INTRODUCTION:

Liver is the major site of intensive metabolic activity. Its dysfunction as a result of infection, toxic chemicals, certain drugs and environmental pollutants has been largely increased in the last few decades¹. Herbs play a major role in the management of various liver disorders. Sansevieria senegambica is one of such plants used in African traditional health care for the treatment of liver diseases.

Sansevieria senegambica belongs to the family Agavaceae (or Ruscaceae). It has upright curved leaves that measure about 24–40 cm in length. The leaves appear flattened toward the tip end with a slim point, and a surface that is a matte-green with faint banding. This good compact waxy leaved species has reddish rhizomes and violet-white floral². It is grown as an ornamental plant³.

In Kaïn, northern Yatenga, Burkina Faso, the dried powder of S. senegambica is used in the preservation of grains⁴. In traditional health care practice, especially in Southern Nigeria, it is used for curing bronchitis, inflammation, cough, boils and gonorrhea⁵. It is also used in arresting the effects of snake bites, in compounding solutions used as hair tonics, and in the management of diabetes mellitus, hypertension and liver problems. The weight reducing, hypoglycemic and hypocholesterolemic activities of the leaves have been reported^6,7. In this study, the ability of an aqueous extract of the rhizomes of Sansevieria senegambica, to protect against carbon tetrachloride induced liver damage was investigated in Wistar albino rats.

MATERIALS AND METHODS:

Preparation of plant extract:

Samples of fresh S. senegambica plants (Figure 1) were procured from a horticultural garden by University of Port Harcourt main gate, Abuja campus and behind Ofrima complex, University of Port Harcourt, Port Harcourt, Nigeria. After due identification at the University of Port Harcourt Herbarium, Port Harcourt, the identity was confirmed/authenticated by Dr. Michael C. Dike of Taxonomy Unit, Department of Forestry and Environmental Management, Michael Okpara University of Agriculture, Umudike, Abia State, Nigeria; and Mr. John Ibe, the Herbarium Manager of the Forestry Department, National Root Crops Research Institute (NRCRI), Umuahia, Nigeria. The rhizome was removed, rid of dirt, oven dried at 55 °C and ground into powder. The resultant powder was soaked in hot, boiled distilled water for 12 h, after which the resultant mixture was filtered and the filtrate, hereinafter referred to as the aqueous extract was stored in the refrigerator for subsequent use. A known volume of this extract was evaporated to dryness, and the weight of the residue used to determine the concentration of the filtrate, which was in turn used to determine the dose of administration of the extract. The percentage recovery of the aqueous crude extract was 22%. The resultant residue was use for the phytochemical study, in order to determine its tannin and phytosterol composition.

Figure 1 Sansevieria senegambica Baker

Determination of the phytochemical content of the crude aqueous extract:

Calibration, identification and quantification: Standard solutions were prepared in methanol for tannins and methylene chloride for phytosterols. The linearity of the dependence of response on concentration was verified by regression analysis. Identification was based on comparison of retention times and spectral data with standards. Quantification was performed by establishing calibration curves for each compound determined, using the standards. A sample chromatogram of the extract is shown in Figure 2.

Figure2 Chromatogram of the flavonoid composition of an aqueous extract of the rhizomes of Sansevieria senegambica

Determination of the phytosterol composition of the aqueous crude extract: Extraction of oil was carried out according to AOAC method 999.02,⁸ while the analysis of sterols was carried out according to AOAC method 994.10.⁹ This involved extraction of the lipid fraction from homogenized sample material, followed by alkaline hydrolysis (saponification), extraction of the non-saponifiables, clean-up of the extract, derivatisation of the sterols, and separation and quantification of the sterol derivatives by gas chromatography (GC) using a capillary column. Chromatographic analyses were carried out on an HP 6890 (Hewlett Packard, Wilmington, DE, USA), GC apparatus, fitted with a flame ionization detector (FID), and powered with HP Chemstation Rev. A 09.01 [1206] software, to quantify and identify compounds. The column was HP INNOWax Column (30 m × 0.25 mm × 0.25 μm film thickness). The inlet and detection temperatures were 250 and 320°C. Split injection was adopted with a split ratio of 20:1. Nitrogen was used as the carrier gas. The hydrogen and compressed air pressures were 22 psi and 35 psi. The oven was programmed as follows: initial temperature at 60°C, first ramping at 10 °C/min for 20 min, maintained for 4 min, followed by a second ramping at 15°C/min for 4 min, maintained for 10 min.

Determination of the tannin composition of the aqueous crude extract: Extraction was carried out according to the method of Luthar¹⁰. The crude aqueous extract were extracted with methanol followed by gas chromatographic analysis. Chromatographic analyses were carried out on an HP 6890 (Hewlett Packard, Wilmington, DE, USA), GC apparatus, fitted with a flame ionization detector (FID), and powered with HP Chemstation Rev. A 09.01 [1206] software, to quantify and identify compounds. The column was HP 5 Column (30 m × 0.25 mm × 0.25 μm film thickness). The inlet and detection temperatures were 250 and 320 °C. Split injection was adopted with a split ratio of 20:1. Nitrogen was used as the carrier gas. The hydrogen and compressed air pressures were 28 psi and 40 psi. The oven was programmed as follows: initial temperature at 120 °C, followed by ramping at 10 °C/min for 20 min.

Experimental design for the hepatoprotective study:

Wistar albino rats (150-200 g) were collected from the animal house of the Department of Physiology, University of Nigeria, Enugu Campus. Studies were conducted in compliance with applicable laws and regulations. All the experiments were conducted in accordance with the internationally accepted principles for laboratory animal use and care as found in the European Community Guidelines (EEC Directive of 1986; 86/609/EEC). The rats were weighed and sorted into eighth groups (Table 1) of five animals each, so that their average weights were approximately equal. The animals were housed in plastic cages in the animal house of the Department of Biochemistry, University of Port Harcourt. After a one-week acclimatization period on guinea growers mash (Port Harcourt Flour Mills, Port Harcourt, Nigeria), the treatment commenced. The extract was administered orally on daily basis for eight days. The dosages of administration were adopted and modified from Ikewuchi⁶ and Ikewuchi et al.⁷. The carbon tetrachloride was prepared 1:5 (v:v) in olive oil, and administered subcutaneously at 1 mL/kg body weight of carbon tetrachloride, on days 4 and 8. The dosage and method of administration of carbon tetrachloride was adopted from Obi and Uneh¹¹, with modification. Twenty four hours after the last administration of carbon tetrachloride, the rats were weighed and anaesthetized by exposure to chloroform. While under anesthesia, they were painlessly sacrificed and blood was collected from each rat into heparin sample bottles, after which their livers were collected and preserved in 10% formalin, for histological studies. The heparin anti-coagulated blood samples were centrifuged at 1000 g for 10 min, after which their plasma was collected and stored for subsequent analysis.

Table 1 Experimental design for the hepatoprotective screening

S/ N	ID	Treatment
1	Normal	Olive oil (1 mL/kg) and Normal saline and Water
2	Test control	Carbon tetrachloride (1 mL/kg) and water
3	Treatment control I (SRC1)	Olive oil (1 mL/kg) and extract (100 mg/Kg)
4	Treatment control II (SRC2)	Olive oil (1 mL/kg) and extract (200 mg/Kg)
5	Treatment control III (SRC3)	Olive oil (1 mL/kg) and extract (300 mg/Kg)
6	Treatment I (SR1)	Carbon tetrachloride (1 mL/kg) and extract (100 mg/Kg)
7	Treatment II (SR2)	Carbon tetrachloride (1 mL/kg) and extract (200 mg/Kg)
8	Treatment III (SR3)	Carbon tetrachloride (1 mL/kg) and extract (300 mg/Kg)

Assay of plasma hepatospecific markers:

The plasma activities of alanine transaminase, aspartate transaminase and alkaline phosphatase, were determined using Randox test kits (Randox Laboratories, Crumlin, England). The activities of alanine and aspartate transaminases were respectively measured by monitoring at 546 nm the concentrations of pyruvate and oxaloacetate hydrazones formed with 2,4-dinitrophenylhydrazine. The activity of alkaline phosphatase was determined by monitoring the degradation of p-nitrophenylphosphate to p-nitrophenol, at 405 nm.

Plasma total bilirubin and protein concentrations were determined using Randox test kits (Randox Laboratories, Crumlin, England). Total bilirubin was determined in the presence of caffeine, which released albumin bound bilirubin, by the reaction with diazotized sulphanilic acid, with intensity of the resultant solution monitored at 578 nm. Plasma total protein was determined by the Biuret method using Randox test kits. Alkaline copper solutions react with peptide bonds in protein to produce a violet color whose intensity at 560 nm, is directly proportional to the amount of protein present.

Determination of percentage protection (% protection):

The percentage protection provided by the extract against carbon tetrachloride induced liver damage was calculated using the following formula adapted from Al-Qarawi et al.¹²

Histopathological study:

The histopathology study was carried out by Professor S.O. Nwosu, of the Department of Anatomical Pathology, University of Port Harcourt Teaching Hospital. Small pieces of liver tissues were collected in 10% formalin for proper fixation. These tissues were processed and embedded in paraffin wax. Sections of 5-6 μm in thickness were cut, mounted on slide and stained with hematoxylin and eosin. The sections were then examined via light microscopy (Opticphot-2; Nikon, Tokyo, Japan) at 100× magnification.

Statistical analysis of data:

All values are reported as the mean ± s.d. The values of the various parameters were analyzed for statistical significant differences between the groups, using the Student’s t-test, with the help of SPSS Statistics 17.0 package (SPSS Inc., Chicago Ill). P<0.05 was assumed to be significant.

RESULTS AND DISCUSSION:

Table 2 shows the phytosterol and tannin composition of an aqueous extract of the rhizomes of Sansevieria senegambica. The sterol extract consisted 100% of sitosterol, while the tannin extract consisted 100% of tannic acid. These two compounds have established antineoplasmic, anticarcinogenic and hepatoprotective properties^13-17.

Table 2 Phytosterol and tannin composition of an aqueous extract of the rhizomes of Sansevieria senegambica

Compounds	Retention time (min)	Composition (mg/kg)
Sterols
Cholesterol	20.590	0.00
Cholestanol	21.392	0.00
Ergosterol	22.393	0.00
Campesterol	23.057	0.00
Stigmasterol	23.210	0.00
5-Avenasterol	24.224	0.00
Sitosterol	24.779	176.72

Tannins
Tannic acid	19.206	6924.06

The effect of an aqueous extract of the rhizomes of Sansevieria senegambica on the plasma markers of liver function and integrity, of normal and carbon tetrachloride treated rats is presented in Figures 3 and 4. The carbon tetrachloride treatment produced significantly high (P<0.05) plasma alanine and aspartate transaminases and alkaline phosphatase activities, and total and conjugated bilirubin levels. The plasma alanine and aspartate transaminases, alkaline phosphatase activities, and total and conjugated bilirubin levels of the treated animals were significantly lower (P<0.05) than that of the test control. Figure 5 shows the ability of the aqueous extract of the rhizomes of Sansevieria senegambica to protect against carbon tetrachloride toxicity. Histopathological studies showed that carbon tetrachloride caused fatty degeneration and necrosis of hepatocytes (Figures 6 and 7). It also revealed that pre-treatment with aqueous extract of the rhizomes of Sansevieria senegambica exhibited protection, which confirmed the results of the biochemical studies. The results of this study indicated that treatment with the plant extract protects the liver against carbon tetrachloride - induced hepatotoxicity.

Figure 3 Effects of an aqueous extract of the rhizomes of Sansevieria senegambica on the activities of plasma hepatospecific marker enzymes in normal and carbon tetrachloride treated rats.

Values are mean ± s.d., n=5, per group. ^a,b,cValues in the same column group with different superscripts are significantly different at P<0.05.

Figure 4 Effects of an aqueous extract of the rhizomes of Sansevieria senegambica on the concentrations of plasma hepatospecific marker molecules in normal and carbon tetrachloride treated rats.

Values are mean ± s.d., n=5, per group. ^a,b,cValues in the same column group, with different superscripts are significantly different at P<0.05.

A: Section of the liver of rats administered olive oil (1 mL/kg) and treated with water, showing normal cells. B: Section of the liver tissue of rats administered carbon tetrachloride (1 mL/kg) and treated with water, showing fatty change. C: Section of the liver of rats administered olive oil (1 mL/kg) and treated with 100 mg/kg extract, showing normal cells. D: Section of the liver of rats administered olive oil (1 mL/kg) and treated with 200 mg/kg extract, showing normal cells. E: Section of the liver of rats administered olive oil (1 mL/kg) and treated with 300 mg/kg extract, showing normal cells. F: Section of the liver of rats administered carbon tetrachloride (1 mL/kg) and treated with 100 mg/kg extract, showing fatty change. G: Section of the liver of rats administered carbon tetrachloride (1 mL/kg) and treated with 200 mg/kg extract, showing normal cells. H: Section of the liver of rats administered carbon tetrachloride (1 mL/kg) and treated with 300 mg/kg extract, showing focal areas of fatty change.

Carbon tetrachloride induced liver damage is probably the best-studied model of liver injury¹⁸. It is characterized by elevated plasma activities of aspartate transaminase, alanine transaminase and alkaline phosphatase. Therefore, the reduction of carbon tetrachloride-induced elevated plasma activities of aspartate transaminase, alanine transaminase and alkaline phosphatase, and total and conjugated bilirubin levels in the animals pretreated with the aqueous extract of the rhizomes of Sansevieria senegambica shows its ability to protect the normal functional and structural integrity of the poisoned liver, and also to protect against subsequent carbon tetrachloride hepatotoxicity. This hepatoprotective activity may have been achieved via any of the following mechanisms. It is possible that β-sitosterol, a constituent of the aqueous extract of the rhizomes of Sansevieria senegambica (see Table 2) is at least partly responsible for the protective activity against carbon tetrachloride hepatotoxicity. This compound had earlier been reported to be the antihepatotoxic principle in Sambucus formosana¹⁹.

Compounds that block or retard the chain reaction of oxidation are reported to prevent oxidative stress-induced hepatotoxicity¹⁶. Various studies have demonstrated that tannins from plants exhibit hepatoprotective efficacy^16,17. Earlier, Mittal et al.¹⁵ had reported the hepatoprotective activity of tannic acid; while Pithayanukul et al.¹⁶ reported that tannic acid was majorly responsible for the hepatoprotective activity of the nut galls of Quercus infectoria against carbon tetrachloride induced liver damage. Therefore, it can be suggested that the tannic acid in the aqueous extract of the rhizomes of Sansevieria senegambica (see also Table 2), could be responsible for its hepatoprotective ability.

CONCLUSION:

This study clearly demonstrated that aqueous extract of the rhizomes of Sansevieria senegambica is an effective agent in the treatment and prevention of carbon tetrachloride-induced hepatic injury. It suggests that the daily oral consumption of the extract was prophylactic to carbon tetrachloride poisoning. This corroborates the use of Sansevieria senegambica in African traditional health care for the management of liver problems.

ACKNOWLEDGEMENT:

We gratefully acknowledge Mr. T. Mark-Balm, manager of the animal house of the Department of Biochemistry, University of Port Harcourt, for his assistance in taking care of the experimental animals.

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Received on 13.09.2011 Modified on 08.10.2011

Asian J. Research Chem. 4(12): Dec., 2011; Page 1854-1860