MEDICINAL PLANTS BIOPROSPECTING AND BIOPRODUCTION Natural World

MEDICINAL PLANTS BIOPROSPECTING AND BIOPRODUCTION

Natural World “The rain forest and the river are my brothers. The heron and the others are my friends and we are all connected to each other in a circle, in a hoop that never ends” Colors of wind

God “ caused the grass for the cattle, and herb for the service of man” Psalms 104: 14 “Nature has everything to meet man’s needs, not his greeds” Gandhiji

World Resource Institute 8 COUNTRIES RETAIN THEIR ORIGINAL FORESTS; BRAZIL, CANADA AND RUSSIA ACCOUNT FOR 2/3 OF GLOBAL ORIGINAL FOREST TRACTS q q 76 COUNTRIES HAVE LOST THEIR ORIGINAL FORESTS COMPLETELY q 28 COUNTRIES INCLUDING INDIA ARE LOOSING ORIGINAL VIRGIN FORESTS AND FACE SEVERE THREAT OF ECOLOGICAL IMBALANCE IF CONSERVATION EFFORTS ARE NOT TAKEN URGENTLY

INDIA’S POPULATION YEAR MILLIONS 1801 1951 1961 1988 1996 2000 2035 207 356 439 683 812 960 1000 1460

INDIA • A MEGA DIVERSITY CENTRE AND A CENTRE OF CROP ORIGIN • 2. 4% LAND AREA HOLDS 8% OF WORLDS BIODIVERSITY; 10 TH AMONG PLANT RICH COUNTRIES OF THE WORLD; 4 TH AMONG COUNTRIES OF ASIA • APPROXIMATELY 9000 HIGHER PLANT SPECIES OUT OF 17, 000 ARE MEDICINAL; 25 -30% ARE ENDEMIC • ABOUT 70% OF INDIA’S POPULATION DEPEND ON PLANTS FOR PRIMARY HEALTHY CARE • GREAT RAW MATERIAL (LOW VALUE) EXPORTER; 75% OF WORLDS PLANT BASED DRUGS COME FROM INDIAN PLANTS • DIVERSITY WITHIN SPECIES (GENETIC DIVERSITY) IS RICH DUE TO DIVERSE SOIL AND ECO-CLIMATIC FACTORES; HABITAT DEGRADATION AND LOSSES COMMON; 27, 500 HECTARES OF FOREST LAND DEPLETED ANNUALLY

INDIA’S RESOURCES Category Percentage share Population 16. 0 Oil 0. 6 Gas 0. 5 Coal 6. 0 Cattle 20. 0 Buffaloes 55. 0 Biodiversity 8. 0 Land area 2. 2

CHALLENGES OF 21 ST CENTURY Bioresources are common resources • Open access and lack of property rights • No control on depletion • Forests mostly under state control • MNCs to privatize genes , gene products and transgenics

If a thing belongs to everyone it really belongs to no one Hardin Tragedy of the Commons

Alice : “Which Direction Should I go? ” Cat : “It Depends a great deal on where you want to go. ” Alice : “But I don’t know where I want to go? ” Cat : “In that case, it really doesn’t matter which direction you take. ” Lewis Carol - Alice In Wonderland

Indian Health Care Challenge 16% of World Population, 20% of World Mortality. 22% of World Morbidity, But only 2% of World’s GDP and 1% of World’s Healthcare. Investment

Plant species used in traditional and modern systems of medicine in India Tribal 7000 Siddha 700 Tibetan 300 Unani 600 Modern 90 Ayurveda 2200

Some important medicinal plants

System Market Size Rs. Cr, Ayurveda US $ M. 3, 500 813 Homeo 600 139 Siddha 5 1 Unani 100 23 Total 4205 976 Market Size E. U 28 Rest Europe 2. 4 Asia 10. 8 Japan 9. 8 N. America 6. 9 Others 4. 1 Total US $ b 62. 0 India’s contribution 0. 5%

Why this small share ? • We export mostly raw materials • No value addition • Mostly collected from the wild

Why bioprospecting and bioproduction important? l. Most medicinal plants are still wild types not upgraded to the level of crops with improved yields l. Unit value of medicinal plants is low; however heightened the demand for raw germplasm may be l. Life span is usually long; harvest size reached after years l. Endogenous concentrations of active principles are low and unpredictable due to uncontrolled conditions of cultivation l. Genes of prospective value not yet identified and used for productive transgenics l. We have to exercise our rights over our genetic resources/ unique genes, gene products, and utilize them in value added form. l. Tissue and cell cultures may be fielded into service for sustainable production of high value compounds

Pharma companies and R&D organizations involved in Bioprospecting Organisation Status of Plant Screening Programme Supplied by Region of origin NCI , USA Large scale screening of plants, marine organisms. Missouri Botanic Garden, NY. Africa, Madagascar Central, South America, S. E. Asia, Botanic Garden, Univ. Illinois, Private contractors. Australia USA Bristol-Meyers Developed Taxol Glaxo Natural Products Discovery Commercial and Academic Dept. institutions, RBG Kew, Singapore Natl Univ. Many therapeutic areas (CNBR) South America, Africa and Asia Merck, Sharp Marine organisms, plants and Microbes South America Dohme Res Labs N. Y. Botanic Garden, work with IN BIO, Costa Rica Shannon Pharma Ethnobotanicals Individuals, institutions and Tropical South America, Govt. Departments Africa, S. E. Asia Smith Kline Marine organisms Beecham Plants and Microbes Individuals and own collectors Malaysia, Asia

Does Global Pharma R&D Deliver The Goods? l. Between 1975 and 1997, an impressive 1223 new compounds were launched in the market. Only 11 of them were designed for Tropical diseases. l. The WHO estimates that $56 Bio. a Year is spent on health research, but less than 10%is directed towards diseases that afflict 90%of World’s population. l. Around $ 2 Bio/Year spent on AIDS Drugs and Only $ 250 Mio. for vaccines; the latter more useful for developing countries where 90% of HIV occurs.

Examples of drugs and new therapeutic agents based on traditional knowledge Species Origin Clinical use Trad. Medicine Developer Cinchona pubescens South America Anti-malarial Europe Coleus forskohlii India, Sri Lanka Anti-metastatic Cardio-vascular respiratory and renal use Germany (Hoechst) Ginkgo biloba China Anti-asthmatic France/Germany Also evaluated graft injection Piper futokadsura China Bronchoasthma stiffness USA (Merck, Sharp & Dohme) Trichopus zeylanicus India Amti-stress Anti-fatigue India (TBGRI) aphrodisiac Azadirachta indica India Pesticidal Preservation of seeds USA (? ) Curcuma aromatica India Anti-bacterial USA (? )

STATISTICS OF IMPORTANCE u u u Only 2% of the 250, 000 described species of vascular plants have been screened for their chemicals Each year the U. S imports more than $ 20 million of rain forest plants valued for their medicinal properties Roughly 119 pure chemical substances extracted from some 90 species o higher plants are used in pharmaceuticals around the world Of the top selling 150 prescription drugs in the United States, 79% have their origin in nature Many synthetic drugs, including aspirin, were first discovered in wild plants and animals In 1960, a child with leukemia had a 1 in 5 chance of remission. Anti-cancer drugs developed from a compound discovered in wild periwinkle plants, same child’s chance of survival has increased to 80%

Success stories Stress tolerant genes u A gene tolerant to extreme cold temperature from a plant species of the Spiti valley of Himachal Pradesh has been identified, isolated, sequenced and cloned Salt tolerant genes Betaine Aldehyde Dehydrogenase (BADH), Superoxide dismutase (SOD), Lipid transfer protein (LTP) and Glyoxalase I (Gly 1) have been isolated from the mangrove species Avicennia marina. Transformation of Tobacco and Brassica already achieved with constructs containing BADH, SOD 1, LTP 1 and Gly 1. Transgenic plants (Ro generation) overexpressing BADH showed increasing tolerance with exogenous supply of betaine aldehyde Novel Enzymes u A novel antioxidant enzyme (SOD enzyme) from a plant species of the Western Himalayas has been identified and purified. It retains its activity after autoclaving and is catalytic at sub-zero temperatures. The enzyme may find applications in medicinal, cosmetic and food industry u

Selection of high yielding genotype

APPROACHES TO INCREASED PRODUCTION IN PLANT CELLS: SLOW GROWTH + LOW PRODUCTIVITY A. Explant Differentiated cells = slow growth B. Explant Cells + Elicitor = limited to specific class of compounds in each species C. Metabolic pathway Isolated genes prospective plant product = technology not yet developed (Metabolic Engineering) • increase the flux through the pathway to the desired product • increase the number of productive cells • decrease catabolism of the desired product

Bioprospecting of Hypericum species Global trade US $ 570 million : US market share : $ 210 million Germany : $ 55 million Retail price $ 100. 6/ mg :

Cell suspension cultures

Why bioproduction using plant tissue culture? Vanilla plants grow mainly in Madagascar and are not cheap. Thus scientists in California are trying to cultivate the essence of vanilla in a laboratory, using living cells taken from the plant. Chocolate without the cocoa plant, orange juice without trees and morphine without the poppy are all contemplated in the race to develop cell culture technology.

BIOPRODUCTION Business rationale : Demand , cost, quality and supply (A product like fruit flavour is too costly to extract from whole fruit sources. Raw material cost $10, 000$100, 000/kg pure flavour material). • A tissue culture process guarantees control over these parameters at the point of usage, from one batch to the next and from one year to another. • Technical rationale : Totipotency of plant cells. Certain cell culture systems produce compounds upto 30% on dry weight basis and 6 -7 g/L culture broth.

MARKET CONSIDERATIONS Selection of potential targets for production via plant tissue culture depends on • Overall market size in dollars and in production volume • Product selling price • Likelihood of penetration or expansion of the market with a • biotechnology based product Specific issues • Natural vs. Synthetic • Positioning of biotechnology based product vs. existing products • Relationship between the product value and the expected annual usage(Volume) • A candidate drug/compound has to be selected based on their relationship

Major Groups of Compounds With Commercial Importance derived From Plants Compound group Types and examples 1 Pharmaceuticals Alkaloids, Steroids, anthraquinones 2 Enzymes Proteases (e. g. Papain) 3 Latex Isoprenoids (e. g. Rubber) 4 Waxes Wax esters (e. g. Jojoba) 5 Pigments Stains and dyes 6 Oils Fatty acids (e. g. seed oils) 7 Agrochemicals Insecticides (e. g. Pyrethrins) 8 Essential oils (e. g. Monoterpenes) 9 Cosmetic substances Food additives 10 Gums Flavour compounds, non-nutritive sweetners (e. g. Thaumatin) Polysaccharides (e. g. Gum Arabic)

Advantages of in vitro cultures 1. Season independent production of compounds occurs. 2. 2. Compared to cultivation in the field, considerable savings in space, time and cost can be realized. 3. 3. The physical and chemical environment of the cell can be controlled and the optimum production of secondary metabolites can be achieved. 4. 4. Selection of high yielding cell lines is relatively easy in plant cells. 5. 5. For organ specific compounds root, Hairy root or shoot cultures can be used. 6. 6. Genetic engineering techniques can be utilized to

CULTURE SYSTEM CHARACTERISTICS SHOOT CULTURE : TRIED IN A FEW SPECIES SLOW GROWTH HIGHLY PRODUCTIVE CALLUS CULTURE : TRIED IN MANY SPECIES MODERATE GROWTH LOW PRODUCTIVITY CELL SUSPENSION : CULTURE TRIED IN MAXIMUM NUMBER OF SPECIES RAPID GROWTH LOW PRODUCTIVITY A FEW CULTURES COMMERCIALISED ROOT CULTURE : TRIED IN A FEW SPECIES SLOW GROWTH HIGH PRODUCTIVITY HAIRY ROOT : CULTURE TRIED IN SOME SPECIES MODERATE GROWTH HIGH PRODUCTIVTY

Process for Scaling up n Biotransformation (Solasodine) n Elicitation (Ajmalicine) n Immobilization (Capsaicin, Ajmalicine)

Products Produced At Industrial Scale By Plant Tissue Cultures Product Shikonin Berberine Digoxin Rosmarinic acid Geraniol Purpurin Ginsengosides Phytovanilla Species Lithospermum erythrorhizon Coptis japonica Digitalis lanata Coleus blumei Company Mitsui Country Japan Mitsui Boehringer Nattermann Japan Germany Geranium spp. Rubia akane Panax ginseng Vanilla planifolia Kenebo Mitsui Nitton. Denki Escergenetics Japan USA

Projected Price of Some Cell Culture Derived Products Product Application Plant source Price $/Kg Ajmalicine Anti-hypertensive Catharanthus roserus 37, 000 Berberine Anti-fertility Coptic japonica 3, 250 Artimisinin Anti-malarial Artimisia annua n/a Codeine Sedative Papaver somniferum 17, 000 Digoxin Heart stimulant Digitalis lanata 3, 000 Diosgenin Steroid Dioscorea deltoidea 1, 000 Jasmine Fragrance Jasminum spp 350 Morphine Drug Papaver somniferum 3, 40, 000 Quinine Anti-malarial Cinchona ledgeriana 500 Sanguinarine Antibiotic Papaver somniferum 4, 800 Shikonin Anti-bacterial Lithospermum erythrorhizon 4, 500 Taxol Anti- cancer Taxus brevifolia 0. 6 m Vanillin Flavour Vanila planifolia 1, 000 Vincristine Anti- leukemic Catharanthus roseus 2 m

Plumbago rosea Biomass production and plumbagin synthesis in variou culture systems Culture system Initial inoculam (mg dw) Biomass in g Plumbagin dw (period- mg/g dw days) Shoot 100 1. 094(45) 0. 0118 Callus 100 1. 671(50) 0. 0505 Cells 100 1. 527(22) 0. 0268 Untransformed roots Transformed roots 100 2. 352(45) 12. 318 100 2. 230(25) 11. 210

Untransformed root cultures of Plumbago rosea

Rooted plant

Strategy for the production of secondary metabolites by hairy roots and p. Ritransformed regenerants

Plant regeneration from hairy roots

Hairy root derived morphovariants

MASS CULTIVATION OF HAIRY ROOTS IN BIOREACTORS Species Reactor used Product Panax ginseng Plug flow reactor Ginseng extract Datura stramonium Stirred tank Hyocyamus Daucus carota Airlift, stirred tank Carotenoids Nicotiana tabacum Stirred tank Nicotine Catharanthus roseus Air lift Ajmalicine Lithospermum erythrorhizon Shikonin Air lift

Root differentiation in cell culture Kettle Reaction culture of roots

Hairy root biomass production on a suspended partition

Nutrient sprinkler 3 Tier root culture

Economics of plant tissue culture –based production process is determined by • Type of process employed • Product yield • Batch Cycle time • Cell growth rates • Nature of product purification

BIOTECHNOLOGY IN DEVELOPING COUNTRIES Countries with good biotechnological competence Brazil, China, India, South Korea and Mexico Countries that have created the Indonesia, Malaysia, necessary frame work for Philippines, Thailand, Ivory applying biotechnology Coast, Argentina and other South American countries Countries that have no research Mainly African countries South infrastructure and are of Sahara, Sahel countries, dependent on finished Ghana, Nepal, Bangladesh biotechnological products

HISTORY OF MEDICINE 2000 BC Here, eat this Root 1000 BC The root is health then. Here, say this Prayer 1085 AD That prayer is superstition. Here, drink this Potion 1940 AD That potion is snake oil. Here, swallow this Pill 1960 AD That pill is ineffective. Here, take this Antibiotic 2000 AD That antibiotic does not work anymore. Here, eat this Root.