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The present study was undertaken to investigate the protective effect of hydroalcoholic extract of leaves of Ipomoea staphylina against carbon tertrachloride- and gentamicin-induce hepatotoxicity and nephrotoxicity in rats respectively. The extract (200 mg/kg, p.o) showed hepatoprotective activity against carbon tetrachloride-induced hepatotoxicity by significantly (p<0.001) reducing the levels of AST, ALT, ALP, total bilirubin and significantly (p<0.001) increasing the serum total protein. The extract also showed nephroprotective activity against gentamicin-induced nephrotoxicity by significantly (p<0.001) reducing the levels of blood urea, blood urea nitrogen (BUN), serum creatinine and significantly (p<0.001) increasing the serum total protein. The extract also improves the histology of the liver and kidney. The total phenolic content and the total flavonoid content of the extract were 94.59 ± 2.54 mg gallic acid equivalent/g and 44.68 ± 2.00 mg rutin equivalent/g respectively.

Introduction

The liver is an important organ responsible for the metabolism, bile secretion, elimination of many substances, blood detoxifications, synthesizes, and regulation of essential hormones. The reasons for the liver diseases in the developing countries are hepatitis viruses, environmental toxins and parasitic disease. Certain drugs such as antibiotics, chemotherapeutic agents, high doses of paracetamol, carbon tetrachloride (CCL4), thioacetamide (TAA) causes hepatotoxicity (Saleem et al., 2010).

Kidney failure describes a medical condition in which the kidneys fail to adequately filter toxins and waste products from the blood. The two forms are acute and chronic; a number of other diseases or health problems may cause either form of renal failure to occur. Chronic kidney disease attacks the kidneys slowly and progressively over a period of time. It can take years for the damage to these organs to be noticeable because there are no symptoms, which is why the disease is often called the “silent killer” (Levey et al., 2007).

Ipomoea staphylina is an extensive climber belonging to the family convolvulaceae. A literature review reveals antiulcer activity (Firdous and Nitesh, 2009) and anti-inflammatory activity (Firdous et al., 2012) of I. staphylina. Two compounds namely sitosteryl-3-O-β-D-glucoside and chiro deoxy inositol were isolated (Kota et al., 2013).

Materials and Methods

Leaves of I. staphylina were collected from forest area of Karnataka near to Bangalore. The plant I. staphylina was taxonomically identified and authenticated by Dr. K. Karthigeyan at Central National Herbarium, Botanic Garden, Howrah, where the voucher specimen for the same is conserved under the reference number SMF-01.

The leaves of I. staphylina were cleaned and dried under shade at room temperature for several days and powdered. Same was defatted using petroleum ether (60-80 GR) for 72 h and then ethyl alcohol and distilled water at the ratio of 7:3 was used for the extraction of the powdered leaves, which resulted with a yield of 10.2%w/w. The obtained dried extracts were stored in airtight container and placed in refrigerator.

Carbon tetrachloride was procured from E. Merck chemicals pvt. Ltd, Mumbai, India, silymarin were obtained from Micro Labs, Bangalore, India and Gentamicin was procured from Piramal Health Care, Ahmedabad, India. ALT, AST, ALP, Total bilirubin, Total protein, Urea, Uric acid and Creatinine kits were obtained from Span Diagnostics, Surat, India. All other chemicals used in this study were obtained commercially and were of analytical grade.

In-breed Wistar rats (150-200 g) of either sex were maintained under controlled conditions of temperature (23 ± 2°C) and humidity (50 ± 5%) and a 12-hour light–dark cycle, were used for the experiment. The animals were housed in sanitised polypropylene cages containing sterile paddy husk as bedding. They had free access to standard rat pellet diet and water ad libitum. The animals were given a week’s time to get acclimatized with the laboratory conditions. All the experimental procedures were performed according to the committee for the purpose of control and supervision of experiments on animals (CPCSEA), ministry of social justice and empowerment Government of India, norms and approved by the Institutional Animal Ethics Committee (IAEC).

Rats were kept overnight fasting prior to drug administration. Animals received a single oral dose (2,000 mg/kg, b.w.) of hydroalcoholic extract of leaves of I. staphylina. After the administration of I. staphylina leaves extract, food was withheld for further 3–4 h. Animals were observed individually at least once during the first 30 min after dosing, periodically during the first 24 hours (with special attention during the first 4 hours) and daily thereafter for a period of 14 days. Once daily cage side observations included changes in skin and fur, eyes and mucous membrane (nasal) and also respiratory rate, circulatory (heart rate and blood pressure), autonomic (salivation, lacrimation, perspiration, piloerection, urinary incontinence, and defecation) and central nervous system (ptosis, drowsiness, gait, tremors and convulsion) changes. Mortality, if any, was determined over a period of two weeks (OECD, 2002).

LD₅₀ was done as per OECD guidelines for fixing the dose for biological evaluation. The LD₅₀ of I. staphylina leaf extract as per OECD guidelines falls under class four values with no signs of acute toxicity at 2,000 mg/kg. The biological evaluation was carried out at doses of 100 and 200 mg/kg body weight.

In brief, a 1 mL of sample (1 mg/mL) was mixed with 1 mL of Folin-Ciocalteu’s phenol reagent. After 5 min, 10 mL of a 7% Na₂CO₃ solution was added to the mixture followed by the addition of 13 mL of deionized distilled water and mixed thoroughly. The mixture was kept in the dark for 90 min at 23°C, after which the absorbance was read at 750 nm. The TPC was determined from extrapolation of calibration curve which was made by preparing gallic acid solution. The estimation of the phenolic compounds was carried out in triplicate. The TPC was expressed as milligrams of gallic acid equivalents (GAE) per g of dried sample (Kim et al., 2003).

In a 10 mL test tube, 0.3 ml of extract, 3.4 mL of 30%methanol, 0.15 mL of NaNO₂ (0.5 M) and 0.15 mL of AlCl₃.6H₂O (0.3 M) were mixed. After 5 min, 1 mL of NaOH (1 M) was added. The solution was mixed well and the absorbance was measured against the reagent blank at 506 nm. The standard curve for total flavonoids was made using rutin standard solution (0 to 100 mg/L) under the same procedure as earlier described. The total flavonoids were expressed as milligrams of rutin equivalents per g of dried fraction (Park et al., 2008).

Carbon tetrachloride-induced hepatotoxicity in rats (Sanjay et al., 2009, Rajib et al., 2009)

Thirty Wistar albino rats of either sex were assigned to five groups (n= 6): Group I: Animals were administered a single dose of water (1 mL/kg, p.o.) daily for 5 days and received liquid paraffin (1 mL/kg, s.c.) on day 2 and 3. Group II: Animals were administered distil water (1 mL/kg, p.o.) once daily for 5 days and received CCl₄: liquid paraffin (1:1, 2 mL/kg, s.c.) on day 1, 2 and 3. Group III: Animals were received silymarin (50 mg/kg, p.o.) once daily for 5 days and received CCl₄: liquid paraffin (1:1, 2 mL/kg, s.c.) on day 2 and 3. Group IV: Animals were received extract of I. staphylina (200 mg/kg, p.o.). Group V: Animals were received extract of I. staphylina (100 mg/kg, p.o.).

Test group animals (Groups IV and V) were administered orally hydroalcoholic extracts once daily for 5 days and received CCl₄: liquid paraffin (1:1, 2 mL/kg, s.c.) on day 2 and 3. Animals were sacrificed 24 h after the last treatment. Blood was collected by retero orbital sinus puncture and serum was separated by centrifugation. Rats were sacrificed; livers were excised, rinsed clean in saline and preserved in 10% formalin for histopathological study.

Twenty four Wistar albino rats of either sex were assigned to four groups (n= 6): Group I: Rats in this group were injected with normal saline, intraperitonealy and served as control; Group II: Rats in this group were injected with gentamicin (100 mg/kg, i.p) for seven consecutive days; Group III: Rats in this group were injected with gentamicin (100 mg/kg, i.p) and administered extract of I. staphylina (200 mg/kg, p.o) for seven consecutive days; Group IV: Rats in this group were injected with gentamicin (100 mg/kg, i.p) and administered extract of I. staphylina (100 mg/ kg, p.o) for seven consecutive days.

After the last dosing of seventh day after 24 h the blood sample were collected by puncturing retro-orbital plexus and serum was separated by centrifugation. Rats were sacrificed; kidneys were excised, rinsed clean in saline and preserved in 10%formalin for histopathological study.

Portions of the liver and kidney from all the experimental groups were fixed in 10% formalin, dehydrated in alcohol and then embedded in paraffin. Microtome sections (5 μm thick) were prepatred from each liver sample and stained with hemtoxylin-eosin (H&E) dye. The sections were examined for the pathological findings.

Results were expressed Mean ± SEM from six animals in each group. Comparison between the groups were made by using one way analysis of variance (ANOVA), followed by Tukey-Kramer Multiple Comparisons Test with the help of INSTAT software. P<0.05 was considered as statistically significant.

Results

The hydroalcoholic extract of leaves I. staphylina was found to contain 94.59 ± 2.54 mg gallic acid equivalent/g of phenolics while the total flavonoid contents were 44.68 ± 2.0 mg rutin equivalent/g.

In CCl₄ induced hepatotoxicity the activities of serum hepatic marker enzymes namely aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP) and total billirubin level showed a significant (p<0.001) increase in CCl₄ treated animals as compared to control group and the total serum protein concentration was significantly (p<0.001) lower in CCl₄ treated group (Table I). Administering I. staphylina (200 mg/kg, p.o) significantly reduced the levels of AST, ALT, ALP and total bilirubin level in CCl₄ treated rats as compared to the group treated with CCl₄ alone and the total serum protein concentration was significantly increased (Table I).

Groups	Treatment	AST IU/L	ALT IU/L	ALP IU/L	Total protein (g/dL)	Total bilirubin (g/dL)
I	Normal Control	58.29 ± 2.68	88.58 ± 2.49	127.64 ± 3.45	7.20 ± 0.09	1.04 ± 0.04
II	CCl4 Treated	260.55 ± 4.87^a	426.79 ± 3.17^a	274.67 ± 4.97^a	3.61 ± 0.17^a	5.52 ± 0.13^a
III	Silymarin (100 mg/kg)	92.95 ± 4.00^b	195.04 ± 6.63^b	135.75 ± 4.17^b	6.37 ± 0.21^b	2.22 ± 0.21^b
IV	Ipomoea staphylina (200 mg/kg)	99.45 ± 4.04^b	226.23 ± 6.01^b	153.21 ± 5.76^b	5.54 ± 0.23^b	2.46 ± 0.14^b
V	Ipomoea staphylina (100 mg/kg)	142.18 ± 4.52^b	299.28 ± 3.66^b	215.87 ± 6.98^b	4.54 ± 0.28	3.39 ± 0.16^b
Values are expressed as mean ± SEM, n=6; ^ap<0.001 considered statistically significant as compared to normal control group; ^bp<0.001 considered statistically significant as compared to CCl4 treated group

In gentamicin-induced nephrotoxicity, gentamicin treated group showed a significant (p<0.001) increase in blood urea, blood urea nitrogen and serum creatinine as compared to control group and the total serum protein concentration was significantly (p<0.001) lower in gentamycin treated group. Administering hydroalcoholic extract of I. staphylina (200 mg/kg, p.o) significantly (p<0.001) reduced the levels of blood urea, blood urea nitrogen and serum creatinine in gentamicin treated rats as compared to the animals treated with gentamicin treated group alone and the total serum protein concentration was significantly (p<0.001) increased (Table II).

Groups	Treatment design	Blood urea (mg/100 mL)	Blood urea nitrogen (mg/100 mL)	Serum creatinine (mg/100 mL)	Total protein (mg/dL)
I	Normal Control	18.04 ± 1.30	8.42 ± 0.60	0.49 ± 0.04	7.32 ± 0.39
II	Gentamicin (100 mg/kg)	70.24 ± 2.55^a	32.79 ± 1.19^a	2.20 ± 0.12^a	2.71 ± 0.23^a
III	Ipomoea staphylina (200 mg/kg)	46.49 ± 1.71^b	21.70 ± 0.80^b	0.75 ± 0.03^b	5.39 ± 0.32^b
IV	Ipomoea staphylina (100 mg/kg)	61.39 ± 2.02^c	28.66 ± 0.94^c	1.52 ± 0.06^b	3.93 ± 0.26
Values are expressed as mean ± SEM, n=6; ^ap<0.001 considered statistically significant as compared to normal control group; ^bp<0.001 and ^cp<0.05 considered statistically significant as compared to gentamicin treated group

The histological examination (Figure 1) shows the effect of silymarin, I. staphylina leaves extract on CCl₄ induced hepatotoxicity in rats. Control group (A) animals showed a normal hepatic architecture. In CCl₄ treated group (B), severe hepatotoxicity was evidenced by kupffer cell hyperplasia, inflammatory cells, apoptosis, microvascular fatty changes and centrilobular necrosis. Treatment with silymarin (C) showed normal architecture with less fatty changes. Hydroalcoholic extract of I. staphylina leaves (D, E) at the dose of 200 and 100 mg/kg, p.o. reduced such changes in kidney histology

In gentamicin induce nephrotoxicity model normal control rats (Figure 2A) showed normal glomerular and tubular histology whereas gentamycin treated group was found to cause distorted tubular shape, cellular infiltration of the tubules (tubulitis), glomerular and blood vessel congestion, and also result in the presence of inflammatory cells in kidney sections (Figure 2B). The concurrent treatment with the extract of leaves of I. staphylina (200 mg/kg, p.o.) reduced such changes in kidney histology (Figure 2C).

Discussion

In hepatoprotective experiment, carbon tetrachloride was used to induce hepatotoxicity by metabolic activation, therefore, it selectively causes toxicity in liver cells maintaining normal metabolic function. Carbon tetrachloride is accumulated in hepatic parenchyma cells and metabolically activated by the cytochrome P-450 dependent mixed oxidase in the endoplasmic reticulum to form trichloromethyl free radical (*CCl₃) which combined with cellular lipids and proteins in the presence of oxygen to induce lipid peroxidation (Krishna et al., 2009). These resulted in changes of structures of the endoplasmic reticulum and other membrane, loss of metabolic enzyme activation, elevated level of serum marker enzymes like AST, ALT, ALP, reduction of protein synthesis, increased lipid peroxidation and destruction of Ca²⁺ homeostasis (Kaplowitz et al., 1986).

The impairment in the transport function of the hepatocytes causes the leakage of enzymes from cells due to altered permeability of membrane, which results in decreased levels of AST, ALT and ALP in the hepatic cells and a raised level in serum. For such assessment of liver damage by CCl₄ hepatotoxin, the enzyme levels such as AST and ALT is largely considered (AL-Howiriny et al., 2004). Serum ALP and bilirubin levels on the other hand, are related to the function of hepatic cell. Increase in serum level of ALP is due to increased synthesis, in presence of increasing biliary pressure. Bilirubin is one of the most useful clinical clues for the severity of necrosis and its accumulation is a measure of binding, conjugation and excretory capacity of hepatocyte(Manokaran et al., 2008). A reduction in total serum protein observed in the CCl₄ treated group may be associated with the decrease in the number of hepatocytes which in turn may result into decreased hepatic capacity to synthesize protein(Wahid et al., 2009). In CCl₄-induced hepatotoxicity in rats, our results suggest that the treatment with I. staphylina extract and its different fractions significantly reduced the enhanced level of serum ALT, AST which seem to offer the protection and maintain the functional integrity of hepatic cells. Effective control of bilirubin level and alkaline phosphatase activity by different doses of the extract and its fractions points towards an early improvement in the secretory mechanism of the hepatic cell. The significant raise in protein levels suggests the stabilization of endoplasmic reticulum leading to protein synthesis(Firdous et al., 2008). These results indicate that the I. staphylina hydroalcoholic extract preserved structural integrity of the hepatocellular membrane and showed dose dependant protective effect.

Nephrotoxicity induced by gentamicin is characterized by a decrease in the glomerular filtration rate and tubular injury due to the formation of reactive oxygen species (ROS), which may be directly involved in membrane lipid peroxidation, mesangial cells contraction, which alters the filtration surface area and modify the ultrafiltration coefficient and decrease the glomerular filtration rate (Poormoosavi et al., 2010). Management of such renal hemodynamic abnormality and reduction of the same are important to prevent the deterioration of normal functions of kidney. Formation of non-protein nitrogenous compound such as urea and creatinine takes place due to degradation of these protein and nucleic acids (Punitha et al., 2005).

Changes in the levels of serum urea, creatinine and uric acid concentrations strongly suggested impairment of kidney function in diabetes. Administration of hydroalcoholic extract of leaves of I. staphylina decreased the levels of serum urea, creatinine, uric acid and protein level in treated groups significantly.

Conclusion

This hepatoprotective and nephroprotective activity of the I. staphylina extracts may be due to antioxidant activity which may be due to the presence of flavanoids and phenolic compounds. The results of our study demonstrate the hepatoprotective and nephroprotective activity of hydroalcoholic extract of I. staphylina leaves. The probable mechanism for its protection against cellular damage may be due to its antioxidant activity.

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