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Ayu. 2012 Jan-Mar; 33(1): 130–135.
PMCID: PMC3456851
Pharmacognostical and phytochemical evaluation of Vara Asanadi Kwatha
Anju P. Ramachandran,1 Shyam M. Prasad,1 S. M. S Samarakoon,2 H. M. Chandola,3 C. R. Harisha,4 and V. J. Shukla5
1Assistant Professor, Department of Kaya Chikitsa, PNPS Ayurveda Medical College and Research Centre, Parakalai, Kasargod, Kerala, India
2Senior Lecturer, Gampaha Wickramarachchi Ayurveda Institute, University of Kelaniya, Yakkala, Sri Lanka
3Professor and Head, Department of Kaya Chikitsa, Institute for Post Graduate Teaching and Research in Ayurveda, Gujarat Ayurved University, Jamnagar, Gujarat, India,
4Head, Pharmacognosy Laboratory, Institute for Post Graduate Teaching and Research in Ayurveda, Gujarat Ayurved University, Jamnagar, Gujarat, India
5Head, Pharmaceutical Chemistry Laboratory, Institute for Post Graduate Teaching and Research in Ayurveda, Gujarat Ayurved University, Jamnagar, Gujarat, India
Address for correspondence: Dr. Anju P. Ramachandran, Assistant Professor, Department of Kaya Chikitsa, PNPS Ayurveda Medical college and Research Centre, Parakalai, Kasargod, Kerala, India. E-mail: anjushyamprasad/at/gmail.com
Vara Asanadi Kwatha (VAK) is a compound Ayurvedic formulation prescribed in the management of obesity. Pharmacognostical study counting both macroscopic and powder microscopy of raw drug exposed the quality and genuineness of all the constituents of VAK. Organoleptic features of coarse powder made out of the crude drugs were within the standard range. Specific gravity of the decoction was 1.0185 and pH was 5.5.Total solid content present in the Kwatha was 4.525% w/v, total ash 0.949% w/v, and acid insoluble ash was 0.052% w/v. Iron assay showed the presence of Fe2O3 as 0.065% w/v. Qualitative scrutiny demonstrated the presence of flavonoids and tannis. Thin Layer Chromatography (TLC) and High Performance Thin Layer Chromatography (HPTLC) were carried out after organizing appropriate solvent system in which maximum three spots were distinguished in TLC and nine spots in HPTLC and most of the Rf values were identical when done with different sample extractive methods. This shows the presence of certain definite constituents in the decoction and is helpful for the easy separation of these constituents.
Keywords: HPTLC, Pharmacognosy, TLC, Vara Asanadi Kwatha
The vital responsibility played by herbal medicine in serving the therapeutic requirements of major fraction of human populace worldwide is identified since ancient times. But the quality control and standardization facets of these herbal drugs stay as a herculean task even in the 21st century. Accurate identification and guarantee of purity through pharmacognosy and pharmaceutical chemistry measures is inescapable ladder needed for the quality assurance and standardization of any of the herbal medicine whether it is single drug or formulation. Vara Asanadi Kwatha is one of the most potential herbo-mineral compound preparations mentioned in Sahasra Yoga[1] which is claimed to be extremely successful in the management of obesity and overweight. But till now no pharmacognostical, pharmacological or phytochemical evaluation of this compound is been conducted. Obesity is considered as a global epidemic and stands as the most established risk factor for number of serious disorders like Cardio Vascular Diseases (CVD), diabetes mellitus, metabolic syndrome etc.[2] Ayurveda, the Indian traditional system of medicine can offer much in this regard, if thousands of unexplored combinations are brought into limelight. Vara Asanadi Kwatha is one among them. Ingredients of this decoction are Haritaki (Terminalia chebula Retz., Bibhitaka (Terminalia belerica Roxb.) Amalaki (Emblica officinalis Gaertn.), Asana (Pterocarpus marsupium Roxb.), Chitraka (Plumbago zeylanica Linn.), Haridra (Curcuma longa Linn.), and Loha Patra (Thin iron foil/flake).
Collection
Dried specimens of mature fruits of Triphala, heart wood of Asana, rhizome of Haridra, roots of Chitraka and Loha Patra were collected from natural environment of Kannur, Kerala. The samples were preserved and transported by placing in separate high quality air tight containers avoiding any kind of infectivity. The samples were made into coarse powder from Dept of Bhaishajya kalpana I P G T and R A, GAU, Jamnagar. Later, the organoleptic and powder microscopy of all the individual drugs were carried out in Pharmacognosy Laboratory of I P G T and R A Jamnagar. All the drugs were confirmed to be authentic and of good quality. The test drug Vara Asanadi decoction was prepared as per the classical reference and phytochemical analysis of the final product was carried out in Pharmaceutical chemistry Laboratory of IPGT and RA.
Microscopy
Powder microscopy of each raw drug was made with the coarse powder of the dried samples before and after staining. Phloroglucinol and HCl were used for the staining purpose.
Physicochemical parameters and qualitative analysis
Kwatha was made out of the ingredients in equal proportion and it was subjected to physicochemical parameters like refractive index, specific gravity, pH value, total solid content, total ash and acid insoluble ash. The percentage of iron present in the Kwatha was assessed by Iron assay method. In qualitative analysis, presence of tannins and flavonoids were assessed. TLC and HPTLC were carried out after making appropriate solvent system with ethyl acetate extract and methanolic extract of VAK.
Thin layer chromatography
Thin layer chromatography (TLC) and high-performance thin layer chromatography (HPTLC) were performed for the normal phase separation of components of ethyl acetate and methanol extracts of VAK. Solvent system was prepared by taking toluene, ethyl acetate, formic acid, and glacial acetic acid in a proportion of 5:5:1:1, respectively.[3] The spots obtained from both the extracts were examined under ultra violet light of wave length 254 nm and 366 nm.
  • Sample preparation: Track 1: Ethyl acetate extract of VAK (E.A)
  • Track 2: Methanol extract of VAK(Me-OH)
  • Solvent system -Toluene:Ethyl acetate:Formic acid:Glacial acetic acid(5:5:1:1)
  • Stationary phase- Silica gel G
  • Visualization-Under long U.V (366nm) and short U.V (254nm)
High-performance thin layer chromatography
HPTLC study of the ethyl acetate extract and Methanol extract was also carried out by using the same solvent system of Toluene+Ethyl acetate+Formic acid+Glacial acetic acid (5:5:1:1).[4] After completion of HPTLC; post chromatographic deprivation was done with methanolic sulphuric acid.
Track 1: Ethyl acetate extract of VAK
Tack 2: Me-OH extract of VAK
Solvent system: Toluene+Ethyl acetate+Formic acid+Glacial acetic acid (5:5:1:1)
The initial purpose of the study was to confirm the authenticity of the drugs used in the preparation of VAK. For that, coarse powder of all the ingredients/drugs was subjected for organoleptic feature estimation.
Detailed pharmacognostical evaluation was carried out for all the ingredients of VAK. Macroscopic characters of the each drug observed were as mentioned in Table 1. The powder microscopy of the roots of Plumbago zeylanica revealed presence of lignified pitted parenchyma [Figure 1], sclerieds, lignified fibres thick walled cork cells [Figure 2], and reticulate vessels [Figure 3]. Phloem fibers [Figure 4], pitted stone cells [Figure 5] and bordered pitted vessels [Figure 6] were present in the Ht.wood of Pterocarpus marsupium. When microscopy of coarse powder of mature fruit of Terminalia chebula was done it showed features like parenchyma [Figure 7] simple and compound starch grains [Figure 8], sclerieds [Figure 9], tannin contents, fibres etc. Features seen in the powder microscopy examination of Emblica officinalis were tannin [Figure 10] oleoresin, mesocarpal cells [Figure 11], fibres [Figure 12] pitted stone cells, and prismatic crystals. Whereas Terminalia belerica presented with abundant number of trichomes [Figure 13], tannins [Figure 14], starch grains, pitted stone cells.
Table 1
Table 1
Macroscopic Characters
Figure 1
Figure 1
Pitted parenchyma
Figure 2
Figure 2
Sclerieds, lignified fibres thick walled cork cells
Figure 3
Figure 3
Reticulate vessels
Figure 4
Figure 4
Phloem fibers
Figure 5
Figure 5
Pitted stone cells
Figure 6
Figure 6
Bordered pitted vessels
Figure 7
Figure 7
Parenchyma
Figure 8
Figure 8
Simple and compound starch grains
Figure 9
Figure 9
Sclerieds
Figure 10
Figure 10
Tannin
Figure 11
Figure 11
Oleoresin, mesocarpal cells
Figure 12
Figure 12
Fibres
Figure 13
Figure 13
Trichomes
Figure 14
Figure 14
Tannins
[Figure 15], sclerieds etc. Curcuma longa on microscopy mainly consist of annular vessels [Figure 16], sceleriform vessels.
Figure 15
Figure 15
Starch grains, Pitted stone cells
Figure 16
Figure 16
Annular vessels
[Figure 17], and epidermal cells [Figure 18]. The diagnostic features obtained by powder microscopy was compared with the standards mentioned in Ayurvedic Pharmacopia of India (API).[5] The details of results obtained in pharmacognostical study are enlisted in the Table 2.
Figure 17
Figure 17
Sceleriform vessels
Figure 18
Figure 18
Epidermal cells
Table 2
Table 2
Results of powder microscopy
Organoleptic parameters: The prepared test drug VAK is liquid in form, brownish – black in color, astringent in both taste and odor.
Physicochemical parameters: The results observed after experiments are mentioned in the Table 3.
Table 3
Table 3
Physicochemical parameters
Preliminary qualitative tests revealed the presence of flavonoids and tannins in the test drug [Table 4].
Table 4
Table 4
Qualitative analysis
The specific gravity of the current sample was 1.0185 showing the sample is denser than water. In the present sample, pH was detected by using pH indicator paper and it was 5.5 showing the mild acidic nature of the solution. In present study the total solid content was 4.525% w/v. Vara Asanadi Kwatha the total ash determined was 0.949%w/v and percentage of acid insoluble ash was 0.052% w/v. The test showed Iron assay result as 0.065% w/v.[6]
Qualitative test was performed mainly to detect the presence of tannins and flavonoids in the sample. The presence of tannins was confirmed by adding 5% of FeCl3 solution to 2-3 ml of aqueous extract of sample resulting in the formation of deep blue – black color showing the abundance of tannin content in the test drug. Methanol extract of VAK was used for the purpose of detection of flavonoid and test was carried out by two methods by adding lead acetate and sodium hydroxide respectively. Both the experiments showed positive result for the presence of flavonoids.
Mobile phase of TLC was Toluene+Ethylacetate+Formicacid+Glacialaceticacid in the proportion of 5:5:1:1, respectively. The sample tracks and mobile phase remained the same for all the experiments related to TLC and HPTLC. The spots produced by TLC were observed in day light, short UV and long UV and Rf value was calculated. Track 1 showed two spots with Rf 0.90 and 0.35 and three spots were available in track 2 with Rf 0.90, 0.35, and 0.1 [Table 5]. The matter of significance is that in both the tracks the Rf value of two compounds are same. So this implies the definite presence of certain constituents in the sample. HPTLC is a more convenient and simple procedure in which finger printing profile is available in the form of graph and densitogram.[7] In the present HPTLC study, four spots obtained in track 1 and 9 spots in track 2 in which once again most of the Rf values were matching when compared between both the tracks [Tables [Tables66,,77 and Figures Figures1919,,2020,,21].21]. After completion of chromatographic procedure spraying of the plate was done with methanolic sulphuric acid and the spots obtained were observed in day light.
Table 5
Table 5
Results of TLC
Table 6
Table 6
Results of HPTLC
Table 7
Table 7
After post chromatographic deprivation with methanolic sulphuric acid Solvent front -6
Figure 19
Figure 19
Densitogram of ethyl acetate extract of VAK at 254 nm
Figure 20
Figure 20
Densitogram of methanolic extract at 254 nm
Figure 21
Figure 21
Densitogram of methanolic extract at 366 nm
Preliminary Organoleptic features and results of powder microscopy were compared with the parameters mentioned in API and all the ingredients were proved to be authentic. In phytochemical analysis specific gravity, pH, total ash, acid insoluble ash, and total solid content were assessed. The percentage of amount of Iron present in the sample was detected and it was within the acceptable range. Qualitative analysis revealed the presence of tannins and flavonoids in the decoction. Though the base work requisites for the standardization of VAK are covered in the current study, additional analysis and investigations are required for the identification of all the active chemical constituents of the test drug to substantiate the clinical efficacy.
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2. Jeffrey S, Marathos-Flier E, Kasper, Braunwald, Fauci, Hauser, Longo, Jameson Harrison's principles of internal medicine. 16th ed. Vol. 1. New York: Mc Graw Hill publications; 2005. p. 422.
3. Wagener H, Bladt S. Plant drug analysis-A thin layer chromatography atlas. Berlin: Sprnger; 1996. pp. 230–1.
4. Heftmann E. Chromatography-A laboratory handbook of chromatographic and electrophoretic methods. 3rd ed. United States: Van Nostarand, Princeton M J; 1975.
5. Ministry of Health and Family welfare. 1st ed. Part 1. Vol. 1. New Delhi: Government of India; 2007. Anonymous. the Ayurvedic Pharmacopea of India.
6. Furman N. Howell, PhD, Robert E., editors. 6th ed. Reprint. Vol. 1. Huntington New York: Krieger Publishing Company; 1962. Standard methods of chemical analysis, The elements, Chap. 25th – Iron:Determination of Iron as Fe2O3, Gravimetric method; p. 535.
7. Liang YZ, Xie P, Chan K. Quality control of Herbal medicines. J Chromatogr B Analyt Technol Biomed Life Sci. 2004;812:53–70. [PubMed]
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