on Potential Biotechnology Programmes using Bioresources of NE - - PowerPoint PPT Presentation

National Workshop

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“Potential Biotechnology Programmes using Bioresources of NE regions”

DBT-NECAB Workshop Assam Agricultural University, Jorhat September 13 -14 , 2019

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• Dr. P.M. Bulakh

Former Dean, B.J. Medical College And Sassoon General Hospital, Pune. Director, Board Of College And University Development, Bharati Vidyapeeth Deemed To Be University.

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Bioprospecting practice in the pharmaceutical industry

• Bioprospecting is the process of discovery and commercialization of new

products based on biological resources.

• These resources or compounds can be important for and useful in many

fields, including pharmaceuticals, agriculture, bioremediation, and nanotechnology, among others.

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• Between 1981-2010, one third of all small molecule new chemical

entities approved by the U.S. Food and Drug Administration (FDA) were either natural products or compounds derived from natural products.

• Despite

indigenous knowledge being intuitively helpful, bioprospecting has only recently begun to incorporate such knowledge in focusing screening efforts for bioactive compounds.

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Bioprospecting practice in the pharmaceutical industry (Contd…)

Need of bioprospecting

• The underlying aim of bio prospecting is to find new resources and

products from nature that can be used by humans.

• Improving human health, through both medicine and better

nutrition are key focal areas.

• It plays a dominant role in discovering leads for drug development,

since existing/known compounds for developing drugs for human use are limited.

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Need of bioprospecting

• A study showed that between 1983 and 2003, almost two

thirds of anti-cancer agents being investigated as drug candidates were derived from natural products.

• Other related sectors, such as crop plant biotechnology,

screen natural resources for useful traits, such as disease resistance.

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Approach to drug developed

• A different approach has been developed to make use of the

structural diversity of isolated natural products: in silico drug discovery or virtual screening.

• In

this, the chemical structures and physico-chemical properties of compounds are gathered in a computerised database that can be searched to find matches either to complement the three-dimensional structure of a drug target

• r the chemical features of a compound with the desired

activity.

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Drug development protocol

R Janna, Q Cassandra, I Gazi. The Four-Sided Triangle of Ethics in Bioprospecting: Pharmaceutical Business, International Politics, Socio-Environmental Responsibility and the Importance of Local Stakeholders. Ethnobiology and Conservation, 2012

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Bioprospecting programmes : Examples from India

• CSIR Coordinated Programme on Drug Discovery (1996)
• Dept. of Biotechnology – Bioprospecting and Molecular

Taxonomy Programme (1998)

• New Millennium Indian Technology Leadership Initiative

(NMITLI) – Planning Commission/CSIR (2002)

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Bioprospecting : Major Areas

Chemical prospecting

• Drug &

Pharmaceuticals

• Pesticides
• Cosmetics
• Food additives
• Other industrially

valuable products

Gene prospecting

• Genetic engineering
• Crop developmnet
• Fermentation
• Cell culture

Bionic prospecting

• Designs
• Sensor technologies
• Architecture
• Bioengineering
• Bio-modeling

Bioprospecting : Major Areas

• P. Pushpangadan. National Botanical Research Institute, Lucknow, IUCN South Asia Regional Training Programme on Bioprospecting, Access and Benefit Sharing,

NBRI, 10-12, January 2005

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Ocimum sanctum: (Holy Tulsi)

Actions of O.santum

❑ Antimicrobial, ❑ Antispasmodic, ❑ Bactericide, carminative, ❑ Biological activity ❑ Luteolin anthelmintic, ❑ Hepatoprotective, ❑ Antiviral, ❑ Larvicidal – ❑ Antinociceptive, ❑ Anti-fungal, ❑ Antiaflatoxigenic, ❑ Anti-diarrheic, ❑ Amylase inhibitory ❑ Insecticidal ❑ Mosquito repellent ❑ Antiinflammatory ❑ Wound healing ❑ Antidiabetic ❑ Antipyretic ❑ Antimelanoma ❑ Rediaoprotective ❑ Antioxident ❑ Anticancer ❑ Anthelmintic ❑ Anticandidal ❑ Hypolipaemic

Research Methodology

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Types of research

* Applied research * Clinical trials

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Scientific Advisory Committee

Chairperson – Outsider. ( Professor/ Scientist from National Institute) Subject Experts

• Internal
• External

Statistician Member Secretary

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Ethics Committee

IEC (Independent Ethics Committee) IRB – Institutional Review Board Constitution

❖ Chairperson (Preferably outside the Institution) ❖ Member Secretary (Within the Institution) ❖ 1-2 Basic Med. Scientists ❖ 1-2 Clinicians ❖ One legal expert / retired judge ❖ One Social Scientist / NGO ❖ One Philosopher ❖ Lay person from Community eg. Housewife

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Nuremberg Code

“Voluntary consent of the human subject is absolutely essential”

• Human experiments must be based on prior

animal experiments

• Anticipated results should justify the experiments
• Only qualified scientists should conduct research
• Physical and mental suffering should be avoided
• There should be no expectation of death or

disabling injury from an experiment

Members of Animal Ethics Committee

❖CPCSEA ( Committee for the purpose of control and supervision of experiments

• n animals)

❖This is a committee formulated by Government of India to control experiments on animals

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Members of Animal Ethics Committee contd…

❖Members of ethics committee are

– Nominated by CPCSEA: Main nominee – Scientist from outside the institute – Socially aware member ( Generally having knowledge about animals) – Nominated from institute : 4 or 5 members – Scientist in charge of animal house – Chairman nominated from amongst the people (Generally Principal) – Scientist from other speciality – Veterinary Doctor – Member Secretary

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Regulatory Authorities

❖Indian /council of Medical Research ❖State Government ❖Central Government ❖Data Safety Monitoring Board.

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DSMB Composition

• Clinical Medicine experts
• Biostatistician, Bioethics
• Pharmacologists
• Pt. Advocate, community representative
• Chairman – previous experience in monitoring
• Ex officio – member secretary, no voting rights.

Functions of DSMB

Initial Meeting 1) Protocol approved by IRB 2) CRF & DM Plans 3) Informed consent forms, Protocol design – capture all efficacy and safety.

Types of research

* Applied research * Clinical trials

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Animal Models

❖Alloxan Induced Diabetes Mellitus

Antidiabetic drugs (catalin), Medicinal plants

❖Cholesterol Induced Atherosclerosis

statins, Garlic, Onion, Vitamin C, Aspirin

❖Glucose Induced Cataract in Goat lenses.

Medicinal Plants : Amala, Bael, Jamun and Garlic

❖Steroids Induced cataracts in goat lenses. Metabolites – Uric acid, Bilirubin, certain amino acids having antioxidant properties. ❖UV and IR Radiation Induced Cataract :

Goat lenses were exposed to UV and IR Radiation.

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Effect of Medicinal Plants on Lens Enzymes in Experimental Diabetic Cataract

by Arti Hajarnavis Under The Guidance of

• Dr. P.M. Bulakh

Department of Biochemistry BVDUMC, Pune.

Cataract

❖ Cataract – opacification of lens of eye is a leading cause of visual impairment. ❖ Accounts for 51 % of global blindness. ❖ Cataractogenesis

• is more pronounced and accelerated in

diabetic individuals ❖ Is one of the earliest secondary complications of Diabetes mellitus. ❖ Risk of cataract advances with duration and severity of diabetes.

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Treatment of cataract

❖A definite pharmacological therapy for cataract is not yet available. ❖Surgical removal of cataractous lens is the

• nly respite to cataract patients.

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Mechanism of Cataractogenesis

• Multiple mechanisms have been proposed in the development of

diabetic cataract.

• Three important and probable

mechanisms are:-

• Non enzymatic glycation of lens

proteins

• Oxidative stress and
• Increased polyol pathway in

hyperglycemia.

In view of this, researchers have proposed a ‘multi target’ strategic approach for a possible prevention / delay

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diabetic cataractogenesis

• This could be achieved by

using substances which could act as – – Antiglycation agents – Antioxidants and – Aldose reductase inhibitors

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Medicinal Plants

❖Phytochemicals from various plants have been found to have such multiple beneficial effects in diabetes and its complications. ❖India has a rich heritage of ancient traditional systems

• f medicine .

❖ Various indigenous plants are used in Ayurveda for the treatment of diabetes and cataract .

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Sample Collection

❖ Goat eyeballs were obtained from the slaughter house and were transported to the laboratory in an ice-box. Lenses were removed from the eyeballs by intracapsular lens extraction method. ❖The weight of the lenses was recorded and lenses were then carefully placed on sterile petri dishes with a dark colored nylon net. ❖The lenses were incubated in tissue culture medium (TC-199) by “Lens Organ Culture Technique” for 72 hours.

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Preparation of Tissue Culture Medium:

❖ Tissue Culture medium -199 (Medium 199- with Earles salt and without sodium carbonate) was supplied in powder form by Hi Media. ❖The powder was reconstituted to one litre with distilled water. Penicillin and mycostatin were added to the medium to prevent bacterial and fungal contamination. ❖The pH of the medium was adjusted and maintained at 7.2 to 7.4 ❖All materials and solutions required for lens organ culture were autoclaved under standard conditions for the elimination of microorganisms including bacterial spores.

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Lens Morphology

❖The lenses were placed on a grid/net and changes in lens transparency were observed by noting the number and characteristics of the squares of the grid/net seen through the lens. Generalized haziness or opacity, intumescence, swelling, disruption and other morphological changes in the lens were also noted.

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Lenses were removed from the culture medium (after 72 hours) and placed on glass slides with grids and visually observed for changes in lens clarity and transparency.

A) Control Transparency, clarity maintained (grids visible) B) Dextrose Opaque, mature cataract, nearing rupture (grids not visible)

Changes in lens morphology

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Lens Morphology

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Preparation of Homogenate

❖At the end of 72 hours, the lenses were removed from the culture media and gently rolled on filter paper to remove adherent water. ❖Each lens was then homogenized in 0.1 M Sodium phosphate buffer, pH 7.4 and w/v was adjusted to 10gm %. ❖The homogenate was centrifuged at 10,000 x g for 30 min at – 40 degree C in a refrigerated centrifuge. ❖The supernatant was collected and stored at –20 degree C until further use.

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Preparation of medicinal plant water extracts

❖The guidelines mentioned in the Ayurved Pharmacopeia of India were followed for preparing the plant water extracts. ❖Dry powders of S.cumini (Jambhul)seeds, Aegle marmelos (Bael) leaf, Embilica officinalis (Amla) fruit respectively were taken and 25% w/v water extract was prepared .

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❖25 gm fresh peeled garlic cloves (Allium sativum) were ground in 100 ml distilled water to prepare the extract. ❖All extracts were analysed for their purity and Total Dissolved Solids at the ‘Indian Drugs Research Association & Laboratory’, Pune. ❖0.25% solution of each extract was used for the study.

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Syzygium cumini (Jambhul)

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• S. Cumini seeds ( Jambhul)

❖Various flavonoids are known to inhibit AR. S.cumini seeds being rich in flavonoids the inhibitory effect on AR could be attributed to the presence of these flavonoids. ❖Jambhul seeds are reported to be rich in flavonoids or Polyphenols due to which they exhibit high free radical scavenging activity, thereby sparing the antioxidant enzymes.

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Aegle marmelos (Bael)

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Aegle Marmelos ( Bael)

❖High amount of flavonoids in a bael leaves act as scavengers of free radicals. ❖The extract has been shown to have membrane stabilizing property which probably prevents the osmotic imbalance related protein damage.

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Emblica officinalis (Amala):

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ß - glucogallin

Amala

❖β-glucogallin is the most prominent tannin found in amla ❖It favourably binds to the active site of the enzyme AR. ❖The glucose moiety present in the structure of this molecule binds to the active site of the enzyme and appears to mimic the natural substrate – thereby preventing sorbitol formation

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Amala contd..

• This helps to maintain osmotic balance and

prevent the excessive formation of advanced glycation end products via this pathway.

• Amla may hence also play an important role in

maintenance of lens proteins and its clarity.

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Allium sativum (garlic)

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Allium Sativum ( Garlic)

❖ Garlic extract brought significant preservation of the soluble proteins in the lens. ❖ It (Allicin- active principle) prevents protein glycation. ❖ Decrease in Lipid peroxidation.

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GROUP TRANSPARENCY & CLARITY B) Dextrose Opaque, mature cataract, nearing rupture (grids not visible) c) Dextrose + S.cumini Transparency, lens integrity maintained (grids visible) D) Dextrose + A. marmelos Opacity, but lens integrity maintained (grids faintly visible) E)Dextrose + A.Sativum Transparency, lens integrity maintained (grid visible) F)Dextrose +E.officinalis Transparency, clarity maintained (grids visible) G)Dextrose + Vit. C Lens integrity maintained , opacity (grids faintly visible)

Effect of Medicinal plants. Changes in lens morphology

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Conclusions

❖All plant extracts caused ❖A decrease in lipid peroxidation, ❖Increased the activity of anti oxidant enzymes and ❖Decrease the activity of Aldose Reductase ❖In this study s. Cumini (Jambhul ) emerged as a promising therapeutic agent against glucose/Hyperglycemic induced cataract.

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Conclusions contd…

❖Use of these plant extracts for formulation of eye drops for topical application as an anti – cataract agents could provide respite to mankind.

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STUDIES ON POTENTIAL BIOLOGICAL IMMUNOMODULATORS IN HIV INFECTED INDIVIDUALS

• Ph. D.

IN MICROBIOLOGY

BY

BALLAL BHARAT BAJARANG

DEPARTMENT OF MICROBIOLOGY BHARATI VIDYAPEETH, DEEMED UNIVERSITY YASHWANTRAO MOHITE COLLEGE OF ARTS,SCIENCE AND COMMERCE, PUNE .

UNDER THE GUIDANCE OF

• Dr. M. G. BODHANKAR & Dr. P. M. BULAKH

Viva voce examination:- 17th February 2017

1) Chlorophytum borivilianum ( Safed Musli ) Family- Agavaceae Genus- Chlorophytum Species- borivilianum Medicinal part- Root

2) Withania somnifera ( Ashwagandha ) Family- Solanaceae Genus- Withania Species- somnifera Medicinal part- Root

3) Wagatia spicata Dalz. {Caesalpinia spp.} (Wakeri) Family- Leguminoceae Subfamily- Caesalpinaceae Genus- Wagatia Species- spicata Medicinal part- Stem 3) A

4) Picrorrhiza kurroa ( Kutki ) { Note: Root powder was collected from Ayurvedic Rasashala, Pune} Family- Scrofulariaa ceae Genus- Picrorrhiza Species- kurroa Medicinal part- Root

Preparation No. (1)

Caesalpinia (crude stem powder)– 100 mgs. Picrorrhiza kurroa (crude root powder) – 50 mgs. (Dosage – 1 blended animal dose in the morning)

Preparation No. (2)

Withania somnifera (crude root powder) – 250 mgs. Caesalpinia spp (crude stem powder)– 50 mg. Spilanthes paniculata Wall. ex DC (crude root powder) – 20mg. (Dosage : 1 blended animal dose in the evening)

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In vivo study of immunomodulating activities of biological immunomodulators: Study of ‘carbon clearance test’ Animal ethics clearance number : NTC/ RP 169/APRIL 2012

Biozzi et.al. Method Control Group No medication Phagocytic index = 1.00 Standard Group FDA approved immunomodulators Cytonini and Cytomaw Test Group Blended doses of selected biological immunomodulators

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Phagocyt ic index = 1.25 Phagocyt ic index = 2.72

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Human clinical trials: Phase - I

Human ethics clearance no. BVDU/YM/Ethics 2012-13/452 dt. 30/06/2012

Treatment of blended doses of biological immunomodulators for 60 days

15 HIV infected individuals 5 healthy individuals

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Phase II Human Clinical Trials

Before treatment Before treatment After treatment After treatment

Mean CD4 count 266.17 Mean CD4 count 399.29

Mean viral load 1,56,178.05 Mean viral load 3,27,881.19

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Research output :

❖ One patent (patent application number 2326/MUM/2009) ❖ Approved by Maharashtra state FDA (Lic no. PD/AYU/005-A/06) ❖ Import export code number 3107020931 (gama sterilized product get exported in foreign countries.)

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