Biochemical and Histopathological Effects on Liver Profile due to Acute and Subacute Oral Toxicity of Somina on Rats

Methodology: Group– I served as control (saline), while other groups (II, III) were daily treated with somina at different doses of 0.285g/kg (group – II), 10g/kg/day (group – III), for 14 (set I), 21 (set II), and 30 (set III) consecutive days. Each group contains 12 rats. During the study period, signs and behavioral changes, mortality, were observed. At the end of study period, blood sample was drawn directly from heart, for the estimation of liver enzymes: Bilirubin (BIL), alkaline phosphatase (ALP), serum glutamic pyruvic transferase (SGPT), aspartate aminotransferase (SGOT), Albumin (ALB) and total protein (TP). The liver was carefully dichotomized, weighed, and further processed for histopathological analysis.

sleep cycle [1]. In Pakistan different herbalist (Hakeem) prescribed somina, for the treatment of mental health disorders. Somina strengthen the memory processes and alertness of brain and increases memory retention power by affecting the neurotransmitters level [2]. Further, the cardiovascular activity of somina showed its hypotensive effect [3], positive inotropic and negative chronotropic effects [4]. Despite the scientific activities and significant effects of Somina, some of the single dose toxicological studies showed that somina is practically non-toxic [5], and did not produce any teratological effect [6]. However, these alternative herbal medicines have a risk of toxicity for human [7]. Previously some traditional herbal medicines were claimed to be harmful or toxic [8].
That's why this study was planned to inspect the acute and subacute toxicity of somina at different doses because all things are poisons, for there is nothing without poisonous qualities. It is only dose and frequency of dose, which makes a thing a poison. The present study was aimed to estimate the effect of somina on main target organs (liver) for its possible toxic effects after acute and subacute oral administration of somina. Saline (0.9% NaCl) was also used as control.

Ethical Considerations
All experimental work (animal handling) was conducted in accordance with relevant national legislation on the use of animals for research [9].
The experimental handling procedures were approved by the Animal Ethics Board of the DRHMIIPHS, Hamdard University, Karachi, Pakistan; which sanctioned the experiments according to regulations that conform to the provisions of the Declaration of Helsinki (1995).

Experimental Animals
Sprague-Dawley rats (200-225g) were randomly selected and accommodated in cages seven days before the start of experimentation for close monitoring of their body weight with free access to food and tap water ad libitum.

Experimental protocol
Following sets of experiments were conducted during present study ( Table 1). All Animals were weighed daily before dosing. They were treated orally and observed in first 2 hour after dosing. At the end of study (24 hours after the last dose) thiopental (40mg/kg i.p.) was injected intraperitoneally to rats [10] for autopsy and dissection of liver.
Biochemical studies (Liver profile) Before dissection, the blood samples were collected directly from heart (cardiac puncture) with sterile disposable syringe. Blood sample (approximately 3 to 4ml) then centrifuged, at 3000 rpm for 15-20min and serum was separated. Different kits (Diagnostica Merck, Germany) were used for the measurement of Liver profile (TP, BIL, cholesterol, glucose, ALP, SGPT, SGOT and uric acid) on the same day [11].

Histopathological studies
For histopathological studies liver was removed immediately after the blood was collected. From each rat, a part of the lobe was sliced, processed and stained (hematoxylin and eosin). Prepared permanent slide was used for the examination of liver morphology [11].

Statistical Analysis
One way ANOVA followed by dunnett's test determined the statistical significance between the control and treated groups (II, III). Results were tabulated as average values and mean standard error (average ± S.E).

R E S U L T S
Rats (male and female) were treated with Somina (285mg/kg and 10g/kg) which is 2 and 70 times greater than human dose (10g/kg powder/70kg) for 14, 21 and 30 consecutive days. Any mortality or morbidity was not observed during/after experimental period as shown in Table 2. All experimental animals were continuously monitored and did not show any sign of toxicity. Some non-significant behavioral changes were noticed in first 120 mins after dosing like grooming, sedation, decreased motor activity, corner sitting, and palpaberal ptosis. All animals were autopsied that revealed that no gross change was observed in liver. There were non-significant differences in body weight and liver weight when compared with the control group ( Table 2). The values are presented as mean+S.E. n = 12. *Indicates the significant difference when compared with control (P<0.05).

Effect of Somina on biochemical parameters
The dose of Somina (285mg/kg) had decreased the serum level of bilirubin, total protein, Glucose, SGOT and uric acid. This change in different biochemical parameters (P>0.05) throughout the study period was non-significant. It increased the serum level of cholesterol and alkaline phosphatase but this increase is non-significant (P>0.05) when compared with their respective control ( Table 3). The nonsignificant data collected after 21 and 30 days were interpreted as biological variability normally observed in rats.
RADS J. Pharm. Pharm. Sci. 79 The values are presented as mean+S.E. n = 12. *Indicates the significant difference when compared with control (P<0.05).

HISTOPATHOLOGICAL EFFECT OF SOMINA ON LIVER
The histopathological study confirmed that somina did not affect liver. Liver histological photograph (Figure 1) confirmed that any type of structural & functional disturbance of liver did not observe after the administration of somina. Hepatocytes are flat and arranged into lobules. The sinusoids are lined by a discontinuous layer of cells. Necrotic lesions were not seen in animal treated with somina (285mg/kg, 10g/kg) and hepatocytes were comparable with the normal control (Figure 1). Figure 1. Histopathology of rat livers following administration of Somina (285mg/kg, 10g/kg: b and c respectively) compared with its control (a). H, hepatocyte; V, central vein; Staining was done using H&E and magnification was ×400.

D I S C U S S I O N
There is growing research interest about the evaluation of toxic effects of alternative medicine used commercially. Although, these medicines were extracted from natural resources but contain different active constituents. The specific and explicit mechanism of action as well as adverse effects are still anonymous [12]. Different herbal medicines are reported to cause severe complication (Severe liver damage: 13), such as Sedum aizoon, Shan Chi (Gynura segetum: 14), germander (Teucriumchamaedrys), chaparral (Larrea tridentate) etc [15]. Hence, biochemical and histopathological RADS J. Pharm. Pharm. Sci. 80 alterations associated with oral acute and subacute toxicity of somina (an alternative medicine used in Pakistan) was conducted.
Previously Ahmed [5] reported that LD50 value of the somina was found to be more than 10g/kg as toxicity sign were not noticed up to 10g/kg and the minimum lethal dose was more than 10g/kg when given orally.
Organ weights of experimental animals are important parameters for the valuation of drug-associated toxicities [16]. Hence the non-significant changes in liver weight in the current investigation even at very high dose (10g/kg for 30 days) of somina indicates its nontoxic potential on liver because significant alterations in liver weight may suggest hepatocellular hypertrophy induced by treatment [17].
For further analysis liver function tests were conducted to interpret the functional state of the liver after the acute and sub-acute administration of somina. These enzyme levels described the functionality (albumin), cellular integrity (transaminases) and biliary tract (alkaline phosphatase) performance [18]. SGPT enzyme level rises during liver damage due to necrotic hepatocytes [19] present results suggest that SGPT level did not show any significant variation even after the administration of somina for 30 consecutive days which indicates the integrity of hepatocytes. The nonsignificant variation in ALP and bilirubin levels after the administration of somina (14 and 30 consecutive days) is usually a peculiar finding in non-cholestatic liver disease [20]. Non-significant variation in Total protein suggests intact liver mass function [20]. These biochemical parameters are the index of liver function, results suggest that the somina (10g/kg/d) does not induce toxicity to the liver. These findings were further confirmed by the histological findings of the liver shown in Figures 1. Somina, when administered in high dose (10g/kg/d) for 30 consecutive days did not produce any significant damage/deterioration in the internal integrity of Liver. Basically somina contain all natural seeds Like Lagenaria vulgaris (calabash or bottle gourd), Prunus amygdalus (Almond) and Sesamum indicum (sesamum) used at homes.

C O N C L U S I O N
In conclusion, somina did not cause either acute or sub-chronic toxicities in rats. It is concluded that Somina is practically non-toxic herbal drug. According to Loomis [21] any drug, which is given 5-15g/kg, did not show any mortality is practically non-toxic drug. The biochemical and histopathological examination also suggest that it did not produce any significant toxic effect.