RESUBMISSION OF PROPOSAL AS PER MODIFICATION

TITLE OF THE PROJECT

ISOLATION AND CHARACTERIZATION OF BIOMOLECULES OF

PHARMACEUTICAL, COSMETIC AND NUTRITIONAL IMPORTANCE FROM

NATIVE. PLANTS OF CHHATTIS6ARH STATE

MODIFICATIONS IN PROPOSAL AS PER DIRECTION OF

CHHATTISGARH STATE MEDICINAL PLANT BOARD, RAIPUR (C.G.)

Submitted by

DR. ANIL KUMAR Asstt. Professor

Deptt. of Biotechnology/Zoology Govt. V.Y.T.P.G. Autonomous College, Durg I

Chhattisgarh

INTRODUCTION

During the last decade, "Biotechnology•· has emerged as a powerful discipline for the

manipulation of life-forms. Plant biotechnology is particularly, arousing a great interest in both

the developed and developing countries because of its vast ramification in agricultural and

medical field. The potential of plant biotechnology is based on the totipotency of plant cells

regeneration of complete plants from cultured cells, and the production of metabolite variants

with useful characters.

Plant tissue cul Lure technique allow isolation of variant cell lines and plants, microtubular

production, the generation of somatic hybrids by protoplast fusion and the regeneration of

genetically engineered plants from single transformed cells. In addition, the potential value of

somaclonal and gametoclonal variation. haploid plant production, in vitro fertilization, embryo

rescue, and in vitro gerrnplasm conservation is now being increasingly recognized.

Medicinal plants are the most important sources for life saving drugs for the majority of

the worlds' population. The biotechnological tools are important to select, multiply and

conserve the critical genotypes of medicinal plants. In vitro regeneration holds tremendous

potential for the production ofhigh-quality plant based medicine.

Cryopreservation is long term conservation method in liquid nitrogen and provides an

opportunity for conservation of endangered medicinal plants. In vitro production of

secondary metabolites in plant cells suspension cultures has been reported from various

medicinal plants. Bioreactors are the key step towards commercial production of secondary

metabolites by plants biotechnology. Genetic transformation may be a powerful tool for

enhancing the productivity of novel secondary metabolites, especially by Agrobacterium

rhizojenes induced hairy roots.

The World Health Organization (WlIO) has recommended that all members' state actively

promote native medicines in their countries. The WHO estimated that some 20,000 species of

higher plants are used medicinally throughout the world (Phillipson, 1994) but over 85% of the

plants await scientific investigation of their biological activity and chemical constituents

(Houghton, 1995). India is endowed with a rich flora, as it is situated on the tropical belt and, of

approximately 15,000 species or higher plant in fndia, medicinal uses are attributed to at least

1500 plants (Husain, 1992; Kirtikar and Basu, 1956). Our Chhattisgarh State is remarkably a

hot spot of biodiversity with very good forest zone. so urgent need is immediate evolution of

plants of medicinal property from Chhattisgarh region for their micropropagation as well as

medicinal isolation and characterization.

CONCEPT

In our Chhattisgarh State the floral diversity 1s extensive and even today

maximum rural populations are dependent on traditional medicine from plant resource. A large

network of Baigga community/villagers (the rural traditional doctors) community are serving

mass of the population especially among the tribe population and they have good knowledge

about traditional floral resource.

Our concept is we have to learn idea from Baigga community for scientific

validation of plant with following goals ­

1. Scientific verification of medicinal property of the plants used by Bagga' s in tribal area.

2. Isolation and characterization of therapeutic agents from plant.

3. Quantity improvement of important therapeutic agents through cell culture method.

4. Identification of antimicrobial and antifungal properties of plant.

5. Development ofprotocol for micro propagation of imp01tant species. (Tissue - Culture).

JUSTIFICATION

Indigenous scientific evaluation of om original medicinal plant is urgent need of

today's scenario of liberalized economy. It is expected that many biomolecules of our

indigenous plant may prove medically significant for global market. Micropropagation is

another aspect by which we can help pharmaceutical industry as well as local people. Once it

will be established it will open opportunity of employment for local people and thus livelihood

of local people will improve.

So many biomolecules have been isolated and characterized from indigenous

plants by many institutes up to til I now. Some imp01iant examples are ­

1. Immunomodulator berberin from Berberis aristata by RRL, Jammu.

2. A novel peptide (8 kDa) has been isolated from seed extract of bitter guard (Karela)

(Momadica charantia) having hypoglycaemic activity by JNU, New Delhi.

3. Anti oxidant property of Terminalia mjuna has been established by AIIMS, New Delhi.

4. Extr~ct from medicinal plants (Holorrhena antidysentrica and Cyperus rotundus) have

been identified to have anti-proliferative activity against Entamoeba hystolylica by Bose

Institute, Kolkata, etc.

Micropropagation protocols was also established by various institute by many

institute. Some of them are ­

1. For Swertia chirayita, Lavendula officinalis and Gymnema sylvestre by KAU, Trissur.

2. For Crataeva magna by BISR, Jaipur.

3. For Garcinia indica by KETs V.G. Vaze College, Mumbai.

We are also working on same line and we feel that a large scale project is required

for both biomolecule isolation & characterization as well as micropropagation for medicinally

significant plants of Cbhattisgarh state. It will be fru itful for national documentation of om

original plant, scientific evaluation of our lrnditional knowledge industrial application of

pharmaceutical property of our plants, conservation of our original indigenous plant, upliftment

of livelihood of local people and finally will be a great boost for forestry of Chhattisgarh as

well as nation.

THE COMMERCIALLY VIABLE SPECIES

The present project is divided into three phase

The commercially viable species considered for the I51 phase project work are as below mentioned ­1. Ocimum gratissium Linn. (Ban Tulsi) 2. Pueraria tuberosa De. (Patal Kuumda) 3. Boerhavia diffusa Linn. (Punarnava) 4. Curculigo orchiodes gaertn. (Kali Musli).

1. Ocimum gratissium Linn. (Ban Tulsi) The plant belongs to Family Labiatae and abundantly available in the forest of Chhattisgarh. Tn

Our Society the plant is known for medicinal use of neurological problems; rheumatic

problems and seminal weakness. Its oil having antibacterial and antifungal property and in

homeopathy the plant leaves are used for constipation, cough, fever etc. Some ingredients

reported from this plants are myrecene and eugenol.

We expect many more ingredients from the plant m different quantity from land of

Chhattisgarh for commercial application.

2. Pueraria tuberosaDe. (Patal Kuumda) This Plant belongs to Family Papilionacae and are reported from Punjab, Western UP and

Central India. In our traditional medicinal Syatem different parts of plants are generally used

for treatment of various disease like - Diuretic problem , Cardiac tonic, Fertility problem, as

refrigerant in feveras cataplasm feor swelling of joints, for protection of hepatic damage , as

anti implantation factor.

Further investigation for separation and characterization of active ingredients from different

parts is urgently required. Another important gap is characterization of Specific ingredients for

specific Pharmaceutical application.

3. Boerhavia diffusa Linn. (Punarnava) The plant belongs to Family Nyctaginaceae and found across the country. lt is generally used

as Diuretic, anti inflammatory, antiarthritic, spasmolytic, antibacterial, anti convulsant,

analgesic, expertorant, CNS depressant, and abortifacient. One active ingredients Quinolizidine

alkaloid has been identified from the plant but still many more active ingredients for specific

pharmaceutical imporatance are yet to be worked out.

4. Curculigo orchiodes gaertn. (Kali Musli).

This plant belongs to Family Amangllidaceae and is reported prominently from subtropical

Himalayan, Western Ghats and Some parts of Central India. Its medicinal importance is

primarily concerned with Nervine, adaptogenic, sedative, anticonvulsive, androgenic, anti­

inflammatory, Jaundice. Urinary disorder. Skin disease, Asthma. The plant is also used for

immunological improvement and sexualabi lity improvement, besides, it is hypoglycemic too.

Some important active ingredients reported from the plant are Saponins and Curcu ligoside. But

keeping in mind the above medical importance we are sure that many more active ingredients

can be isolated and characterized from different parts of plants.

lo the second phase following 10 plants will be considered ­

I. Apamarg (Achyrenthes aspera)

2. Wedelia (Wedelia wal/ichi)

3. Hadjor ( Cissw; quadragularis)

4. Nirgundi (Vitex nirgundo)

5. Gudmar (Gymnema sylvestres)

6. Dhatura (Datura slramonium)

7. Chirayata (Swertia angw;tifolia)

8. Bel (Aegle marmelos)

9. Baheda (Tenninalia bellerica)

I0. Harra (Terminalia chebula)

In the third phase following 11 plants will be considered ­

I. Aswagandba (Witlwnia somnifera)

2. Amaltas (Cassia.fistula)

3. Adusa (Adathoda vasica)

4. Chhota Chirayata (Enicostemma liolrale)

5. Kali Haldi (Curcuma zedoria)

6. Arjuna (Terminalia mjuna)

7. Ghrit Kumari (Aloe indica)

8. Amla Bhui (Phyllanthus amarussclwm)

9. Kalihari (Glorioso superba)

10. Dronpushpi (Leucas ceph/atus)

II Vaividang (Embelica ribes)

Further plants shall be considered for the experiment as per advice of Medicinal Plant Board and

feasibility.

PROTOCOL FOR STANDRIZATION OF ACTIVE INGREDIENTS (METHODOLOGY)

STEP- I- STERLIZATION • Different parts of plants (lea( root, stem, flower/inflorescence) shall be collected and

properly steri I ized.

STEP - II - EXTRACTION

• Extraction of all the parts will be obtained by 'Soxhlet Extraction Method ' in different

solvent (Aquous, Methanolic, Ethanolic etc.)

STEP - III - FTIR - ANALYSIS

• Samples from each part will be analyzed by 'Fourier Transfer Infrared Spectroscopy' (FT­

IR- Spectroscopy).

• An - IR spectrum represent a fingerprint of a sample with absorption peaks which

correspond to the frequencies of vibrations between the bonds of the atoms making up the

material. Because each different material is a unique combination of atoms no two

compounds produce the same IR spectrum. Therefore, IR spectroscopy can result a positive

identification (quantitative analysis) of every different kinds of material. In addition the size

of the peaks in the spectrum is a direct indication of the amount of material present

(Quantitative analysis)

• Before FT-IR- spectroscopy, extract will be lyophilized.

• We have both FT-IR spectroscopy and Lyophilizer.

STEP - IV - HPTLC ANYL YSIS

• All he samples shall processed for HPTLC analysis.

• HPTLC generate a chromatographic fingerprint of the drug sample in the form of a unique

sequence of peaks or zones due to the components of the sample. The fingerprint of

botanically authenticated raw material serve as a primary reference against which unknown

material can be characterized. Both samples and reference material are chromatographed

side by side on the sample plate. The resulting fingerprints are then compared with respect

to the number, sequence, position and color of separated zone.

• We do not have HPTLC - so the park of experiment will be performed by hiring service.

STEP- V-HPLC-ANALYSIS

• Some chemical compounds needs HPLC analysis.

• lt is high performance liquid - chromatography which is a from of column chromatography used frequently in biochemistry and analytical chemistry to separate, identity & quantify compounds.

• This part of the project work shall be performed if required as per development of project result.

• We do not have HPLC, so hiring service will be required.

STEP - VI - NMR -ANALYSIS • Nuclear magnetic resonance spectroscopy is a technique which exploits the magnetic

properties of certain nuclei. In this project work the required application may be proton

NMR and Carbon - 13 NMR spectroscopy. In principle it is applicable to any nucleus

possessing spin.

• NMR analysis will be need based as per development of project result.

• We do not have NMR, so if needed, this part of experiment will be performed by hiring

service.

STEP - VII-HEAVY METAL ANALYSIS BY ATOMIC ABSORPTION SPECTROPHOTOMETER

• Presence of heavy metal in the plant parts is very common which may causes adverse effect

on health and ham pie prospect ofmedicinal application of plants. So analysis of heavy metal

both qualitatively & quantitatively is very essential.

• The analysis of heavy metal shall be performed by atomic absorption spectrophotometer.

• We have Atomic Absorption Spectrophotometer in our Lab.

STEP - VIII - MICROPROPAGATION • Micropropagation of all the plants is another important part of the project with the aim to

enhance active ingredients (Secondary metabolites).

• Micropropagation shall be performed in Lab.

• We have all essential for this part ofexperiments like Tissue Culture Lab and supplementary

Apparatus.

EXISTING FACILITY FOR BIOMOLECULAR STUDY

We have FTIR, ATOMIC ABSORPTION SPECTROPHOTOMETER, GAS

CHROMATOGRAPHY, UV- VIS SPECTROPHOTOMETER, LYOPHJLIZER, PCR, DNA

SEQUENCER, GEL DOCUMENTATION SYSTEM AND TISSUE CULTURE LAB WITH ALL

SUPPLEMENTARY APPARATUS.

But we do not have

•!• HPTLC

•!• HPLC

•!• NMR

FINANCIAL REQUIREMNT FOR BASTAR ZONE (DURG)

1. Man power

a. Research Scholar (4 nos.)

b. Class IV (2 nos.)

2. Recurring

3. Non Recurring

a. HPTLC

b. Green House & Mist House

4. Field and Travel

5. Hiring Charges

6. Overhead and Lab Requirements

Total

17 ,28,000=00

2, 16,000=00

50,00,000=00

25,00,000=00

10,00,000=00

8,00,000=00

10,00,000=00

8,00,000=00

1,30,44,000=00

Rs. One Crore Thirty Lakh Forty Four Thousand

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PHASE WISE REQUIREMENTS OF BASTAR ZONE

PHASE-I

I. Man Power ­

a. Research Scholar (4 nos.) 17 ,28,000=00

b. Class IV (2 nos.) 2, 16,000=00

2. Recurring 30,00.000=00

3. Non Recurring ­

25,00,000=00 a. HPTLC

b. Green House I Mist House 10.00.000=00

3.00.000=00 4. Field & Travel

5. Hiring Charge 4,00,000=00

6. Overhead & Lab Requirement 4,00.000=00

Total 95, 44,000=00

PHASE- II

1. Recurring 10,00,000=00

2. Field & Travel 2,50,000=00

3. Hiring Charge 3,00,000=00

4. Overhead & Lab Requirement 2,00,000=00

Total 17, 50,000=00

PHASE- III

10,00.000=00 l. Recurring

2.50.000=00 2. Field & Travel

3. Hiring Charge 3.00.000=00

4. Overhead & Lab Requirement 2.00.000=00

Total 17, 50,000=00

Explanation ­

l. Phase I is establisl11Tient phase oflhe project, so require maximum expenditure.

2. Out of total duration of three year the Phase l \\ ilJ be completed in 151 year.

3. Man power will be appointed for complete duration or the project.

4. Due to establishment phase maximum basic and specilic chemicals and glassware etc. \\·ill be

required in Phase I. so out of Rs. 50 LakJ1s recurring grant. total Rs. 30 Lakhs will be required

in initiaJ stage.

5. Non recurring grant will be required in !'1 phase onl}. Both HPTLC and Green/ Mist House

will be essentially required from very beginning of U1c project operation.

6. Extensive field work is required before initiation of the chemical analysis. so out Rs. 8 Lakhs.

Rs. 3 Lakbs is proposed in 1~1 phase.

7. Out of Rs. 10 Lakhs , total Rs. 4 Lakhs is proposed in first phase as hiring charge for NMR

like analysis.

8. Out of Rs. 8 Lakhs for over head & lab requirement. total Rs. 4 lakhs is required in early

stage to meet the initial requirement of the lab.

9. ln Phase 11 the lab wi ll require only support of chemicals & glassware from rccu1Ting head, so

Rs. 10 Lakhs is proposed. Similar!) from field & travel. hiring charge and Overhead charge

Rs. 2.5 Lakhs, Rs. 3 Lakhs and R~. 2 Lakhs respccti, d) are proposed to meet the running

requirement of the lab.

10. In Phase IIl, for fLuther analysis again recurring for d1emical & glassware will cost Rs. JO

Lakhs and field & travel, hiring and over head will require further Rs. 2.5 Lakhs. Rs. 3 Lakhs

and Rs. 2 Lakhs respectively to supplement the need or running lab.

11. If further plants are considered after completion of third phase than a n~v: financial suppo11

will be essential as per the need of the experiment.