxyethylcellulose and Carbopol-934 in ratio 1:2.

Khanna R. et al., have prepared and evaluated the mucoadhesive buccal films of

clotrimazole for oral candida infections. Mucoadhesive buccal films of clotrimazole

for local delivery of the drug to the oral cavity were formulated by the solvent

casting technique. A number of different bioadhesive and film- forming polymers

were evaluated. Propylene glycol was used as the plasticizer while the solvents

depended on the type of polymer chosen. The films were evaluated on the basis of

their physical characteristics, biadhesive performance, release characteristics,

surface ph, folding endurance and strechability. A combination of carbopol-934P

and hydroxy propyl cellulose- M in the ratio of 1:5 and using ethanol (95%) as the

solvent was found to give satisfactory results. The film exhibited an in vitro

adhesion time of 4 hours and maintained the concentration of clotrimazole in the

dissolution medium(isotonic phosphate buffer ph 6.6) above the MIC of candida

albicans for upto 4 hours. A maximum concentration of 21.1 µg/ml was obtained in

the dissolution medium after 2 hours. The drug release from the formulation was

found to be microbiologocally active.



Mizrahi B.et al., developed a bioadhesive erodable patch for treating mouth ulcers.

Adhesive patches were prepared by compression molding of mixed polymeric

powders, absorbed with citrus oil and magnesium salt .A clinical trial was conducted

on 248 volunteers suffering from mouth ulcers. Patches gradually erode for eight

hours releasing the citrus oil in a zero order pattern while the magnesium is released

during a period of two hours. Patches were effective in reducing pain and decreasing

healing time (p<0.05) without adverse side effects . The mucoadhesive patch was

found to be highly effective for pain reduction and healing time in both single ulcer

and recurrent aphthous stomatitis patients.

Kim TH et al., have prepared and Characterized the novel mucoadhesive polymer

blend film consisting of Carbopol, poloxamer, and hydroxypropylmethylcellulose

(HPMC). Triamcinolone acetonide (TAA) was loaded into

Carbopol/poloxamer/HPMC polymer blend film. Swelling ratio of

Carbopol/poloxamer/HPMC films was lowest in Carbopoll poloxamer/HPMC at

mixing ratio of 35/30/35 (wt/wt/wt). Adhesive force of Carbopol/poloxamer/HPMC

films increased with increasing HPMC content in Carbopol/poloxamer/HPMC

polymer blend film and increasing hydroxypropyl group content in HPMC due to

hydrophobic property of HPMC although bioadhesive force was highest at mixing

ratio of 35/30/35 (wt/wt/ wt). Release of TAA from TAA-loaded

Carbopol/poloxamer/HPMC polymer blend film in vitro increased with increasing

loading content of drug.

Dhiman M. et al., prepared and evaluated in vitro the bioadhesive gels of 5-

Fluorouracil (FU) for the treatment of oropharyngeal cancer. The gel formulations

containing FU were prepared by using Poloxamer 407, HPMC K 15 M, and Gantrez

S-97 (polymethylvinylether-co-maleic anhydride). The formulations contained

Poloxamer 407 (16-18% w/w) either alone or in combination with HPMC K 15 M and

Gantrez S-97. The bioadhesiveness of the gels was found to increase with increasing

proportion of HPMC K 15 M and Gantrez S-97. In vitro release studies indicated that

release could be sustained up to 8 hr. Increasing temperature increased the drug

release by increasing drug diffusion despite increase in viscosity. The pH of the

release medium showed a very slight effect on the release of FU.

Ali J. et al., designed and characterized Buccoadhesive erodible disk for treatment of

oro-dental infections. The optimized disk containing 5.0 mg of cetylpyridinium

chloride, 2.0 mg of magnesium stearate and 6.0 mg of mannitol along with sodium


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carboxy methyl cellulose DVP and hydroxypropylmethylcellulose K4M in the ratio of

1:3 was found to release the drug for a period of over 6.0 h without getting dislodged.

Maximum in vitro drug release was found to be 94.78% in 6.0-h study. The

bioadhesive performance and the surface pH of the disks were satisfactory.

Cetylpyridinium chloride disks were tested against microorganisms commonly found

in oro-dental infections namely Candida albicans, Staphylococcus aureus,

Escherichia coli and Streptococcus mutans. The disk as well as the in situ samples

showed inhibition of growth of microorganisms.

Varshosaz J. et al., developed a localized drug delivery system that offers prolonged

administration of metronidazole into the periodontal pocket, muccoadhesive gel

formulations containing 5% w/w metronidazole were prepared using the bioadhesive

polymers: carboxymethylcellulose, methylcellulose, hydroxyethylcellulose,

polyvinylpirrolidone, and carbopol. Increased concentrations of the polymers

decreased the drug release rate and enhanced syringeability, yield value, and

adhesiveness but decreased the spreadability. The bioadhesive properties of the gels

were affected by pH and Ca2+ concentration. The gel containing 20%

hydroxyethylcellulose, 20% polyvinylpirrolidone, and 1% carbopol exhibited zero-

order drug release kinetics and suitable physical properties for drug delivery to the

periodontal pocket.



References:

1. Mahdi AB, Coulter WA, Woolfson AD, Lamey PJ, Journal of oral pathology

& medicine, 1996 Sep;25(8):416-9

2. Sang-Chul Shin ; Ja-Young Kim ; In-Joon Oh, Drug development and industrial

pharmacy, 2000 Mar;26(3):307-12

3. Mizrahi, Boaz; Domb, Abraham J., Recent Patents on Drug Delivery &

Formulation, Volume 2, Number 2, June 2008 , pp. 108-119(12)

4. Chien, Yie, W.; Nair, Mona, Mucosal adhesive device for long-acting delivery of

pharmaceutical combinations in oral cavity, Patent no.WO/1995/015137

5. Salamat-Miller Nazila ; Chittchang Montakam ; Johnston Thomas P. , Advanced

drug delivery reviews , 2005, vol. 57, no 11 (171 p.)

6. M.V.Raman, C Nagda, M Himaja, Indian Journal of Pharmaceutical Science, Year

: 2007 , Volume : 69 , Issue : 4 , Page : 515-518

7. R.Khanna, S.P.Agarwal, Alka Ahuja, Indian Journal Of Pharmaceutical

Science,1997,59(6),page.299-305

8. http://www.cankercover.comram

9. Kim TH , Ahn JS, Choi HK, Choi YJ, Cho CS. Arch Pharm Res. 2007

Mar;30(3):381-6

10. Dhiman M , Yedurkar P, Sawant KK. Pharmaceutical development and

technology, 2008; 13(1):15-25

11. J. Ali, R. Khar, A. Ahuja and R. Kalra, International Journal of Pharmaceutics,

Volume 238, Issues 1-2, 15 May 2002, Pages 93-103

12. Jaleh Varshosaz ; Nasser Tavakoli ; Sharona Saidian , Drug Delivery, Volume 9,

Issue 2 April 2002 , pages 127 - 133


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http://www.ingentaconnect.com/content/ben/ddf;jsessionid=3bu2tqsrqsqu9.alice

http://www.ingentaconnect.com/content/ben/ddf;jsessionid=3bu2tqsrqsqu9.alice

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Chapter: 2.5 Review of Literature on Mouth Ulcer

Moghadamnia A. A. et. al. studied the efficacy of the bioadhesive patches containing

licorice extract in the management of recurrent aphthous stomatitis. This study

evaluated the efficacy of licorice bioadhesive hydrogel patches to control the pain and

reduce the healing time of recurrent aphthous ulcer. According to the results of this

study, licorice bioadhesive can be effective in the reduction of pain and of the

inflammatory halo and necrotic center of aphthous ulcers.

Stuart (1979) has referred the use of the boiled and strained aqueous extract of the

heartwood as an astringent for inflamed conditions of the throat, gums, and mouth and

also externally for boils and chronic ulcers.

Mahdi A. B. et al., evaluated the efficacy of bioadhesive hydrogel patches, made of a

pharmaceutical grade cellulose derivative, in the control of pain and as an aid to

healing of aphthous ulceration. A significant reduction in stimulated pain was

recorded following application of the patches to the ulcers (P<0.01). The patches

were found to adhere longer to large ulcers in the early stages of ulceration, when they

achieved their maximum protective and pain-attenuating effects. The ulcer size was

recorded daily by the patient and patients claimed a reduction in healing time

following patch therapy.

Mizrahi B. et al., developed a bioadhesive erodable patch for treating mouth ulcers.

Adhesive patches were prepared by compression molding of mixed polymeric

powders, absorbed with citrus oil and magnesium salt. A clinical trial was conducted

on 248 volunteers suffering from mouth ulcers. Patches gradually erode for eight

hours releasing the citrus oil in a zero order pattern while the magnesium is released

during a period of two hours. Patches were effective in reducing pain and decreasing

healing time (p<0.05) without adverse side effects. The mucoadhesive patch was

found to be highly effective for pain reduction and healing time in both single ulcer

and recurrent aphthous stomatitis patients.

Hau; Kee Hung patented the invention that provides a medicament for topically

treating acute bacterial infections in the oral mucosa, and methods of use. The

medicament comprises a dry dosage (such as a troche or powder) of one or more

antibacterial agents and, preferably, one or more polyvalent metal compounds. The

medicament is directly applied to the site of the infection and dissolves in saliva,

within about 5 to about 15 minutes, thereby directly delivering a supratherapeutic

dosage of the antibacterial agent to the infected oral tissue. Further, in a preferred

embodiment the medicament directly delivers a therapeutically high concentration of


Chapter: 2.5 Review of Literature on Mouth Ulcer

a polyvalent metal compound in suspension to the infected area, thereby forming a

protective barrier over the infected oral tissue.

Campbell, Phillip patented Lesion and ulcer medication .The invention is based on

the discovery that certain combinations of antimicrobial agents, anti-inflammatories,

mid antihistamines, along with an accompanying mucoadhesive provide unexpected

and highly effective intraoral ulcer medications. The present invention provides an

intraoral ulcer medication which prevents both secondary infection and promotes

healing while simultaneously providing immediate relief from pain. This ulcer

medication comprises an antimicrobial, an anti-inflammatory, an antihistamine, and

optional antifungal and anesthetic components along with a mucoadhesive or

equivalent.

References:

1. Moghadamnia A. A. , Motallebnejad M. , Khanian M., The efficacy of the

bioadhesive patches containing licorice extract in the management of recurrent

aphthous stomatitis, Phytotherapy Research,2008, 23(2), 246 – 250

2. Stuart M, Reference section. In: Stuart M (Ed.) The Encyclopedia of Herbs and

Herbalism. 1979 pp. 141-283. London: Orbis Publishing

3. Mahdi AB, Coulter WA, Woolfson AD, Lamey PJ, Journal of oral pathology

& medicine, 1996 Sep;25(8):416-9

4. http://www.cankercover.comram

5. Hau; Kee Hung, Lesion-directed dry dosage forms of antibacterial agents for the

treatment of acute mucosal infections of the oral cavity, United States Patent

6,248,718, June 19, 2001.

6. Campbell, Phillip, Lesion and ulcer medication, US Patent 6352711


http://www3.interscience.wiley.com/journal/12567/home

http://www.cankercover.comram/

http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22Lamey%20PJ%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstract

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Chapter: 3 Aim and Plan of the Work

Aim of the work:

The prime aim of this investigation is to develop the Gel and Patch formulations of

the Glycyrrhiza glabra extract, Acacia catechu extract and clove oil for the mouth

ulcer and to evaluate them for in vitro release efficiency. These formulations are also

evaluated for various formulation parameters.

Plan of the work:

The plan of the work is divided in the following parts.

Procurement of Raw materials

Evaluation of Raw materials

Preparation of Extracts

Standardization of extracts by HPTLC

Preparation and Evaluation of Gel Formulations

Preparation and Evaluation of Patch Formulations

The evaluation of developed formulation is carried out as:

Evaluation of various physicochemical parameters of the gel like pH,

Viscosity, Spreadibility and Drug content.

Evaluation of various physicochemical parameters of the patch like Folding

endurance, Surface pH, In vitro Residence time and Drug content.

In vitro Drug Release study using cellophane membrane in modified diffusion

cell.

Antimicrobial Activity of Selected Formulations.


Chapter: 4 Evaluation of Raw Materials


4.1 Experimental:

4.1.1 Procurement of Plant Materials

The whole material of Glycyrrhiza glabra (G. glabra) and Acacia catechu (A.

catechu) was procured from LVG and SONS and powder was made in laboratory and

passed through sieve no. 60. The clove oil was procured from Saraiya Chemicals,

Ahmedabad. The standard compounds, 18-β-glycyrhhetinic acid and Catechin was

procured from Sigma Aldrich Ltd., Banglore.

4.1.2 Evaluation of G.glabra whole Material and Powder

(1) Macroscopic study of Whole material and Powder

The morphology of G. glabra root was examined and photographed using camera.

The powder of G. glabra was tested for color, odor and taste. The preliminary

evaluation for foreign matter was also done to remove the admixture material if

present in sample may be as adulterant or substitute by visualization method.

(2) Microscopy study 1

The stained and unstained slide of G. glabra was prepared and the characters were

examined photographed using CCD camera.

(3) Chemical Identification1

Test for Saponins and Flavanoids:

Foam test:

Mixed the powder with water. After shaking stable foam was produced due to

presence of saponins.

Liebermann-Burchard test:

Mixed 2 ml of extract with chloroform,1-2 drops of acetic anhydride and 2 drops of

conc. H2SO4 was added. Red color was produced due to the presence of triterpenois.

The drug powder with 80% v/v sulphuric acid shows orange yellow color due to

presence of flavanoids.

(4) Identification by TLC 2

TLC of G. glabra was carried out in the following conditions.

Stationary phase : Aluminium-backed silica gel 60F254 plate (E. Merck)

Mobile phase : Ethyl Acetate: Ethanol: Water: Ammonia (65:25:9:1)

Chamber saturation : 30 minutes

Standard solution : 18-β-glycyrhhetinic acid dissolved in methanol

Test sample : Dry aqueous extract of G. glabra was dissolve in methanol



Detection : (1) In U.V.254 nm

(2) After spraying with Anisaldehyde sulphuric acid

Sample ID : A. G. glabra extract

B. 18-β-glycyrhhetinic acid

(5) Physicochemical parameters

To ensure good quality of G. glabra powder, various physicochemical parameters was

performed as per WHO guidelines 5.

a) LOD at 105° C

b) Ash value

Total ash

Acid insoluble ash

Water soluble ash

c) Extractive value

Alcohol soluble extractive value

Water soluble extractive value

(6) Estimation of 18-β-glycyrhhetinic acid (18-β-G.A.) in G. glabra powder by

HPTLC

Preparation of std. stock solution: 100 µg per ml stock solution was prepared by

dissolving 1 mg 18-β-glycyrhhetinic acid in10 ml chloroform

Preparation of test solution: 1 gm of G. glabra powder was mixed with 50 ml of water

and extracted with 25 ml chloroform. The chloroform was evaporated and residue was

dissolved in 10 ml chloroform.

Test solution of 3 µl was applied along with 3, 4, 5, 6, 7 and 8 µl standard 18-β-

glycyrhhetinic acid solution on Aluminium- backed silica gel 60F254 plate (E.

Merck) (10 x 10 cm). It was followed by development of plate up to 85 mm at 25º C

and the chomatogram was recorded by scanning at 254 nm on a CAMAG TLC

scanner. The Amount of 18-β-glycyrhhetinic acid was determined using the

calibration curve plotted between concentration and peak area of standard 18-β-

glycyrhhetinic acid.



Chromatogrhaphic condition:

Stationary phase : 10 x 10 cm Aluminium-backed silica gel 60F254 plate

(E.Merck)

Mobile phase : Ethyl Acetate: Ethanol: Water: Ammonia (65:25:9:1)


Band with : 5 mm

Distance of band : 7 mm

Rate of spotting : 10 sec/ µl

Distance run : 85 mm

Scanning wavelength : 254 nm

Scanning speed : 5mm/ sec

Slit dimension : 5.0 x 0.45 mm

Temperature : 25º C

4.1.3 Evaluation of A. catechu whole material and Powder


The morphology of A. catechu root was examined and photographed using camera.

The powder of A. catechu was tested for color, odor and taste. The preliminary

evaluation for foreign matter was also done to remove the admixture material if

present in sample may be as adulterant or substitute by visualization method.

(2) Chemical Identification 1

Test for Phenolic compounds and tannins

Ferric chloride test:

To 1 ml of extract, 5% ferric chloride solution added, formation of dark blue or

greenish black color shows the presence of the tannins.

Lead acetate test:

The test solution mixed with lead acetate solution. Formation of white precipitates

indicates the presence of tannins.

Match stick test:

A matchstick was dipped in decoction of black catechu, dried in air and dipped it in

concentrated hydrochloric acid and warmed it near the burner. Magenta or purple

color was produced.

Vanillin –Hydrochloric acid test:

(Vanillin 1: Alcohol 10: dil. hydrochloric acid 10) catechu showed pink or red color.




TLC of A. catechu was carried out in the following conditions.

Stationary phase : Aluminium-backed silica gel 60F254 plate (E. Merck)

Mobile phase : Toluene: Ethyl Acetate: Methanol: Glacial Acetic Acid

(2.7:6:1:0.3)


Test sample : Dry aqueous extract of acacia catechu was dissolved in

methanol.


(2) After spraying with vanillin sulphuric acid


To ensure good quality of A. catechu powder, various physicochemical parameters

was performed as per WHO guidelines 5.

d) LOD at 105° C

e) Ash value

Total ash

Acid insoluble ash

Water soluble ash

f) Extractive value

Alcohol soluble extractive value

Water soluble extractive value

(5) Estimation of Catechin in A. catechu powder by HPTLC

Preparation of std. stock solution : 100 µg per ml stock solution was prepared by

dissolving 1 mg catechin in10 ml methanol

Preparation of test solution: 1 gm of acacia catechu powder was mixed with 20 ml of

water, filtered and evaporated to dryness. The residue was dissolved in 50 ml

methanol and filtered. From this 1 ml of solution was taken and diluted up to 10 ml

with methanol. From this 1 ml of solution taken and mixed with 1 ml of methanol.

Test solution of 6 µl was applied along with 5, 6, 7, 8 and 9 µl standard Catechin

solutions on a Aluminium- backed silica gel 60F254 plate (E. Merck) (10 x 10 cm).

Then the plate was developed up to 85 mm at 25º C and the chomatogram was

recorded by scanning at 254 nm on a CAMAG TLC scanner. The Amount of catechin



was determined using the calibration curve plotted between concentration and peak

area of standard catechin.



(E.Merck)

Mobile phase : Toluene: Ethyl Acetate: Methanol: Glacial Acetic Acid

(2.7:6:1:0.3)


Band with : 5 mm








(6) Estimation of total tannins in Acacia catechu 4

1 gm of Acacia caetchu powder was accurately weighed and introduced into a 250 ml

glass stopered flask, 100 ml of water was added and shaken for 1.0 hr. and kept

overnight. The material was allowed to settle and the liquid was filtered through a

filter paper, discarded first 20 ml of filtrate.10 ml of filtrate was transferred to a 1 liter

conical flask. To this filtrate 750 ml water and 25 ml of Indigosulphonic acid solution

were added. This solution was titrated with 0.1 N Potassium Permanganate and

shaken vigorously till a golden yellow end point (T2) was reached. A blank

determination (T1) was also performed.

Each ml of 0.1 N Potassium Permanganate is equivalent to 0.004157 g of total

tannins.

Quantity of total tannins (%) = [(T2-T1) x actual normality x 0.004157 x1000]

W x 0.1

Where W = the total weight of the plant material



4.1.4 Evaluation of Clove oil

(1) Description

The clove oil was tested for its appearance, color, odor and taste.

(2) Determination of Refractive Index 6

The refractive index of clove oil was measured using Cyber Abbe Refractometer at

25º C (±0.5) with reference to the wavelength of the D line of sodium (Ψ =589.3 nm).

The temperature was carefully adjusted and maintained since the refractive index

varies significantly with temperature. To achieve accuracy, the apparatus was

calibrated against distilled water: which has a refractive index of 1.3325 at 25 º C.

(3) Determination of Specific Gravity 6

Specific gravity of clove oil was determined using specific gravity bottle. The specific

gravity bottle was calibrated by filling it with recently boiled and cooled water at

25°C and weighing the contents. It was assumed that the weight of 1 ml of water at

25°C when weighed in air of density 0.0012 gm per ml is 0.99602 gm; calculate the

capacity of the specific gravity bottle. The temperature of clove oil was adjusted to

about 20° C and fills the specific gravity bottle with it and weighed. The tare weight

of the specific gravity bottle was substracted from the filled weight of specific gravity

bottle. The specific gravity was determined by dividing the weight in air, in g, of the

quantity of liquid which fills the specific gravity bottle at the specified temperature,

by the capacity expressed in ml, of the specific gravity bottle at the same temperature.


TLC of clove oil was carried out in the following conditions.

Stationary phase : Aluminium- backed silica gel 60F254 plate (E. Merck)

Mobile phase : Toluene: Ethyl Acetate (93:7)


Standard solution : Eugenol diluted with toluene

Test sample : Clove oil diluted with toluene


(2) After spraying with Anisaldehyde sulphuric acid

Sample ID : A. Diluted Clove Oil

B. Eugenol



(5) Estimation of Eugenol in clove oil by HPTLC

Preparation of std. stock solution: 50 µl eugenol was diluted up to 50 ml with

methanol. From this 4 ml of solution was taken and diluted up to 50 ml with

methanol. Preparation of test solution: 100 µl of clove oil was diluted up to 10 ml

with methanol.

Test solution of 1.5 µl was applied along with 2.5, 5, 7.5,10 and12.5 µl standard

eugenol solution on a precoated silica gel 60 plate (10 x 10 cm). After that the plate

was developed up to 85 mm at 25º C and the chomatogram was recorded by scanning

at 254 nm on a CAMAG TLC scanner. The Amount of eugenol was determined using

the calibration curve plotted between concentration and peak area of standard

eugenol.



(E.Merck)

Mobile phase : Toluene: Ethyl Acetate (93:7)


Band with : 5 mm








4.1.5 Preparation of extracts

Preparation of aqueous extracts of G. glabra powder and A. catechu powder

100 gm of G. glabra and A. catechu powder was extracted with 500 ml of water by

cold maceration for 24 hrs. The solution was filtered and evaporated to dryness in

Rotary evaporator at 60 ºC. The resulting extract was dried in dessicator.



4.1.6 Standardization of extracts by HPTLC

(1) Standardization of extracts of G. glabra


dissolving 1 mg 18-β-glycyrhhetinic acid in10 ml chloroform

Preparation of test solution: 10 mg of aqueous extract of G. glabra was dissolved in

50 ml of chloroform.

Test solution of 7 µl was applied along with 3, 4, 5, 6, 7 and 8 µl standard 18-β-

glycyrhhetinic acid solution on Aluminium-backed silica gel 60F254 plate (E. Merck).

(10 x 10 cm). It was followed by development of plate up to 85 mm at 25º C and the

chomatogram was recorded by scanning at 254 nm on a CAMAG TLC scanner as per

the chromatographic condition mentioned in section 4.1.2.(6). The Amount of 18-β-

glycyrhhetinic acid was determined using the calibration curve plotted between

concentration and peak area of standard 18-β-glycyrhhetinic acid.

(2) Standardization of extracts of A. catechu


dissolving 1 mg 18-β-glycyrhhetinic acid in10 ml methanol.

Preparation of test solution: 10 mg of aqueous extract of Acacia catechu was

dissolved in 50 ml of methanol.

Test solution of 14 µl was applied along with 5, 6, 7, 8 and 9 µl standard Catechin

solution on an Aluminium-backed silica gel 60F254 plate (E.Merck) (10 x 10 cm).

After that the plate was developed up to 85 mm at 25º C and the chomatogram was

recorded by scanning at 254 nm on a CAMAG TLC scanner as per the

chromatographic condition mentioned in section 4.1.3.(5). The Amount of catechin

was determined using the calibration curve plotted between concentration and peak

area of standard catechin.



4.2 Results and Discussion:

The result of various standardization parameters performed for ensuring the quality of

samples collected from market was as noted below

4.2.1 Evaluation of G.glabra whole Material and Powder


Figure: 4.1 Morphology of Glycyrrhiza glabra

The morphology of the sample was showed that outer surface was yellowish brown or

dark brown in colour, externally longitudinally wrinkled with patches of cork. fracture

was coarsely fibrous in bark and splintery in wood. This morphological characters

was giving identity of G. glabra.

The G. glabra powder having pale yellow to brown color with characteristic odor and

sweet taste. The foreign matter determination by visualization method was showing

absence of any admixture or any other obnoxious materials. The particle size of the

samples was characterized to 60 # by sieve analysis.

(2) Microscopy study 1

Cork cells Crystal fibres



Vessel Ca-oxalate crystal

Figure: 4.2 Microscopic characters of G. glabra powder sample

Microscopic characterization showed the presence of calcium oxalate crystals, cork

cells, crystal fibres and vessels when observed under stained and unstained slide. The

presence of this well defined diagnostic characters was giving identity of G. glabra

species.

(3) Chemical Identification

G. glabra extract shows positive result for Foam test, Liebermann-burchard test and

with 80% v/v sulphuric acid. So it contain triterpenoidal saponins and flavanoid

glycosides.

(4) Identification by TLC

In the TLC identification , violet spot was observed at Rf = 0.46 in extract of G.

glabra and in std. 18-β-glycyrhhetinic acid solution in U.V 254 nm and after spraying

with Anisaldehyde sulphuric acid reagent which confirm the identity of the G. glabra.

A B A B

(1) (2)

A: Std. 18-β-glycyrhhetinic acid, B: test solution of G. glabra powder extract ,

(1): In U.V. 254 nm , (2): After spraying with Anisaldehyde sulphuric acid

Figure: 4.3 TLC profile of Glycyrrhiza glabra powder




The various physicochemical parameters performed for G. glabra was mentioned in

Table 4.1.

Table: 4.1 Physicochemical parameters of G. glabra powder

Sr. no. Physicochemical

parameters

Observation* Reference value

1 LOD at 105 ºC 5.3 ± 0.4 NMT 8 %

2 Ash value

A. Total ash 7.22 ± 0.09 NMT 10 %

B. Acid insoluble ash 2.21 ± 0.26 NMT 2.5%

C. Water soluble ash 0.77 ± 0.025 -

3 Extractive value

A. Alcohol extractive value 11.4 ± 0.52 NLT 10 %

B. Water extractive value 22.43 ± 0.51 NLT 20 %

* = Mean ± Standard Error Mean ( n = 3)

The G. glabra powder shown loss on drying within the pharmacopoeial limit. The

results of ash values were also within the pharmacopoeial limit as mentioned in the

table. The results of water soluble extractives and alcohol soluble extractives of

samples are mentioned in the table. High values in cases of of water soluble

extractives indicate the presence of good amount of water soluble chemical

constituents in the drug.

(6) Estimation of 18-β-glycyrhhetinic acid in G. glabra powder by HPTLC

The quantification of of 18-β-glycyrhhetinic acid in G. glabra powder was carried

out by HPTLC as per the procedure mentioned in Section 4.1.2 (6).



1 2 3 4 5 6 T 1 2 3 4 5 6 T

A B

A: In U.V. 254 nm, B: After spraying with Anisaldehyde sulphuric acid reagent

Figure: 4.4 HPTLC profile of G. glabra powder

Figure: 4.5 HPTLC chromatogram of standard 18-β-G.A.



Figure: 4.6 HPTLC chromatogram of G. glabra extract

Table: 4.2 Observation table for calibration curve of std. 18-β- G.A.

(Using HPTLC method)

Standard solution Test

solution

Spot no. 1 2 3 4 5 6

µl of spot 3 4 5 6 7 8 3

Concentration

(mcg/µl)

0.3 0.4 0.5 0.6 0.7 0.8 -

Peak area 2172.2 2700 3197.7 3570.5 3766.4 4195.5 3178.9

% 18-β-G.A. - - - - - - 0.18 %

y = 3975.x + 1073.1R² = 0.977

0

1000

2000

3000

4000

5000

0 0.2 0.4 0.6 0.8 1

AU

C

Concentration (mcg/µl )

Figure: 4.7 Calibration curve of 18-β- G.A. by HPTLC method



The concentration of 18-β-glycyrhhetinic acid in G. glabra extract was calculated

using regression equation obtained from the standard curve which was found to be

0.18 %.

4.2.2 Evaluation of A. catechu whole material and Powder


Figure: 4.8 Morphology of A. catechu

The morphology of the sample was showed that it occurs as square blocks or irregular

pecies black to blackish brown in color. Outer surface was hard and brittle; when

broken fractured surface showed small cavities and soft. This morphological

characters was giving identity of A. catechu.

The A. catechu powder having brownish black color with astringent taste.

The foreign matter determination by visualization method was showing absence of

any admixture or any other obnoxious materials. The particle size of the samples was

characterized to 60 # by sieve analysis.

(2) Chemical Identification

Acacia catechu extract shows positive result for Match stick test, Vanilin

Hydrochloric acid test, 5% FeCl3 test and lead acetate test. So, it contain catechin and

tannins.


In the TLC of A. catechu extract, grey spot at Rf = 0.6 in U.V. 254 nm and after

spraying with vanillin sulphuric acid reagent respectively in both extract and std.

solution.



A B A B

(1) (2)

A: Std. catechin, B: Test solution of A. catechu powder extract ,

(1): In U.V. 254 nm, (2): After spraying with vanillin sulphuric acid reagent

Figure: 4.9 TLC profile of A. catechu powder


The various physicochemical parameters performed for A. catechu was mentioned in

Table 4.3.

Table: 4.3 Physicochemical parameters of A. catechu powder

Sr. no. Physicochemical

parameters

Observation* Reference value

1 LOD at 105 ºC 4.38 ± 0.46 -

2 Ash value

A. Total ash 11.13 ± 0.32 NMT 15%

B. Acid insoluble ash 0.6 ± 0.26 NMT 2%

C. Water soluble ash 1.4 ± 0.46 -

3 Extractive value

A. Alcohol extractive value 43.53 ± 0.53 NLT 40 %

B. Water extractive value 50.5 ± 0.71 NLT 50 %

* = Mean ± Standard Error Mean ( n = 3)



The Acacia caetchu powder shown loss on drying within the pharmacopoeial limit.

The results of ash values, water soluble extractives and alcohol soluble extractives

were also within the pharmacopoeial limit as mentioned in the table.

(5) Estimation of Catechin in A. catechu powder by HPTLC

1 2 3 4 5 T 1 2 3 4 5 T

A B

A: In U.V. 254 nm, B: After spraying with vanillin sulphuric acid reagent

Figure: 4.10 HPTLC profile of A. catechu powder

Figure: 4.11 HPTLC chromatogram of standard catechin



Figure: 4.12 HPTLC chromatogram of standard A. catechu extract

Table: 4.4 Observation table for calibration curve of std. catechin


Standard solution

Test

solution

Spot no. 1 2 3 4 5

µl of spot 5 6 7 8 9 6

Concentration

(mcg/µl)

0.5 0.6 0.7 0.8 0.9 -

Peak area 3143.8 3524.1 3822.8 4299.8 4788.5 4230.1

% catechin - - - - - 12.8 %

y = 4065.x + 1070.R² = 0.991

0

1000

2000

3000

4000

5000

6000

0 0.4 0.8 1.2

AU

C

Concentratuion (mcg/µl )

Figure: 4.13 Calibration curve of catechin by HPTLC method



The concentration of catechin in A. catechu extract was calculated using regression

equation obtained from the standard curve which was found to be 12.5 %.

(6) Estimation of total tannins in A. catechu powder

The total tannins content estimated in A. catechu was 25.36% and it reveals that the

A. catechu powder sample contain total tannins within pharmacopoeial limit which is

25-30 % .

4.2.3 Evaluation of Clove oil

(1) Description

The clove oil was transparent with pale yellow color, aromatic odour and pungent

taste.

(2) Determination of Refractive Index

The Refractive index of clove oil was measured as per IP procedure and it was within

the pharmacopoeial limit as mentioned in Table 4.5.

(3) Determination of Specific Gravity

The Specific Gravity of clove oil was measured as per IP procedure and it was within

the pharmacopoeial limit as mentioned in Table 4.5.


In the TLC of clove oil, pink spot at Rf = 0.52 and greenish yellow spot at Rf = 0.52

was observed in U.V. 254 nm and after spraying with Anisaldehyde sulphuric acid

reagent respectively in both extract and std. solution.

Table: 4.5 Results of performed physical parameters of clove oil

Sr. No. Physical parameter Observation Reference value

1. Refractive index 1.534 1.527 - 1.535

2. Specific gravity 1.039 1.38 -1.060



A B A B

(1) (2)

A: Test solution of clove oil, B: std. solution of eugenol ,

(1): In U.V. 254 nm , (2): After spraying with Anisaldehyde sulphuric acid

Figure: 4.14 TLC profile of clove oil

(3) Estimation of Eugenol in clove oil by HPTLC

1 2 3 4 5 T 1 2 3 4 5 T

A B

A: In U.V. 254 nm, B: After spraying with Anisaldehyde sulphuric acid reagent

Figure: 4.15 HPTLC profile of clove oil



Figure: 4.16 HPTLC chromatogram of standard Eugenol

Figure: 4.17 HPTLC chromatogram of clove oil

Table: 4.6 Observation table for calibration curve of std. eugenol


Standard solution Test

solution

Spot no. 1 2 3 4 5

µl of spot 2.5 5 7.5 10 12.5 6

Concentration

(mcg/µl)

0.2 0.4 0.6 0.8 1.0 -

Peak area 13350.9 15673 17123 18435 19200 13355.6

% Eugenol - - - - - 58.34 %



y = 7409.1x + 12335R2 = 0.9792

0

5000

10000

15000

20000

25000

0 0.2 0.4 0.6 0.8 1 1.2

AU

C

Concentration (mcg/µl )

Figure: 4.18 Calibration curve of Eugenol by HPTLC method

4.2.4 Standardization of extracts by HPTLC

The extracts of G. glabra and A. catechu was showed 9.71% of 18-β-glycyrhhetinic

acid and 41.14 % of catechin , respectively when standardized by HPTLC method.

4.3 Conclusion

Intended raw materials are subjected here for various quantitative and qualitative

parameters like morphology, microscopy, TLC, ash and extractive value and HPTLC.

The observations were showing satisfactory outcomes for various standardization

parameters.



References:

1. Khandelwal K.R., Preliminary phytochemical screening, Practical

Pharmacognosy, Nirali Prakashan, Page no. 125, 149-153.

2. Wagner H. and Bladt S., Plant Drug Analysis, A Thin Layer Chromatography

Atlas ,second edition, page no.170,326

3. Anonymous, “Quality standards of Indian medicinal plants”, Vol -III, Published

by Indian council for medicinal research, 2006, 73.

4. Anonymous, “Indian Herbal Pharmacopoeia”, Indian drug Manufacture’s

Association, Mumbai, 2002, page no.1,89,146.

5. Anonymous, “Quality control methods for medicinal plant materials”, WHO,

Geneva, 2002.

6. Anonymous, “ Indian pharmacopoeia”, , Government of India, Ministry of Health

and family welfare, published by the controller of the publications, Delhi, vol. II,

1996A-96, A-99.

Chapter: 5 Formulation and Evaluation of Gel

5.1 Experimental

5.1.1 Materials

Carbopol- 934P

Distilled water

Triethanolamine

Propylene glycol

Sodium dihydrogen phosphate

Sodium hydroxide

Aqueous extracts of Glycyrrhiza glabra and Acacia actechu

Clove oil

5.1.2 Instruments

Linomat V HPTLC spotter and scanner

Modified Diffusion cell

pH meter

Brookfield viscometer

5.1.3 Preparation of Gel Formulations

The Carbopol-934P gels of Glycyrrhiza glabra, Acacia actechu and clove oil was

prepared by cold method described by schmolka1.The weighed amount of carbopol-

934P (0.8%, 1%, 1.2%, 1.4 %) was placed in a beaker and sufficient amount of water

containing weighed amount of Glycyrrhiza glabra, Acacia catechu extract was added

to obtain a homogenous viscous mixture and kept at room temperature for 24 hours.

After formation of carbopol gel, weighed amount of clove oil was mixed in it with

continuous agitation and obtained gel, stored at ambient temperature prior to its use.

Table: 5.1 Preparation of Gel Formulations



Ingredients A1 A2 A3 A4 A5

G .glabra extract 1.5 % 1.5 % 1.5 % 1.5 % 1.5 %A.catechu extract 1.5 % 1.5 % 1.5 % 1.5 % 1.5 %Clove oil 1.5 % 1.5 % 1.5 % 1.5 % 1.5 %Carbopol -934P 0.6 % 0.8 % 1 % 1.2 % 1.4 %Propylene glycol 10 % 10 % 10 % 10 % 10 %Tirethanolamine 0.5 % 0.5 % 0.5 % 0.5 % 0.5 %Water (q. s.) 100 ml 100 ml 100 ml 100 ml 100 ml

5.1.4 Preparation of standard calibration curves

(1) Preparation of standard calibration curve of 18-β-glycyrhhetinic acid

1 mg of 18-β-glycyrhhetinic acid was dissolved in 25 ml pH Phosphate buffer (pH

6.8) and extracted with 25 (10+10+5) ml of ether. The ether extract was evaporated

up to 10 ml and filtered. The volume of filtrate was adjusted up to 10 ml with ether.

Standard solution of 2, 3, 4, 5, 6, 7 and 8 µl was applied on a Aluminium-backed

silica gel 60F254 plate (E. Merck) (10 x 10 cm). The plate was developed up to 85 mm

in Toluene: Ethyl acetate : Methanol: Glacial Acetic Acid (2.7:6.0:1.0:0.3) as mobile

phase at 25º C and the chromatogram was recorded by scanning at 254 nm on a

CAMAG TLC scanner.

(2) Preparation of standard calibration curve of Catechin

1 mg of Catechin was dissolved in 25 ml Phosphate buffer (pH 6.8 ) in and extracted

with 25 (10+10+5) ml of ether. The ether extract was evaporated up to 10 ml and

filtered. The volume of filtrate was adjusted up to 10 ml with ether. Standard solution

of 5, 10, 15, 20, 25, 30 and 35 µl was applied on a Aluminium-backed silica gel 60F254

plate (E. Merck) (10 x 10 cm). The plate was developed up to 85 mm in Toluene:

Ethyl acetate: Methanol: Glacial Acetic Acid (2.7:6.0:1.0:0.3) as mobile phase at 25º

C and the chromatogram was recorded by scanning at 254 nm on a CAMAG TLC

scanner.

5.1.5 Evaluation of gel formulations 2-9

a) pH



1 gm of gel was accurately weighed and dispersed in 10 ml of distilled water. The pH

of these dispersions was measured using pH meter (Systronics digital- DI- 707).

b) Viscosity and Rheological studies

Viscosities of gels were determined using Brookfield Viscometer. Gels were tested

for their Rheological characteristics at 25oC using Brookfield Viscometer (DV-III

programmable Rheometer). The Measurement was made over the whole range of

speed setting from 10 rpm to 100 rpm with 30 seconds between two successive speeds

and then in descending order.

c) Spreadibility

For the determination of spreadibility excess of sample was applied in between two

glass slides and was compressed to uniform thickness by placing 1000 gm weight for

5 minutes. Weight (50 gm) was added to the pan. The time required to separate the

two slides i.e. the time in which the upper glass slide moves over the lower plate was

taken as measure of spreadibility (S).

S= m × l/t

Where,

m = weight tide to upper slide

l = length moved on the glass slide

t = time taken

d ) Drug Content

For the determination of drug content , 1 gm of gel from each formulation was taken

and dissolved in 25 ml Phosphate buffer (pH 6.8) and extracted with 25 (10+10+5) ml

of ether. The ether extract was evaporated up to 10 ml and filtered. The volume of

filtrate was adjusted up to 10 ml with ether. 5 µl of this solution was applied on a

Aluminium-backed silica gel 60F254 plate (E. Merck) (10 x 10 cm). The plate was

developed up to 85 mm in Toluene: Ethyl acetate: Methanol: Glacial Acetic Acid

(2.7:6.0:1.0:0.3) as mobile phase at 25º C and the chromatogram was recorded by

scanning at 254 nm on a CAMAG TLC scanner. The concentration of 18-β-

glycyrhhetinic acid and catechin in the gel was determined using the calibration curve

of standard 18-β-glycyrhhetinic acid and catechin respectively.



e) In vitro Release study

In-vitro diffusion studies were carried out in fabricated diffusion tube of surface area

1.5 cm2 through cellophane membrane. The cellophane membrane was hydrated for

24 hours with 0.1N NaOH and 1 hour with distilled water, 1gm of gel was applied on

it and fixed to the one end of the tube which act as a donor compartment. The

assembly was placed in the beaker contained 50 ml of Phosphate buffer (pH 6.8). The

teflon coated magnetic bead was placed in the beaker and rotated at 100 rpm using

magnetic stirrer and the temperature was maintained at 37 1 0 C.

Samples of 3 ml were withdrawn at regular intervals of every one hrs up to 8

hrs and replace the volume with same buffer and maintained sink condition through

the studies. The samples were extracted with 5 ml of ether and filtered. The volume of








5.2 Results and Discussion

5.2.1 Preparation of gel formulations



Gels prepared with carbopol were found to be translucent and homogeneous with

characteristics colour of extracts of Glycyrrhiza glabra and Acacia catechu.

Figure 5.1: Prepared Gel Formulation


The calibration curves of 18-β-glycyrhhetinic acid and catechin were prepared as per

the procedure given in section 5.1.4 and are mention below.

y = 7720.2x - 1163

R2 = 0.9934

0

1000

2000

3000

4000

5000

6000

0 0.2 0.4 0.6 0.8 1

Concentration (mcg/µl)

AU

C

Figure: 5.2 Calibration curve of 18-β-G.A. by HPTLC method



y = 4697.2x + 953.1

R2 = 0.9932

0

4000

8000

12000

16000

20000

0 0.5 1 1.5 2 2.5 3 3.5 4


AU

C

Figure: 5.3 Calibration curve of Catechin by HPTLC method

5.2.3 Evaluation of Gel Formulations

Evaluation parameters of prepared gels have been carried out as per the procedure

given in section 5.1.5 and show in Table 5.2 and 5.3.


Table: 5.2 Evaluation Parameters of Gels

Formulation

codepH

Viscosity

(cps)

Spreadibility

(gm*cm/s)Drug content (%)

18-β-G.A. CatechinA1 6.65 587.33 × 103 3.21 84.65 71A2 6.47 747.3 × 103 1.19 80.0 73A3 6.35 124 × 104 0.93 79.5 70A4 6..27 136.33 × 104 0.81 83.2 72.65A5 6.10 145.66 × 104 0.74 81.7 68


1 2 3 4 Catechin Eugenol 18-β.G.A

1, 2, 3, 4 :spot of gel

Figure: 5.4 HPTLC profile of Gel

Figure: 5.5 HPTLC chromatogram of gel




Figure: 5.7 HPTLC chromatogram of standard catechin




In the prepared gel formulations, drug content of 18-β-glycyrrhitinic acid was within

79.5-84.65 and of catechin was 68-73 % (Table 5.2) in the formulations. Viscosity,

Spreadibility and pH were measured for different formulation are show in Table 5.2.

The Formulation A4 had viscosity 136.33 × 104 which was easy to spread.

In vitro release profile from gel formulations

The data obtained from in vitro release (Q1 and Q6) are show in Table 5.3.

Table: 5.3 In vitro Release profile of gel formulationsFormulation

code18-β-G.A. Catechin

Q1 Q6 Q1 Q6

A1 6.4 71.2 4.1 71A2 8.6 77.4 4.6 73A3 9.2 80.1 6.3 70A4 10.1 85.6 7.2 72.65A5 10.5 86.8 8.1 68

Q1 : In vitro Release of Drug in 1 hr

Q6 : In vitro Release of Drug in 6 hr



0

20

40

60

80

100

0 2 4 6 8 10

% CPR of 18

-18-β.

G.A.

Time (hr)

A1 A2 A3 A4 A5

Figure: 5.9 In vitro release profile of 18-β-G.A. in gel

in phosphate buffer (pH 6.8 ) at 254 nm

0

20

40

60

80

100

0 2 4 6 8 10

% CPR for Catechin

Time (hr)

A1 A2 A3 A4 A5

Figure: 5.10 In vitro release profile of catechin in gel

in phosphate buffer (pH 6.8)at 254 nm

5.3 Conclusion:

From this study it was revealed that as the concentration of the polymer increase , the

various parameters such as viscosity, spreadibility, pH, drug content and in vitro

release was also changed. The viscosity of the formulation was increased and

spreadibility decrease with increasing the concentration of polymer in the

formulations. The formulations were shown maximum in vitro release at 6 hr and

after that the release was constant. According to data shown in Table 5.2 for pH,



viscosity, spreadibility, Drug content and In vitro Release study, it was concluded that

the formulation A4 was better than other formulations in respect to in vitro release

profile and viscosity.

References:

1. Schmolka, I.S, J. Biomed. Maater. Res., 1972, 6, 571.

2. Saleem, M. A., Sanaullah, S. and Purohit, M. G., Antimicrobial and wound

healing activity of prepared gatifloxacin topical gels, Indian Drugs, 2005, 43(4),

292-295.

3. Kikwai, L., Babu, Jayachandra, Prado, R., Kolot, A., Cheryl A., Armstong, Ansel,

John, C. and Singh Mandeep, Invitro and invivo evaluation of topical formulations

of spantide II, AAPS PharmSciTech, 2005, 6(4), EF 65-72.

4. Nayak, S. H., Nakhat, P. D. and Yeole, P. G., Development and evaluation of

cosmeceutical hair styling gels of ketoconazole, Indian J. Pharm. Sci., 2005, 313-

316.

5. Kavitha, K., Sivaramakrishnan, M. and Nalini, C. N., Formulation and evaluation

of topical drug delivery system of fluconazole, Indian Drugs, 2003, 40 (12), 720-

723.

6. Djordjevic, J., Michniak, B. and Uhrich, Kathryn E., Amphiphilic star like

macromolecules as novel carriers for topical delivery of non steroidal anti-

inflammatory drugs, AAPS PharmSciTech, 2003, 5(4), 1-12.

7. Ota, Yusuke, Hamada, A., Nakano. M. and Saito, H., Evaluation of percutaneous

absorption of midazolam by terpenes, Drug Metab. Pharmacokin, 2003, 18(4),

261-266.



8. Uma devi, S., Ganesan, M. and Mohanta, G. P., Design and evaluation of

tetracycline hydrochloride gels, Indian Drugs, 2002, 39(10), 552-554.

9. Raghuramana.S. et.al., In Design and evaluation of propranolol hydrochloride

buccal films. Indian J. Pharm. Sci., 2002, 64 (1): 32-36.


Chapter: 6 Formulation and Evaluation of Patch

6.1. Experimental:

6.1.1. Materials:

HPMC K15M

Distilled water

Methanol

Glycerine

Sodium dihydrogen phosphate

Sodium hydroxide

Aqueous extracts of Glycyrrhiza glabra and Acacia actechu

Clove oil

6.1.2. Instruments:

Linomat V HPTLC spotter and scanner

Modified Diffusion cell

pH meter

USP disintegration apparatus

Digital vernier caliper

6.1.3. Preparation of patch formulations 1

The patches were prepared by solvent evaporation technique in Petridish. The

weighed amount of Glycyrrhiza glabra and Acacia catechu extracts were dissolved in

sufficient amount of distilled water. The weighed amount of clove oil was mixed with

sufficient amount of methanol and mixed with above mixture. The weighed amount of

HPMC K15M (0.5%, 1 %, 1.5%) was added in the mixture under stirring condition

with a magnetic stirrer. Glycyrine was added as plasticizer. The dispersion was

poured on a petridish and dried at room temperature for 24 hrs. the prepared patches

were packed in aluminum foil.

Table 6.1 : Preparation of patch formulations



Ingredients B1 B2 B3

G.glabra extract 2 mg 2 mg 2 mgA.catechu extract 2 mg 2 mg 2 mgClove oil 6 µl 6 µl 6 µlHPMC K15M 0.5 % 1 % 1.5 %Glycerine 0.2 ml 0.2 ml 0.2 mlWater 5 ml 5 ml 5 mlMethanol 5 ml 5 ml 5 ml

6.1.4. Preparation of standard calibration curve:

(1) Preparation of standard calibration curve of 18-β-glycyrhhetinic acid

1 mg of 18-β-glycyrhhetinic acid was dissolved in 25 ml Phosphate buffer (pH 6.8) in

and extracted with 25 (10+10+5) ml of ether. The ether extract was evaporated up to

10 ml and filtered. The volume of filtrate was adjusted up to 10 ml with ether.

Standard solution of 0.2, 0.3, 0.4, 0.5, 0.6, 0.7 and 0.8 µl was applied on a


developed up to 85 mm in Toluene: Ethyl acetate : Methanol: Glacial Acetic Acid

(2.7:6.0:1.0:0.3) as mobile phase at 25º C and the chomatogram was recorded by

scanning at 254 nm on a CAMAG TLC scanner.

(2) Preparation of standard calibration curve of catechin

1 mg of catechin was dissolved in 25 ml Phosphate buffer (pH 6.8) in 100 ml beaker

and extracted with 25 (10+10+5) ml of ether. The ether extract was evaporated up to

10 ml and filtered. The volume of filtrate was adjusted up to 10 ml with ether.

Standard solution of 0.5, 1, 1.5, 2, 2.5 ,3 and 3.5 µl was applied on a Aluminium-

backed silica gel 60F254 plate (E. Merck) (10 x 10 cm). The plate was developed up to

85 mm in Toluene: Ethyl acetate : Methanol: Glacial Acetic Acid (2.7:6.0:1.0:0.3) as

mobile phase at 25º C and the chomatogram was recorded by scanning at 254 nm on a

CAMAG TLC scanner.

6.1.5 Evaluation of gel formulations 2,3,4

a) Patch Thickness



Thickness of patches of was measured using digital vernier caliper at three different

places and the mean value was calculated.

b) Determination of Surface pH

For determination of surface pH, the patches (14 mm) of each formulation were kept

in contact with 1 ml of distilled water for 2 hr, in fabricated glass tubes. Excess of

water from the tubes was drained and the pH was noted by bringing the electrodes

near the surface of the formulation and allowing it to equilibrate for 1 min.

c) Folding Endurance:

The patches of each formulation of size (2× 2 cm) were cut by using sharp blade.

Folding endurance was determined by repeatedly folding a small strip of patch at the

same place till it broke. The number of times the patch could be folded at the same

place without breaking gave the value of folding endurance.

d) In vitro Residence time

The in vitro residence time was determined using USP disintegration apparatus. The

disintegration medium was 800 ml of Phosphate buffer (pH 6.8) maintained at

37±2°C. The Segment of rat intestinal mucosa, each of 3 cm length, was glued to the

surface of glass slab, which was then vertically attached to the apparatus. Three

mucoadhesive patches of each formulation were hydrated on one surface using pH 6.8

PB and the hydrated surface was brought into contact with the mucosal membrane.

The glass slab was vertically fixed to the apparatus and allowed to move up and

down. The patch was completely immersed in the buffer solution at the lowest point

and was out at the highest point. The time required for complete erosion or

detachment of the patch from the mucosal surface was recorded.

e) Drug Content

For the determination of drug content , 1.5 × 1.5 cm of patch from each formulation

was taken and dissolved in 10 ml Phosphate buffer (pH 6.8) and extracted with 10

(5+5) ml of ether. The ether extract was evaporated up to 5 ml and filtered. The

volume of filtrate was adjusted up to 5 ml with ether. 20 µl of this solution was

applied on a Aluminium-backed silica gel 60F254 plate (E. Merck) (10 x 10 cm). The



plate was developed up to 85 mm in Toluene: Ethyl acetate: Methanol: Glacial Acetic

Acid (2.7:6.0:1.0:0.3) as mobile phase at 25º C and the chromatogram was recorded

by scanning at 254 nm on a CAMAG TLC scanner. The concentration of 18-β-



f) In vitro Release study

In-vitro diffusion studies were carried out in fabricated diffusion tube of surface area

1.5 cm2 through cellophane membrane. The cellophane membrane was hydrated for

24 hours with 0.1N NaOH and 1 hour with distilled water, 0.5 × 0.5 cm of patch from

each formulation was applied on it and fixed to the one end of the tube which act as a

donor compartment. The assembly was placed in the beaker contained 50 ml of

Phosphate buffer (pH 6.8). The teflon coated magnetic bead was placed in the beaker

and rotated at 100 rpm using magnetic stirrer and the temperature was maintained at

37 1 0 C.

Samples of 3 ml were withdrawn at regular intervals of every one hrs up to 8

hrs and replace the volume with same buffer and maintained sink condition through

the studies. The samples were extracted with 5 ml of ether and filtered. The volume of








6.2 Results and Discussion

6.2.1 Preparation of patch formulations



Patches prepared with HPMC were found to be translucent, smooth and with

characteristics color of extracts of Glycyrrhiza glabra and Acacia catechu.

Figure 6.1: Prepared Patch Formulation


The calibration curves of 18-β-glycyrhhetinic acid and catechin were prepared as per

the procedure given in section 6.1.4 and are mention below.

y = 7720.2x - 116.3

R2 = 0.9934

0

100

200

300

400

500

600

0 0.02 0.04 0.06 0.08 0.1

Concentration (mcg/ µl)

AU

C

Figure: 6.2 Calibration curve of 18-β-G.A.by HPTLC method



y = 4697.2x + 95.309

R2 = 0.9932

0

400

800

1200

1600

2000

0 0.1 0.2 0.3 0.4


AU

C

Figure: 6.3 Calibration curve of Catechin by HPTLC method

6.2.3 Evaluation of Patch Formulations

Evaluation parameters of prepared patches have been carried out as per the procedure

given in section 6.1.5 and show in Table 6.2 and 6.3.

1 2 3 4 Catechin Eugenol 18-β.G.A


Table: 6.2 Evaluation Parameters of PatchesFormula

-tion

code

Patch

Thickness

(mm)

Surface

pH

In vitro

residence

time (hr)

Folding

Endurance

Drug content

(mg/cm2)

18-β-G.A. catechinB1 0.10 ± 0.015 6.15 2.50 150 4.08×10-3 4.08×10-3

B2 0.14 ± 0.021 6.37 2.75 159 6.12×10-3 6.12×10-3

B3 0.16 ± 0.015 6.42 3.15 168 7.64×10-3 7.64×10-3


1, 2, 3, 4:spot of patch

Figure: 6.4 HPTLC profile of Patch

Figure: 6.5 HPTLC chromatogram of Patch




Figure: 6.7 HPTLC chromatogram of standard Catechin


In the prepared patch formulations, Drug content of 18-β-glycyrrhitinic acid was

within 4 × 10-3 -7.7 × 10-3/ cm2 and of catechin was 0.016-0.033/ cm2 (Table 6.2).

Patch Thickness, Surface pH , In vitro residence time and Drug content were

measured for different formulation was shown in Table 6.2.

In vitro release profile from patch formulations

The data obtained from in vitro release study are show in Table 6.3.

Table: 6.3 In vitro Release profile of patch formulationsFormulation

code18-β-G.A. Catechin

Q0.5 Q3 Q0.5 Q3

B1 8.9 75.8 7.6 71.8



B2 13.5 79.3 8.6 75.1B3 15.3 83.2 12.22 80.0

Q0.5: In Vitro Release of drug in 0.5 hr

Q3: In Vitro Release of drug in 3 hr

0

20

40

60

80

100

0 1 2 3 4 5 6

% CPR of18

-β.G.A

Time (hr)

B1 B2 B3

Figure: 6.9 In vitro release profile of 18-β-G.A. in patch

in Phosphate buffer (pH 6.8) at 254 nm

0

20

40

60

80

100

0 1 2 3 4 5 6

% CPR of Catechin

Time (hr)

B1 B2 B3

Figure: 6.10 In vitro release profile of catechin in patch

in Phosphate buffer (pH 6.8) at 254 nm

6.1 Conclusion:



From this study it was revealed that the as the concentration of the polymer , the

various parameters such as surface pH, in vitro residence time, drug content and in

vitro diffusion was also changed. The In vitro residence time of the formulation was

increased with increasing the concentration of polymer in the formulations. The

formulations were shown maximum in vitro diffusion at 3 hr and after that the

diffusion was constant. The selection of best formulation depends on the In vitro

residence time and In vitro Release study. The in vitro Release was higher in the

formulation B3 as compared to other formulations. The In vitro residence time of

formulation B3 was also higher as compared to other formulations and it was retain

on the mucosal surface up to the maximum diffusion of the drug . So, formulation B3

was selected as the best formulation.

References:

1. Ting Li et. Al., Optimized preparation and evaluation of Indomethacin

Transdermal Patch, Asian Journal of Pharmaceutical Sciences, 2007, 2 (6): 249-

259.

2. Semalty M., Semalty A., Kumar G., Formulation and Characterization of

Mucoadhesive Buccal Films of Glipizide, Indian Journal of Pharmaceutical

Sciences, Jan-Feb 2008, 43-48.

3. Sahni J., Raj S., Ahmad F.J. Khar R.K., Design and In Vitro Characterization of

Buccoadhesive Drug Delivery System of Insulin, Jan-Feb 2008, 61-65.

4. Raghuramana.S. et.al., In Design and evaluation of propranolol hydrochloride

buccal films. Indian J. Pharm. Sci., 2002, 64 (1): 32-36.




Chapter: 7 Antimicrobial Activity of selected Formulations

7.1 Introduction 1

Pharmaceutical compositions are provided which comprise effective amounts of

antimicrobials, anti-inflammatories, and antihistamines, to provide an ulcer

medication which prevents secondary infections and promotes healing while

providing immediate relief from pain. Antimicrobial compounds can be used to

prevent the ulcer from spreading and relief.

In some cases there are infectious agents that are both bacterial and viral in nature that

are considered as causes of mouth ulcers. The various chemical compounds that are

found within the infectious agents are perhaps one of the reasons for mouth ulcers

forming.

7.2 Material and method 2-6

Test organisms and Inoculums:

Gram positive:- Bacillus subtilis, Staphylococcus aureus

Gram negative :- Escherichia coli, Pseudomonas aeruginosa

Standard:- Antimicrobial Nitrofurazone gel (500mg).

Media: - Dehydrated nutrient agar media was used and was prepared in distilled

water. The composition of the media was as given below.

Composition of nutrient agar medium

1) Agar 15.0%

2) Peptic digest of animal tissue 5.0%

3) Sodium chloride 5.0%

4) Beef extract 1.5%

5) Yeast extract 1.5%

6) pH 7.4 ± 0.2 at 25oC

7) distilled water 1000 ml

The medium was autoclaved at 15 lbs per square inch pressure at 121oC

Preparation of media: - Dehydrated nutrient agar media (28gm) was

accurately weighed and suspended in 1000 ml of distilled water in a conical

flask. It was heated on a water bath to dissolve the medium completely.



Sterilization of media: - The conical flask containing the nutrient agar

medium was plugged with the help of non-absorbent cotton bung. The mouth

of the conical

flask and the cotton bung were properly covered with aluminum foil. The medium

was then sterilization by autoclaving at 15 lbs per square inch pressure for 20 minutes.

Method: - Cup and Plate method. The sterile nutrient agar medium at a

temperature between 40o to 50o C was immediately poured into the sterile Petri

plates to give a depth of 3 to 4 mm, by placing the plates on a level surface.

The plates were then allowed to solidify. Each plate was then inoculated with

0.1ml of the solution of test organisms prepared in water for injection. The

wells in each plate were bored in the centre that was filled with 500 mg plain

gel, 300,500 and 700 mg of selected gel (A4) and 0.5 cm2 plain patch , 0.5 cm2

and 1.0 cm2 of selected patch (B3). The plates were then incubated at 37o for

24h. After incubation, zonal inhibition (inhibition around each well) was

measured and this value was taken as an indicator for the antimicrobial

activity.

7.3 Result and Discussion

7.3.1 Result of Antimicrobial activity of selected Gel Formulations

(a) (b)



(c) (d)

Figure: 7.1 Antimicrobial Activity of gel formulation; (a) Bacillus subtilis ,

(b) Staphylococcus aureus, (c) Pseudomonas aeruginosa , (d) Escherichia coli

The zone of inhibition of gel formulations was mentioned below in Table 7.1

7.3.2 Result of Antimicrobial activity of selected Patch Formulations

(a) (b)

(c) (d)


Table: 7.1 Zone of Inhibition (cm) of gel formulationNitrofurazone

gel

Plain

gel0.3 mg gel 0.5 mg gel 0.7 mg gel

Bacillus subtilis 2.2 0 1.8 1.7 2.5Escherichia coli 2.0 0 2 2.4 2.7Pseudomonas

aeruginosa2.7 0 1.9 2.2 2.6

Staphylococcus

aureus2.0 0 2.5 2.7 2.9


Figure: 7.2 Antimicrobial Activity of patch formulation; (a) Bacillus subtilis ,

(b) Staphylococcus aureus, (c) Pseudomonas aeruginosa , (d) Escherichia coli

The zone of inhibition of patches formulations was mentioned below in Table 7.2

7.4 Conclusion :

The selected formulation of gel and patch was evaluated for antimicrobial activity

against various gram positive and gram negitive bacteria. the plain gel and patch

without the herbal extracts did ot shown any zone of inhibition .The formulations

were shown the significant antimicrobial activity against various bacteria and which

would be benificial in the Mouth ulcer.

References:

1. Campbell, Phillip, Lesion and ulcer medication ,United States Patent 6352711

2. Anonymous, “British pharmacopoeia”, 2002, Pharmaceutical press, London, 796.

3. Anonymous, “Indian pharmacopoeia”, 1996, The Controller of Publications,

Delhi, Vol. II, A-105.

4. Vivek K. Gupta, Atiya Fatima, Uzma Faridi, Arvind S. Negi, Karuna Shanker,

J.K. Kumar, , Journal of Ethnopharmacology, 5 March 2008,116( 2) , 377-380.

5. G.A. Ayoola, African Journal of Microbiology Research. July 2008, 2 , 162-166

6. Li ZhongXing, Wang XiouHua, Yue YunSheng, Zhao BaoZhen, Chen JingBo, Li

JiHong, Chinese Journal of Information on Traditional Chinese Medicine.


Table:7.2 Zone of Inhibition (mm) of patch formulationPlain patch 0.5cm2 patch 1.0 cm2 patch

Bacillus subtilis 0 1 1.6Escherichia coli 0 1.2 1.2Pseudomonas aeruginosa 0 0.8 0.9Staphylococcus aureus 0 0.9 1

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