Boiled, sprouted, and raw cowpea‐incorporated diets modulate high‐fat diet‐induced hypercholesterolemia in rats

Abstract This study was carried out to investigate the effect of processed (boiled and sprouted) cowpea‐incorporated experimental diets on serum cholesterol and serum antioxidant capacity in high‐fat diet (HFD)‐fed Wistar rats. Seven weeks old male Wistar rats were fed 20% fat as a control (CD), for comparison with 20% fat‐enriched diets containing 20% whole raw cowpea diets (Bombay Raw Diet; BRD and MI35 Raw Diet; MRD), boiled cowpea diets (Bombay Boiled Diet; BBD and MI35 Boiled Diet; MBD) and sprouted cowpea diet (Bombay Sprouted Diet; BSD) for 6 weeks. The increase in serum total cholesterol as a result of high‐fat diet was significantly countered by boiled and raw cowpea‐incorporated diet‐fed rats. Increased serum non‐HDL‐C level caused by HFD was significantly (p < 0.05) countered by raw, boiled, and sprouted cowpeas, while HDL–C was increased by raw MI and boiled Bombay incorporated diets. Boiling has improved the hypocholesterolemic ability of Bombay cowpea and BBD has significantly (p < 0.05) modulated serum HDL‐C level and liver weight in rats. These findings were supported significantly high soluble fiber content in processed cowpea powder than that in raw cowpea powder. The decrease in serum antioxidant activity as a result of HFD was significantly countered by BRD. Processing has reduced the antioxidant activity in cowpeas and serum antioxidant activity in rats. Cecal lactobacilli population was significantly high in all cowpea diet‐fed groups compared to control. Modulated serum cholesterol level in cowpea diet‐fed rats was accompanied by dietary fiber composition, antioxidant activity in cowpeas and fecal weight, cecal weight and cecal lactobacilli population in rats compared to control. Both processed and raw cowpea‐incorporated diets have modulated HFD‐induced hypercholesterolemia by modulating serum antioxidative capacity, cholesterol metabolism, and cecal fermentation.


| INTRODUC TI ON
Legume consumption is considerably high in developing countries and is used as a main source of protein compared to the developed world. With the shift in dietary pattern, throughout the world legume consumption has reduced dramatically during the past few decades. Many studies have revealed a clear relationship between diet and noncommunicable diseases.
International Diabetes mellitus guidelines recommended the consumption of legumes for controlling of the glycemic index.
Legumes are generally consumed after various processes and with processing, the antinutritional factors are improved.
Germination is one of the most common techniques used to reduce most of the antinutritional factors in legumes (Alonso, Orúe, & Marzo, 1998). It has been shown that cooking modulates nutritional and biochemical parameters of the legumes (Nergiz & Gökgöz, 2007;Nisha, Singhal, & Pandit, 2005;Ramesh & Tharanathan, 1999). However, there has been very little research reported on the effect of processing on functional properties of legumes (Dahiya et al., 2015). Hence in this study, effect of processed cowpea-incorporated experimental diets on in vivo hypocholesterolemic effect and serum antioxidant activity were studied in an animal experimental model. This will provide useful information to the industrialists and others alike on the subsequent incorporation of the studied samples into food products by producing natural, cheap, and adaptable functional foods.

| Preparation of raw cowpea powder
Cowpea cultivars "Bombay", and "MI35" were purchased from government seeds farm Palwehera, Dambulla, Sri Lanka. Dry cowpea seeds were visually inspected and defective seeds were discarded before the preparation of powder. Cowpea seeds were washed and oven dried in drying oven (YAMATO IC600, Yamato Scientific Co., Ltd., Japan) at 60°C until a constant weight was obtained and ground using a grinder (MX-151SG1, Panasonic Co., Ltd, China) to a fine consistency.

| Preparation of boiled cowpea powder
Cowpea seeds were washed and soaked overnight in distilled water in room temperature and boiled for 30 min in distilled water. Boiled seeds were then air dried for 24 hr and oven dried in drying oven (YAMATO IC600, Yamato Scientific Co., Ltd., Japan) at 60°C until a constant weight was obtained and ground using a grinder (MX-151SG1, Panasonic Co., Ltd, China) to a fine consistency.

| Preparation of sprouted cowpea powder
Cowpea seeds were soaked in distilled water overnight and allowed for germination on wet kitchen towels in room temperature for 2 days. They were then air dried for 24 hr and oven dried using YAMATO IC600 drying oven (Yamato Scientific Co., Ltd., Japan) at 60°C until a constant weight was obtained and ground using a grinder (MX-151SG1, Panasonic Co., Ltd, China) to a fine consistency.

| Animals and diets
Male Wistar rats (7 weeks old) were purchased from the Medical Research Institute, Colombo, Sri Lanka. They were housed individually in cages 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 relative humidity of 60 ± 5%. The rats were randomly assigned into five groups (n = 5). There were no significant (p < 0.05) differences in body weights and serum total cholesterol concentrations among groups at the beginning of the experiment. Proximate composition of the cowpea cultivars in raw and processed forms was determined by the procedure of the Association of Official Analytical Chemists (AOAC, 1990). The insoluble dietary fiber (IDF), soluble dietary fiber (SDF), and total dietary fiber (TDF) content of the cowpea powder were analyzed using a combination of enzymatic and gravimetric procedures of Prosky, Asp, Schweizer, DeVries, and Furda (1988). Antioxidant activity and phenol content of cowpea powders were measured. Antioxidant activity was measured by FRAP method (Al-Farsi, Alasalvar, Morris, Baron, & Shahidi, 2005) and phenol content was measured by Folin-Ciocalteu assay (Singleton & Rossi, 1965). Experimental diets were prepared according to AIN 93G semipurified rodent diets (Table 1). The experimental rats were fed for 6 weeks, with 20% fat as a basal diet (HFD), for comparison with 20% fat-enriched diets containing 20% whole raw Bombay and MI35 cowpea powder (BRD and MRD), boiled Bombay and MI35 cowpea powder (BBD and MBD) and sprouted Bombay cowpea powder (BSD).
There was no significant difference (p < 0.05) in food intake and final body weight among groups at the end of the experimental period.
The blood samples (1 ml) were taken at the beginning and at the end of the 6 weeks between 09.00 and 10.00 in the morning from the jugular vein of fasting rats after anesthetizing. The samples were taken into tubes without any anticoagulant. After the samples were allowed to stand at room temperature for 2 hr, the serum was separated by centrifugation at 1,500 × g for 20 min. At the end of the 6week experimental period, all feces excreted during last 3 days were collected. The rats were anesthetized and killed, and the livers and cecum 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 Faculty of Veterinary Medicine and Animal Science, University of Peradeniya, Sri Lanka. All animal procedures conformed to standard principles described in Guide for the Care and Use of Laboratory Animals (The National Research Council, 2011).

| Statistical analysis
Data are presented as the mean and standard deviation for serum TC, HDL-C and, non-HDL-Cat the prescribed times. The significance of difference among treated groups was determined by ANOVA with Duncan's multiple range test using SAS statistical software (SAS Institute, Cary, NC, USA).

| Dietary fiber content, antioxidant activity, and phenol content in cowpea powder
Soluble dietary fiber content was significantly high (p < 0.05) in processed cowpea powder than that in raw cowpea powder, while insoluble dietary fiber content was significantly low (p < 0.05) in processed cowpea powder (Table 1). Total dietary fiber content in raw MI35 was significantly higher (p < 0.05) than MI35 boiled, Bombay raw and Bombay sprouted powder. In this study, boiling and sprouting have improved the soluble dietary fiber content in cowpea compared to raw cowpea and these findings were supported by findings of our study on mung beans (Liyanage et al., 2018). Antioxidant activity (AOA) was significantly lower (p < 0.05) in processed cowpea powders than the raw cowpea powder (Table 2). Phenol content was significantly lower (p < 0.05) in boiled cowpea powders than that in raw cowpea powders. These findings were supported by previous findings showing that boiling reduced the total phenol content and antioxidant actvity in mung beans (Liyanage et al., 2018). Phenol content (Table 2) in sprouted Bombay cowpea powder was not significantly different (p < 0.05) compared to that in raw Bombay cowpea powder (Table 3).  experimental diets for 6 weeks. The serum TC level was significantly lower (p < 0.05) in rats fed BRD, BBD, MRD, and MBD diets than that in the control, showing that both boiled and raw cowpea powderincorporated diets significantly reduced the HFD induced serum TC level in Wistar rats. The serum non-HDL-C level was significantly (p < 0.05) lower in all cowpea-incorporated diet-fed rats compared to control. Serum HDL-C level was significantly high (p < 0.05) in BBD, MRD-fed group compared to that in BRD and CD-fed groups.

| Serum cholesterol level in rats fed experimental diets
Dietary fibers exert hypocholesterolemic effects by increasing fecal excretion of steroids (Sembries, Dongowski, Mehrländer, Will, & Dietrich, 2004). The Higher soluble fiber content in processed cowpeas may have favorably modulated serum non-HDL-C level in rats as shown previously (Pande, Platel, & Srinivasan, 2012;Rideout, Harding, Jones, & Fan, 2008). The impact of various soluble fibers on lipid metabolism has been well established both in humans and animal models (Aller et al., 2004;Levrat, Texier, Régerat, Demigné, & Rémésy, 1993). The results were in good accordance with the study on whole cowpea seeds and its protein isolates (Frota et al., 2008(Frota et al., , 2015. Legumes are also good sources of saponins and phytosterols which may assist in decreasing the absorption of cholesterol from the gut. Clinical studies have suggested that saponins in legumes protect the human body from cancers and high blood cholesterol (Shi et al., 2004). Boiling has improved the hypocholesterolemic ability of Bombay cowpea and BBD has significantly (p < 0.05) modulated serum HDL-C level and liver weight (Table 4) in rats than those in raw Bombay cowpea diet-fed rats. Significantly low TC level (p < 0.05) in rats fed both raw and boiled cowpea-incorporated diets was supported by significantly lower serum (p < 0.05) non-HDL-C F I G U R E 1 Serum Total cholesterol, High-Density Lipoprotein cholesterol (HDL-C), Non-HDL-cholesterol (Non-HDL-C), and triglyceride (TG) concentration in rats fed experimental diets for 6 weeks. Values are expressed as mean ± SD. Mean values within a group with different superscript letters are significantly different at p < 0.05 TA B L E 4 Initial body weight, final body weight, body weight gain, fecal weight, and cecal weight of rats fed experimental diets for 6 weeks  F I G U R E 2 Serum antioxidant activity in rats fed experimental diets for 6 weeks. Values are expressed as mean ± SD. Mean values within a group with different superscript letters are significantly different at p < 0.05 level, significantly higher fecal weight (Table 4) and significantly high dietary fiber content in cowpeas as shown previously (Bazzano et al., 2011).

| Serum antioxidant activity in rats fed experimental diets
Serum antioxidant activity was significantly high (p < 0.05) in BRD-fed group compared to that in BBD, MBD, and CD ( Figure 2).
Significantly low (p < 0.05) serum TC and non-HDL-C level in BRDfed rats was supported by significantly higher (p < 0.05) serum antioxidant activity compared to control diet, and this was in agreement with previous studies showing that significantly lower serum lipid level may be due to higher antioxidant activities preventing lipid peroxidation (Jemai et al., 2008). However, the BBD and MBD-fed rats had significantly (p < 0.05) lower serum antioxidant activity compared to that in BRD and MRD-fed rats and these data were accompanied by significantly low (p < 0.05) antioxidant activity and phenol content in boiled Bombay cowpea powder (BB) and boiled MI35 powder than raw cowpeas. Thus, processing has reduced the antioxidant activity in cowpeas and serum antioxidant activity in rats.

| Cecal fermentation in rats fed experiental diets
There was no significant (p < 0.05) difference in anaerobe bacterial population among groups (data not shown). Cecal Lactobacillus population (Figure 3) was significantly high (p < 0.05) in all cowpea diet-fed groups compared to control group followed by significantly lower (p < 0.05) cecal pH and significantly higher ceceal weight (Table 4)  diet as shown previously (Jenkins, Kendall, & Vuksan, 2000).

| CON CLUS ION
The increase in TC and non-HDL-C level as a result of HFD was significatly encountered by boiled, raw cowpea diets and boiled, sprouted, raw cowpea-incorporated diets, respectively. Lower serum cholesterol level in cowpea-fed rats was supported by higher serum antioxidant activity and higher cecal lactobacilli population compared to control diet. Both processed and raw cowpea-incorporated diets have modulated HFD-induced hypercholesterolemia by modulating serum antioxidative capacity, cholesterol metabolism, and cecal fermentation. Thus, boiled, sprouted, and raw cowpea may be effective in the treatment of hypercholesterolemia in humans and further studies in human volunteers need to be done to substantiate these findings.

ACK N OWLED G M ENT
We gratefully appreciate the financial support of National Science Foundation, Sri Lanka (Grant No. RG/2011/AG/09).

CO N FLI C T O F I NTE R E S T
None declared.