Fingerprinting of Herbal Drugs using Advanced Bioanalytical Techniques

Dr. Bhaswat S. Chakraborty

104/11/23

204/11/23

Herbal Drugs as Traditional Medicine

“…the medicine that refers to health practices, approaches, knowledge and beliefs incorporating plant, animal and mineral based medicines, spiritual therapies, manual techniques and exercises, applied singularly or in combination to treat, diagnose and prevent illnesses or maintain well-being …”

WHO

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Some Indian Medicinal Plants

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Use of Traditional Medicine

• Worldwide 40-70% people use traditional medicine (of herbal, animal or mineral origin)– Exclusively, alternately or terminally

• The quality of herbal medicines is often poor– Production of herbals is not controlled or regulated

• Herbal medicines usually contain a range of pharmacologically active compounds– Often not known which of these constituents produces the therapeutic

effect. – Testing for efficacy is more complex than with synthetic drugs

• Complex safety issues– Toxicity of natural herbal constituents– Presence of contaminants or adulterants– Herb-herb or herb-prescription drug interactions

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Renewed Interest in Herbal Medicine

• The effectiveness of plant medicines.

• The preference of consumers for natural therapies, a greater interest in alternative medicines and a commonly held erroneous belief that herbal products are superior to manufactured products.

• A dissatisfaction with the results from synthetic drugs and the belief that herbal medicines might be effective in the treatment of certain diseases where conventional therapies and medicines have proven to be inadequate.

• The high cost and side effects of most modern drugs.

• Improvements in the quality, efficacy, and safety of herbal medicines with the development of science and technology.

• Patients’ belief that their physicians have not properly identified the problem; hence they feel that herbal remedies are another option.

• A movement towards self-medication

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But the Problem is

• Herbal drugs are usually mixtures of many constituents.

• The active principle(s) is (are), in most cases unknown.

• Selective analytical methods or reference compounds may not be available commercially.

• Plant materials are chemically and naturally variable.

• The same species varies in various drug contents depending on locality, cultivation process, etc.

• The source and quality of the raw material are variable.

• The methods of harvesting, drying, storage, transportation, and processing (for example, mode of extraction and polarity of the extracting solvent, instability of constituents, etc.) have an effect.

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QC of Herbal Drugs• Identity

• Purity

• Content or assay – very challenging for herbal drugs

• Parameters for Quality Control of Herbal Drugs– Microscopy

– Foreign matter determination

– Determination of ash

– Determination of heavy metals

– Determination of Microbial Contaminants and Aflatoxins

– Determination of Pesticide Residues

– Determination of Radioactive Contamination

– Analytical method and its validation

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Why QC Herbal Drugs?• Accurate identity and specifications

• Content uniformity

• Reduced batch to batch variability

• Efficacy related to quality

• Safety– Some sub-species can be very toxic

• Raw drug and dosage form stability

• Control of shelf life decreasing contaminations

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Sample Preparation for Chromatographic Fingerprinting

• Follows the same priciples as those for any HPLC (or other chromatographic) analysis

• Herbs may require very specific additional steps prior to chromatographic analysis– Grinding and pulverizing

– Extraction • Organic solvent

• Organic solvent free

• Super-critical fluid

• Co-solvent super-critical

– Hydro-distillation of essential oils1004/11/23

Instead of chemical solvent, compressed CO2 is used. It is a fine extracting solvent and doesn’t contaminate the herb, environment or our body.

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LC-MS-MS

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MS vs. MS/MS

InletInlet DetectDetectMass

AnalyzeMass

AnalyzeIonizeIonize

MSMS

InletInlet FragmentFragmentMass Analyze

Mass AnalyzeIonizeIonize

MassAnalyze

MassAnalyze

DetectDetect

MS1MS1 CollisionCell

CollisionCell

MS2MS2

MS/MSMS/MS

HPLC

Separation Identification

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Chromatographic Fingerprinting of Herbs

• These are chromatograms representing characteristics of the herbs– Identity, consistency and authenticity of samples

• Hyphenated techniques (GC/MS, LC/MS and CE/MS) show greatly improved performances– retention time shift, selectivity, chromatographic separation abilities

and measurement precision

• Chromatographic fingerprinting is a highly accurate, meaningful quality control method of herbal medicines

• Since it is usually difficult to distinguish herbal drugs by different manufactures visually, principal components analysis (PCA) is used.

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Curcumin

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Parent Ion of Curcumin

Total Ion Current of Parent Ion

HPLC Fingerprint of Ashwagandha

1704/11/23 Dr. Quazi et al (downloaded), CSIR

Glycowithanolides and Withanolidesfrom Ashwagandha

1804/11/23 Dr. Quazi et al (downloaded), CSIR

LC-MS Fingerprint of Ashwagandha

1904/11/23 Dr. Quazi et al (downloaded), CSIR

Fingerprint of Formulations

2004/11/23Dr. Quazi et al (downloaded), CSIR

Total Ion Chromatogram of Ginseng Sample

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Chr

omat

ogra

ms

of D

iffe

rent

Bat

ches

of

Gin

seng

in S

henm

ai in

ject

ion

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Similarity of Fingerprints

• Equivalence of herbal drugs of different sources – “Phytoequivalence”– Very good, reproducible and accurate chromatograms are needed

• For good comparison, correction of retention time shifts of the component drugs is necessary

• So many components from so many sources– Visual comparison is extremely difficult

– May even be misleading

• Chemometric techniques,e.g., Principal Component Analysis (PCA) are used to confirm similarity or dis-similarity

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TIC of essential oils of Cortex cinnamomi from four different sources

04/11/23 24Liang et al (2004), J Chromat B, 812, 53-70

Phytoequivalence

Chromatograms of Ginko biloba samples by two different HPLC instruments

04/11/23 25Liang et al (2004), J Chromat B, 812, 53-70

Correction for RT Shift

Principal Component AnalysisHPLC UV of G. biloba extracts from 17 different sources

04/11/23 26Liang et al (2004), J Chromat B, 812, 53-70

Principal Component AnalysisHPLC UV of G. biloba extracts from 17 different sources

04/11/23 27Liang et al (2004), J Chromat B, 812, 53-70

Different fingerprint from those below

Principal Component AnalysisHPLC UV of G. biloba extracts from 17 different sources

04/11/23 28Liang et al (2004), J Chromat B, 812, 53-70

On-Line Chemometric ComparisonHPLC-MS TIC of Schisandra extracts by 6 different methods

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Liang et al (2004), J Chromat B, 812, 53-70

DNA Microarray of Herbal Drugs

04/11/23 30Wang M et al (2005), Phytotherap Res 19, 173-182

Thank You

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