1. INTRODUCTION:

Now a days, traditional medicine is revealed by an extensive activity of research on different plant species and their therapeutic principles all over the world^1-2. Free radicals of different forms are constantly generated for specific metabolic requirement and quenched by an effective antioxidant network in the body.

When the generation of these species exceeds the levels of antioxidant mechanism, it leads to oxidative damage of tissues and bio molecules, eventually leading to disease conditions, especially degenerative diseases³. As plants produce a lot of antioxidants to control the oxidative stress caused by sun beams and oxygen, they can represent a source of new compounds with antioxidant activity⁴. Some of the non-nutritive antioxidants of plants are phenolic compounds, flavonoids, coumarins, benzylisothiocyanate etc⁵. Epidemiological evidence indicates an inverse relationship between the intake of food rich in phenolic compounds and the reduction of certain chronic diseases and coronary heart disease mortality⁶.

Ashwagandharishta is a polyherbal hydro alcoholic preparation and is used as rasayana. Rasayanas are used to promote health and longevity by increasing defense against disease, arresting the ageing process and revitalizing the body in debilitated conditions⁷. The chief ingredient of Ashwagandharishta is roots of Ashwagandha, Withania somnifera, commonly known for its usefulness in the treatment of hypercholesterolemia, arthritis in combination with other drugs, is also credited to be hypoglycemic and diuretic⁸. The pharmacological effect of the roots of Withania somnifera is attributed to withanolides, a group of steroidal lactones⁹.

Besides Withania roots, the other ingredients of Ashwagandharishta as arjuna (bark of Terminalia arjuna), liquorice (roots of Glycyrrhiza glabra), majith (roots of Rubia cordifolia), rasna (roots of Alpinia chinensis), taj (inner bark of Cinnamomum zeylanicum), nagarmotha (rhizomes of Cyperus rotundus), haritaki (fruits of Terminalia chebula), turmeric (rhizomes of Curcuma longa), nagakesara (stamens of Mesua ferrea) etc. contain a rich quantity of polyphenolic compounds and flavonoids and possess significant antioxidant activity^10-11.

Therefore, we undertook the present investigation to estimate the total phenolic content as well as total flavonoids and to evaluate the antioxidant potential of Ashwagandharishta-T and Ashwagandharishta-M prepared by traditional and modern methods respectively and its marketed preparation on two different in vitro antioxidant activity models as super-oxide radical scavenging activity and lipid per-oxidation assay.

2. MATERIAL AND METHODS:

2.1. Preparation of Ashwagandharishta-T:

This was prepared by the method as given in the Ayurvedic Formulary of India⁷. The ingredients of Ashwagandharishta were procured from local market, Jamnagar. Identification of all the individual plant material was done as per Ayurvedic Pharmacopoeia of India. Authentication of all these ingredients was done by Dr. G D Bagchi, Scientist, Department of Taxonomy and Pharmacognosy, Central Institute of Medicinal and Aromatic Plants, Lucknow. Prepared herbarium has been deposited in the CIMAP for future reference.

According to this method, coarsely powdered ashwagandha roots (Withania somnifera) with prescribed ingredients were placed in polished vessel of brass along with prescribed quantity of water (24.576 l), and allowed to steep. After 12 h of steeping, this material was warmed at medium flame until the water for decoction reduced to one eighths of the prescribed quantity (3.072 l), then the heating was stopped and it was filtered in cleaned vessel and after that honey was added. Then, dhataki flowers (Woodfordia floribunda) and prakshepa dravyas as sonth, marich, pippali, tvak, tejpatra, priyangu and nagakesara were added and this sweet filtered material was placed for fermentation in incubator for fifteen days at 33^oC±1^oC. After 15 days, completion of fermentation was confirmed by standard tests¹². The fermented preparation was filtered with cotton cloth and kept in cleaned covered vessel for further next seven days. Then, the preparation was poured in amber colored glass bottles, packed and properly labeled.

2.2. Preparation of Ashwagandharishta-M:

Method of preparation was same as followed with Ashwagandharishta-T only dhataki flowers were replaced with yeast for inducing fermentation¹³.

2.3 Chemicals:

Folin-Ciocalteu and thiobarbituric acid were obtained from Loba Chemie, India. Nitroblue tetrazolium (NBT) and gallic acid were obtained from Sigma chemicals, St. Louis, USA. Quercetin was purchased from Yucca Enterprises, Bombay. Ferrous sulphate, trichloroacetic acid, potassium dihydrogen phosphate, phenazine methosulphate , sodium carbonate, aluminium chloride, ethanol and methanol etc were of analytical grade and obtained from Ranbaxy Fine Chemicals.

2.4 Estimation of total phenolic content:

Total phenolic content was determined in both types of Ashwagandharishta as Ashwagandharishta-T and Ashwagandharishta-M prepared by traditional and modern methods respectively and in its marketed preparation by using Folin Cio-calteu’s reagent¹⁴. For the preparation of calibration curve, 1 ml of each of the different concentration of standard gallic acid solution in ethanol as 100, 50, 25, 10, 5, 2.5 and 1 µg/ml was mixed with 5 ml Folin-Ciocalteu reagent (diluted ten fold) and 4 ml of sodium carbonate solution (7.5 g/ml). The absorbance of the blue colored solution was measured after 30 min at 20ºC at 765 nm in Schimadzu 1700 UV-Visible spectrophotometer and the calibration curve was constructed between concentration versus absorbance.

The same procedure was applied for both types of test formulations of Ashwagandharishta as Ashwagandharishta-T and Ashwagandharishta-M respectively and for its marketed preparation, 1 ml of each of the test preparation of Ashwagandharishta as Ashwagandharishta-T (1g/100ml), Ashwagandharishta-M (1g/100ml) and its marketed preparation (1g/100 ml) was mixed with the same reagents as did in the construction of calibration curve and after 1 h, the absorbance of blue colored solution formed was measured for the determination of total phenolic content in both types of Ashwagandharishta as Ashwagandharishta-T, Ashwagandharishta-M and in its marketed preparation. All determinations were performed in triplicate. The total phenolic content was determined in terms of gallic acid equivalent (GAE) as milligram per gram of the test formulation and expressed as percentage weight by weight (%w/w).

2.5 Estimation of total flavonoid content:

Total flavonoid content was estimated in both types of Ashwagandharishta as Ashwagandharishta-T and Ashwagandharishta-M prepared by traditional and modern methods respectively and in its marketed preparation by aluminium chloride method¹⁵. For the preparation of calibration curve, 1 ml of each of the different concentration of standard quercetin solution in methanol as 10, 20, 40, 60, 80 and 100 µg/ml was added to the 10 ml capacity volumetric flask containing 4 ml of distilled water. To the above mixture, 0.3 ml of 5% sodium nitrite (NaNO₂) was added. After 5 min, 0.3 ml of 10% aluminium chloride (AlCl₃) was added. After 6 min, 2 ml of 1 M NaOH was added and the total volume was made up to 10 ml with distilled water. The solution was mixed well and the absorbance was measured against a separately prepared reagent blank at 510 nm.

The same procedure was applied for both of the test formulations of Ashwagandharishta as Ashwagandharishta-T and Ashwagandharishta-M prepared by traditional and modern methods respectively and its marketed preparation, 1 ml of each of the test preparation of Ashwagandharishta as Ashwagandharishta-T (1g/10ml), Ashwagandharishta-M (1g/10ml) and marketed Ashwagandharishta (1g/10ml) was mixed with the same reagents as did in the construction of calibration curve, and then the absorbance was measured for the determination of total flavonoid content in both the test formulations of Ashwagandharishta and in its marketed preparation. All determinations were performed in triplicate. The total flavonoid content was measured in terms of quercetin equivalent as milligram per gram of the test formulation and expressed as percentage weight by weight (%w/w).

2.6. In vitro determination of antioxidant activity:

2.6.1 Super-oxide radical scavenging activity:

The super-oxide radical scavenging activity of both types of Ashwagandharishta as Ashwagandharishta-T and Ashwagandharishta-M prepared by traditional and modern methods respectively and marketed Ashwagandharishta was measured by NBT method¹⁶. About 1 ml NBT solution containing 156µM NBT dissolved in 1 ml 10mM phosphate buffer, pH 7.4 and 0.1 ml of different concentration as 100, 150, 200, 250 and 300 µg/ml of each of the test preparation of Ashwagandharishta as Ashwagandharishta-T, Ashwagandharishta-M and marketed Ashwagandharishta respectively and standard antioxidant Vitamin-E was mixed and the reaction was started by adding 100 µl of phenazine methosulphate in 100 mM phosphate buffer having pH 7.4. The reaction mixture was incubated at 25ºC for 5 min, and absorbance at 560 nm was measured against control sample. Percentage inhibition was determined by comparing the results of test and control as per the formula mentioned below-

Inhibition (%) =	(Control absorbance- Test absorbance)	x 100
	Control absorbance

2.6.2 Assay of lipid per-oxidation:

The extent of lipid per-oxidation in goat liver homogenate was measured in vitro in terms of formation of thiobarbituric acid reactive substance (TBARS) by using standard method with the help of spectrophotometer¹⁷. Goat liver was purchased from local slaughter house. Its lobes were dried between blotting paper and were cut into small pieces with a heavy duty blade. They were then homogenized in glass-teflon homogenizing tubes in cold phosphate buffer saline (pH 7.4). It was centrifuged at 2000 rpm for 10 minutes, and supernatant was diluted with phosphate buffer saline up to final concentration of protein 0.8-1.5mg/0.1ml. Protein concentration was measured by using standard method¹⁸. To study the comparative response, five different concentrations as 100, 150, 200, 250 and 300 µg/ml of each of the test preparation of Ashwagandharishta as Ashwagandharishta-T, Ashwagandharishta-M prepared by traditional and modern methods respectively and marketed Ashwagandharishta were taken in this experiment. Liver homogenate was aliquoted to seventeen different glass petri dishes. The first two groups were treated as control and standard where buffer and Vitamin-E were added respectively. From the 3^rd group upto 7^th group, different concentration as (100, 150, 200, 250 and 300 µg/ml) of Ashwagandharishta-T, from 8^th group upto 12^th group different concentration as (100, 150, 200, 250 and 300 µg/ml) of Ashwagandharishta-M while from 13^th to 17^th group various concentration as (100, 150, 200, 250 and 300 µg/ml) of marketed Ashwagandharishta were added.

Lipid per-oxidation was initiated by adding 100 μl of 15 mM ferrous sulphate solution to 3 ml of liver homogenate. After 30 minutes, 100 μl of this reaction mixture was taken in a tube containing 1.5 ml of 10% trichloro acetic acid. After 10 minutes, tubes were centrifuged and supernatant was separated and mixed with 1.5 ml of 0.67% thiobarbituric acid. The mixture was heated in a water bath to complete the reaction. The intensity of pink colored complex formed was measured at 535 nm. The percentage of inhibition of lipid per-oxidation was calculated by the following formula –

Inhibition (%) =

(Control absorbance-

Test absorbance)

x 100

Control absorbance

3. RESULTS:

3.1. Results of estimation of total phenolics and flavonoids

Total phenolic content and flavonoids have been estimated in both types of Ashwagandharishta as Ashwagandharishta-T and Ashwagandharishta-M prepared by traditional and modern methods respectively and in its marketed preparation. The calibration curve of standard gallic acid for the estimation of total phenolics has been shown in Fig. 1 while calibration curve of standard quercetin for the quantification of total flavonoids has been shown in Fig. 2. A good linear relationship was observed between absorbance and concentration of standard gallic acid in the range of 1 to 100 µg/ml while for quercetin in the range of 10 to 100 µg/ml. Total phenolic content was measured in the terms of gallic acid equivalent (GAE) in milligram per gram of test formulation and was expressed as percentage weight by weight (% w/w) while total flavonoid content was measured in terms of quercetin equivalent in milligram per gram of the test formulation and was expressed as % w/w. Total phenolic content and flavonoids were found present in rich concentration in both types of Ashwagandharishta as Ashwagandharishta-T, Ashwagandharishta-M and in its marketed preparation. Results of total phenolic content and total flavonoids in both types of Ashwagandharishta as Ashwagandharishta-T, M and marketed Ashwagandharishta have been shown in Table 1.

3.2. Results of in vitro antioxidant activity

Both types of Ashwagandharishta as Ashwagandharishta-T and Ashwagandharishta-M prepared by traditional and modern methods respectively were evaluated for their antioxidant potential and showed dose dependent antioxidant activity in super oxide radical scavenging activity as well as in lipid per oxidation assay.

3.2.1. Super oxide radical scavenging activity:

Effect of both types of Ashwagandharishta as Ashwagandharishta-T and Ashwagandharishta-M prepared by traditional and modern methods respectively on super-oxide radical scavenging activity has been shown in Fig.3. Both types of Ashwagandharishta as Ashwagandharishta-T and Ashwagandharishta-M showed significant scavenging of super oxide radical in dose dependent manner and showed inhibitory concentration (IC₅₀) 91.32 µg/ml and 99.39 µg/ml respectively. Marketed Ashwagandharishta also showed significant scavenging of super oxide radical in dose dependent manner and showed IC₅₀ 94.21 µg/ml.

3.2.2. Lipid per oxidation assay:

Effect of both types of Ashwagandharishta as Ashwagandharishta-T and Ashwagandharishta-M prepared by traditional and modern methods respectively on lipid per oxidation assay has been shown in Fig.4. Both types of Ashwagandharishta as Ashwagandharishta-T and Ashwagandharishta-M inhibited the ferrous sulphate induced lipid per oxidation in dose dependent manner and showed inhibitory concentration (IC₅₀) 181.88 µg/ml and 191.05 µg/ml respectively. Marketed Ashwagandharishta also significantly inhibited the ferrous sulphate induced lipid per oxidation in dose dependent manner and showed IC₅₀ 187.24 µg/ml.

Table 1. Total phenolic content and flavonoids in Ashwagandharishta-T, M and marketed Ashwagandharishta

Sample	Total phenolic content (%w/w)	Total flavonoids (%w/w)
Ashwagandharishta-T	0.1068	0.01366
Ashwagandharishta-M	0.1065	0.01315
Marketed Ashwagandharishta	0.1066	0.01332

4. DISCUSSION:

Plants contain a large variety of phyto-nutrients, many having antioxidant properties. Antioxidant compounds include vitamins, carotenoids, flavonoids and phenolics. Among them, phenolics and flavonoids are the most important and exhibit substantial antioxidant activity^19-20. Thus, on the basis of presence of rich concentration of total phenolics and flavonoids, both of the test formulations of Ashwagandharishta as Ashwagandharishta-T and Ashwagandharishta-M prepared by traditional and modern methods respectively were evaluated for their antioxidant potential on two different in vitro models as super-oxide radical scavenging activity and lipid per-oxidation assay.

In vitro superoxide radical scavenging activity

Superoxide radical is a highly toxic species and is generated by numerous biological and photochemical reactions. Both aerobic and anaerobic organisms possess super oxide dismutase enzymes that catalyze the breakdown of super oxide radical²¹.

Reduced phenazine methosulphate assay was used to measure the super oxide dismutase activity of Ashwagandharishta-T, M and its marketed preparation and all these test preparations of Ashwagandharishta exhibited dose dependent antioxidant activity.

In vitro assay of lipid per oxidation

Lipids are widely involved in oxidative reactions and these reactions can be induced by some radicals, called reactive oxygen species (ROS). Oxidative stress caused by ROS in the living cell is associated with numerous diseases as coronary heart disease, atherosclerosis, inflammation, cancer, anaemia, age related muscular degeneration and ageing. Use of antioxidants can reduce the problems caused by reactive oxygen species and thus they retard the oxidative process²².

The results presented in Fig.4, showed that both types of Ashwagandharishta as Ashwagandharishta-T, Ashwagandharishta-M and its marketed preparation inhibited the ferrous sulphate induced lipid per-oxidation in a dose dependent manner. The inhibition could be caused by the absence of ferryl-perferryl complex or by changing the ratio of ferric to ferrous or by reducing the rate of conversion of ferrous to ferric or by changing the iron itself or combination thereof²³. Thus, Ashwagandharishta-T, M and its marketed preparation showed potent antioxidant activity and evidenced that the free radical scavenging potential helps in ameliorating disease process. Therefore, both types of Ashwagandharishta as Ashwagandharishta-T, Ashwagandharishta-M and its marketed preparation can be recommended for the in vivo pharmacological activities based on their antioxidant potential as cardioprotective activity, hepatoprotective, antidiabetic and many others. Enzyme modifying action of antioxidants could account for their pharmacological activities.

5. REFERENCES:

1. Richards RT and Sharma HM. Free radicals in health and disease. Industrial Journal of Clinical Practice 1991; 2(7): 15-26.

2. Niwa Y. Effect of Maharishi four and Maharishi five on inflammatory mediators with special reference to their free radical scavenging effect. Industrial Journal of Clinical Practice 1991; 1(8): 23-27.

3. Gutteridge JMC. Free radicals in disease processes: A compilation of cause and consequence. Free Radical Research Communication 1995; 19: 141.

4. Ester S and Paolo S. Review on some plants of Indian traditional medicine with antioxidant activity. Journal of Ethnopharmacology 2000; 71: 23-43.

5. Joyce DA. Oxygen radicals in disease. Advances in Drug Research Bulletin 1987; 127:476.

6. Hertog MGL, Kromhout D and Aravanis C. Flavonoid intake and long term risk of coronary heart disease and cancer in seven countries study. Archives of Internal Medicine 1995; 155: 381-386.

7. The Ayurvedic Formulary of India Part-I. Controller of Pubications, Delhi, 2000; 8-9.

8. Andallu B, Radhika B. Hypoglycemic, Diuretic and Hypocholesterolemic effect of winter cherry (Withania somnifera, Dunal) root. Indian Journal of Experimental Biology 2000; 38:607-9.

9. Budhiraja RD, Sudhir S. Review of biological activity of withanolides. Journal of Scientific and Industrial Research 1987; 46:488-91.

10. Jadhav PD, Laddha KS. Estimation of gallic and ellagic acid from Terminalia chebula Retz. Indian Drugs 2004; 41(4):200-206.

11. Tabu AK, Ilhami G. Antioxidant and free radical scavenging properties of curcumin. Chemico-Biological Interactions 2008; 174:27-37.

12. Mishra S. Bhaisazya Kalpana Vigyan, Chaukambha Surbharati Prakashan. Varanasi. 2005; 253-54.

13. Alam M, Radhamani S, Ali U, Purushottam KK. Microbiological screening of dhataki flowers. Journal of Research in Ayurveda and Siddha 1984; 2(4):371-5.

14. Singleton VL and Rossi JA. Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. American Journal of Enology and Viticulture 1965; 116:144-158.

15. Kumar S. Antioxidant free radical scavenging potential of Citrullus colocynthis (L.) Schard. methanolic fruit extract. Acta Pharmaceutica 2008;58: 215-220.

16. Nishimik M, Rao NA, Appaji N and Yagi K. The occurrence of superoxide anion in there action of reduced phenazine methosulphate and molecular oxygen. Biochemical and Biophysical Research Communication 1972; 46:849.

17. Ohkawa H, Oshishi N and Yagi K. Assay for lipid peroxidation in animal tissues by thiobarbituricacid. Analytical Biochemistry 1979; 95:351.

18. Lowery OH, Rosenbrough NJ, Farr AL and Randall RJ. Protein estimation with Folin phenol reagent. Biological Chemistry 1951; 193: 265.

19. Cao G, Sofic E and Prior RL. Antioxidant capacity and pro-oxidant behaviour of flavonoids: structure activity relationships. Free Radical Biology and Medicine 1997; 22: 749-760.

20. Wang H, Cao G and Prior RL. Oxygen radical absorbing capacity of anthocyanins. Journal of Agriculture and Food Chemistry 1997; 45: 304-309.

21. Govindrajan R, Vjaykumar M, Rawat AKS and Mehrotra S. Free radical scavenging potential of Picrorrhiza kurroae. Indian Journal of Experimental Biology 2003; 3(41), 875.

22. Purohit A and Vyas KB. Hypolipidemic efficacy of Capparis deciduas fruit and shoot extracts in cholesterol fed rabbits. Indian Journal of Experimental Biology 2005; 43:863-866.

23. Bragghler JM, Duncan CA and Chase IR. The involvement of iron in lipid peroxidation. Importance of ferrous to ferric ion in initiation. Journal of Biological Chemistry 1986; 261:10282-89.