A COMPARATIVE ACUTE TOXICITY EVALUATION OF ASHOKA KSHEERAPAKA PREPARED FROM TWO DIFFERENT SPECIES OF SARACA (S. ASOCA & S. THAIPINGENSIS)

Abstract

Ashoka (Saraca asoca) is an important Ayurvedic drug for treating gynecological disorders. Hence it is economically important. There were reports that it has become quite scarce in several localities and reported to be threatened in North Eastern Region of India. Ashoka bark widely adulterated with other barks & or from same genus of different species. Ashoka Ksheerapaka is one among clinical formulations of the plant Ashoka. Literature review revealed that no toxicity studies have been undertaken on this formulation especially on Ashoka Ksheerapaka made from S. asoca 8l S. thaipingensis. Because of this the present study was designed to evaluate Ashoka Ksheerapaka made from two species for acute toxicity in Wistar strain albino rats as per OECD (Organization for Economic Co-operation and Development) guideline 425 with 2000 mg/kg as limit test. On 1st day test formulations were administered & observed for any toxicity changes for next 14 days. On 15th day serum biochemical and hematological parameters were estimated. In all the three groups normal weight gain was observed. Both the formulations did not produce any mortality up to the dose of 2000 mg/kg on oral administration. S. asoca increased the Monocyte percentage & Blood Sugar Level (BSL) significantly, while in S. thaipingensis significant increase in Monocyte percentage & significant decrease in the Platelet count in comparison to Normal Control group.The significant increase in Monocyte percentage is in accordance with the property of estrogens to mediate its effect through estrogen receptor in monocytes. [PUBLICATION ABSTRACT]

Headnote

ABSTRACT

Ashoka (Saraca asoca) is an important Ayurvedic drug for treating gynecological disorders. Hence it is economically important. There were reports that it has become quite scarce in several localities and reported to be threatened in North Eastern Region of India. Ashoka bark widely adulterated with other barks & or from same genus of different species. Ashoka Ksheerapaka is one among clinical formulations of the plant Ashoka. Literature review revealed that no toxicity studies have been undertaken on this formulation especially on Ashoka Ksheerapaka made from S. asoca 8l S. thaipingensis. Because of this the present study was designed to evaluate Ashoka Ksheerapaka made from two species for acute toxicity in Wistar strain albino rats as per OECD (Organization for Economic Co-operation and Development) guideline 425 with 2000 mg/kg as limit test. On 1st day test formulations were administered & observed for any toxicity changes for next 14 days. On 15th day serum biochemical and hematological parameters were estimated. In all the three groups normal weight gain was observed. Both the formulations did not produce any mortality up to the dose of 2000 mg/kg on oral administration. S. asoca increased the Monocyte percentage & Blood Sugar Level (BSL) significantly, while in S. thaipingensis significant increase in Monocyte percentage & significant decrease in the Platelet count in comparison to Normal Control group.The significant increase in Monocyte percentage is in accordance with the property of estrogens to mediate its effect through estrogen receptor in monocytes.

KEY WORDS: Ashoka, Ashoka Ksheerapaka, Acute toxicity, Saraca asoca, Saraca thaipingensis Cite this article:

INTRODUCTION

Ashoka [Saraca asoca (Roxb.) Willd.] is one of the most important Ayurvedic drug for the treatment of various feminine disorders especially in menorrhagia (Kirtikar & Basu, 2001). The word Ashoka means "without sorrow", a reference to reputation of it's bark for keeping a woman healthy and youthful (Shashikant Patwardhan, 2013). The natives and traditional healers of Chhattisgarh use Sita- Ashoka (the name given to Saraca asoca) mainly in treatment of gynecological disorders (Nalin, 2005). Its bark is bitter, astringent and sweet in taste. It has stimulating effect on endometrial and the ovarian tissue. It is useful in internal bleeding, hemorrhoids, ulcers, uterine affections, menorrhagia especially due to uterine fibroids, meno-metrorrhagia, leucorrhoea and pimples (Govind Das, 1970). Dried stem bark of Saraca asoca (Roxb.) Willd. is a genuine drug collected from wild or cultivated trees, found in Central and Eastern Himalayas, Western Ghats and Deccan (Anonymous, 1986).

Number of studies has shown the adulteration of Ashoka bark with barks of other trees, but less on the trees of the same genus. Ashoka bark is widely adulterated with barks of Polyalthia longifolia, ocassionally bark of Ashoka is mixed with Rohitaka bark (Aphanamixis polystachya (Wall.) R.Parker) and Caesalpinia pulcherrima (L.) Sw (Pradhan Ρ et al, 2009). Raw material from wild is mostly collected by local people. There are high and unintentional chances for a mistaken identity of various other species of the same Genus in the name of Ashoka. The drug from the same genus but of different species is difficult to identify. Such close resemblances of Saraca asoca plant is observed with Saraca thaipingensis. So it is imperative to carry out the toxicological study of these drugs (spp.) before being used in therapeutics. Since ancient times, Ashoka is being used in Ayurvedic preparations but comprehensive data on majority of them is not available. Till date no reports on the toxicological study on Ashoka Ksheerapaka (a medicament prepared with milk, water & plant drug) made from S. asoca & S. thaipingensis Prain. are available. Hence, this study was designed to evaluate Ashoka Ksheerapaka made from two species of Saraca i.e. S. asoca 8l S. thaipingensis for acute toxicity in Wistar strain albino rats.

MATERIAL AND METHODS

Plant material

The dried bark of S. asoca and S. thaipengensis were procured from Orissa from authentic source and also correct identification was made in Pharmacognosy laboratory attached to the Institute. The herbarium samples of these two species were deposited in the laboratory (S. asoca- voucher specimen no.6024 & S. thaipingensis- voucher specimen no.6023). Dried barks were coarsely powdered and stored in dry air tight container.

Preparation of Ksheerpaka

The Ksheerapaka (a medicament prepared with milk, water & plant drug) was prepared according to Acharya Sharangdhara by taking one part drug material, adding cow's milk 8 times of bark then adding water 32 times of bark, (i.e. 1:8:32). Ashoka bark powder was weighed. Then, in stainless steel pot weighed coarsely powdered bark of Ashoka was taken as one part, then eight times of the bark cow milk was added & 32 times of bark, water was added in it. Then it was boiled till only milk remained & water was evaporated (Tripathi Brahmanand, 2004).

Animals

Twenty Wistar strain female albino rats, weighing 160 ±20 g were taken from the animal house attached to the institute (Institute of PG Teaching and Research in Ayurveda, Jamnagar). They were housed in polypropylene cages with stainless steel cover meshes, at 22 ± 3°C with relative humidity of 50-60 %, on a 12 h natural day and night cycle. They were fed with Amrut brand rat pellet feed supplied by Pranav Agro Industries and with tap water ad libitum. The experiments were carried out in accordance with the norms of the Institutional Animal Ethics Committee (IAEC), after obtaining its permission (IAEC -04/09- 10/PhD- 1).

Study protocol

Acute oral toxicity study for both the samples was carried out as per OECD (Organization for Economic Co-operation and Development) guideline 425 with 2000 mg/kg as limit test. Out of twenty animals 6 animals were allotted to normal control (NC) group. In both the test drug groups (i.e. S. asoca & S. thaipingensis), single animals were dosed in sequence usually at 48 h intervals. Using the default progression factor, doses were selected from the sequence 175, 550, and 2000 mg/kg (because no estimate of the substance's lethality was available, dosing was initiated at 175 mg/kg) as recommended in OECD Guidelines 425. Food, but not water was withheld for overnight before the experiment and further 2 h after administration of test drug. As there was no mortality observed even at 2000 mg/kg, additional 4 more animals were dosed with 2000 mg/kg and observed for 14 days with different parameters. The animals were observed continuously for 6 hours after the dosing. The careful cage side observation was done without disturbing the animal attention and at the end of every hour the animals were individually exposed to open arena for recording the behavioural changes. On 14th day evening the rats were kept in metabolic cages for fasting. On 15th day body weight of each animal was recorded. Blood was collected by supra-orbital puncture with the help of micro capillary tubes under mild ether anesthesia for estimation of serum biochemical and hematological parameters.

To estimate haematological parameters 0.08 ml blood was mixed with 0.02 ml of EDTA (33.33 mg/ml) and fed to the auto analyzer (Sismes KX-21, Trans Asia). The parameters measured were; Total WBC count, differential leucocyte count, Total RBC count, haemoglobin content, PCV, MCV, MCH, MCHC and platelet count.

For estimation of biochemical parameters, serum was separated from collected blood and requisite quantity of serum was fed to the auto analyzer (Fully automated Biochemical Random Access Analyzer, BS-200; Lilac Medicare Pvt. Ltd., Mumbai) which was automatically drawn in to the instrument for estimating different parameters. Biochemical parameters like blood sugar (BSL) (Pennock CA et al, 1973), serum cholesterol (Roeschlau Ρ et al, 1974), serum triglyceride (Fossati Ρ & Prencipe L, 1982), HDL cholesterol, blood urea, serum creatinine (Slot C, 1965), serum glutamic pyruvic transaminase (SGPT) (Burtis CA & Ashwood ER, 1999), serum glutamic oxaloacetic transaminase (SGOT) (Tietz NW, 1995), serum total protein (Tietz NW, 1986), serum albumin and serum globulin (Doumas BT, 1972), serum alkaline phosphatase (Wilkinson JH, 1969), total billirubin (Pearlman PC & Lee RT, 1974), uric acid (Kabasakalian P, 1973), were estimated.

Statistical analysis

The results are presented as Mean ± SEM. The generated data were analyzed by employing student's t test for unpaired data. One way ANOVA was also employed with Dunnets' multiple t test (DMTT) as post-hoc test. For this purpose Sigma-stat software (version 3.1) was employed.

RESULTS

In all the three groups normal weight gain was observed (Table no. 1). Data pertaining to the effect of test drug on WBC related parameters are given in Table no.2. Both the test drugs increased the Monocyte percentage significantly in comparison to Normal Control group. While a non-significant increase in Total WBC count, Neutrophils percentage & decrease in Lymphocyte percentage, Eosinophils percentage was observed.

The effect of test drugs on RBC related parameters is shown in Table no. 3. Both the test drug treated groups did not show any significant changes in RBC related parameters. Test drug ? decreases the Platelet count significantly in comparison to NC group, while the test drug S. asoca did not show any significant changes.

Effect of test drug on BSL, lipid profile is presented in Table no. 4. Animals from S. asoca group exhibit significant elevation in the BSL while non-significant up & downs were observed in lipid profile in comparison to NC group. Test drug S. thaipingensis did not produce any significant changes in these parameters.

Both the test drugs did not produce any significant change in Blood Urea, S. Creatinine, S.G.P.T., S.G.O.T. activity, Total Protein, Albumin, Globulin, A/G ratio, Alkaline Phosphatase activity & Bilirubin. (Table no. - 5)

DISCUSSION

Ashoka, which is an economically important plant, has become quite scarce in several localities and is reported to be threatened in North Eastern Region of India (Sharma PC et al., 2005; Alok Sharma, 2008). Hence, it has become the target for adulteration of its bark. A drug is taken for the beneficial effect so it must not produce any toxicological changes in the recipient's body. But till date no study has been reported on the safety aspect of Ashoka Ksheerapaka of S. asoca and S. thaipingensis. 15 days after drug administration, no mortality was observed hence both the drugs are not lethal even at the limited test dose. Normal weight gain shows there are no serious effects on the body weight which was common side effect in synthetic estrogenic compound which were mostly used in menstrual disorders (Olinda rola, 2012). Both the drugs elevate the WBC count non- significantly and no significant changes were observed in Neutrophils percentage in comparison to NC group values i.e. both the drugs restricted the increase in WBC count and Lymphocyte percentage which was observed significantly in earlier studies on oral contraceptive pills. Further, Monocyte percentage in S. asoca group (41.64%) & S. thaipingensis group (33.33%) was significantly increased which is also in accordance with property of estrogens, this finding suggests that test drugs modulate the monocyte numbers and its effect may be mediated through estrogen receptor in monocytes (Sajida SH et al, 2006).

Both the test groups did not produce any significant change in RBC related parameters i.e. RBC count, Hb, MCV, MCH, MCHC. Platelet count in S. thaipingensis group was decreased significantly (36.83%), while in S. asoca group no significant changes were observed in comparison to NC group values. It can be suggested that observed no significant effect on Hb in both the test drugs was in accordance with the earlier studies on oral contraceptive pills (Sajida SH etal., 2006).

In S. asoca group significant increase in BSL (24.69%) was observed in comparison to NC group value while on lipid profile no significant changes were observed. S. thaipingensis did not produce any significant changes on glycemic control (BSL) value and on lipid profile. S. asoca lowers S. cholesterol (2.76%) and HDL cholesterol (5.15%) non- significantly in comparison to NC group values. Both the test formulations did not produce any significant changes in Blood urea, S. creatinine, SGPT, SGOT, Total protein, Albumin, Globulin, A/G ratio, Alkaline phosphatase, Bilirubin levels. This indicates that they do not have any toxicological implication for acute administration even at very high dose levels.

CONCLUSION

Both the species of Saraca i.e. S. asoca 8l S. thaipingensis are safe at limited test dose when administered orally in the form of Ksheerapaka. However further toxicological evaluation like chronic toxicity studies etc. are required to provide complete safety profile. Both the drugs modulate the Monocytes percentage which may produce its estrogenic effect by affecting the estrogenic receptor on Monocytes which may be evaluated only after detailed efficacy related study on these plants.

ACKNOWLEDGEMENTS

The authors are thankful to the authorities of IPGT and RA, Gujarat Ayurved University for providing facilities to carry out the research work. One of the authors extends his deep gratitude to Director General, CCRAS, New Delhi & Dr. Sudesh Gaidhani, Dy. Director (Pharmacology) CCRAS, New Delhi, for providing fellowship.

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Cite this article:

Chavan Sulakshan S, Garnit R V, Ashok ? Κ, Shukla V J, Das P, Ravishankar ? (2013), A COMPARATIVE ACUTE TOXICITY EVALUATION OF ASHOKA KSHEERAPAKA PREPARED FROM TWO DIFFERENT SPECIES OF SARACA (S. ASOCA & S. THAIPINGENSIS), Global J Res. Med Plants & Indigen. Med., Volume 2(4): 270-277

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