Crataeva nurvala (C. nurvala) Buch-Ham (Family: Capparidaceae) commonly known as Varuna, is an evergreen tree indigenous to India ⁶. It is a medium sized branched deciduous plant distributed throughout the river banks of southern India and other tropical, sub-tropical countries of the world, wild or cultivated ⁷. It requires dry, hot climate and shady places to grow effectively. Vedic literatures described its potentiality as blood purifier and to maintain homeostasis ⁸. Its bark is hot, bitter at first and then sweet sharp taste, easy to digest, stomachic, laxative, anthelmintic, expectorant and anti-pyretic ⁹. Moreover, pharmacological study reveals the potentiality of C. nurvala extract and its active principle, particularly lupeol as diuretic, anti-inflammatory, antioxidant, cardio-protective, hepatoprotective, lithonotriptic, anti-rheumatic, anti-periodic, contraceptive, anti-protozoal, rubifacient and vesicant ¹⁰. With this back ground, an attempt has been taken to establish toxic profile for C. nurvala through acute toxicity study on Wistar rats based on OECD-425 guideline.

MATERIALS AND METHODS:

Collection of plant material:

The stem bark of C. nurvala was collected from the stream sides of Western Ghat, India in September, 2012. The plant was identified and authenticated by Dr. K.V. Nagalakshamma, Professor and Head, Department of Biotechnology (UG) of St. Aloysius College, Mangalore, India and herbarium (voucher specimen no. NGSMIPS/Hb-04/2011) was preserved in the institutional department

Extraction:

100 gm coarsely powdered raw material of C. nurvala was extracted by cold maceration with ethanol for 3 days, with frequent shaking for first day and occasional stirring for the remaining days at room temperature. The process was repeated once more in order to increase the yield. The extract was concentrated by rotary flash evaporator at 40⁰C under reduced pressure and stored in deep freezer at -20⁰C ¹¹. The yield of ethanolic extract was found to be 17 % w/w.

Experimental animals:

Healthy young female Wistar rats (nulliparous and non-pregnant) aged between 8-12 week old and weighing between 120-150 gm obtained from the institutional animal house were used for the acute toxicity study. The animals were housed in a cross-ventilated room under standard environmental condition (25⁰C and 50-70% relative humidity) of 12/12 h light/dark cycle and fed with standard rat pellet and water ad-libitum ¹². The composition feed are tabulated in table 1. Animals were allowed to acclimatization for 5 days to the laboratory conditions before the experiment. The experiment was performed in accordance with the guidelines established by Institutional Animal Ethical Committee (IAEC).

Table 1: Composition of Feed

Name	Percentage
Crude Protein	20 - 21 % minimum
Ether Extractive	04 - 05 % minimum
Crude Fiber	04 % maximum
Ash	08 % maximum
Calcium	1.2%
Phosphorus	0.6 % minimum
NFE	54 %
ME Kcal/Kg	3600
Pallet Size	12 mm

Acute toxicity study:

The acute toxicity study was performed to establish the therapeutic index i.e. the ratio between the pharmacological effective dose and the lethal dose to perform preliminary screening. The acute oral toxicity study was carried out as per OECD guidelines 425, where doses of extract of plant materials ranging from 175-2000 mg/kg were administered to animals ¹³.

Healthy female Wistar rats weighing between 120-150 g maintained under standard laboratory conditions were used for the acute toxicity test according to the Organization for Economic Cooperation and Development (OECD) guidelines 425. A total of five female rats were used and each received a single oral dose of 2000 mg kg^-1 body weight of ethanolic extract of C. nurvala stem bark. Animals were kept overnight fasting prior to drug administration by oral gavage. After administration of drug sample, food was withheld for further 3-4 h. Animals were observed individually at least once during first 30 min after dosing, periodically during first 24 h (with special attention during the first 4 h) and daily thereafter for a period of 14 days. Daily observations on the changes in skin and fur, eyes and mucus membrane (nasal), respiratory rate, circulatory signs (heart rate and blood pressure), autonomic effects (salivation, lacrimation, perspiration, piloerection, urinary incontinence and defecation), central nervous system (ptosis, drowsiness, gait, tremors and convulsion) changes, body weight changes, cage side parameters and mortality rate were noted (OECD, 2002)¹⁴. The study protocol is summarized in table 2

Experimental design:

Twenty healthy female Wistar rats were used for sub-chronic toxicity study. They were divided into four groups of five rats each. Group I served as control and was fed with standard diet and water only. Group II was fed with standard diet, water and 175 mg/kg of extract, while Group III was fed with standard feed, water and 550 mg/kg of extract and Group IV was fed with standard feed, water and 1750 mg/kg of extract of C. nurvala stem bark (Table 3). The drugs were administered using a curved, ball-tipped stainless steel feeding needle for a period of 14 days ¹⁵.

Table 2: Study protocol for acute toxicity study

Name of the study	Acute toxicity study
Test material	Ethanolic extract of stem bark of C. nurvala
Physical status of test material	Semisolid extract
Species/strain used	Wistar rat
Animal procured from	Institutional animal house, NGSM Institute of Pharmaceutical Sciences
Sex	Female
Weight range	120-150 g
Age	8-12 weeks
Route of administration	Oral with help of gavage with stomach tube, volume: 1 ml/dose
Vehicle	Distilled water
No. of administration	Single
Concentration of doses	2000 and 175, 550, 1750 mg/kg body weight
Study duration	14 days
Parameters observed	Cage side observation, body weight and mortality rate
Housing condition	Air conditioned room with 10-15 air changes per hour temperature between 190-250 C, relative humidity 30-70%, and illumination cycle set to 12 hours light and 12 hours dark
Accommodation	Individual animals of similar sex in polypropylene cages with stainless steel grill tops, facilities for food and water bottle, and bedding of clean husk

Table 3: Feeding study

Group	Treatment	Duration (days)	No. of animals
Group I (Control group)	Normal feed + water	14	5
Group II	Feed +175 mg/kg bwt C. nurvala ethanolic extract	14	5
Group III	Feed + 550 mg/kg bwt C. nurvala ethanolic extract	14	5
Group IV	Feed +1750 mg/kg bwt C. nurvala ethanolic extract	14	5

RESULTS AND DISCUSSION:

Cage side observations:

Cage side observations were reported by recording general observations of each animal on a daily basis from the stage of dosing to the end of the study. Any changes or abnormalities recorded could be an indication of toxicity. The test animals at all dose levels of the extract showed no significant changes in behavior. The observations for all parameters studied are summarized in Table 4.

Mortality rate:

Mortality is the main criteria in assessing the acute toxicity study (LD₅₀) of any drug. There was no mortality recorded even at the highest dose level i.e. 2000 mg/kg body weight of the plant extract.

Body weight changes:

Body weight changes are an important factor to monitor the health of an animal. Loss of body weight is frequently the first condition of the onset of adverse effects. A dose, which causes 10% or more alteration in body weight, is considered to be toxic dose. All the animals from treated groups did not show any significant alteration in body weight for 14 days. Moreover, there was no significant change in food and water intake of the rest animals at all dose levels of the extract of C. nurvala stem bark.

Table4: Effect of ethanolic extract of C. nurvala on acute oral toxicity test in Wistar rats

Parameters	Observation
Condition of the fur	Normal
Skin	Normal
Sub-cutenous swelling	Nil
Abdominal distension	Nil
Eye-dullness	Nil
Eye-opacities	Nil
Pupil diameter	Normal
Ptosis	Nil
Colour and consistency of the faces	Normal
Condition of teeth	Normal
Breathing abnormalities	Nil
Gait	Normal

CONCLUSION:

The non-toxic nature of the ethanolic extract of C. nurvala stem bark is evident from the acute oral toxicity conducted as per OECD 425 guidelines. The normal behavior of the test animals during a period of 14 days suggests the non-toxic nature of the foresaid extracts. Moreover, there were no significant changes in body weight observed and mortality rate even at highest dose (2000 mg/kg body weight) was found to be nil. Hence, C. nurvala could be safe up to the dose of 2000 mg/kg body weight of the animal. Further studies are required for determining chronic toxic symptoms.

ACKNOWLEDGMENTS:

The authors acknowledge the financial support of NGSM Institute of Pharmaceutical Sciences and Nitte University, Mangalore, India for the research work (Grant no. NU/PhD/Pharm/Res-10/2011).

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Received on 09.10.2013

Modified on 30.10.2013

Accepted on 05.11.2013

Research Journal of Pharmacognosy and Phytochemistry. 5(6): November –December 2013, 293-296