Abstract: The study was carried out to investigate the safety profile as well as the effect of “Rohitakarista” (RHT) on various biochemical parameters of rats` plasma after chronic administration. RHT, a classical Ayurvedic preparation used in hepatosplenic disorders, was administered per oral route at a dose of 100 mg kg-1 body weight, once daily, up to 46 days for all the experiments. Forty albino rats (Rattus novergicus: Sprague-Dawley strains), equally of both sexes, were randomly grouped into four where each group had ten animal/sex. One male and one female group were used as control and other groups were used as test. In the male, rats there was a statistically insignificant increase (p = 0.763) in the total protein but there was a statistically significant increase (p = 0.022) in the total protein content of the plasma of female rats. Statistically very high significant increase (male: p = 0.001 and female: p = 0.001) in the albumin content of the plasma was noted in both sexes. In case of bilirubin, interestingly it was decreased very high significantly (p = 0.001) in plasma of male rats but increased very high significantly (p = 0.001) in the plasma of female rats. In the male rats, statistically there was a very high significant decrease (sGPT: p = 0.001, sGOT: p = 0.001 and ALP: p = 0.001) in the sGPT, sGOT and ALP activities in the plasma. On the other hand, statistically there was a very highly significant increase (sGPT: p = 0.001, sGOT: p = 0.001 and ALP: p = 0.001) in the sGPT, sGOT and ALP activities in the plasma of female rats. Very high significant decrease (male: p = 0.001 and female: p = 0.001) in creatinine in plasma of both sexes were observed after chronic administration of RHT. Urea in the plasma was decreased very high significantly (p = 0.001) in plasma of male rats but increased very high significantly (p = 0.001) in the plasma of female rats. There was high significant increase (p = 0.002) in uric acid in male rats. On the contrary, no significant increase (p = 0.324) of uric acid was observed in female rats.
INTRODUCTION
“Rohitakarista” is an Ayurvedic OTC drug and traditionally
used in pliha (diseases of spleen); udara (diseases of the abdomen); gulma
(localised abdominal swelling or tumour); grahani (malabsorption syndrome
or sprue) and asthila (enlarged prostate). Basically it is the preparation
of Tecomella undulate (Family: Bignoniaceae) stem and bark but
some other medicinal plants are also used in small proportions (Table
1). T. undulata, commonly known as Rohitaka or Rohida, is an
important deciduous, ornamental tree found in the Thar Desert. It is also
indigenous to western part of India and southeastern parts of Pakistan
(Bhau et al., 2007). Plant parts are effective against syphilis
and eczema and the bark have mild relaxant, cardio tonic and chloretic
activities (Bhau et al., 2007). Azam (1999) reported that T.
undulata leaves contain oleanolic acid, ursolic acid and betulinic
acid which are strong HIV prohibitors. B sitosterol, triacontanol, cirsimaritin,
cirilineol, pentatriacontanol and 4,5-dihydroxy-3,6,8-trimethoxy flavone
are other compounds isolated form the leaves of T. undulata. It`s
antibacterial activities (Parckh and Chanda, 2007) and analgesic properties
(Ahmad et al., 1994) are also reported. Among other plants used
in this formulation, Embelica officinalis (Euphorbiaceae), Terminalia
chebula (Combretaceae) and Terminalia beleracia (Combretaceae)
are medicinally important. Their equi-proportional mixture, known as Triphala
in Ayurvedic system, is reported by Jagetia et al. (2004) for free
radical scavenging and radio-protective activity. But no report on the
biochemical study of this formulation is available. Considering the widespread
use of Ayurveda as the popular form of TM in Bangladesh, one cannot emphasize
enough the need for establishing the safety profiles of Ayurvedic drugs.
Keeping in mind the present scenario, this research work on Ayurvedic
formulation, Rohitakarista (RHT) explores a spectrum of its toxicological
aspects utilizing experimental animals. The objective is to have a better
understanding of the possible toxicological profile of the drug under
study and to some degree, to decide how justifiable the use of this drug
is under the stated circumstances. The project will eventually result
in supplementing and complementing the existing health care facilities
and in the long run, will ensure total coverage of the population in terms
of public health.
| Table 1: | The plants and ingredients used in the formulation of Rohitakarista (RHT) and their therapeutic uses |
MATERIALS AND METHODS
Dose and route of administration: For the biochemical study, Rohitakarista (RHT) was collected from Sri Kundeswari Aushadhalaya Ltd., Chittagong in November, 2006. The liquid were administered at a volume such that it would permit optimal dosage accuracy without contributing much to the total increase in the body fluid. The drugs were administered per oral route at a dose of 100 mg kg-1 body weight for all the experiments. Ketamine were administered intra-peritoneally (500 mg kg-1 i.p.).
Experimental animal: Forty eight-week old albino rats (Rattus novergicus: Sprague-Dawley strain) of both sexes, bred and maintained at the Animal House of the Department of Pharmacy, Jahangirnagar University were used in the toxicological experiment. These animals were apparently healthy and weighed 50-70 g.
The animals were housed in a well ventilated hygienic experimental animal house under constant environmental and adequate nutritional conditions throughout the period of the experiment. All of the rats were kept in plastic cages having dimensions of 30x20x13 cm and soft wood shavings were employed as bedding in the cages. Feeding of animals was done ad libitum, along with drinking water and maintained at natural day night cycle. They were fed with mouse chow (prepared according to the formula developed at BCSIR, Dhaka). All experiments on rats were carried out in absolute compliance with the ethical guide for care and use of laboratory animals.
Controls: A group of equal number of rat as the drug treated group was simultaneously employed in the experiment. They were administered with distilled water as placebo as par the same volume as the drug treated group for the same number of days and this group served as the control. Prior to the experiment, they were randomly divided into 4 groups of 10 animals/sex. Thus 10 rats were taken for each group for both control and the experimental group.
After acclimatization, administration of the Ayurvedic medicinal preparation was done by intra-gastric syringe. Administration of the extract was between the hours of 10 am and noon.
Blood samples preparation: At the due of the 46 day treatment period, the animals were fasted for 18 h and also 24 h after the last administration, the animals were anaesthetized using i.p. Ketamine (500 mg kg-1 i.p.). Blood samples were collected from post vena cava and transferred into heparinised tubes immediately. Blood was then centrifuged at 4,000 g for 10 min using bench top centrifuge (MSE Minor, England) to remove red blood cells and recover plasma. Plasma samples were separated and were collected using dry Pasteur pipette and stored in the refrigerator for analyses. All analyses were completed within 24 h of sample collection.
Determination of biochemical parameters: Biochemical analysis was carried out on plasma to assess the state of the liver and kidney. Biochemical studies involved analysis of parameters such as total protein, serum albumin, blood urea nitrogen (BUN), bilirubin (total and direct), creatinine and liver enzymes such as aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP).
Total protein content of the samples was assayed by the Biuret method (Plummer, 1971). Serum albumin concentration was determined using the method of Doumas et al. (1971). The method of Evelyn and Malloy (1938) was employed to determine the serum bilirubin concentration of the samples. The procedure of Tietz et al. (1994) was used to determine serum creatinine concentration while the serum urea concentration was determined by the method of Kaplan (1965). Alkaline phosphatase activities were determined using the method as described by King and King (1954).
The absorbance of all the tests was determined using spectrophotometer (UV-Visible Spectrophotometer Model No. UV-1601 PC.). The obtained data were analysed using unpaired t-test according to Glasnapp and Poggio (1985) and presented as Mean ± SEM (Standard Error of the Mean). SPSS (Statistical Package for Social Science) for windows was applied for the analysis of data. p = 0.05 was taken to be level of significance.
RESULT AND DISCUSSION
In the male rats there was a statistically insignificant increase (p = 0.763) in the total protein but there was a statistically significant increase (p = 0.022*) in the total protein content of the plasma of female rats (Table 2). Statistically a very high significant increase (male; p = 0.001 and female; p = 0.001) in the albumin content of the plasma was noted in both sexes which indicating abnormal liver function (Naganna, 1989). This may be a result of increase synthetic function of liver (Naganna, 1989). In case of bilirubin, useful in differential diagnosis of jaundice from bilirubin overproduction, interestingly it was very high significantly decreased (p = 0.001) in plasma of male rats but very high significantly increased (p = 0.001) in the plasma of female rats. This result may be sex dependent. The observed elevation in bilirubin in plasma of female rates indicates either abnormal liver function due to jaundice or decrease excretion to bile. In the male rats, there was a statistically very high significant decrease (sGPT: p = 0.001, sGOT: p = 0.001 and ALP: p = 0.001) in the sGPT, sGOT and ALP activities in the plasma. On the other hand, there was a statistically very highly significant increase (sGPT: p = 0.001, sGOT: p = 0.001 and ALP: p = 0.001) in the sGPT, sGOT and ALP activities in the plasma of female rats. Alkaline phosphatase is the marker enzyme for plasma and endoplasmic reticulum (Wright and Plummer, 1974; Shahjahan et al., 2004). The increased alkaline phosphatase activity may be resulted from either de novo synthesis of enzymes molecules or loss of other protein from tissue (Wright and Plummer, 1974; Umezawa and Hooper, 1982). Such increase in alkaline phosphatase activities may results abnormal cellular damage. However, decrease of alkaline phosphatase indicating increased synthetic activity of liver.
There was very high significant decrease (male; p = 0.001 and female;
p = 0.001) in creatinine in plasma of both sexes after chronic administration
of RHT. Creatinine is the kidney function parameter which providing a
rough approximation of glomerular filtration (Table 3).
This very high significant decrease might have results from the decrease
synthesis or increased functional capacity of tabular excretion (Mitchell
et al., 1972; Zilva et al., 1991). Urea in the plasma was
very high significantly decreased (p = 0.001) in male rats but was very
high significant increased (p = 0.001) in of female rats. There was high
significant increase (p = 0.002) in uric acid in male rats. On the contrary,
no significant increase (p = 0.324) of uric acid was observed in female
rats.
| Table 2: | Effect of chronic administration of RHT (100 mg kg-1 body weight) on various parameters of liver functions of rats` plasma |
| *Significant, ***Very high significant | |
| Table 3: | Effect of chronic administration of RHT (100 mg kg-1 body weight) on various parameters of kidney functions of rats` plasma |
| **High significant, ***Very high significant | |
The result of the present investigation has shown that RHT has altered the biochemical parameters investigated. It has found that albumin was increased very high significantly and creatinine was decreased very high significantly in both of the sexes. But interesting sex dependency has found in case of bilirubin, sGPT, sGOT, ALP and urea. All these parameters were very high significantly decreased in male rats but very high significantly increased in female rats. Further study may be carried out to identify the insight of this sex dependency.