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Biological effect of high plant protein legumes intake on hyperuricemic rats Soheir Ahmed Al-Masri Food Sciences and Nutrition Dept, Faculty of Food Sciences and Agriculture, King Saud University, Riyadh, Saud Arabia almasrisoheir@yahoo.com Abstract: This study was conducted to investigate gross chemical composition of high plant protein legumes as lupine, lentil and fenugreek seeds and the biological effect of feeding diet supplemented with experimental seeds on hyperuricemia in rats.10 rats served as a normal control (ve) group, fed on standard diet only while other 50 rats were fed on standard diet containing 2% potassium oxonate for six weeks before the start of the experiment to induce hyperuricemia. Hyperuricemic rats were classified into control (+ve) c, lupine (10% lupine in the standard diet), lentil (10% lentil in standard diet), lupine with fenugreek (5% from both in the standard diet) and lentil with fenugreek (5% from both in the standard diet). Rats were fed on experimental diets for 60 days, sacrificed and blood samples were taken. The obtained results showed that hyperuricemic rat groups which fed on diet supplemented with lupin with fenugreek and lentil with fenugreek showed non significant difference in nutritional results compared with control-ve group. Hyperuricemic rat groups which fed on diet supplemented with lupine , lentil, lupine with fenugreek and lentil with fenugreek seeds powder showed significant decrease in uric acid, creatinine, urea and nitric oxide (NO) but significant increase in total protein, albumin and globulin, antioxidant enzymes (SOD, catalase, and GPX) and immunoglobulin (Ig M and Ig G) when compared with control +ve group. It is concluded that diet supplemented with two high protein legumes as lupine with fenugreek or lentils with fenugreek had improvement effect on nutritional and biochemical results. These legumes increase the immunity and antioxidant activity. [Soheir Ahmed Al-Masri. Biological effect of high plant protein legumes intake on hyperuricemic rats. Life Sci J 2014;11(1):325-331] (ISSN: 1097-8135). http://www.lifesciencesite.com. 50 Keywords: Lupines- fenugreek lentils-liver- kidney-immunoglobulin 1.Introduction Protein is the major functional and structural component of all the cells of the body; all enzymes, membrane carriers, blood transport molecules, serum albumin, keratin, and collagen are proteins, as are many hormones and a large part of membranes. Moreover, the constituent amino acids of protein act as precursors of many coenzymes, hormones, nucleic acids, and other molecules essential for life (Millward 1999). Legume seeds contain 20-25% protein, which is 2-3 times higher than the content in cereals, and have therefore been considered as leading candidates for protein supply to malnourished areas of the world. Legumes with cereals are the main plant source of proteins in human diet. They are generally rich in dietary fiber and carbohydrates. Legumes contain several phenolic compounds, in addition to glutathione, soluble proteins, and tocopherols, which are considered to be natural antioxidants (Chavan et al., 1989 and Rochfort and Panozzo 2007). Lupin is a legume used as human food and animal feed since early Roman times. Lupins albus is known as sweet lupins due to their low levels of bitter-tasting and potentially toxic alkaloids without risk of toxicity for animals and humans. Lupinus sp. is an important plant of leguminous crops for both animal and human nutrition because of their high seed protein and oil content (Martinez-Villalluenga et al., 2009). Lentils (Lens culinaris L) are a member of the legume family, and have been an important component of the human diet for many centuries. Lentil seeds are rich sources of proteins and bioactive peptides. Lentils have gained recent attention for their many healthful characteristics, including being a low glycemic index food, nutrient and protein rich, and a natural source of phytochemicals and antioxidants (Combe et al., 2004 and Mitchell et al., 2009). Fenugreek, (trigonella foenum-graecum L.) belongs to the plant family fabaceae or leguminosae. It grows in most of the countries around world. Powdered fenugreek seed has a beautiful golden yellow color due to its coloring agent called coumadine. That is why fenugreek seeds were used for a yellow dye by ancient Indians and Egyptians. Seeds of fenugreek contain many active biological chemicals may it a potent medicinal and nutraceutical supplement. Fenugreek seeds are bitter in taste and has a strong and quite peculiar odor, hence, used in a very small quantity as a spice. The The health-promoting property of fenugreek has been long documented when it is taken as vegetables, food supplements or medicinal remedies. Fenugreek seed powder can

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prevent obesity and diabetes beside lower glucose and cholesterol (Lu et al., 2008 and Kassaian et al., 2009) So, the objective of this work was to investigate the gross chemical composition of high plant protein seeds as lupines, lentil and fenugreek and to study the effect of diet supplemented with lupines and lentil alone or combined with fenugreek in hyperuricemic rats. 2. Materials and Methods I- Materials: The experimental seeds were lupines (Lupin albus), lentil (Lens esculenta) and fenugreek (Trigonella foenum graceum) were purchased from the local market in Riyadh. BioMeriuex Kits were purchased from Alkan Co. for Chemicals and Biodignostics. The standard diet was prepared according to NRC (1995). Potassium oxonate is a white powder purchased from El-Nil Company for chemical industries for inducing hyperuricemia. Sixty Sprague Dawley strain rats weighing 100 8 g. provided from experimental animals center in Medicine collage of King Saudi University in Riyadh. B- Methods: Lupines, lentil and fenugreek seeds were crushed to fine powder. Approximate crude protein, crude fat, crude fiber and ash of experimental seeds were determined according to the methods of the A.O.A.C. (1995). Total carbohydrates were estimated according to Nilsen (1994) as follows: Carbohydrates% = 100 - (% moisture+ % protein+ %fat +%ash). After adaptation period (one week), 10 rats served as a normal control (ve) group, fed on standard diet only while other 50 rats were fed on standard diet containing 2% potassium oxonate for six weeks before the start of the experiment according to previous studies (Stavric and Nera 1978). Random blood samples from orbital canthus of the eye were taken for estimation of uric acid to ensure incidence of hyperuricemia. At the beginning of the study, the average weight of rats was 160 8g. hyperuricemic rats were classified into four groups which were control (+ve) and four groups that fed on lupines (10% in the standard diet), lentil(10% in standard diet), lupines with fenugreek(5% from both in standard diet) and lentil with fenugreek(5% from both in standard diet). Daily food intake (FI) and the weekly body weight gain (WG) were recorded. Food efficiency ratio (FER), protein intake (PI) and protein efficiency ratio (PER) was calculated (Chapman et al., 1950 and Hsu et al., 1978). After 60 days, the rats were sacrificed to obtain blood for biochemical analyses. Serum uric acid, creatinine, urea, total protein and albumin were determined according to Fossati et al., (1980) Bonsens and Taussky (1984),

Kanter (1975), Henry (2001) and Bartholomev and Delany (1966), respectively. Serum superoxide dismutase (SOD), catalase, glutathione peroxidase (GPX), glutathione transferase (GST) and nitric oxide (NO) were determined by enzymatic colorimetric procedures according to Dechatelet et al., (1974), Sinha (1972), Habig et al., (1974), Moran et al., (1979) and Kirima et al., (2003), respectively. Hemoglobin (HG) and packed cell volume (PCV) were estimated in heparinized blood according to Drabkin (1949) and Mc Inory (1954), respectively while immunoglobulins G and M (IgG & IgM) were determined by direct ELISA according to Engvall and Perlmann1971. All obtained data was statistically analyzed by SPSS computer soft ware. The calculated occur by analysis of variance ANOVA and follow up test LSD by SPSS ver. 11 according to Abo-Allam (2003). 3. Results Data in table 1 showed that lupines seeds have higher levels of crude protein, fat and fiber compared with the content in lentils seeds while lentils seeds contain higher levels of ash and carbohydrate. Fenugreek seeds are rich in carbohydrate and protein. Hyperuricemic non treated group (control +ve) group showed highly significant decrease in WG, WG %, FER and PER at p<0.001 compared with control-ve group. Hyperuricemic rat groups which fed on diet supplemented with lupines and lentil seeds powder showed significant decrease in WG, WG %, FER and PER at p<0.01 compared with control-ve group but showed significant increase compared with control +ve group. Food intake and protein intake were within normal values in all experimental groups. Hyperuricemic rat groups which fed on diet supplemented with lupines with fenugreek and lentil with fenugreek showed non significant difference in WG %, FER and PER compared with control-ve group as shown in table2. Table 3 showed that control +ve group had significant higher values of uric acid, creatinine and urea at p<0.001 compared with control-ve group. Hyperuricemic rat groups which fed on diet supplemented with lupines and lentil seeds powder showed significant increase in uric acid, creatinine and urea at p<0.001&0.05 compared with control-ve group but showed significant decrease compared with control +ve group. Rat groups which fed on diet supplemented with lupines with fenugreek and lentil with fenugreek showed non significant difference in uric acid and significant increase in creatinine and urea at p<0.05&0.01 compared with control-ve group and significant decrease compared with control +ve group.

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Table 4 illustrated that control +ve group had significant lower values of total protein and globulin at p<0.01 but showed non significant increase in albumin compared with control-ve group. All hyperuricemic rat groups which fed on diet supplemented with lupines, lentil, Lupines with fenugreek and lentil with fenugreek showed non significant difference in total protein, albumin and globulin at p>0.05 compared with control-ve group but showed significant increase compared with control +ve group. Table 5 showed that control +ve group had significant lower values of serum SOD, catalase, GST and GPX at p<0.001and significant higher value of NO compared with control-ve group. Hyperuricemic rat groups which fed on diet supplemented with lupines , lentil, lupines with fenugreek and lentil with fenugreek seeds powder showed non significant difference in serum SOD, catalase, and GPX compared with control-ve group but showed significant increase compared with control +ve group. They also showed non significant difference in serum NO compared with control-ve group but showed significant decrease compared with control +ve group. Hyperuricemic rat groups which fed on diet supplemented with lupines and lentil showed Crude protein 37.86 27.81 34.01 Crude fat 11.96 1.86 6.55

significant decrease in GST compared with control-ve group and significant increase compared with control +ve group. Table 6 showed that control +ve group had significant lower values of HG, PCV, Ig M and Ig G at p<0.01&0.001 compared with control-ve group. Hyperuricemic rat group which fed on diet supplemented with lupines showed significant decrease in PCV, Ig M and IgG at p<0.05&0.01 compared with control-ve group but significant increase in HG, Ig M and IgG compared with control +ve group. Hyperuricemic rat group which fed on diet supplemented with lentil showed non significant difference in serum HG, PCV, and IgG at p>0.05compared with control-ve group but showed significant increase compared with control +ve group. Also, this group showed significant lower value of Ig M compared with control-ve group and significant increased compared with control +ve group. Hyperuricemic rat groups which fed on diet supplemented with lupines with fenugreek and lentil with fenugreek seeds powder showed non significant difference in HG, PCV, Ig M and Ig G at p>0.01compared with control-ve group and significant increase in compared with control +ve group. Crude fiber 10.91 4.22 6.08 ash 3.67 4.01 2.9 carbohydrate 35.60 62.10 50.46
Lentils with Fenugreek 83.213.14ab 50.732.75a 18.551.2a 0.0450.005a 3.710.33a 0.3730.07ab

Table (1): Gross Chemical composition of lupinese, lentile and fenugreek (100g/dry weight)

Lupine Lentile Fenugreek

Table (2):Mean values SD of WG, WG %, FI ,FER,PI and PER of the experimental rat groups Groups Control -ve Control +ve Lupines Lentils Lupines with Variables Fenugreek WG(g) 89.413.14a 48.772.11c*** 70.812.77b** 72.703.25b** 78.143.57b* a c*** b** b** WG% 55.192.13 29.921.19 43.172.15 44.873.01 47.932.18ab a a a a F I(g/d) 17.351.10 16.811.07 17.211.05 18.141.22 17.801.11a FER 0.0530.001a 0.0290.003c*** 0.0410.002b** 0.0420.007b** 0.0440.004ab a a a a P I (g/d) 3.470.31 3.360.30 3.440.2 3.620.21 3.560.42a a d*** bc** c** PER 0.4290.01 0.2410.01 0.3430.03 0.3340.04 0.3650.05ab Significant with control (-ve) group * P<0.05 ** P<0.01 *** P<0.001 Values with the same letters in raw indicate non- significant difference (P<0.05) and vice versa Table (3): Mean values SD of uric acid, creatinine and urea of the experimental rat groups

Groups Variables Uric acid (mg/dl) Creatinine (mg/dl) Urea ( /mg)

Control -ve Control +ve 2.550.34c 8.641.14a***

Lupines 4.250.64b** 0.900.01b*

Lentils 4.170.73b** 0.910.02b*

Lupines Fenugreek 3.220.52bc 0.820.03c*

with Lentils Fenugreek 3.190.29bc 0.800.02c* 27.112.91bc*

with

0.700.04d 1.770.22a***

20.161.71d 53.714.22a*** 31.0153.18b** 30.143.19b** 28.812.75b**

Significant with control (-ve) group * P<0.05 ** P<0.01 *** P<0.001 Values with the same letters in raw indicate non- significant difference (P<0.05) and vice versa

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Table (4): Mean values SD of total protein, albumin and globulin of the experimental rat groups Groups Control -ve Control +ve Lupines Lentils Lupines with Variables Fenugreek Total protein(g/dl) 7.140.51ab 6.110.62c** 7.660.72a 7.510.63a 7.870.59a a a a a Albumin(g/dl) 3.350.41 3.500.30 3.610.42 3.540.41 3.700.31a a b** a a Globulin (g/dl) 3.790.23 2.610.08 4.050.30 3.970.27 4.170.25a Significant with control (-ve) group * P<0.05 ** P<0.01 *** P<0.001 Values with the same letters in raw indicate non- significant difference (P<0.05) and vice versa Table (5): Mean values SD of serum SOD, catalase, GST,GPX, and NO of the experimental rat groups Groups Control -ve Control +ve Lupines Lentils Lupines with Variables Fenugreek SOD(/mg) 3.170.33ab 1.570.25c*** 3.190.39ab 3.240.39ab 4.210.48a a c*** ab ab Catalase( /l) 5.671.02 1.250.13 4.430.41 4.400.45 4.590.50a a c*** b* b* GST(/mg) 4.210.59 1.870.29 3.050.73 3.110.45 3.770.43a a b*** a a GPX(/mg) 2.140.24 0.650.03 2.140.26 2.010.27 2.220.30a bc a*** b b NO(mOl/l) 2.490.44 7.861.03 3.820.72 3.740.63 3.610.77b Significant with control (-ve) group * P<0.05 ** P<0.01 *** P<0.001 Values with the same letters in raw indicate non- significant difference (P<0.05) and vice versa Table (6): Mean values SD of HG, PCV, Ig M and Ig G.of the experimental rat groups Groups Control -ve Control +ve Lupines Lentils Lupines with Variables Fenugreek HG 12.771.11a 10.671.21b** 11.251.20a 11.411.40a 11.211.31a a c*** bc* ab PCV 38.663.44 29.652.19 32.963.14 33.663.29 34.713.22ab a c*** b* b* Ig M 101.368.66 65.176.19 85.618.19 89.148.60 98.119.41a (mg/l) Ig G 202.7723.61a 129.6610.21e*** 155.6112.13cd** 163.6114.03c* 190.2115.19a (mg/l) Significant with control (-ve) group * P<0.05 ** P<0.01 *** P<0.001 Values with the same letters in raw indicate non- significant difference (P<0.05) and vice versa

Lentils with Fenugreek 7.680.68a 3.660.22a 4.020.11a

Lentils Fenugreek 4.160.43a 4.960.42a 3.820.62a 2.110.22a 3.510.84b

with

Lentils with Fenugreek 1.331.29a 35.613.11a 99.668.67a 185.1414.19ab

4. Discussion Although proximate composition of lupines, lentil and fenugreek have been widely documented in many literature, the data are not always comparable due to differences in genotypes, environments, cooking and dehulling process and methods of analysis (Wang et al., 2009). The obtained results of gross composition of lentil powder were agreed with Costa et al. (2006) and USDA (2009). The genus lupinus typically contains 36-52% protein, 5-20% oil, and 30-40% fiber (Fudiyansyah et al., 1995). The proximate analysis showed that lentil seed has a protein and carbohydrates concentration of 20.67g/100g and 60.47 g/100g, respectively. The moisture, ash and lipids content in flour were 9.57, 0.97 and 3.22 g/100 g sample respectively. Carbohydrates represent a major component of lentils, which include soluble and insoluble dietary fiber as well as prebiotic oligosaccharides (Vidal-Valverde et al., 2002). Potassium oxonate is uricase inhibitor associated with renal insufficiency and used for induction of chronic insufficiency. The oxonatetreated rat can serve as a useful animal model not only in investigation of the uric acid nephropathy, but also

in a number of other toxicologic evaluations connected with uric acid. This model has been used to evaluate drugs that affect uric acid excretion, to determine which dietary factors affect serum urates, or to evaluate possible therapeutic agents in certain disorders associated with uric acid (Stavric and Nera 1978). Lupines seeds are employed as a protein source for animal and human nutrition in various parts of the world. Lupines are also a good source of lipids, dietary fiber, minerals and vitamins (Sujak et al., 2006). There are variations in the protein content between species and cultivars as a result of the characteristics of the growing conditions and soil types from 28% in to 48% (Rodrguez-Ambriz et al., 2005 and Martnez-Villaluenga et al., 2009). Dietary fiber represents 40% of the kernel weight of sweet lupines, which is a higher level than in most other legumes. Lupines seeds have a good balance of essential amino acids and also have a considerable amount of oil in its seeds. It is a legume that contains approximately 5 20% of crude oil in the whole seed. The seed contains minute amounts of starch (5% to 12%) and higher levels of soluble non-starch polysaccharides as 30 40% (Hall et al., 2005 and Uzun et al., 2007).

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Legumes, including lentils and beans, are significant sources of resistant starches, which have been reported to be fermented by colonic bacteria to short chain fatty acids, and thus for improving the colonic health (Monsoor and Yusuf 2002). It was found that lupin kernel fibres have appetite suppression and cholesterol lowering properties, that they lower blood glucose and insulin levels, bowel health as a faecal bulking agent. However, little is known about their photochemistry and antioxidant activity (Wang and Daun 2006 and Hall et al., 2005). Fenugreek (Trigonella foenum-graecum) is used as a spice, vegetable and a medicinal plant. Fenugreek seeds were separated into husk and endosperm. The proximate composition of fenugreek seeds, husk and cotyledons showed that endosperm had the highest saponin (4.63 g/100 g) and protein (43.8 g/100 g) content. In contrast, husk had higher total polyphenols. At 200 g concentration, extracts of husk, fenugreek seed, and endosperm exhibited 72%, 64%, and 56% antioxidant activity respectively by free-radical scavenging method. Due to its strong flavour and aroma fenugreek in one of such plants whose leaves and seeds are widely consumed as in other oriental countries as a spice in food preparations, and as an ingredient in traditional medicine. It is rich source of calcium, iron, and carotene (Madhava et al., 2011). It is known that the end products of protein metabolism are urea and ammonia arising from the partial oxidation of amino acids. Uric acid and creatinine are indirectly derived from amino acids as well. Protein deficiency has also been shown to adversely affect kidney function, where it has adverse effects on both glomerular and tubular function. In addition, urinary creatinine excretion has been used as a reflection of muscle mass (Benabe and MartinezMoldonado, 1998 and Corish and Kennedy, 2000). The growth rate of males fed sweet lupine flour containing 41.7% protein and 0.025% lupanine was significantly higher than that of the controls with normal haematological parameters and tests of liver function. Protein deficiency has been shown to have adverse effects on the immune system, resulting in a higher risk of infections (Ballester et al., 1984 and Amarowicz et al., 2008). The obtained antioxidant results were attributed to their constituents. Lentils are also good sources of phenolic and nonphenolic antioxidants as catechins and procyanidins.Total antioxidant measurements for lentil seeds were reported to be significantly higher than those of other legumes by using different antioxidant capacity indicators (Xu et al., 2008 and Martnez-Villaluenga et al., 2009). Fenugreek also contains a biologically significant level of saponins that are known to have improve liver and kidney function. The seeds of fenugreek contain lysine and L-tryptophan rich

proteins, mucilaginous fiber and other rare chemical constituents such as saponins, coumarin, fenugreekine, nicotinic acid, sapogenins, phytic acid, scopoletin and trigonelline, which are thought to account for many of its presumed therapeutic effects, may inhibit cholesterol absorption and thought to help lower sugar levels (Syeda et al., 2008 and Abd El-Ghany et al., 2011). The main phenolic compounds found in fenugreek seed and their extracts were 4 hydroxy- isoleucine, pbenzoic acid, caffien, salycilic acid, p-cumaric acid and chlorogenic acid and Dihydroxy cinnamic acid.The main phenolic compounds in lupin are catechin and caffien (Hadriche et al., 2013) The hepatic levels of antioxidant enzymes such as GST were significantly greater in rats fed chemopreventive diets than in rats fed the control diet implies an increased antioxidant capacity to protect from oxidative stress. The induced GST in the livers of rats fed split lentils could be ascribed partly to the presence of appreciated quantities of phytic acid that elevating hepatic GST levels. Extracts from seeds of lupinus have antioxidant activities and contain total phenolic compounds, phenolic acids and flavonoid (Khatiwada et al., 2006 and Siger et al., 2012). Recommendation: The need for leguminous seeds as source of alternative animal protein for the nutritional supplementation is increased. Technological improvement of traditional plant-derived nutrients is needed with increasing awareness of the impact of dietary habits on human. Lupines, lentils and fenugreek seeds alone or in combination are recommended for renal disease patient, and are also recommended to be used for the manufacture of different bakery products as supplement for plant protein especially in categories that need protein for growth as children. Many researches are needed for investigate the effect of different cocking methods on the acceptability and bioavailability of these legumes. Acknowledgement This research project was supported by a grant from the Research Center of the Female Scientific and Medical Colleges, Deanship of Scientific Research, King Saud University. Reference A.O.A.C ,1995. Official methods of analysis (16 ed), Association of official Analytical Chemists, Washington, DC. Abd El-Ghany, M., A. Ramadan and S. Hassanan, 2011. Antioxidant activity of some agroindustrial peels on liver and kidney of rats exposed to oxidative stress. World Journal of Dairy & Food Sciences, 6(1): 105-114.

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