Antihyperlipidemic And Antioxidant Effects Of Phaseolus Vulgaris Linn In Wistar Rats

Antihyperlipidemic And Antioxidant Effects Of Phaseolus Vulgaris Linn In Wistar Rats

Abstract

In this study, the antihyperlipidemic and antioxidants activities of the extract and fractions of Phaseolus vulgaris L. were evaluated. The crude extract (PVE) of dried pulverized plant material was obtained by maceration in methylene chloride/methanol (1:1) while the solvent fractions were obtained by successive solvent-solvent partition in separating funnel between the crude extract suspended in aqueous medium and solvents of increasing polarity to obtain the n-hexane fraction (PVHF), ethylacetate fraction (PVEF), and butanol fraction (PVBF) in that order. Antihyperlipidemic effects of the extracts and fractions were investigated using acute, subacute and chronic models. In all three models, treatment with (PVE) caused significant reduction (P<0.05) in the lipid profile parameters with the most significance seen in the sub acute study where total cholesterol was decreased by 38.44%. Triglycerides level was significantly decreased (P<0.05) by 18.18% in the acute study model. Similarly, very low density lipoprotein (VLDLC)and low density lipoprotein (LDL-C) was respectively decreased by 18.18% and 48.73% in the acute antihyperlipidemia model. In contrast, high density lipoprotein (HDL-C) level was significantly (P<0.05) increased in the acute phase by 20.85%.In all study protocols involving the various fraction there were significant increase(P<0.05) in lipid profile. PVEF (400 mg/kg) produced- the most significant reduction in total cholesterol level in the acute study with percentage decrease of 22.10% compared to the control treatment. Triglycerides level was similarly reduced by 21.59% and 15.91% at PVHF (200 mg/kg) and PVEF (200 mg/kg) with the acute study. The sub-acute protocol showed significant decrease at PVBF 200 mg/kg percentage decrease of 17.28%. VLDL-C level for the fraction study showed significant decrease (P<0.05) at PVHF 200 mg/kg and PVEF 200 mg/kg with percentage decrease of 21.59% and 15.91% during the acute protocol, the sub-acute protocol showed significant decrease at PVBF 200 mg/kg percentage decrease of 17.28. LDL-C level for fraction extract study showed dose dependent significance decrease (P<0.05) seen at PVHF 100 mg/kg, PVEF100 mg/kg, and 200 mg/kg with percentage decrease of 47.19%, 41.62% and 53.39% respectively during the acute protocol, Finally in the chronic protocol a significant decrease was seen with PVHF 200mg/kg, PVEF 100 mg/kg, and PVBF 200 mg/kg with percentage decrease of 26.03%, 24.82% and 20.05% respectively. HDL-C level for extract study showed dose dependent significant increase (P<0.05) seen at PVHF 100 mg/kg, PVEF 100 mg/kg and 200 mg/kg with percentage increase of 30.44%, 28.88% and 30.86%. DPPH reduction and nitric oxide scavenging assays were used in the investigation of the extract and fractions for the in vitro antioxidant activities study, the antioxidant activities of the extract and fractions were further determined in vivo in rats. Antioxidant enzymes and factors such as catalase, glutathione peroxidase, and lipid peroxidation activities were measured in carbon tetrachloride-treated rats treated with or without the extract and fractions studies. The highest percentage reduction of DPPH was 80.61% seen with PVEF and PVBF fraction at 400 mg/kg. The highest percentage reduction of nitric oxide was 75.86% seen with PVHF at 200 mg/kg. The in vitro study showed significant increased (P<0.05) scavenging activity with the PVHF and PVEF having scavenging activity comparable with ascorbic acid. In in-vivo antioxidant assay showed that the Lipid peroxidation levels estimated by thiobarbituric acid reaction showed no significant (P>0.05) increase or decrease in the serum MDA of both the treated and untreated group, while in catalase activity estimation showed significant (P<0.05) increase was seen with PVE 100 mg/kg of 71.05%, Glutathione peroxidise activity showed the most significant percentage (P<0.05) increase of 76.19% for PVBF 100 mg/kg.The results of the study showed that the extracts and fractions of Phaseolus vulgaris posses anti-hyperlipidemic and antioxidant with the PVEF showing better and more consistent effects with all protocols used in the investigation.

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Chapter Four

Discussion and Conclusion

4.1 Discussion

This study evaluated the antihyperlipidemic and antioxidant properties of extract and various fractions of Phaseolus vulgaris L.In this study, hyperlipidemic animals received extract and various fractions of Phaseolus vulgaris L. for different models of experimental studies while an in-vivo and in-vitro antioxidant studies were carried out using various fractions of Phaseolus vulgaris L.

There was no death recorded during both phase of acute toxicity study at all doses up to 5000 mg/kg. Theabsence of death observed showed that the LD50 of the extract was greater than 5000 mg/kg body weight, indicating their relative safety. The phytochemical screening results revealed that both extract and fractions showed positive reactions for the presence of tannins, saponins and flavonoids while moderate amounts of carbohydrates was seen with all the extract. There was a link between flavonoids and atherosclerosis, this was based partially on evidence that some flavonoids possessed antioxidant properties and have been shown to be potent inhibitors of LDL oxidation invitro (Frankel et al., 1993). Flavonoids had also been shown to inhibit platelet aggregation and adhesion (Gryglewski et al., 1987) which might be another way they lower the risk of heart disease, Iso-flavones in soy foods have been reported to lower plasma cholesterol and also to possess estrogen like effects (Dwyer et al., 1994).
The qualitative HPLC-fingering and comparison with various database revealed the presence of various compounds such as derivatives of catechin 4’-methylcatechin and 3’,4’-dimethylcatechin. Catechin a plant secondary metabolite belongs to the group of flavan-3-ols (or simply flavanols), part of the chemical family of flavonoids (Brown and Goldstein, 1986). High concentrations of catechin which could be found in red wine, black grapes broad beans etc. and its consumption had been associated with a variety of beneficial effects including increased plasma antioxidant activity (ability of plasma to scavenge free radicals, fat oxidation and resistance of LDL to oxidation (Brown et al.,1981).

In all models of hyperlipidemic study, there was a significant decrease in serum levels of lipid profile parameters when compared with the untreated groups. In the acute study models, the decrease in serum triglycerides, total cholesterol, LDL-C and VLDL-Cwas in order of PVEF > PVHF > PVE > PVBF with the PVBF showing a non-significant decrease, in sub-acute study serum TG, TC and VLDL-C showed a decrease in this order PVEF >PVBF > PVE > PVHF with the PVHF showing a non-significant decrease, while all the fractions showed a non-significant decrease in LDL-C and an increase in HDL-C level. Finally in the chronic study there was a decrease in serum TG, TC, LDL-C and VLDL-C in these order PVEF > PVBF > PVE > PVHF levels. The results of this present hyperlipidemic study is consistent with the findings of RomanRamos et al.,1995 that reported that administration of Phaseolus vulgaris L. resulted in hypolipidemic effects.

The administration of triton and high fatty diet resulted in increase in cholesterol and triglycerides level, this was due to an apparent increased of LDL secretion by the liver, catabolism of LDL and a strong reduction in VLDL (Otway and Robinson, 1967), elevated levels of these lipoproteins plays a crucial role in atherosclerotic lesions that progresses from fatty streaks to ulcerated plaque. HDL-C transports cholesterol from peripheral tissues to the liver thereby reducing the amount stores in tissue anddecreasing the likelihood of getting atherosclerotic plagues, HDL-Cholesterol is considered to have anti-atherogenic properties, since there is negative correlation between HDL-cholesterol and risk of cardiovascular disease (Eder, 1982).

In all models there were a significant decrease in total cholesterol, triglycerides, low density lipoprotein and very low density lipoprotein with increase in high density lipoproteins, this suggest some interferences in the catabolism and increase rate of transport of cholesterol by HDL-C.

The animals treated with various Phaseolus vulgaris L. fractions, was seen to show a nonsignificant difference in the percentage body weight gain in the acute and sub-acute models while there was a significant decrease (P<0.05) in the chronic study, since the feeding patterns of the animals were normal, this might suggest the extract is unlikely to cause obesity (Obi et al., 2012) and that long term administration can be effective in managing obesity. .
Atherogenic index of plasma (AIP), a logarithmically transformed ratio of molar concentrations of triglycerides to HDL-cholesterol, it is an indices used for the diagnosis and prognosis of cardiovascular diseases. There is a strong correlation of AIP with lipoprotein particle size and this might explain its high predictive value (Dobiasova, 2006). Results from this study suggests that Phaseolus vulgaris L. might havesignificant activity against cardiovascular as seen with the atherogenic indices study especially when used for a long period of time as seen with the chronic study.

The anti-oxidant activities were determined by in-vivo and in-vitro studies. In the in-vitro studies free radical scavenging assay was determined using DPPH radical scavenging activity and nitric oxide scavenging activity while in-vivo activities was determined by measuring tissue estimates of catalase, glutathione-peroxidase and lipid peroxidation activities.

Enzymes such as catalase, MDA, glutathione peroxidases are amongst several other compounds/enzymes in which there primary expression is an indication of the body defense mechanism (Rajesh & Latha 2004; Paramavsivan 2012 et al., 2012). In this study Lipid peroxidation levels estimated by thiobarbituric acid reaction showed no significant increase or decrease in the serum MDA of both the treated and untreated group, while there was an increase in catalase activities, catalase is an anti-oxidant enzyme that is presence almost everywhere, it catalyzes the degradation of hydrogen peroxides, which is a reactive oxygen species (ROS) which is a toxic product of both pathogenic ROS production and normal aerobic metabolism (Kohen and Nyska, 2002). The increase in catalase activities revealed that the Phaseolus vulgaris L. has anti-oxidant activities there was also a non-significant increase in glutathione peroxidase activity was observed with extracts treated with the crude extract when compared with the untreated groups, while there was a significant increase for other groups.

The in-vitro antioxidant activity was assayed using DPPH and nitric oxide, DPPH which is stable nitrogen centered free radical exhibits discoloration when it undergoes reduction from a compound. Substances which are able to perform this reaction can be considered as antioxidants, results of this study shows that all test extract and fractions showed significant percentage reduction of DPPH molecules with PVEF and PVBF showing the highest rate at 80.61%. In the study involving nitric oxide, all test extract and fraction showed significant nitrite inhibition with the PVHF showing the highest inhibition at 75.86% sustained level of nitric oxide production leads to vascular collapse and septic shock and its toxicity increases greatly when it reacts with superoxide radical to form the highly reactive peroxy nitrate anion. Antioxidants inhibit formation of nitrite by directly competing with oxygen in the reaction with nitric oxide. Free radicals causes an imbalance in homeostatic environment between antioxidants and oxidants in the body (Tiwari, 2001), thus leading to oxidative stress that is been linked to the advent of coronary heart diseases (Doughan et al.,2008), Hence the ability of the extract and fractions of Phaseolus vulgaris to inhibit the formation of free radical is indicative of an antioxidant effects. The present study showed that Phaseolus vulgaris L. has antioxidant activities albeit not at a significant level which is supported by works of Cadador-Martinez et al., 2002 but which showed significant antioxidant activity as seen with the in-vitro scavenging assay.
Antioxidant therapy may inhibit atheroscelosis which has hyperlipidemia has a risk factors and thereby preventing clinical complications of disease. Hence Phaseolus vulgaris L. can be exploited as an alternative anti-hyperlipidemic and antioxidant therapeutic agent or adjuvant in existing therapy for the treatment of hyperlipidemia. The present study showed that Phaseolus vulgaris L. has antioxidant activities albeit not at a significant level which is supported by works of Cadador-Martinez et al., 2002 but which showed significant antioxidant activity as seen with the in-vitro scavenging assay.

4.2 Conclusion

Results of these study shows that the various extract of Phaseolus vulgaris L. has antihyperlipidemic and antioxidant effects with the ethylacetate fractions showing substantially significant activity in both short terms, sub-chronic and chronic models of hyperlipidemia. The results also showed consistent free radical scavenging activity in both in-vivo and in-vitro antioxidant models.Further studies are encouraged in elucidating the exact mechanism of action of the ethylacetate fractions of Phaseolus vulgaris L.,in possessing antihyperlipidemic and antioxidant properties in albino-Wistar rats.