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Pharmacognostical and Phytochemical Evaluation of a Polyherbal Ayurvedic Formulation Trikatu Churna

Authors:
  • Captain Srinivasa Murthy Central Ayurveda Research Institute
  • Central Council for Research in Ayurvedic Sciences-CCRAS(CSMCARI)

Abstract and Figures

Trikatu Churna is an Ayurvedic polyherbal formulation useful in wide range of diseases and disorders. Efficacy of formulation depends on their genuineness of herbs used. Authentication of herbs by anatomical studies is first and fundamental step for standardization of herbal formulation. In this paper Pharmacognostic investigations like macroscopic, microscopic and chemical studies like preliminary phytochemical, physico- chemical constants and TLC/HPTLC fingerprint of Trikatu Churna were studied. Methods: Findings of the study is helpful in standardization of polyherbal Ayurvedic formulation Trikatu Churna, which will promote global acceptance of the formulation and reputation of the Ayurveda system
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Ayurveda
Medical
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Journal for Rapid Publication of Ayurveda and Other
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©Journal of Ayurveda Medical Sciences HRGS’ Ayurveda Journal
J Ayu Med Sci | 2016 | Vol 1 | Issue 1 (Jul Sep)
Nartunai et al. J Ayu Med Sci 2016:1(1);34-40.
34
HRGS’ Ayurveda Journal
Journal of Ayurveda Medical Sciences
Peer Reviewed Journal of Ayurveda and other Traditional Medicines
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Pharmacognostical and Phytochemical Evaluation of a Polyherbal
Ayurvedic Formulation Trikatu Churna
Nartunai Govindarajan,1* Arunachalam Chinnapillai,1 Maheswari Balasundaram,3 Cheemalapati Venkata Narasimhaji,1 Kusuma Ganji,2
Ilavarasan Raju4
1Research officer, 2Research officer (S-II), 3Lab Technician, 4Assis tant Director (S-III), Captain Srinivasa Murthy Regional Ayurveda Drug Development
Institute, Arignar Anna Government Hospital Campus, Arumbakkam, Chennai 600 106
*Correspondence: E-mail: natrunai@yahoo.co.in, Mobile: +919894154514
ABSTRACT
Introduction: Trikatu Churna is an Ayurvedic polyherbal formulation useful in wide range of diseases and disorders. Efficacy of formulation
depends on their genuineness of herbs used. Authentication of herbs by anatomical studies is first and fundamental step for standardization of
herbal formulation. In this paper Pharmacognostic investigations like macroscopic, microscopic and chemical studies like preliminary phyto-
chemical, physico- chemical constants and TLC/HPTLC fingerprint of Trikatu Churna were studied. Methods: The standard methods recommended
in Quality Control Methods for Medicinal Plant Materials by WHO, 1998 was followed. Results: Macro-microscopic, preliminary phyto-chemical
studies and TLC/HPTLC studies of the formulation has been documented. Conclusion: Findings of the study is helpful in standardization of
polyherbal Ayurvedic formulation Trikatu Churna, which will promote global acceptance of the formulation and reputation of the Ayurveda
system.
KEYWORDS
Trikatu Churna, Pharmacognosy, Phytochemistry, Standardization, HPTLC fingerprint
Received: 15.09.2016
Accepted: 21.09.2016
DOI: 10.5530/jams.2016.1.5
Trikatu Churna is an Ayurvedic polyherbal formulation consisting of fine powders of Pippali (Piper longum Linn (Fruit), Marica
(Piper nigrum Linn. (Fruit) and Sunthi (Zingiber officinale Rosc. (Rhizome) in equal proportion. [1] In preparation of Churna the
ingredients are collected, dried, powdered individually and passed through sieve number 80/85 to prepare a fine powder.[1,2] In
Ayurveda, Trikatu Churna is used in the treatment of Agnimandya (digestive impairment), Gala roga (throat diseases), Svasa
(dyspnea), Kushtha (skin diseases), Pinasa (sinusitis), Kasa (cough) and Slipada (filariasis).[1] In Siddha, Tirikatugu Churanam having
same composition is used in the treatment of loss of appetite, rumbling in the abdomen, stomach pain, cough and fever.[3] A small
quantity of Trikatu Churna is mixed with water and dropped into the nostrils in coma and drowsiness.[4] Polyherbal formulation in
powdered form where the botanical ingredients are not more than ten can be identified microscopically.[5] Pharmacognostic
characters of herbal drugs play an important role since particular macro-microscopic features are unique for each plant. The
macroscopic and microscopic studies of the herbs should be the first and fundamental step to authenticate the botanical source.
Proceeding for chemical methods of standardization, preclinical and clinical evaluations will bear no value if authentic drugs are
not used. Macro-microscopic evaluation is simple and cost effective. TLC/HPTLC is one of the most effective and common
chromatographic technique because of its simplicity of use and cost effectiveness. The accuracy and precision of HPTLC with low
uncertainty emerge this technique as simple powerful separation technique and widely adopted in many Pharmacopoeias as an
identification method. Another advantage of HPTLC is being used by personnel with minimum of technical training and under
reasonable laboratory facility.[9] Preliminary phytochemical evaluation provides information about presence of phytoconstituents in
the extract. Physico-chemical constants indicate the purity and identity of the formulation. In the present investigation macro-
microscopic, preliminary phyto-chemical, physico-chemical constants and TLC/HPTLC fingerprint of the formulation were carried
out.
MATERIALS AND METHODS
The ingredients of Trikatu Churna were purchased from local raw material traders and the raw materials were authenticated by
comparing with the in-house standards of Botany/Pharmacognosy department, Captain Srinivasa Murthy Regional Ayurveda Drug
Development Institute, Arignar Anna Government Hospital Campus, Arumbakkam, Chennai 600 106. The dried cleaned samples
Nartunai et al. J Ayu Med Sci 2016:1(1);34-40.
35
were powdered and passed through sieve No. 80. Each one of the powder is weighed separately and equal parts of each powder
are mixed together. Powders of individual ingredients of Piper longum (Fruit), Piper nigrum (Fruit), Zingiber officinale (Rhizome)
(Table 1 and Figure 1.1-1.3), and the compound formulation (Figure 1.4) were analysed microscopically after clearing them in
chloral hydrate solution. A few milligram of powder treated with iodine in potassium iodide solution and mounted in glycerine for
observation of starch. A few milligram of powder treated with solution of phloroglucinol, allowed to dry, added a few drops of
hydrochloric acid and mounted in glycerine to observe lignified tissues. Quantitative analysis for total ash, acid insoluble ash, water
and alcohol soluble extractive values and loss on drying at 105⁰c were carried out in triplicate for the polyherbal Ayurvedic
formulation Trikatu Churna according to the method recommended in Quality Control Methods for Medicinal Plant Materials by
WHO, 1998.[6] Preliminary phytochemical analysis, Fluorescence analysis and TLC/HPTLC fingerprint were also carried out.[7,8]
Table 1. Ingredients of Trikatu Churna
Figure 1. Ingredients of Trikatu Churna
1.1 Pippali
1.2 Marica
1.3 Sunthi
1.4 Trikatu Churna
Preparation of extracts for TLC/HPTLC
4 g of the each drug sample were soaked in aqueous alcohol (10%) for overnight, refluxed for 30 minutes on water bath and filtered.
The filtrates were concentrated on water bath and made up to 10 ml in a standard flask separately.
Method for developing TLC/HPTLC
Chromatographic separation was achieved on TLC/HPTLC fs pre-coated with silica gel 60 F254 TLC plate (E-Merck) of 0.2 mm
thickness with aluminium sheet support. Samples were spotted using CAMAG Linomat IV Automatic Sample Spotter (Camag
Muttenz, Switzerland) equipped with syringe (Hamilton, 100µL). Plates were developed in a glass twin trough chamber (CAMAG)
pre-saturated with mobile phase. Scanning device used was CAMAG TLC scanner II equipped with CATS 3 software. The
Ayurvedic names
Botanical names
Part used
Quantity
Pippali
Piper longum Linn.
Fruit
All ingredients in
equal parts
Marica
Piper nigrum Linn.
Fruit
Sunthi
Zingiber officinale Rosc.
Rhizome
Nartunai et al. J Ayu Med Sci 2016:1(1);34-40.
36
experimental condition was maintained at 20+ 2⁰C. The aqueous alcoholic extract was applied on pre-coated silica gel 60 F254 TLC
plate (E-merck) as absorbent and the plate was developed using solvent system toluene: ethyl acetate (5: 1.5). After developing, the
plates were dried and observed the colour spots at UV-254, UV-366 nm and vanillin-sulphuric acid spraying reagent and
Dragendorff’s reagent.[9,10]
RESULTS AND DISCUSSION
Macroscopy: Trikatu churna is a yellowish green fine powder with aromatic odour, taste is pungent producing numbness on the
tongue (Figure 1.4).
Microscopy: Under microscope the following characteristics were observed in various mounts; Pippali fragments of thick walled,
lignified, in different shapes and sizes of stone cells with wide lumen, a few fragments of pointed multicellular trichome, a few
fragments of perisperm embedded with aleurone grains and oil globule, a few yellowish brown content cells, numerous simple,
oval to rounded, starch grains measuring upto 8µ in diameter; Marica fragments of thick walled, lignified, in different shapes and
sizes of stone cells with wide lumen, a few fragments of perisperm embedded with aleurone grains and oil globule, a few
yellowishbrown content cells, numerous simple, oval to rounded starch grains measuring upto 40µ in diameter; Sunthi - a few
septate fibres, a few fragments of rectangular, thin walled, cork cells in sectional view, a few fragments of lignified vessels with
spiral thickenings, a few fragments of thin walled parenchyma with starch grains, a few yellowishbrown content cells, numerous
simple, oval to rounded starch grains measuring upto 60µ in diameter (Figure 2).
Figure 2. Microscopy of Trikatu Churna
a. Cork cells (Sunthi); b. Starch grains (Sunthi, Marica and Pippali); c. Yellowish-brown content (Sunthi, Marica and Pippali); d. Perisperm with aleurone
grains (Marica and Pippali); e. Multi-cellular pointed trichome (Pippali); f. Vessel with spiral thickenings (Sunthi); g. Parenchyma cells with starch
(Sunthi); h. Stone cells (Pippali and Marica); i. Septate fibre (Sunthi); j.Perisperm embedded with oil globules (Marica and Pippali).
Physico-chemical analysis
Physico-chemical analysis shows 11.36 % of moisture content. Ash content of the drug was 4.22 % and 0.72 % of acid in-soluble ash
shows the siliceous matter in the plant. Alcohol soluble extractives 9.59 % represent the extraction of polar constituents like
phenols, tannins, glycosides, alkaloids and flavonoids. The water soluble extractive 11.38 % denotes the presence of inorganic
contents (Table 2).
Nartunai et al. J Ayu Med Sci 2016:1(1);34-40.
37
Table 2. Physico-chemical analysis of Trikatu Churna
Parameters
Mean Value (n=3) ± S.D
Ash value
a. Total ash (%)
b. Acid-insoluble ash (%)
4.22 + 0.18
0.72 + 0.20
Extractive value
a. Water soluble extractive (%)
b. Alcohol soluble extractive (%)
11.38 + 0.24
9.59 + 0.21
Loss on drying at 105⁰C (%)
11.36 + 0.10
Preliminary Phyto-chemcial test
Preliminary phyto-chemical test of the aqueous alcoholic extract of Trikatu churna shows presence of alkaloid, sugar, phenol,
quinone, tannin and triterpenoids and the absence of coumarin, flavanoid, steroid, saponin and acid (Table 3).
Table 3. Preliminary phyto-chemical analysis of Trikatu Churna
Test
Presence/Absence in aqueous alcoholic
extract
Alkaloid
+
Coumarin
-
Flavonoid
-
Sugar
+
Phenol
+
Quinone
+
Steroid
-
Tannin
+
Triterpenoid
+
Saponin
-
Acid
-
Fluorescence Analysis
Fluorescence analysis of the Trikatu churna with different chemical reagents shown in Table 4.
Table 4. Fluorescence Analysis of Trikatu Churna
Reagents with powder
UV-254 nm
UV- 366 nm
Visible light
Powder as such
Black
Pale greyish colour
Yellowish brown
n-hexane
Black
Pale greyish colour
Dark brown
Chloroform
Black
Pale greyish colour
Black
Ethyl acetate
Black
Pale greyish colour
Yellowish brown
Ethanol
Black
Pale greyish colour
Brown
Acetone
Black
Pale greyish colour
Yellowish brown
Water
Brown
Pale greyish colour
Brown
1N Sodium hydroxide (Aqueous)
Black
Yellowish green
Yellowish brown
1N Sodium hydroxide (Alcohol)
Black
Yellowish green
Yellowish brown
1N Hydrochloric acid
Black
Yellowish green
Yellowish brown
50 % Nitric acid
Black
Black
Yellowish brown
50 % Sulphuric acid
Brown
Yellow
Yellowish brown
Conc. Sulphuric acid
Black
Yellowish green
Black
TLC
Among the various solvent systems tested, the mixture containing toluene: ethyl acetate (5:1.5) gives the best resolution. In UV 254,
366 nm, visible light and Derivatization with Dragendorff’s reagent Piper longum, Piper nigrum, Trikatu Churna and Zingiber officinale
aqueous alcoholic extracts were shown Figure 3.
Nartunai et al. J Ayu Med Sci 2016:1(1);34-40.
38
Figure 3. TLC Photo-documentation of Trikatu Churna
UV 254 nm
UV 366 nm
Derivatization with Vanillin-sulphuric acid
Derivatization with Dragendorff’s reagent
Track 1: Piper longum 2: Piper nigrum 3: Trikatu churna 4: Zingiber officinale
HPTLC
The HPTLC densitometric scan of Piper longum at 366 nm showed 15 peaks. The Rf values 0.51, 0.60 and 0.79 were major peaks and
the Rf values 0.02, 0.07, 0.16, 0.19, 0.22, 0.28, 0.30, 0.34, 0.36, 0.41, 0.88 and 0.99 were smaller peaks. Piper nigrum showed 15 peaks
with the Rf values 0.04, 0.07, 0.13, 0.19, 0.24, 0.27, 0.31, 0.33, 0.37, 0.46, 0.60, 0.66, 0.73, 0.84 and 0.98 of which Rf values 0.13, 0.31,
0.33, 0.37, 0.46, 0.60 & 0.66 were the major peaks (Figure 4).
The HPTLC fingerprint profile of Trikatu Churna showed 13 peaks. The Rf values 0.33, 0.38, 0.45, 0.50, 0.60 and 0.66 were major
peaks whereas the Rf values 0.02, 0.07, 0.19, 0.23, 0.79 and 0.87 were smaller peaks. HPTLC fingerprint of Zingiber officinale showed
11 peaks with the Rf values 0.02, 0.08, 0.19, 0.24, 0.30, 0.33, 0.43, 0.50, 0.58, 0.65 and 0.76 of which Rf values 0.43 and 0.76 were the
major peaks (Figure 4).
Pharmacognostic characters of herbal drugs play an important role since particular macro-microscopic features are unique for each
plant. The macroscopic and microscopic studies of the herbs should be the first and fundamental step to authenticate the botanical
source. TLC/HPTLC profile of aqueous alcohol extracts provides a suitable method for monitoring the identity, purity and also
standardization of the drug.
1 2 3 4
1 2 3 4
1 2 3 4
1 2 3 4
Nartunai et al. J Ayu Med Sci 2016:1(1);34-40.
39
Figure 4. HPTLC fingerprint of Trikatu Churna at 366 nm
Piper longum
Piper nigrum
Trikatu Churna
Zingiber officinale
CONCLUSION
The present study analyzed the macro-microscopic characters, preliminary phyto-chemical, physico-chemical constants and
TLC/HPTLC finger print of the formulation. The results obtained will help in standardization of Ayurvedic polyherbal formulation
Trikatu churna.
ACKNOWLEDGEMENT
The authors are very grateful to the Director General, Central Council for Research in Ayurvedic Sciences, New Delhi, for his
encouragement and providing opportunity to conduct this study.
CONFLICT OF INTEREST
Nil
REFERENCES
1. Anonymous, The Ayurvedic Formulary of India. Part-I, Second Revised
English Edition, Ministry of Health & Family Welfare, Govt. of India,
Dept. of Indian Systems of Medicine & Homoeopathy. p. 110; 2003.
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Family Welfare, Department of AYUSH, New Delhi: p.79; 2008.
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India, New Delhi: p.19; 1991.
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Medicines). Fourth Edition, IMPCOPS, Madras: p.130; 1994.
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Formulations. Central Council for Research in Ayurveda and Siddha.
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Nartunai et al. J Ayu Med Sci 2016:1(1);34-40.
40
ABOUT AUTHORS
Mr. Nartunai Govindarajan is a Research Officer (Pharmacognosy) at Captain Srinivasa Murthy Regional Ayurveda Drug Development Institute, Chennai, Under
CCRAS, Ministry of AYUSH, Govt. of India. His doctoral research focused on developing quality standards for medicinal plants used in Ayurveda. He has projects in
Intra Mural and Extra Mural Research Schemes. Has experience in the area of Pharmacognosy. Developed monographs for Ayurvedic Pharmacopoeia of India and
Quality Standards for Indian Medicinal Plants by ICMR.
Mr. Arunachalam Chinnapillai is a Research Officer (Botany) at Captain Srinivasa Murthy Regional Ayurveda Drug Development Institute, Chennai, Under CCRAS,
Ministry of AYUSH, Govt. of India. Has vast experience in the field of developing monographs for Ayurvedic Pharmacopoeia of India and published many research
papers in the field of herbal research. Editor for Recent Progress in Medicinal Plants.
Ms. Maheswari Balasundaram is a Laboratory Technician (Chemistry) at Captain Srinivasa Murthy Regional Ayurveda Drug Development Institute, Chennai, Under
CCRAS, Ministry of AYUSH, Govt. of India. Experienced in the Phytochemical standardization of single drugs and compound formulations of herbal origin.
Dr. Ch. Venkata Narasimhaji is working as Research officer [Chemistry] at Captain Srinivasa Murthy Regional Ayurveda Drug Development Institute, Chennai, under
CCRAS, Ministry of AYUSH, Govt. of India. Handling projects under Intra Mural Research and Extra Mural Research Schemes and h ave experience in the field of area of
chemistry such as isolation of phyto-chemical reference standards, their analysis and Drug standardization of herbs and herbo-minerals. Developed monographs for
Quality Standards of Indian Medicinal Plants sponsored by ICMR.
Dr. G. Kusuma, a doctorate from Banaras Hindu University, is Research Officer (Ayurveda)/Scientist II at Captain Srinivasa Murthy Regional Ayurveda Drug
Development Institute under Central Council for Research in Ayurvedic Sciences, M/o AYUSH, Govt. of India. She is associated with Drug Standardisation and
developing standard operating procedures (SOP’s) for Ayurvedic formulations and is also Principal Investigator of Intra Mural Research project on Ethno-medicinal
survey and co-investigator of Intra Mural Research & Extra Mural Research projects.
Dr. Ilavarasan Raju is an Assistant Director (S-III) in-charge at Captain Srinivasa Murthy Regional Ayurveda Drug Development Institute, Chennai, Under CCRAS,
Ministry of AYUSH, Govt. of India. He has projects in Intra Mural and Extra Mural Research Schemes. H as vast experience in the area of Pharmacology and Herbal Drug
Standardization. Guiding students for Ph.D., studies of various universities.
GRAPHICAL ABSTRACT
Cite this article as: Nartunai Govindarajan, Arunachalam Chinnapillai, Maheswari Balasundaram, Cheemalapati Venkata Narasimhaji, Kusuma Ganji,
Ilavarasan Raju. Pharmacognostical and Phytochemical Evaluation of a Polyherbal Ayurvedic Formulation Trikatu Churna. J Ayu Med Sci 2016: 1(1);34-40.
DOI: 10.5530/jams.2016.1.5
©Journal of Ayurveda Medical Sciences
Herbal Research Guidance and Solutions’ (HRGS) Ayurveda Journal
... Macro-microscopic evaluation is simple and cost effective. 11,12 From the results of this study it is well established that the dried flowers of Hibiscus rosa-sinensis can be easily identified morphologically in the crude drug form and microscopically in the powdered form. The findings of the comparative study on dried flowers of Hibiscus rosa-sinensis and Rhododendron arboreum will be helpful in differentiating their identity. ...
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Macro-microscopic characterization of Hibiscus rosa-sinensis and its differentiation from adulterant Rhododendron arboreum Sm. The dried flowers of R. arboreum are morphologically looking similar to flowers of H. rosa-sinensis and used as adulterant in the raw drug market. The findings of present study is helpful in standardization of formulation containing flowers of H.rosa-sinensis as ingredient in their powdered form and also for authentication/identification of dried flowers of H.rosa-sinensis.
The Ayurvedic Formulary of India. Part-I, Second Revised English Edition, Ministry of Health & Family Welfare, Govt. of India, Dept. of Indian Systems of Medicine & Homoeopathy
  • Anonymous
Anonymous, The Ayurvedic Formulary of India. Part-I, Second Revised English Edition, Ministry of Health & Family Welfare, Govt. of India, Dept. of Indian Systems of Medicine & Homoeopathy. p. 110; 2003.
The Ayurvedic Pharmacopoeia of India. Part-II, Volume-II, First Edition, (Formulations), Govt. of India, Ministry of Health and Family Welfare, Department of AYUSH
  • Anonymous
Anonymous, The Ayurvedic Pharmacopoeia of India. Part-II, Volume-II, First Edition, (Formulations), Govt. of India, Ministry of Health and Family Welfare, Department of AYUSH, New Delhi: p.79; 2008.
Quality Control Standards for Certain Siddha Formulations. CCRAS, Ministry of Health and Family Welfare
  • Anonymous
Anonymous, Quality Control Standards for Certain Siddha Formulations. CCRAS, Ministry of Health and Family Welfare, Govt. of India, New Delhi: p.19; 1991.
  • Vaidya Yoga Anonymous
  • Ratnavali
Anonymous, Vaidya Yoga Ratnavali (Formulary of Ayurvedic Medicines). Fourth Edition, IMPCOPS, Madras: p.130; 1994.
Laboratory guide for the analysis of Ayurveda and Siddha Formulations. Central Council for Research in Ayurveda and Siddha
  • Anonymous
Anonymous, Laboratory guide for the analysis of Ayurveda and Siddha Formulations. Central Council for Research in Ayurveda and Siddha.
  • Anonymous
Anonymous, Quality Control Methods for Medicinal Plant Materials. World Health Organisation, Geneva: p. 25-28; 1998.
Isolation Purification and Preliminary Observation in Elucidation of structures by
  • K H Overton
Overton KH, Isolation Purification and Preliminary Observation in Elucidation of structures by Physical and Chemical Methods. Bently KH, (Ed.), Inter Science Pub., New York: p. 34; 1963.
High Performance Thin Layer Chromatography. 1 st Edn
  • P D Sethi
Sethi PD, High Performance Thin Layer Chromatography. 1 st Edn., Vol.X, CBS Publishers and Distributors, New Delhi: 1996.
Plant Drug Analysis A Thin Layer Chromatography Atlas
  • H Wagner
  • S Bladt
Wagner H, Bladt S, Plant Drug Analysis A Thin Layer Chromatography Atlas. 2nd Edn, Germany: Springer-Verlag;
Balasundaram is a Laboratory Technician (Chemistry) at Captain Srinivasa Murthy Regional Ayurveda Drug Development Institute, Chennai, Under CCRAS, Ministry of AYUSH, Govt. of India. Experienced in the Phytochemical standardization of single drugs and compound formulations of herbal origin
  • Ms
  • Maheswari
Ms. Maheswari Balasundaram is a Laboratory Technician (Chemistry) at Captain Srinivasa Murthy Regional Ayurveda Drug Development Institute, Chennai, Under CCRAS, Ministry of AYUSH, Govt. of India. Experienced in the Phytochemical standardization of single drugs and compound formulations of herbal origin.