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PHARMACEUTICAL STANDARDIZATION
Year : 2012  |  Volume : 33  |  Issue : 1  |  Page : 130-135  

Pharmacognostical and phytochemical evaluation of Vara Asanadi Kwatha


1 Assistant Professor, Department of Kaya Chikitsa, PNPS Ayurveda Medical College and Research Centre, Parakalai, Kasargod, Kerala, India
2 Senior Lecturer, Gampaha Wickramarachchi Ayurveda Institute, University of Kelaniya, Yakkala, Sri Lanka
3 Professor and Head, Department of Kaya Chikitsa, Institute for Post Graduate Teaching and Research in Ayurveda, Gujarat Ayurved University, Jamnagar, Gujarat, India
4 Head, Pharmacognosy Laboratory, Institute for Post Graduate Teaching and Research in Ayurveda, Gujarat Ayurved University, Jamnagar, Gujarat, India
5 Head, Pharmaceutical Chemistry Laboratory, Institute for Post Graduate Teaching and Research in Ayurveda, Gujarat Ayurved University, Jamnagar, Gujarat, India

Date of Web Publication28-Aug-2012

Correspondence Address:
Anju P Ramachandran
Assistant Professor, Department of Kaya Chikitsa, PNPS Ayurveda Medical college and Research Centre, Parakalai, Kasargod, Kerala, India

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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0974-8520.100330

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   Abstract 

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 Fe 2 O 3 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 R f 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


How to cite this article:
Ramachandran AP, Prasad M S, Samarakoon S, Chandola H M, Harisha C R, Shukla V J. Pharmacognostical and phytochemical evaluation of Vara Asanadi Kwatha . AYU 2012;33:130-5

How to cite this URL:
Ramachandran AP, Prasad M S, Samarakoon S, Chandola H M, Harisha C R, Shukla V J. Pharmacognostical and phytochemical evaluation of Vara Asanadi Kwatha . AYU [serial online] 2012 [cited 2020 Jul 12];33:130-5. Available from: http://www.ayujournal.org/text.asp?2012/33/1/130/100330


   Introduction Top


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 21 st 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).


   Materials and Methods Top


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)


   Observations and Results Top


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.
Figure 1: Pitted parenchyma

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Figure 2: Sclerieds, lignified fibres thick walled cork cells

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Figure 3: Reticulate vessels

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Figure 4: Phloem fibers

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Figure 5: Pitted stone cells

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Figure 6: Bordered pitted vessels

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Figure 7: Parenchyma

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Figure 8: Simple and compound starch grains

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Figure 9: Sclerieds

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Figure 10: Tannin

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Figure 11: Oleoresin, mesocarpal cells

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Figure 12: Fibres

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Figure 13: Trichomes

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Figure 14: Tannins

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Table 1: Macroscopic Characters

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[Figure 15], sclerieds etc. Curcuma longa on microscopy mainly consist of annular vessels [Figure 16], sceleriform vessels.
Figure 15: Starch grains, Pitted stone cells

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Figure 16: Annular vessels

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[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: Sceleriform vessels

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Figure 18: Epidermal cells

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Table 2: Results of powder microscopy


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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: Physicochemical parameters

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Preliminary qualitative tests revealed the presence of flavonoids and tannins in the test drug [Table 4].
Table 4: Qualitative analysis


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   Discussion Top


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 R f value was calculated. Track 1 showed two spots with R f 0.90 and 0.35 and three spots were available in track 2 with R f 0.90, 0.35, and 0.1 [Table 5]. The matter of significance is that in both the tracks the R f 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 R f values were matching when compared between both the tracks [Table 6], [Table 7] and [Figure 19], [Figure 20], [Figure 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.
Figure 19: Densitogram of ethyl acetate extract of VAK at 254 nm

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Figure 20: Densitogram of methanolic extract at 254 nm

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Figure 21: Densitogram of methanolic extract at 366 nm

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Table 5: Results of TLC

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Table 6: Results of HPTLC


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Table 7: After post chromatographic deprivation with methanolic sulphuric acid Solvent front -6

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   Conclusion Top


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.

 
   References Top

1.Anonymous, Sahasra yoga, Staulya chikitsa/68, with sujana priya vyakhya, 23 rd ed. Alappuzha - Kerala: Vidhyrambham publication; 2000.p. 544.  Back to cited text no. 1
    
2.Jeffrey S, Marathos-Flier E, Kasper, Braunwald, Fauci, Hauser, Longo, Jameson; Harrison's principles of internal medicine, 16 th ed, vol.1. New York: Mc Graw Hill publications; 2005. p. 422.  Back to cited text no. 2
    
3.Wagener H, Bladt S. Plant drug analysis-A thin layer chromatography atlas, Berlin: Sprnger-1996.p. 230-1.  Back to cited text no. 3
    
4.Heftmann E. Chromatography-A laboratory handbook of chromatographic and electrophoretic methods, 3 rd ed. United States: Van Nostarand, Princeton M J; 1975.  Back to cited text no. 4
    
5.Anonymous. the Ayurvedic Pharmacopea of India, Part I, 1 st ed,vol.1. Ministry of Health and Family welfare, New Delhi: Government of India; 2007.  Back to cited text no. 5
    
6.Standard methods of chemical analysis, The elements, Chap. 25 th - Iron: Determination of Iron as Fe 2 O 3 , Gravimetric method , In: N. Howell Furman PhD; Robert E. editor. 6 th ed, vol.1. Huntington, New York: Krieger Publishing Company; 1962, Reprint -1975. p 535.  Back to cited text no. 6
    
7.Liang YZ, Xie P, Chan K. Quality control of Herbal medicines. J Chromatogr B Analyt Technol Biomed Life Sci 2004;812:53-70.  Back to cited text no. 7
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10], [Figure 11], [Figure 12], [Figure 13], [Figure 14], [Figure 15], [Figure 16], [Figure 17], [Figure 18], [Figure 19], [Figure 20], [Figure 21]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7]


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