Bio-assay guided fractionation of plant extracts linked to chromatographic separation techniques

Standardization & Quality Control of Herbal DrugsAbhisheak SharmaPhytochemistNational Institute of Ayurveda, Jaipur (Raj)

Pharma Industry750 billion (US $)Growth rate 7%6 billion (US $) 8 % by volume 13 % in terms of valueAnnual growth >10% 30000 registered unitsTop 35 companies contribute 75%Market leader 7%50000 branded formulationsGlobalIndian

Drug Development

Costs US $ 1 billion> 10 years, to develop a drug(1 molecule becomes drug out of 5000 new molecules)Average life of drug ~ 3 years60% of the drugs fails in the market

Over 80 % of world population depends upon traditional medicinesPlant based medicines 62 billion US$ (Global market) Plant based medicines 1.0 billion US$ (Indian market)Annual growth up to 15%Herbal MedicineMEDICINES ACCOUNT FOR 3040% OF HEALTH EXPENDITURE

India: 8th largest country > 47000 Plant sps. > 7500 sps.cited as Medicinal Plants 800 sps. claimed to be used 120 sps. used in large scale (1.65% of Med. Plants, 0.25 total sps.)HUGE UNTAPPED POTENTIAL, UNEXPLORED SCIENTIFICALLY

Herbal export: > Rs.1000 crores Target: Rs. 5000 crores (2010) Rs.10000 crores (2012)STANDARDIZATION & QUALITY CONTROL AS PER INTERNATIONAL NORMSINEVITABLE

Advantages/Interest in Natural ProductsUsed as such as Phytomedicine, dietary supplements, food/ beverage ingredientsImportant source of new drug discovery (Broader distribution of molecular properties viz. mass, partition coefficient, ring systems)

Quality Control of Herbal MedicinesHeterogeneous composition: whole plant, parts, extract Crude drug scenario: unorganized, wild sources, unsystematically collected by poor/illiterateNo authentication/identification Intentionally/unintentionally adulterated products Complex tasks

8

IdentificationBotanical identification: Controversy >500 phyllanthus sps. P. amarus & P. debilis hepatoprotective activity (phyllanthin & hypophyllanthin) many sps. devoid of lignansAdulteration: similar in morphology when dried (stem bark of Saraca indica adulterated with polyalthia longifolia) Foreign organic & inorganic matter to increase wt.

Phytochemical VariationsSeasonal changes: temperature, rainfall, photo-periodis etc. (Nicotiana rustica (20), Cassia angustifolia (drought))Geographical variation: Gentiana lutea, Aconitum napalus, N. inflataAge of the plant: Comphor accumulates in heartwood, as trees ready for collections after 40 yearGenetic factors: Acorus calamusEdaphic factors: soil Ph, soil composition, macro/micro nutrientsEnvironmental factors

STANDADIZATION / QUALITY CONTROL OF HERBAL DRUGSBOTANICALORGANOLEPTICPHYSICALBIOLOGICALCHEMICALMacroscopicMicroscopicShapeExternalMarking Qualitative Quantitative SEM Studies Powder Studies

Color Odor Taste Fracture

QualitativeQuantitativeChromatography / Spectroscopy etc. Sec. Metabolites DNA Finger printing Heavy metal Pesticide residue Mycotoxin

QualitativeQuantitativeChromatography / Spectroscopy etc. Sec. Metabolites DNA Finger printing Heavy metal Pesticide residue Mycotoxin

Microbial ContaminationToxicological studiesPharmacological activities

Moist. Cont. Extrac. Values Ash Values Fluores. Analy.

Process of delivering a product with a specified minimum level of one or more plant constituents

To establish consistent potency & to ensure full spectrum of bioactive constituents (markers) from batch to batch

EXTRACTION OF PLANT MATERIALSEPARATION AND ISOLATION OF CONSTITUENTSQUALITATIVE AND QUANTITATIVE ANALYSISCHARACTERIZATION OF ISOTATED COMPOUNDS

CarbohydrateProtein & Amino AcidsLipidAlkaloidGlycosideTerpenoidTannins

ChromatographyGroup of biphasic Separation techniquesFastest growing analytical techniquesAdsorption: TLC, PLC, Column, HPLC, HPTLCPartition: Paper, Electrophoresis, column, HPLC, GLCIon exchange, Size exclusion

HPLC INSTRUENTATION

ALUMINASILICA GELTIME (Minutes)TIME (Minutes)ULTRA VIOLET ABSORBANCE

ColumnSilica column, 5 m, 50 x 2.1 mmMobile PhaseA: 0.1 % Formic acidB: MeOH + 0.1 % Formic acidGradient5 to 100% B in 15 minFlow rate0.3 mL/minDetection ESITemprature 30 C

Ginkgo biloba1). Bilobalide, 2). Unknown (bilobalide), 3). Ginkgolide C, 4). Unknown (ginkgolide C) , 5). Unknown (ginkgolide A), 6). Ginkgolide A, 7). Ginkgolide B, 8). Unknown (ginkgolide A), 9). Quercetin, 10). Kaempferol, 11). Unknown Kaempferol

PesticidesColumnSilica based C18 Column, 5 m, 150 x 4.6 mmMobile PhaseA: H2O2 + 0.1% TFAB: MeOH + 0.1% TFAIsocratic5 to 100% B in 20 minFlow rate1mL/minDetection UV at 254 nmTemprature 25 C

1). Promentryn, 2). Tebuthiuron, 3). Atrazine, 4). Propazine, 5). Propanil, 6). Chlorthaldimethyl

HPTLC imagesLane 1, ginsenosides reference substances mixture (from bottom to top): ginsenoside-Rb1, -Re, -Rg1, -Rf, pseudoginsenoside-F11;Lane 2, White Panax ginseng root; Lane 3, Red Panax ginseng; Lane 4, American ginseng (P. quincefolius) Lane 5, Tienchi ginseng (P. notoginseng).

ConclusionsHerbs possess enormous healing power & only a part is known to mankindCan generate employment & revenueGreat market potentialSuperior quality of crude drugs &finished products neededIt is essential to ensure quality Control at all levels for standardization, efficacy, safety and consistency

Questions?

Thank You