Modulating effects of cowpea incorporated diets on serum lipids and serum antioxidant activity in Wistar rats

The effect of incorporation of four Sri Lankan cowpea cultivars in experimental diets on serum lipids and serum antioxidant activity (AOA) in Wistar rats was investigated. Seven-week-old male Wistar rats (five groups, five per group) were fed with 20 % fat as a control diet (CD) in comparison with 20 % fat enriched diets containing 20 % whole cowpea powder from cowpea cultivars [Bombay (BO), Waruni (WA), Dawala (DA) and MI 35 (MI)] for six weeks. A lower serum total cholesterol concentration (TC) (p < 0.05) and a higher (p < 0.05) serum AOA in WA, BO, and MI diet fed rats were observed compared to the CD fed group. Higher serum AOA in WA and BO fed rats was accompanied by the higher AOA and total phenolic content (TPC). A lower (p < 0.05) serum low density lipoprotein concentration (LDL-C) was observed in all cowpea diets fed rats, and lower (p < 0.05) serum triacylglycerol (TG) and higher (p < 0.05) high density lipoprotein (HDL-C) levels were observed in WA and MI fed groups, respectively compared to CD fed groups. Cowpea incorporated experimental diets modulate serum lipids and serum AOA in Wistar rats.


INTRODUCTION
There appears to be a universal shift towards diets dominated by higher intakes of animal and partially hydrogenated fats, and lower intakes of fibre.Due to this change in dietary habits, the whole world faces a problem of growing prevalence of non-communicable diseases (NCDs) such as obesity, diabetes, cardiovascular diseases (CVD)

Modulating effects of cowpea incorporated diets on serum lipids and serum antioxidant activity in Wistar rats
It has been reported that legumes lower the serum LDL-C (Duane, 1997).Although most of the studies have been carried out using soybean (Sugano et al., 1988;Beynan, 1990), other legumes such as kidney beans, peas and chickpeas have also shown hypocholesterolemic effects.Similar to other vegetables and fruits, legumes are an excellent source of many essential nutrtients, including vitamins, minerals, fibre, phytochemicals, and antioxidants, and is associated with health promoting benefits, such as lowering the risk for chronic diseases including coronary heart disease (CHD) (Macarulla et al., 2001;Darmadi-Blackberry et al., 2004;Rochfort & Panozzo, 2007;Pastor-Cavada et al., 2009).Several studies have demonstrated that lowering LDL-C diminishes both cardiovascular and overall mortality; on the contrary higher levels of high density lipoprotein cholesterol (HDL-C) have been shown to lower the risk of coronary heart disease (Ajayi & Ajayi, 2009).The cholesterol lowering mechanism of legumes may be due to the presence of phytic acid, dietary fibre, saponins, phytosterols, proteins, peptides and their amino acid profiles (Reynold et al., 2006).

March 2016
Journal of the National Science Foundation of Sri Lanka 44 (1) Higher intake of legumes was associated with lower body mass index (BMI), blood pressure, serum total cholesterol (TC), and a lower incidence of diabetes mellitus, compared with lower intake of legumes (Darmadi-Blackberry et al., 2004).
Cowpea (Vigna unguiculata L. Walp), considered a grain legume or pulse is a rich source of proteins, dietary fibre, micronutrients and bioactive phytochemicals.Functional and physicochemical properties of legumes vary with the cultivar, and accurate and credible information on functional properties of commonly consumed cowpea cultivars is important as a primary step in promoting wide consumption of cowpea.This would provide useful information to industrialists and others alike for the subsequent incorporation of cowpea cultivars into food products to produce natural, cheap and adaptable functional foods.The present study investigates the effect of commonly consumed Sri Lankan cowpea cultivars, namely, 'Dawala', 'Waruni', 'Bombay', and 'MI 35' on serum lipids, serum antioxidant activity and ceacal bacterial population in Wistar rats.
Male Wistar rats (7 wks old) were purchased from the Medical Research Institute, Colombo, Sri Lanka and were housed individually in cages (25 W x 18 H x 34.5 L cm) with free access to food and water.The animal facility was maintained on a 12 light/dark cycle at a temperature of 23 ± 1 °C and a relative humidity of 60 ± 5 %.Twenty five rats were randomly assigned into five groups (n = 5).
After 1 wk of acclimatisation, the experimental rats were fed for 6 wks with either 20 % fat as a basal diet (CD), and compared with 20 % fat enriched diets containing 20 % whole cowpea powder from the four cowpea cultivars (BO, WA, DA, and MI).
Blood samples (1 mL) were taken at the beginning and at the end of the 6 wk period between 09.00 and 10.00 hrs from the jugular vein of fasting rats anesthetised with sodium pentobarbital.The samples were collected without any anticoagulant and the serum was separated by centrifugation at 1500 g for 20 min.Faecal material excreted during the last 3 d of the experiment was collected.The rats were anesthetised with sodium pentobarbital and killed, and the livers and ceacum were quickly removed, washed with cold saline (9 g NaCl/L), blotted dry on filter paper and weighed before freezing for storage.This experimental design was approved by the Animal Experiment Committee of the Faculty of Veterinary Medicine and Animal Science, University of Peradeniya, Sri Lanka.All animal procedures conformed to the standard principles described in the Guide for the Care and Use of Laboratory Animals (National Research Council, 1985).

Proximate analysis
Dry cowpea seeds were visually inspected and defective seeds were discarded at the beginning.The seeds were oven dried using Yamato IC600 incubator (Yamato Scientific Co., Ltd., Japan) at 60 o C for 13 hrs and finely ground using ZM 100 ultra centrifugal mill.Proximate composition of the 4 cowpea cultivars was determined by using the procedures of the Association of Official Analytical Chemists (AOAC, 1995).Experimental diets were prepared after considering the proximate analysis data for the 4 cowpea cultivars.

Sample preparation
Seeds were washed, air dried and oven dried (UFE 400, Memmert, Germany) at 60 o C until a constant weight was obtained and ground using a grinder (MX-151SG1, Panasonic Co., Ltd., China) to a fine consistency.
Cold water extracts were prepared dissolving 0.1 g of the dried powder in 10 mL of distilled water.Hot water extracts were prepared by boiling 0.1 g of dried powder in a 10 mL distilled water containing tube for 30 min in a closed water bath (WNB22, Memmert, Germany).

Radical DPPH scavenging assay
All

DPPH scavenging % = ((A control -A sample )A control )×100
where, A sample is the absorbance of the sample and A control is the absorbance of control.

FRAP assay
Working

Total phenol content
The total phenolic content in seeds was determined by Folin-Ciocalteu assay.Cowpea seed extract (50 μL) was reacted with 500 μL of Folin-Ciocalteu's reagent solution.
The samples were vortexed and after 3 min 7.5 % Na 2 CO 3 (400 μL) was added.Finally the mixture was vortexed and incubated for 30 min at room temperature.The absorbance was measured at 765 nm (UV-VIS-2460, Shimadzu, Kyoto, Japan) against distilled water as a blank and the standard used was 0.1 g L -1 tannic acid (Singleton & Rossi, 1965).

TPC (mg/mL) = (A sample /A standard ) × Concentration of standard
where, A sample is the absorbance of the sample, A standard is the absorbance of standard and the concentration of standard is 0.1 g L -1 .

Serum lipid estimation
Blood samples obtained from experimental animals were centrifuged at 1,500 × g for 15 min to separate the serum.TC, HDL-C, and triglyceride (TG) concentrations in the serum were determined enzymatically using commercially available reagent kits (ProDia Internationals, Germany).Absorbance of the samples was read against respective standard solutions.Zero adjustment was made against the blank reagent.
The LDL-cholesterol (LDL-C) concentration was calculated as follows:

Estimation of serum antioxidant activity
Serum AOA was measured by FRAP method as previously described (Benzie & Strain, 1996).

Bacterial count in ceacal content
The ceacal content was taken into tubes containing peptone water just after sacrificing the animals.Total anaerobes, lactobacillus and coliform counts were determined by inoculating diluted ceacal content on Wilkins Chalgren anaerobe agar (Oxoid Ltd., England), lactobacillus MRS agar (Oxoid Ltd., England) and MacConkey agar (Himedia, India), respectively and incubating for 5 days at 37 °C by the Gas Pak method (Mitsuoka et al., 1964;1965;1976).

Statistical analysis
Completely randomized design (CRD) was conducted and the data were analysed by one-way analysis of variance (ANOVA) using the general linear model (GLM) procedure of SAS (SAS Institute Inc., 2000) software Significant differences among means were separated by the Duncan's multiple range test (DMRT).Pearson correlation coefficients were calculated to test for a linear relationship between the variables.Differences at p < 0.05 were considered as significant.

Antioxidant activity and total phenolic content in cowpea cultivars
antioxidant activity (Table 3) in Waruni cultivar, measured by FRAP and DPPH assays in hot and cold extracts was higher (p < 0.05) than the other three cultivars.Total phenolic content in cold and hot water extracts of Waruni was higher (p < 0.05) than those in the other three cultivars.

Body weight, faecal and liver weight of rats fed with experimental diets for 6 weeks
There was no difference in the initial and final body weight among the groups (Table 4).Faecal weight (Table 5) was higher (p < 0.05) in WA, MI and DA fed groups compared to that in the CD fed group.Liver weight (Table 5) was lower (p < 0.05) in WA and DA fed groups compared to the CD fed group.

Serum lipids, atherogenic index and serum antioxidant activity in rats fed with experimental diets for 6 weeks
Table 6 shows the serum TC, HDL-C, LDL-C and TG concentrations (mmol/L) and AI in rats fed with experimental diets for 6 weeks.The serum TC concentration was lower (p < 0.05) in rats fed with BO, MI, and WA diets than that in the CD fed rats.The serum LDL-C level was lower (p < 0.05) in all cowpea incorporated diet fed rats compared to the CD.Serum TG level was lower (p < 0.05) in WA fed group compared to the CD fed group.Serum HDL-C level was (p < 0.05) in MI fed group compared to that in WA and CD fed groups.Serum AI in rats fed with cowpea diets was lower (p < 0.05) than that in the CD fed group.MI fed group had the lowest AI compared to all the other experimental groups.Serum AOA (Table 7) was high (p < 0.05) in WA, BO and MI fed groups compared to that in the CD and DA fed groups.

Ceacal weight and ceacal bacterial population in rats fed with experimental diets for 6 weeks
Ceacal weight (Table 5) was higher (p < 0.05) in WA, BO and MI fed groups compared to the CD fed group.There were no differences in the anaerobe bacterial population (Table 8) among groups.Ceacal lactobacilli population was high (p < 0.05) in all cowpea diet fed groups compared to the CD.Coliform population was lower in MI fed group than that in the BO fed group.There were no differences in coliform populations in cowpea fed groups compared to the CD fed group.coefficients being r = 0.66 and r = 0.90 (p < 0.05), respectively] compared to CD.This is also similar to the results of previous studies, showing that the lower serum lipid level may be due to higher antioxidant activities preventing lipid peroxidation (Jemai et al., 2008).Higher serum AOA in cowpea diet fed rats also showed higher AOA and TPC in cowpeas.Thus, antioxidant rich cowpeas have modulated hyperlipidemia induced oxidative stress by modulating serum AOA in rats.Dark coloured cowpea, Waruni with higher (p < 0.05) AOA and phenol content modulated the serum AOA (p < 0.05) and serum TG level (p < 0.05) compared to the other experimental diet groups.The reason for the higher serum HDL-C level in MI fed group was unknown, but it could be speculated that the dietary fibre content may be atleast partially responsible (Levrat et al., 1993;Aller et al., 2004).This is further supported by the positive correlation observed between the serum HDL-C level and faecal weight in rats fed with experimental diets, the correlation coefficient being r = 0.99 (p < 0.05).Favourable serum lipid levels in rats fed with cowpea diets had a lower AI compared  to the CD fed group.Apart from antioxidants and dietary fibres, saponins and other bioactive components present in cowpea also could be responsible for the lipid modulation ability (Frota et al., 2008).The lower TG level in WA fed group was further accompanied by lower (p < 0.05) liver weight (Table 5) compared to the CD fed group, showing that dark coloured cowpeas with higher AOA and phenolic content have higher lipid lowering ability compared to light coloured cowpeas.
The reason for the higher ceacal lactobacilli population and higher ceacal weight in cowpea fed groups remains unclear.However it could be speculated that undigested cowpea proteins or dietary fibre could have been used as substrates by lactobacilli and produced a higher propionic acid concentration, which may have modulated the serum lipids in rats fed with cowpea incorporated diets as shown previously (Jenkins et al., 2000).

CONCLUSION
Waruni, MI 35 and Bombay cowpea cultivars incorporated into high fat diets modulated the serum TC and serum LDL-C concentration, and serum antioxidant activity in Wistar rats compared to those fed with a control diet.

Table 2 :
Physicochemical properties of Cowpea (Vigna unguiculata L. Walp.) cultivar seeds (1)ple is the absorbance of the sample, A standard is the absorbance of standard and the concentration of working FRAP was 1000 μmol L -1 .March 2016Journal of the National Science Foundation of Sri Lanka 44(1)

Table 3 :
Antioxidant activity (AOA) and total phenolic content (TPC) of cold and hot water extracts of the cowpea powder Values are expressed as means ± SD.The values in each column with different superscripts a to d are significantly different (p < 0.05).Values in each row with different superscripts Q to R for FRAP and S to T for DPPH for cold and hot extracts are significantly different (p < 0.05).TPC in cold and hot extracts do not differ.

Table 4 :
Feed intake and body weight of rats fed with experimental diets for 6 weeks Values are expressed as means ± SD.The values in each raw with different superscripts a and b are significantly different (p < 0.05).

Table 5 :
Faecal, ceacal, and liver weight of rats at the end of the experimental period

Table 6 :
Cholesterol and triglyceride concentrations (mmol/L), and atherogenic index in rats fed with experimental diets for 6 weeks

Table 8 :
Ceacal bacterial population in rats fed with experimental diets for 6 weeks (log 10 cfu/g content)