Comparative Assessment of the Total Lipid Profile , Faecal Cholesterol , Very Low Density Lipoprotein Cholesterol ( VLDL-C ) and Altherogenic Index ( A . I . ) of the Aqueous Fruit Extract of Solanum macrocarpum , α-solanidine and Antihyperlipidaemic Drugs on Triton-Induced Hyperlipdaemic Rats

Studies were undertaken on the effect of 50mg/kg each of the aqueous fruit extract of Solanum macrocarpum, αsolanidine, (a steroidal glycoalkaloid found in the Solanaceae), three antihyperlipidaemic drugs namely nicotinic acid, simvastatin and cholestyramine) on forty two (42) rats made hyperlipidaemic by treating them with 400 mg/kg triton-X for 7 days. The rats were divided into 7 groups of 6 rats each. At 24hrs, 48hrs and 72hrs respectively, the rats in each group were humanely sacrificed and blood samples collected for analysis of total lipid profile [total cholesterol, triglyceride, high density lipoprotein-cholesterol (LDL-C) and low density lipoprotein cholesterol (LDL-C)]. Very low density lipoprotein cholesterol (VLDL-C) and atherogenic index (A.I.) were also calculated. Faecal samples were also collected, homogenized and extracted with chloroform/methanol in the ration 2:1 for faecal cholesterol determination. The extract, α-solanidine and the three hypolipidaemic drugs all significantly decreased (P<0.05) LDL-C and triglycerides at 48hrs and 72hrs, whilst HDL-C increased significantly (0.05) throughout the period of study (24hrs, 48hrs and 72hrs). There was no change in total cholesterol (P>0.05) for all the five substances tested throughout the period of study, whilst VLDL-C and A.I. decreased significantly (P<0.05) at 24hrs, 48hrs and 72hrs. These results imply that under the condition of this study, the aqueous fruit extract of S. macrocarpum compared favourably with α-solanidine and the three hypolipidaemic drugs in reducing the risk of development of heart diseases.


INTRODUCTION
In the traditional North East Arid zone of Nigeria, the unripe fruit of S. macrocarpum (Synonyms: S. macrocarpum L. and S. daysphyllum Schumach and Thonn) called "Gorongo" in Kanuri, (Solanaceae) is know for its laxative, antihypertensive and hypolipidaemic effects.The fruit and flowers are also used in cleaning the teeth (Bokhari and Ahmed, 1980;Grubben and Denton, 2004).Experimental support for the ethnopharmacological use of this plant in folk medicine has been reported (Sodipo et al., 2008a, b;2009a).Recently, we have documented hepatoprotective effects of aqueous fruit extract of S. macrocarpum in diet-induced hypercholesterolaemic rats (Sodipo et al., 2009b), acute and chronic triton-induced hyperlipidaemic rats respectively (Sodipo et al., 2011c;2012a).However, the mechanism of hypolipidaemia has not been extensively studied.Sodipo et al. (2009c;2011a;2012a) observed a favourable lipid profile in diet-induced hypercholesterolaemic, acute and chronic triton-induced hyperlipidaemic rats administered with the aqueous fruit extract, the exact mechanism of cholesterol lowering and hypolipidaemia was not known.
The current drugs that lower hyperlipidaema have renal and hepatotoxicities and are usually expensive (Sodipo et al., 2011c).In an attempt to find alternative in plants which are less expensive and probably less toxic than the existing antihyperlipdaemic drugs, the present study compared the ability of the aqueous fruit extract of S. macrocarpum to lower hyperlipidaemia with that of α-solanidine (a glycoalkaloid said to lower hyperlipidaemia in the Solanaceae), [ANONa, 2007] and three hypolipidaemic drugs (nicotinic acid, simvastatin and cholestyramine) in triton-induced hyperlipidaemic rats.

Plant collection and identification
The plant material (Solanum macrocapum Linn.) used in this study was obtained from Alau in Konduga Local Government, Borno State, Nigeria, between October and November, 2007.The plant was identified and authenticated by Prof. S.S. Sanusi of the Department of Biological Sciences, University of Maiduguri, Nigeria.Specimen voucher No. 548 was deposited at the Research Laboratory of the Department of Chemistry.

Extraction
The fruit of S. macrocarpum with the calyx removed was air dried and pulverized by using pestle and mortar.The 2.2kg of the ground fruit was subjected to exhaustive Soxhlet-extraction in distilled water at 100 o C to give the extract yield 15.3% w / w (Mittal et al., 1981;Fernado et al., 1991;Lin et al., 1999).The resultant solution was concentrated in vacuo and it was stored in specimen bottle and kept in a desicator at room temperature until when required.

Animals and Treatment
Forty two (42) male albino rats of Wistar strain weighing 160-200g were used in this study.The animals were obtained from the Animal House Unit of the Department of Veterinary Physiology and Pharmacology, University of Maiduguri.The animals were housed under standard laboratory condition in plastic cages.They were fed with commercial grower's mash feed (ECWA, Feeds, Jos, Nigeria) and water was provided ad libitum.All the animals were handled according to the International Guiding Principles for Biomedical Research Involving Animals (CIOMS, 1985) as certified by the Animal Ethics Committee of the Faculty of Veterinary Medicine, University of Maiduguri (Approved on October 15 th , 2008 at its 12 th Ethical Committee Meeting).A total of forty two (42) male albino rats weighing between 160 and 200g were used for the work.They were randomly distributed into seven groups of 6 rats per group.

Group one:
Rats in this group served as the negative control.They were fed with normal feed diet and given water ad libitum Group two: Rats in this group served as the positive control.They were fed with normal diet and given water ad libitum.They were also administered 400mg/kg triton-X orally (p.o) for 1 week to make them hyperlipidaemic.

Group three:
Rats in this group were fed with normal feed diet and given water ad libitum; administered 400mg/kg triton-X p.After administration of the extract, α-solanidine and the three hypolipidaeic drugs given to the rats in groups threeseven respectively, every 24hrs for 3 consecutive days, 2 rats from each group (Groups one-seven) were humanely sacrificed and blood samples were collected for total lipid profile VLDL-C and A.I. analysis.(Adapted from Williamson et al., 1996).
All the rats in the 7 groups were weighed at day zero (i.e.before administration of triton-X) and 1 week after before they were sacrificed.

Determination of Total Lipid Profile
Two rats from each of the groups were humanely sacrificed after 24hrs, 48hrs and 72hrs respectively of the effect of the extract on chronic hyperlipidaemic rats by cutting their throat with a sterile blade.Blood was collected into a clean, sterile, labelled centrifuge tubes without an anticoagulant and centrifuged at a rate of 12,000 revolutions per minute (rpm) for 10 minutes.The clear, yellow serum was then separated from settled cellular elements and subjected to determination of total lipid profile.

Determination of Faecal Cholesterol
Faeces from two rats in each of the groups were collected after 24hrs, 48hrs and 72hrs respectively that the extract had acted on the hyperlipidaemic rats into clean, cellophane bags and deep frozen.They were homogenized and extracted with chloroform/methanol in the ratio 2:1 (Folch et al., 1957) for analysis of faecal cholesterol.The faecal cholesterol determination was like that of the serum total cholesterol (Tindar's reaction) [Evans and Stein, 1986;NIH, 1990] using commercial kits from Fortress Diagnostic Ltd; Antrim.

Calculation of Atherogenic Index (A.I)
Although index was calculated with the formula given below (Williamson et al., 1996)

Statistical Analysis
Data were expressed as the mean ± S.D. The results obtained were subjected to Analysis of Variance (ANOVA) and Student t-test using Graph Pad Software (1998).

Change in Mean Body Weight of Male Albino Rats Wistar strain) after being Administered Orally with Triton-X for 7 Days
The effect of triton-X on mean body weight of albino rats fed orally with triton-X is shown in Table 1.There was increase in body weight of the rats in groups one, two and five (p < 0.05) when compared to day zero (i.e. when no triton-X was administered).
Table 1: Change in body weight of male albino rats after being administered orally with triton-X (400 mg/kg) for 7 days Within rows, means with different superscripts are statistically significant (p < 0.05) when compared to zero (0) using student t-test 0 day = before triton-X administration n = 6 rats per group-Group One* = Rats fed with normal diet and had free access to water, but were not administered triton-X

Effect of the Aqueous Fruit Extract of Solanum macrocarpum, α-Solanidine, Nicotinic Acid, Cholestyramine and Simvastatin on Total Lipid Profile of Hyperlipidaemic Rats Administered Triton-X Orally for 7 Days
The effect of the aqueous fruit extract of S. macrocarpum, α-solanidine and the three hypolipidaemic drugs on total lipid profile of hyperlipidaemic rats are shown in Table 2: Table 2: Effect of the aqueous fruit extract of S. macrocarpum, α-solanidine, nicotinic acid, cholestyramine and simvastantin on total lipid profile of hyperlipidaemic rats administered triton-X orally for 7 days ve control = Rats fed with normal feed diet and had free access to water +ve control = Rats fed with normal feed diet and triton-X Within columns, means with different means are statistically significant (p <0.05)There was no change in the total cholesterol (p> 0.05), triglycerides and LDL-C decreased significantly (p <0.05) at 24 and 48 hrs and 48 and 72 hrs respectively, whilst HDL-C increased significantly (p < 0.05) throughout the study.The triglycerides and LDL-C were all lower than that of the positive control, but they were not as high as those of the negative control throughout the period of study.The lowest value of triglycerides at 24 hrs was recorded for nicotinic acid, 0.85 ± 0.07 mmol/L, at 48, it was both α-solanidine and nicotinic acid had the lowest triglyceride level, 0.95 + 0.07mmol/L.The lowest level of LDL-C was recorded at 24 hrs with α-solanidine and simvastatin 100 ± 0.14 mmol/L, whilst at 48 hrs, α-solanidine and nicotinic acid that had the lowest triglyceride level, 0.95 ± 0.07 mmo/L.The lowest level of LDL-C was recorded at 24 hrs with α-solanidine and simvastatin, at 48hrs, with simvastatin and α-solanidine, 0.90 ± 0.28mmol/Land 0.90 ± 0.00 mmol/L respectively.At 72hrs, however, there was no change in the LDL-C (p> 0.05).At 24, 48 and 72hrs, the values of HDL-C were significantly higher (p<0.05)than those of the positive control, but they were not as high as those of the negative control.The highest value of HDL-C at 24hrs was recorded with cholestyramine, 1.45±0.07mm/Lfollowed by those of α-solanidine and simvastatin, 1.30±0.14mm/L,nictotinic acid and the aqueous extract recorded 1.15±0.07mm/L.At 48hrs, simvastatin recorded a HDL-C value of 1.20±0.71mm/L;α-solanidine and cholestyramine had the same value, 1.15±0.07mm/Land 1.15±0.21mm/Lrespectively; and the aqueous extract had a HDL-C value of 1.05±0.21mm/L.At 72 hrs, the HDL-C values of the aqueous extract, and αsolanidine remained at the same value as that recorded at 48 hrs i.e. 1.15 ±0.07mm/L and 1.15±0.07mm/Lrespectively; whilst the value for nicotinicacid, cholestyramine and simvastatin rose slightly i.e. 1.20±0.14mm/L,1.25±0.07mm/Land 1.25±0.07mm/Lrespectively.

Effect of the Aqueous Fruit Extract of Solarn.wz macrocarpum, aSolanidine, Nicotinic Acid, Cholestyramine and Simvastatin on VLDL-C and Atherogenic Index (A.I.) of Hyperlipidaemic Rats Administered Triton-X Orally for 7 Days
The effects of the aqueous extract of S. macrocarpum, α-solanidine, nicotinic acid, cholestyramine and simvastatin on VLDL-C and atherogenic index (A.I.) are shown in Table 3.
Effect of the Aqueous Fruit Extract of Solamum macrocarpum, α-solanidine, Nicotinic Acid, Cholestyrainine and Simvastatin on Faecal Cholesterol Of Hyperlipidaemic Rats Administered Triton-X Orally for 7 Days The effect of the aqueous fruit extract of Solanum macrocarpum on faecal cholesterol of hyperlipidaemic rats are shown in Table 4.There was no change in the faecal cholesterol throughout the study (p> 0.05).
Table 4: Effect of the aqueous fruit extract of S. macrocarpum, α-solanidine, nicotinic acid, cholestyramine and simvastatin on faecal cholesterol of hyperlipidaemic rats administered triton-X orally for 7 days -ve control = Rats fed with normal feed diet and had free access to water +ve control = Rats fed with normal feed diet and triton-X Among groups, means with the same superscripts are not statistically significant (p>0.05).

DISCUSSION
The increase in mean body weight of rats after Triton-X administration for 7 days (Table 1 was statistically significant (p <0.05) in Groups one, two and five when compared to day zero (i.e.before Triton-X administration).This probably implies that Triton-X at the dosage employed, 400 mg/kg or for the length of time given, induced hyperlipidaemia, even though differences in the rats' metabolism may account for the differences in the statistics exhibited in their significance.
The result from the administration of 50 mg/kg each of the aqueous fruit extract of S. macrocarpum, α solanidine, nicotinic acid, cholestyramine and simvastatin on triton-X induced hyperlipidaemic rats revealed a significant reduction in the level of triglycerides at 24 hrs and 48 hrs, LDL-C at 48 hrs and 72 hrs and an elevation in HDL-C throughout the study (Table 3) when compared to the positive control (rats administered only Triton-X).There was no change in cholesterol level (p > 0.05).Clinical studies in humans have shown that lowering levels of serum cholesterol (especially LDL-C) with diet or drugs decreases the incidence of coronary heart disease (Gotto et al., 1990).In the present study, the LDL-C levels decreased.The implication of this is that the use of the aqueous fruit extract of S. macrocarpum, α-solanidine and the three hypolipidaemic drugs (nicotinic acid, cholestyramine and simvastatin will ameliorate the occurrence of heart disease by lowering cholesterol level.It has been shown that solasodine (C 27 H 42 0 2 N), a nitrogen analogue of diosgenin (a steroidal glycoalkaloid like solanidine found in the Solanaceae), reduced serum cholesterol and LDL-cholesterol by 73.3% ad 73.5% respectively and prevented atherogenesis whilst the HDL ratio was raised significantly.Solasodine treatment prevented the accumulation of cholesterol in the liver and aorta and regressed plaque size in the thoracic and abdominal aorta.Faecal excretion of cholesterol was significantly increased suggesting that modulation of absorption was affected (ANONc, 2008;Sodipo et al., 2012a).Nicotinic acid has been shown to lower serum cholesterol and triglycerides in triton-inducedhyperlipidaemic rats and the mechanism had been attributed to either inhibition of lipid biosynthesis or stimulation of lipid catabolism (Schurr et al., 1972).Results with standard hypolipidaemic agents are reproducible and conform well to performance levels of the screen predicted from statistical analysis (Schurr et al., 1972).Simvastatin is a lipidlowering agent synthesized from a fermentation product of Aspergillus terreus.It raises HDL-C and therefore lowers the ratio of LDL-C to HDL-C and that of total cholesterol to HDL-C (ANONb, 2007).The active form of simvastatin is a specific inhibitor of HMG-CoA reductase, the enzyme that catalyses the conversion of HMG-CoA to mevalonate (Hardman and Limbird, 2001).It is well established that rats injected with Triton WR-1339 show an increase in hepatic cholesterol synthesis (Kuroda et al., 1977); hence drugs with an inhibitory action on HMG-CoA reductase are effective in lowering hyperlipidaemia (Endo et al., 1979).Thus, the extract like simvastatin, might exhibit hypolipidaemic effect due to suppression of endogenous cholesterol biosynthesis in the liver.Because the conversion of HMG-CoA to mevalonate is an early step in the biosynthetic pathway of cholesterol, therapy with simvastatin would not be expected to cause an accumulation of potentially toxic sterols.In addition, HMG-CoA is also metabolized readily back to acetyl-CoA, which participates in many biosynthetic processes in the body (ANONb, 2007).In a study of 12 healthy volunteers, the pharmacokinetics of single (80 mg) and multiple doses of simvastatin showed that no accumulation of medicine occurred after multiple dosing and the maximum plasma concentration of inhibitors occurred 1.3 to 2.4 hrs post dose (ANONb 2007).Nicotinic acid lowers plasma triglyceride and cholesterol concentration.It acts as an antilipolytic agent in adipose tissue, reducing the supply of non-esterified free fatty acids and hence the availability of substrate for hepatic triglyceride synthesis (Hardman and Limbird, 2001).The extract from phytochemistry contains saponins (Sodipo et al., 2008a(Sodipo et al., , 2012b)).Saponins according to MacDonald et al., (2005) are cholesterol-lowering agents.They do this by causing a depletion of body cholesterol by preventing its reabsorption, thus increasing its excretion in much the same way as other cholesterol-lowering drugs such as cholestyramine (a bile acid sequestrant).Saponins have been found to be useful in cholesterol lowering, so the cholesterol cannot be reabsorbed into the system, and is therefore excreted from the body (MacDonald et al., 2005), Cholestyramine (an anion exchange resin or a bile acid sequestrant), being negatively charged, binds positively charged acids.Because of the large size, resins are not absorbed and the bound bile acids are excreted in the stool Hardrnan and Limbird, 2001).The results of the study show then that the aqueous fruit extract of S. macrocarpum, just like α-solanidine, nicotinic acid, cholestyramine and simvastatin lowered hyperlipidaemia.
The hyperlipidaernia induced by Triton-X in rats was reduced by 50 mg/kg each of the aqueous fruit extract of S. macrocarpum, α-solanidine, nicotinic acid, cholestyramine and simvastatin as shown in the significant reduction (p < 0.05) in VLDL-C and atherogenic index (A.I.) throughout the study (Table 3).The values for the positive control i.e. hyperlipidaemic rats administered only Triton-X, were high; these are atherogenic and undesirable.These values reduced in groups of rats treated with the extract, α-solanidine and the three hypolipidaernic drugs.This shows the extract (in which the plant contains falvonoids which lower hyperlipidaemia [Sodipo et al., 2012c]), just like the other four substances has the potential to reduce the risk of development of heart diseases since low VLDL-C and low A.I. have been shown to be beneficial and indicative of a lower risk of coronary heart diseases (Williamson et al., 1996;Chander et al., 2005;Sodipo et al., 2012a).
The faecal cholesterol in the hyperlipidaemic rats treated with 50 mkg each of the extract, α-solanidine, nicotinic acid, cholestyrarnine and simvastatin did not change (p > 0.05) when compared to the untreated hyperlipidaemic rats (positive control) [Table 4].The faecal cholesterol in hyperlipidaemic rats administered simvastatin did not change (p > 0.05) throughout the period of study.Drugs with an inhibitory action on HMG-CoA reductase like simvastatin may exhibit their hypolipidaemic effect due to suppression of endogenous cholesterol biosynthesis in the liver because it is well established that rats injected with Triton WR-1339 showed an increase in hepatic cholesterol synthesis (Kuroda et al., 1977;Sodipo et al., 2011aSodipo et al., , 2012a)).Thus it is not surprising that the faecal cholesterol of simvastatin remained constant i.e. unaffected throughout the period of study.

CONCLUSION
The present study shows that the aqueous fruit extract of Solanum macrocarpum compares favourably with αsolanidine, nicotinic acid, simvastatin and cholestyramine in reducing lipids in acute-triton induced hyperlipidaemic rats probably by reducing absorption of lipids and increasing faecal cholesterol excretion, thus reducing hyperlipidaemia, thus buttressing the claim in traditional medicine that the fruit lowers hyperlipidaemia.Also, the extract, just like the other four substances has the potential to reduce the risk of development of heart diseases, since low VLDL-C and low A.I. have been shown to be beneficial and indicative of a lower risk of coronary heart diseases.
is very lower density lipoprotein cholesterol LDL-C is low density lipoprotein cholesterol HDL-C is high density lipoprotein cholesterol.