|Year : 2014 | Volume
| Issue : 3 | Page : 165-171
Physicochemical standardization, HPTLC profiling, and biological evaluation of Aśvagandhādyariṣṭa: A comparative study of three famous commercial brands
Mandeep Singh1, Navdeep Kaur1, Atish Tulsiram Paul2
1 Department of Pharmacognosy and Phytochemistry, School of Pharmaceutical Sciences, Lovely Professional University, Phagwara, Punjab, India
2 Department of Pharmacognosy and Phytochemistry, School of Pharmaceutical Sciences, Lovely Professional University, Phagwara, Punjab; Department of Pharmacy, Birla Institute of Technology and Science, Pilani, Rajasthan, India
|Date of Web Publication||17-Nov-2014|
Atish Tulsiram Paul
Department of Pharmacy, Birla Institute of Technology and Science, Pilani 333 031, Rajasthan
Source of Support: None, Conflict of Interest: None
Background: Aśvagandhādyariṣṭa is a polyherbal formulation that is available commercially as an over the counter drug. There are three famous brands that are available in the market. However, there are no comparative reports on the physicochemical, chromatographic, and biological profiles of Aśvagandhādyariṣṭa manufactured by these famous companies.
Aims: The present study deals with the physicochemical standardization, high performance thin layer chromatography (HPTLC) profiling, and biological evaluation of Aśvagandhādyariṣṭa.
Materials and Methods: Aśvagandhādyariṣṭa manufactured by three leading companies were purchased from Jalandhar, Punjab. The physicochemical standardization of the samples was carried out in accordance with the Ayurvedic Pharmacopoeia of India (API). Authentified Eisenia foetida were procured from Ujjwal Ujala Vermiculture Group, Amritsar. The anthelmintic activity, 1,1-diphenyl-2-picrylhydrazyl scavenging, and hydrogen peroxide scavenging ability of Aśvagandhādyariṣṭa was determined.
Statistical Analysis Used: The data of anthelmintic activity were expressed as mean ± standard error of the mean of six earthworms in each group. The statistical analysis was carried out using one-way analysis of variance, followed by Dunnet t-test. The difference in values at P < 0.05 was considered to be statistically significant.
Results: Most of the physicochemical standardization parameters mentioned as per the API were found to be within limit. HPTLC profiling showed the presence of withanolide D in commercial samples. Out of three commercial brands, ASA-DAB was the most active as compared to the ASA-BDN and ASA-AVP at the concentration of 200 mg/ml for anthelmintic activity against E. foetida. ASA-DAB showed the best antioxidant activity in both the in vitro assay at the concentration of 100 μg/ml.
Conclusions: The ability of this formulation to scavenge free radicals supports its medical claim of antistress formulation. The anthelmintic potential of this formulation helps us conclude that it can also be considered as a general tonic because it provides relief from helminths.
Keywords: Anthelmintic, AŚvagandhādyariṣṭa, 1,1-diphenyl-2- picrylhydrazyl, hydrogen peroxide, high-performance thin layer chromatography
|How to cite this article:|
Singh M, Kaur N, Paul AT. Physicochemical standardization, HPTLC profiling, and biological evaluation of Aśvagandhādyariṣṭa: A comparative study of three famous commercial brands. Ancient Sci Life 2014;33:165-71
|How to cite this URL:|
Singh M, Kaur N, Paul AT. Physicochemical standardization, HPTLC profiling, and biological evaluation of Aśvagandhādyariṣṭa: A comparative study of three famous commercial brands. Ancient Sci Life [serial online] 2014 [cited 2020 Apr 9];33:165-71. Available from: http://www.ancientscienceoflife.org/text.asp?2014/33/3/165/144621
| Introduction|| |
The major portion of the population of India uses a variety of medicinal plants and their products for health care purposes.  According to World Health Organization (WHO), in much of the developing world, especially in Asia, Africa, Latin America and the Middle East, 70-95% of the population rely on these traditional medicines for primary health care.  The extensive use of this traditional medicine by the public gives rise to the need to evaluate the health claims of these medicinal agents and to develop standards for their manufacturing and quality.  WHO formulates suitable guidelines and methodologies for research and evaluation of herbal medicines. This includes a literature review, botanical verification, quality considerations, research and evaluation of safety and efficacy. 
Standardization is a process of ensuring the quality of the finished product. A physicochemically standardized product ensures that the good manufacturing practices are followed in the production units. Various chromatographic techniques are used to ensure the presence of active marker compounds in the formulations. The techniques such as high performance thin layer chromatography (HPTLC), high performance liquid chromatography (HPLC), gas-liquid chromatography, and other hyphenated techniques such as liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry are used for chemical marker based standardization of the formulations and plants. For example, a validated HPTLC method has been developed for quantification of withaferin-A and withanolide-A; HPLC method for analysis of 27-hydroxy withanone, 17-hydroxy withaferin-A, 17-hydroxy-27-deoxy withaferin-A, withaferin-A, withanolide-D, 27-hydroxy withanolide-B, withanolide-A, withanone, and 27-deoxywithaferin-A. ,
Aśvagandhādyariṣṭa is a fermented liquid preparation prepared using Withania somnifera as the main ingredient. It is an official formulation included in the Ayurvedic Pharmacopoeia of India (API). It contains 5-10% of alcohol that is self generated in the preparation over a period. It is a clear, dark brown liquid without frothing, significant sedimentation and has an astringent taste. It is therapeutically used in syncope, epilepsy, cachexia, mania/psychosis, emaciation, piles, digestive impairment and neurological disorders.  Aśvagandhādyariṣṭa has also reported showing antiepileptic activity in rats. 
As a continuing interest in quality evaluation of botanicals using hyphenated chromatographic technique,  we have standardized a commonly used official polyherbal formulation, Aśvagandhādyariṣṭa. Also in pursuit of exploring new therapeutic application for Aśvagandhādyariṣṭa, the anthelmintic activity has been evaluated as a majority of the plants used as ingredients in Aśvagandhādyariṣṭa are reported to possess the above said activity. ,,,,,,,[ 17] The present study also investigates the potential of Aśvagandhādyariṣṭa for antioxidant activity. This is the first ever comparative study of three popular commercial brands of Aśvagandhādyariṣṭa available in Indian market.
| Materials and methods|| |
Aśvagandhādyariṣṭa manufactured by three leading companies of India, that is, Dabur (Batch number BD0249), Shree Baidyanath Ayurved Bhawan Pvt. Ltd. (Batch number 06), and Arya Vaidya Pharmacy Limited (Coimbatore) (Batch number 74051) were purchased from the local market of Jalandhar, Punjab. These were coded as ASA-DAB, ASA-BDN, and ASA-AVP, respectively.
The physicochemical standardization of the samples was carried out in accordance with the API for the parameters: Total solids, pH, specific gravity, alcohol content, total phenolics, microbial limits, reducing and nonreducing sugars and presence of methanol.
The specific gravity of a liquid is the weight of a given volume of the liquid at 25°C (unless otherwise specified) compared with the weight of an equal volume of water at the same temperature, all weighing being made under normal conditions. The specific gravity of Aśvagandhādyariṣṭa was obtained by dividing the weight of the liquid contained in the pycnometer by the weight of water contained in the pycnometer, both determined at 25°C unless otherwise directed in the individual monograph.
The pH value of an Aśvagandhādyariṣṭa was measured potentiometrically.
Total solid content
A volume of 50 ml of the clear ariṣṭa was transferred to an evaporable dish, which had been dried to a constant weight and evaporated to dryness on a water bath, and then dried at 105°C for 3 h. After cooling the dish containing the residue in a desiccator for 30 min, it was weighed immediately.
Total phenolic content
I. Folin-Ciocalteu reagent (1 N): Commercially available Folin-Ciocalteu reagent (2 N) was diluted with an equal volume of distilled water. It was transferred in a brown bottle and stored in a refrigerator (4°C). It was golden in color
II. Sodium carbonate (20%): 40 g of sodium carbonate was dissolved in about 150 ml distilled water and volume were made up to 200 ml with distilled water.
Calibration curve equation (tannic acid): y = 0.0324x + 0.0314.
Analysis of total phenols
Suitable aliquots of the Aśvagandhādyariṣṭa were taken in test tubes, the volume was made up to 0.5 ml with distilled water, and 0.25 ml of the Folin-Ciocalteu reagent was added and then 1.25 ml of the sodium carbonate solution. Tubes were vortexed, and absorbance was recorded at 725 nm after 40 min. The amount of total phenols as tannic acid equivalent from the above calibration curve equation was calculated. Total phenolic content was expressed in (x% w/v).
Total alcoholic content
The ethanol content of a liquid is expressed as the amount of ethanol contained in 100 volumes of the liquid, the volume being measured at 24.9-25.1°C. This is known as the "percentage of ethanol by volume."
A volume of 25 ml of sample, accurately measured at 24.9-25.1°C, was transferred into a distillation flask. It was diluted with 150 ml of water, and a little pumice powder was added. Distillation head and condenser were attached. 95 ml of the distillate was collected into a 100 ml volumetric flask. The temperature was adjusted to 24.9-25.1°C and was diluted to volume with distilled water. Relative density was determined at 24.9-25.1°C. The values indicated in API in column 2 of table 17 were multiplied by 4 in order to obtain a percentage of ethanol by volume contained in the preparation. If the specific gravity was found to be between two values, the percentage of ethanol was obtained by interpolation. After calculation of the ethanol content, result was reported to one decimal place.
Absence of methanol
To one drop of the sample in a test tube, one drop of water with one drop dilute phosphoric acid (10% w/v of water) was added followed by one drop of potassium permanganate solution (1% w/v of water). Sodium bisulfate solution was added dropwise until the permanganate color is discharged. If brown color remains one drop of dilute phosphoric acid was added followed by 5 ml of chromatic acid solution (5 mg chromotropic acid sodium salt in 10 ml mixture of 9 ml sulfuric acid and heated to 60°C for 10 min). If no color is produced indicates the absence of methanol.
In microbial limit tests, the number of viable aerobic micro-organisms present are estimated. In this study, the presence of Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Salmonella typhimurium was estimated. The protocol followed for this test was as per the API.
Authentified Eisenia foetida were procured from Ujjwal Ujala Vermiculture Group, Village Ibban Kalan, Amritsar (reference number is UU/0512/LU-125).
Sample preparation for anthelmintic activity
About 50 ml of each sample was evaporated to dryness over a water bath then dried at 105°C for 3 h respectively and dried extract was stored in a desiccator. The dried sample was weighed accurately for preparing the test solution in the concentration of 50 mg/ml, 100 mg/ml, and 200 mg/ml using normal saline as vehicle.
The anthelmintic activity of the samples was evaluated by the method reported by Reddy et al.  E. foetida was divided into 20 groups, each group consisting of six worms. Group 1 earthworms were released in 25 ml normal saline in petri plate. Groups 2, 3, and 4 were released in 50, 100, and 200 mg/ml of ASA-DAB respectively. Groups 5, 6, and 7 were released in 50, 100, and 200 mg/ml of ASA-BDN respectively. Groups 8, 9, and 10 were released in 50, 100, and 200 mg/ml of ASA-AVP respectively. Groups 11, 12, and 13 were released in 50, 100, and 200 mg/ml of ASA-IH respectively. Group 14 were released in normal saline containing standard drug piperazine citrate (20 mg/ml). Time for paralysis was analyzed when no movement of any sort could be observed except when the worms were shaken vigorously. Time of death of worms was noted after assuring that earthworms neither moved when shaken vigorously nor when dipped in warm water (50°C), with faded body color.
Sample preparation for antioxidant activity
From the stock solution of 10 mg/ml of formulation in methanol, the dilutions of 20, 40, 60, 80, and 100 μg/ml were prepared. Each dilution was centrifuged in order to remove any sort of solid matter.
1,1-Diphenyl-2-picrylhydrazyl radical scavenging assay
The 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging was evaluated by method of Chen et al.  An aliquot of 5.4 ml of 0.1 mM DPPH solution in methanol and 0.6 ml of the samples at various concentrations was mixed. The mixture was shaken vigorously and allowed to stand for 30 min in dark at room temperature. The decolorization of DPPH was determined by measuring the absorbance at 517 nm with a UV spectrophotometer. The scavenging of the radical was calculated as follows:
where Ac is the absorbance of control and At is the absorbance of DPPH in the presence of samples and standards. The ascorbic acid was used as a standard.
Hydrogen peroxide scavenging assay
The hydrogen peroxide (H 2 O 2 ) scavenging ability of the various samples of Aśvagandhādyariṣṭa was determined according to the method of Gόlηin et al.  A solution of H 2 O 2 (40 mM) was prepared in phosphate buffer (pH 7.4). H 2 O 2 concentration was determined spectrophotometrically at 230 nm. Samples (4 ml, 20-100 μg/ml) in distilled water were added to an H 2 O 2 solution (0.6 ml, 40 mM). Absorbance of H 2 O 2 at 230 nm was determined after 10 min against a blank solution containing in phosphate buffer without H 2 O 2 . The percentage of scavenging of H 2 O 2 of samples and standard was calculated as follows:
Ac is the absorbance of the control, and At is the absorbance in the presence of the sample and standard.
High performance thin layer chromatography
The HPTLC analysis for the chloroform extract of the Aśvagandhādyariṣṭa using different commercial brands was carried out. The samples from three manufacturers were coded as ASA-DAB, ASA-BDN, and ASA-AVP. The formulations were partitioned with hexane and chloroform successively. The fractions were concentrated resulting in a solid residue. The Chromatography was performed on 10 × 10 cm thin layer chromatography (TLC) plates coated with 0.2 mm layers of silica gel F 254 (Merck). The samples were applied to the plate as 6 mm wide bands by means of a Linomat IV sample applicator (CAMAG, Switzerland). The plate was developed to a distance of 8.0 cm with chloroform: Methanol: Ethyl acetate: Benzene (70:4:8:24 v/v/v/v) as mobile phase  in a CAMAG twin-trough chamber saturated with mobile phase vapor. The plate was then dried and scanned at 227 nm (lmax for withanolide D) by use of a CAMAG TLC scanner 3 using winCATS 4 software (CAMAG, Switzerland).
The data of anthelmintic activity was expressed as mean ± standard error of mean of six earthworms in each group. The statistical analysis was carried out using one-way analysis of variance (ANOVA), followed by Dunnet t-test. The difference in values at P < 0.05 was considered as statistically significant. An ANOVA was performed using Graphpad Prism 5 (Version 5.03) software (GraphPad Software, Inc. USA) to determine the mean and standard error of paralysis and death time of the earthworms.
| Results and discussion|| |
The physicochemical standardization of the each sample was carried out as per API and the standard values were also taken from API.  The total solid contents of the ASA-DAB and ASA-AVP were less than the limits described in the API, which indicates that lesser quantity of the ingredients may have been used during the manufacture of the formulation. This is also correlated with the lower values of specific gravity. The reducing sugar content of the ASA-BDN sample was lesser than specified limit and also as compared to the other two samples. This may be due to excessive fermentation of jaggery, which is also evident because of higher alcohol content than the prescribed limit (5-10%). All the other parameters falls within their respective ranges [Table 1]. It is interesting to note the variation in physicochemical parameters as all the three formulation labels claim to use the same formula and method of preparation as per the classical text of Bhaiśajya Ratnāvalî. Thus, the variations observed are not due to use of different formula or method of manufacturing but are due to the various reasons cited above.
|Table 1: Physicochemical standardization of ASA-DAB, ASA-BDN, and ASA-AVP |
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No presence of microbial load was observed in the formulations [Table 2].
In the present study, the anthelmintic potential of Aśvagandhādyariṣṭa was evaluated using Eisenia foetida, because of its anatomical and physiological resemblance with the intestinal roundworm parasite of human beings.  The results clearly indicate that ASA-DAB was most active when compared to other samples at all the concentrations (50, 100, and 200 mg/ml) with paralysis time of 20.10 ± 0.59, 10.75 ± 0.81 and 4.30 ± 0.25 min [Figure 1] and death time of 28.31 ± 0.60, 15.36 ± 0.92 and 7.34 ± 0.37 min [Figure 2]. The results of each sample were better than the standard drug piperazine citrate (20 mg/ml), which showed the paralysis time of 30.78 ± 1.09 and death time of 44.98 ± 1.64 min.
|Figure 1: Comparative effect of different batches of Aśvagandhādyariṣṭa on paralysis time of Eisenia foetida|
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|Figure 2: Comparative effect of different batches of Aśvagandhādyariṣṭa on death time of Eisenia foetida|
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These results clearly indicate that besides the main uses of this traditional medicine, it also has an anthelmintic potential. This can be inferred from the dose of this formulation that is 15-30 ml.  When calculated, this volume of formulation equals dry formulation weighing 2.1-4.2 gm. This means if a person takes a dose of 15-30 ml of Aśvagandhādyariṣṭa twice in a day, he consumes an actual dose which is higher than that required for getting relief from the helminths. However, detailed studies need to be done to prove this hypothesis.
Another important mechanism by which the medicinal plants and their polyherbal formulations act are by quenching free radicals. In this regard, the antioxidant activity was performed using DPPH method that is reckoned as a short time-consuming method for determining the antioxidant activity. The stable DPPH radical shows maximum absorbance at 517 nm. The reaction between antioxidant and DPPH radical cause decrease in the absorbance of DPPH radical, which is characterized by discoloration from purple to yellow.  The antioxidant activity of the three brands indicated that the ASA-DAB had the highest DPPH scavenging activity (88%) at the concentration of 100 μg/ml [Table 3]. The results were comparable to the standard ascorbic acid which showed 90% inhibition at 100 μg/ml. This result of higher antioxidant activity is clear from the higher phenolics content of ASA-DAB as compared to the other brands.
|Table 3: Percentage inhibition of DPPH radical scavenging of ASA samples |
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To investigate the antioxidant potential, the H 2 O 2 scavenging activity of all the samples was also performed. The reaction between the antioxidant and H 2 O 2 cause release of water molecules. The result showed that ASA-DAB caused concentration-dependent inhibition of H 2 O 2 [Table 4].
Withanolides are the major phytoconstituents present in W. somnifera. Hence, there is great possibility for their presence in Aśvagandhādyariṣṭa, as W. somnifera is the major ingredient. After the extraction and separation process using different organic solvents namely hexane, chloroform, ethylacetate, and butanol in a sequential manner, the presence of withanolide D was detected in the chloroform extract using HPTLC [Figure 3]a. Withanolide D was detected in UV light at 254 nm in ASA-DAB and ASA-BDN. It was not clearly visible in ASA-AVP due to overlapping of the bands. Since the mere presence of single phytoconstituent does not represent the quality of the entire formulation, the more accepted technique of profiling was performed [Figure 3]b. The HPTLC profiling showed four matching peaks in all the three brands, with slight variation as shown in [Figure 3].
|Figure 3: (a) High performance thin layer chromatography (HPTLC) overlay of ASA-DAB (groups 1, 2, and 3), ASA-BDN (groups 5, 6, and 7), ASA-AVP (groups 9, 10, and 11), and Marker (groups 4 and 8). (b) Photograph (taken under UV light at 254 nm) of developed HPTLC plate showing bands obtained from samples and marker|
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| Conclusion|| |
Aśvagandhādyariṣṭa is used as a nervine tonic and antistress formulation. The ability of this formulation to scavenge free radicals supports its medical claim of being an antistress formulation. The anthelmintic potential of this formulation reveals the fact that it can also be considered as a general tonic because it provides relief from helminths. The physicochemical and chromatographic standardization showed slight to moderate variation in the quality control parameters, which seems to affect the antihelmintic and antioxidant profile of Aśvagandhādyariṣṭa. Hence, more scientific studies of commercial brands need to done. Also based on these studies, modification of limits of quality controls parameter in the API may be proposed if necessary.
| Acknowledgment|| |
The authors acknowledge the help of Dr. Ananya Arjuna, Department of Paramedical Sciences, Lovely Professional University for her help in performing the microbial limit tests.
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[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3], [Table 4]