Evaluation of the In Vitro and In Vivo Antioxidant Potentials of Sudarshana Powder

Sudarshana powder (SP) is one of the most effective Ayurveda powder preparations for paediatric febrile conditions. The objective of the present study was to evaluate the in vitro and in vivo antioxidant potentials of SP. The in vitro antioxidant effects were evaluated using ABTS radical cation decolourization assay where the TROLOX equivalent antioxidant capacity (TEAC) was determined. The in vivo antioxidant activity of SP was determined in Wistar rats using the Lipid Peroxidation (LPO) assay in serum. The in vitro assay was referred to as the TROLOX equivalent antioxidant capacity (TEAC) assay. For the in vivo assay, animals were dosed for 21 consecutive days and blood was drawn to evaluate the MDA level. The in vitro antioxidant activity of 0.5 μg of SP was equivalent to 14.45 μg of standard TROLOX. The percentage inhibition against the radical formation was 50.93 ± 0.53%. The SP showed a statistically significant (p < 0.01) decrease in the serum level of thiobarbituric acid-reactive substance in the test rats when compared with the control group. These findings suggest that the SP possesses potent antioxidant activity which may be responsible for some of its reported bioactivities.


Introduction
Sudarshana powder (SP) is the most effective antipyretic Ayurvedic preparation, widely used in Sri Lanka as well as in India from the inception of Ayurveda treatment. SP is mentioned in the Sri Lankan Ayurvedic Pharmacopeia [1] complied under Sec. 41(2) (c) of the Ayurveda Act no. 31 of 1961. The powder is prepared using different parts of plant materials and therefore is a 100% herbal product. SP contains 53 [1] bitter ingredients and has the capability to treat fever associated symptoms such as dyspepsia, anorexia, fatigue, and nausea. It does not cause constipation and produces a mild laxative effect. It promotes the flow of bile and in gastrointestinal disorders it is used as a digestive.
Initially the main ingredient of the SP in Sri Lanka was Swertia chirata (Roxb. ex Fleming) which was later replaced by Andrographis paniculata (Burm. f.) Wall. ex Nees.
Although literature suggests that SP is the most effective and popular Ayurvedic medicine, no scientific evidence is available for its antioxidant potential. Antipyretic effect [3][4][5][6], anti-inflammatory effect [7], analgesic effect [8][9][10], antihistamine effect [11], and long term administration effect [12][13][14][15] of the SP were established in Wistar rats in our recent experimental studies. Thus, in the present study, in vitro and in vivo antioxidant potentials of SP were evaluated.   All the 53 Ingredients were thoroughly cleaned to remove any contaminated materials using tap water. Washed ingredients were air-dried. Some herbal ingredients (i.e., Plumbago indica Linn.) were purified using purification methods, mentioned in Ayurveda authentic texts, prior to being powdered. All ingredients were powdered at the mesh size of 80 under the Ayurveda concepts. Andrographis paniculata (Burm. f.) Wall. ex Nees. (2600 g) was mixed with 50 g each of the rest of the ingredients (50 × 52) to obtain the Sudarshana powder.

Animals.
Healthy adult male Wistar rats (200-250 g) were used in the in vivo study. The animals were kept in plastic cages (two per cage) under standardized animal house conditions (temperature, 28-31 ∘ C; photoperiod, approximately 12 h natural light "per day"; relative humidity, 50-55%) at the Faculty of Medical Sciences, University of Sri Jayewardenepura, with continuous access to pelleted feed and tap water.
All experiments in rats were carried out in accordance with the guidelines for care and use of laboratory animals and the project proposal was approved (No. 591/11) by the Ethics Review Committee of the Faculty of Medical Sciences of the University of Sri Jayewardenepura, Sri Lanka (http://medical.sjp.ac.lk/index.php/ethics-review-committeeintroduction).

In Vitro Antioxidant
Activity. The in vitro antioxidant free radical scavenging activity of SP was determined by using ABTS radical cation decolourization assay [16].
In this assay, 2,2 -azinobis-(3-ethylbenzothiazoline-6sulfonic acid) or ABTS (C 18 H 18 N 4 O 6 S 4 ) is converted to its radical cation. This radical cation is dark green in colour and absorbs light at 734 nm. [16]. The reaction was monitored spectrophotometrically. This assay is often referred to as the TROLOX equivalent antioxidant capacity (TEAC) assay.

Preparation of Extracts.
The SP (5 g) was extracted with 100 ml ultrapure water, at 80 ∘ C for 20 min in a water bath shaker. After cooling, the extract was centrifuged at 5000 rpm for 10 min. The solution was filtered using No. 1 Whatman filter paper and used for ABTS analysis. The sample was diluted 1 : 10 (100 l sample + 900 l H 2 O) [17].

Formation of ABTS Radical Solution.
The ABTS solution (7 mmol) stock solution, 2.6 ml, was mixed with 11.5 ml of potassium persulfate (K 2 S 2 O 8 ) solution and kept in a dark place at room temperature (23 ∘ C) for 16 hours for free radical formation. The generated ABTS radical cations (ABTS + ) solution was dark green in colour. The stock solution was diluted with ultrapure water until the absorbance reached 0.700 (±0.02) at 734 nm. The prepared sample (10 l) was added to ABTS + solution (2990 l) with phosphate saline buffer until total volume was reached (3 ml). The absorbance reading was taken 6 min after initial mixing. All determinations were performed in triplicate. The stock solution of TROLOX was prepared by dissolving 0.0161 g in 50 ml deionized water. From the prepared stock solution, 5 l (1.56 g TROLOX), 10 l (3.12 g TROLOX), 15 l (4.68 g TROLOX), 20 l (6.24 g TROLOX), and 25 l (7.8 g TROLOX) were taken and reacted with ABTS + solution with initial absorbance of 0.700 at 734 nm against a phosphate saline buffer blank. Absorbance of reaction mixture was taken till the absorbance came to a plateau. The reduction of absorbance was calculated from the initial and final absorbance. The reduction of absorbance relevant to each TROLOX concentration was performed six times. Standard curve was drawn for mean reduction of absorbance versus quantity of TROLOX in g. The antioxidant activity was expressed as TROLOX equivalent antioxidant capacity (TEAC).

In Vivo Antioxidant Activity.
In vivo antioxidant activity of SP was analysed using the method of determination of the Lipid Peroxidation (LPO) in serum. The level of thiobarbituric acid-reactive substance (TBARS) and malondialdehyde (MDA) production was measured in serum by the modified method by Draper and Hadly, 1990 [18].
Wistar rats were randomly divided into two groups of six animals each. On Day 0, blood samples were collected to assess the baseline serum malondialdehyde (MDA) level. The control group received distilled water and test group received hot water extraction of SP (0.5 g/kg). The animals were dosed for 21 consecutive days and were observed daily for signs of toxicity and death throughout the period of study. Body weights were recorded. Twenty-four hours after the last treatment, blood was obtained through direct cardiac puncture to evaluate the MDA level.
The serum (200 L) was deproteinized by adding 1 ml of 14% trichloro acetic acid and 1 ml of 0.6% thiobarbituric acid. The mixture was heated in a water bath for 30 min at 95 ∘ C to complete the reaction and then cooled on ice for 5 min. Following centrifugation at 2000 rpm for 10 min, the absorbance of the coloured product (TBARS) was measured at 535 nm with a UV spectrophotometer.
The TBARS concentration was calculated using the following formula: where is absorbance, Σ is molar coefficient (1.56 × 10 5 L/mol/cm), is concentration of sample, is path length (1 cm).

Statistical Analysis.
The results were analysed using "Student's -test." Values of < 0.05 were considered statistically significant.

In Vitro Antioxidant Activity.
The standard curve equations = 0.040 + 0.060 and 2 = 0.9962 were obtained from the standard curve for TROLOX. The antioxidant activity of SP 0.5 g was equivalent to 14.45 g of standard TROLOX. The percentage inhibition against the radical formation was 50.93 ± 0.53%.
The findings of the study in the rats tested: the serum MDA levels of the control and test group (SP), respectively, were 3.9 ± 0.21 mol/L and 2.07 ± 0.08 mol/L ( < 0.01) and

Discussion
An antioxidant is a substance that is able to protect a substrate susceptible to oxidation, being itself present at fairly low concentrations in relation to the substrate. The SP possesses significant therapeutic effects but scientific evidence for these benefits is scarce. Therefore the in vitro and in vivo antioxidant activity of SP were evaluated in this study using ABTS and TBARS assays, respectively. The ABTS radical method is one of the most frequently used assays for the determination of the concentration of free radicals. The method is applicable to the study of both watersoluble and lipid-soluble antioxidants, pure compounds, and food extracts. The results of the ABTS assay done with SP explained without doubt the potent ability to neutralize the radical spontaneously. The antioxidant activity of 0.5 g of SP was equivalent to 14.45 g of standard TROLOX. The percentage inhibition against the radical formation was 50.93 ± 0.53% and confirms the strong antioxidant power of the SP.
The thiobarbituric acid-reactive substances (TBARS) assay is a widely used method to quantify the concentration of MDA in serum, plasma, or tissue homogenates. At low pH and elevated temperature, MDA readily participates in a nucleophilic addition reaction with 2-thiobarbituric acid (TBA), generating a pink and fluorescent 1 : 2 MDA : TBA adduct. It is a simple, reliable, and a reproducible fluorometric method for measuring TBARS in samples [19]. In this study, the SP showed the significant scavenging activity against MDA formation in rats providing evidence for the potent antioxidant activity of the SP.

Conclusion
The present investigation suggests that poly herbal preparation of Sudarshana powder possesses good antioxidant potential and it can be a useful therapeutic agent for the diseases associated with oxidative stress.

Conflicts of Interest
The authors declare that they have no conflicts of interest regarding the publication of this manuscript.
Evidence-Based Complementary and Alternative Medicine 5