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BIOCHEMICAL AND HEMATOLOGICAL RESPONSES OF RATS FED WITH DETOXIFIED JATROPHA CURCAS SEED MEAL.

Byline: S. Sharif, F. Masood, R. Naseer, S. Khawar, S. Naveed, L. Ahmad and G. Saleem

Abstract

Jatropha curcas, has a great potential to replace soya bean. The main problems are the anti-nutritional factors, which hinder its use as an alternative protein source in the animal feed. The present study was focused on the detoxification of J. curcas seed. Detoxification was carried out using a mixture of aqueous methanol (9:1) and NaOH (0.15%) at 60 C for 30 min. subsequent washing with methanol and then oven dried. Results showed significant detoxification (P>0.05) of J. curcas. The impact of feeding detoxified J. curcas to rats was studied. The detoxified J. curcas seed meal (DJM) at the rate of 25, 50, 75 and 100% was offered to albino rats (n=36) of almost equal weights (45+-0.76gm).

On blood and serum analysis the white blood cells (WBC) (7.23+-0.29, 10.56+-0.16, 8.29+-0.30, 5.94+-0.17, 5.6+-0.177 and (7.07+-0.27), red blood cells (RBC) count (6.73+-0.109, 7.61+-0.18, 7.00+-0.056, 6.68+-0.96, 6.28+-0 and 5.83+-0.18), hemoglobin (11.94+-0.11, 12.29+-0.19, 12.66+-0.27, 11.78+-0.14, 10.97+-0.177 and 10.97+-0.177), total protein (5.33+-0.09, 5.17+-0.86, 4.6+-0.096, 4.41+-0.109, 4.1+-.096 and 3.82+-0.13), uric acid (32.25+-0.82, 34.83+-0.32, 54.83+-0.84, 61.75+-0.68, 62.36+-0.7135 and 26+-0.34), creatinine (0.841 +-0.017, 801+- .018, 0.841+-0.027, 0.833+-0.032, 0.801+- 0.14 and 1.44+-0.049) and LFT (liver function test) including alkaline transaminase (29.2+- 0.42, 24.13+-0.37, 21.133+-0.48 , 32.006+-1.39, 75.93+- 2.16 and 24.75+-0.377), Aspartate transaminase (3.20+-1.01 2.08+-1.03, 1.25+-1.4, 1.33+-0.71, 1.82+-0.72 and 1.81+-1.47) and Alkaline phosphatase (4.96+-1.09, 4.14+-0.71, 4.79+-1.47, 5.30 +-1.5, 5.6+-2.3 and 2.08+-2.3) didn't differ signicantly among the experimental groups.

A significant increase (P>0.05) in red blood cells and hemoglobin level was observed in 25 (7.61+-0.18) and 50% (7.0+-0.056) DJM fed groups as compared to control group (6.73+-0.0.109). There was an increase in the value of PER (Protein efficiency ratio), TD (True digestibility), NPU (+-Net protein utilization) and BV (Biological value) of 25% (0.32+-.009, 77.66+-.21, 65.33+-.17 and 83.42+-0.16) and 50% DJM fed rats (0.42+-.007, 88.08+-.26, 75.5+-0.18, 85.10+-0.073) as compared to control group (0.36+-.004, 90.5+-0.22, 75.25+-0.17, 83.0+-0.14) . Present study indicated that 50% DJM protein fed group has the same performance as the control group and better than other experimental groups, that showed a better efficiency of detoxified J. curcas meal as an alternative livestock feed.

Keywords: Jatropa curas, Anti-nutritional factors, Detoxification, Serum and Digestibility.

INTRODUCTION

Jatropha curcas, commonly known as physic nut, is a tropical plant of the Euphorbiaceae family. It is native to Mexico and Central America, however widely distributed in wild and semi cultivated lands of Latin America, Africa, India and South East (Pandey et al., 2012). J. curcas is a small tree or a large shrub with a height of 8 to 10 meters (Kumar and Sharma, 2008).

The J. curcas seed weighs from 0.53 - 0.86 g (Singh et.al 2008) and the kernel contains 57 - 63% oil. Meal obtained after oil extraction contains 60-66 % crude protein. Conventionally, J. curcas seed is used in fertilizers, briquettes, candles, soaps, lubricants and for illumination. The seed kernels are used as fuel or as direct substitute to diesel. The seed cake after extraction is used as a good source of protein for humans as well as livestock (Abou-Arab and Abu-Salem, 2010).

It has been investigated that increasing value byproducts of oil extraction are important for the viability of the crop for the farmers (Francis et al., 2013). The J. curcas plant can yield upto 4 tons seed annually (Kumar et al., 2010).

Major toxic compounds present in J. curcas seeds are cursin and phorbol ester. Anti-nutritional factors such as Trypsin inhibitor, lectin, phytate, tannis, phenols, and saponins are also present, posing a major hindrance in utilizing curcas seeds in animal feed (Makkar et al., 2008).

The J. curcas seeds can be detoxified using combination of heat and chemical treatments (Azzaz et al., 2011).

Major purpose of detoxification is to prevent the plausible health hazards thought to be associated with its toxic compounds (Kumar et al., 2010b). So, the present study was to determine the potential of heat and chemicals to detoxify J. curcas (Azzaz et al., 2011) and to evaluate the biochemical and hematological responses of albino rats fed with detoxified J. curcas seed meal.

MATERIALS AND METHODS

Raw material: Apparently healthy J. curcas seeds were repeatedly washed, shade dried at high temperature (above 40AdegC) and powdered by using mechanical grinder (Akbar et al., 2009).

Proximate analysis: Proximate analyses of seeds were carried out to determine the percentage of moisture, ash, crude protein, nitrogen free extract, crude fiber and crude fat (AOAC, 1990).

Estimation of the anti-nutritional factors: The phytate contents of seeds were determined by spectrophotometric procedure using phytic acid sodium salt as a standard (Makkar et al., 2008).

Total saponin contents were determined by spectrophotometric method using diosgenin as a standard (Hiai et al., 1976). Total phenolics and tannins were determined by spectrophotometric methods using Folin- Ciocalteu reagent as described by (Harinder et al., 2008). Lectin contents of curcas seed were analyzed by haemagglutination assay using trypsinized cattle washed erythrocytes in a phosphate buffer saline (10%) (Makkar et al., 2008). Trypsin inhibitory activity was determined by colorimetric method using trypsin enzyme and Azocasein as a substrate (Robin et al., 2011).

Detoxification: Defatted seed sample (10g) was mixed with aqueous methanol (90ml methanol+10 mL distilled water) and NaOH (0.4g) in a 250 ml flask. Flask was capped and placed in water bath at 60AdegC for 30 min. Filtrate residue was washed with 20ml methanol (2 times of the weight of the initial defatted seeds). The filtrate was dried in the oven (50AdegC) and stored in air tight jar. Pre and post detoxification the nutritional and anti-nutritional contents were determined according to the methods described by Aregheore et al., (2003).

Feed formulation: The diet formulated contained skimmed milk, ground nut oil, vitamin mixture, salt mixture, corn starch and sucrose. The experimental diets were prepared by replacing 25%, 50%, 75% and 100% skimmed milk casein with detoxified curcas seed as shown in table. In addition, a negative control diet was also prepared without protein. The diets were offered randomly to the rats (Rakshit et al., 2008).

Table-1. Feed formulations for trial on rats.

Ingredients###Control###Group A###Group B###Group C###Group D###Group E

###Group###25% J.C###50% J.C###75% J.C###100% J.C###Protein Free

###Sucrose###15%###15%###15%###15%###15%###15%

###Canola oil###10%###10%###10%###10%###10%###10%

###Potato starch###5%###5%###5%###5%###5%###5%

Vitamin and minerals###2%###2%###2%###2%###2%###2%

###Casein protein###10%###7.5%###5.0%###2.5%###5.0%###-

###Corn starch###58%###58%###58%###58%###58%###68%

###Jatrpha Curcas###-###2.5%###5.0%###7.5%###5.0%###-

Experimental design: Thirty six (n=36) weanling rats were divided into 6 groups randomly. One group was positive control and the other as negative control. The experiment were conducted in duplicate (Azzaz et al., 2011). The experimental parameters studied were body weight, feed intake, protein efficiency ratio (PER), true digestibility (TD), net protein utilization (NPU) and biological evaluation (Miller and Bender 1955).

Biochemical and hematological analysis: Liver function tests (LFTs), lipid profile, TP (total protein), glucose, urea and creatinine were determined. The total erythrocyte count (RBC's) and the total leucocyte count (WBC's) were determined using method described by Azzaz et al., (2011)

Statistical analysis: To determine the significant differences in each experiment analysis of variance (ANOVA) was applied using SPSS version 17.0

RESULTS AND DISCUSSION

Proximate analysis: Proximate composition refer to crude protein, total fat, total fiber and ash. The proximate composition of defatted and detoxified J. curcas seeds was calculated according to A.O.A.C ( 1990) and is presented (Table 2).

Table-2: Proximate composition and Anti-nutritional Factors present in defatted and fatted Jatropha curcas seed meal

###Defated###Detoxified

Parameters###P-value###P-value

###Mean###Std. Error###Mean###Std. Error

Dry matter###96.7000###.32146###.000###96.2233###.12706###.000

Moisture###5.9333###.08819###.000###4.0633###.03180###.000

Crude protein###34.9800###.07937###.000###31.8200###.09866###.000

Crude Fiber###37.0333###.14530###.000###35.3233###.05925###.000

Total Fat###5.7933###.02028###.000###3.2000###.05774###.000

Ash###3.3000###.05774###.000###3.4833###.04410###.000

NFE###12.4333###.05608###.000###22.0500###.01732###.000

Saponin###.1700###.00577###.001###.1400###.00577###.002

Tanin###.0530###.00115###.000###.0177###.00088###.002

Trpsin###.1800###.00577###.001###.0500###.00577###.013

Phytic acid###.3467###.00882###.001###.153###.03977###.019

Phytate###.3100###.00577###.000###.1100###.00577###.003

Phenolics###.1767###.00882###.002###.0467###.00333###.005

Lectin###4.2633###.02404###.000###2.5167###.01764###.000

By proximate analysis estimated contents of crude protein, crude fiber and total fat were 34.98+-0.07, 37+-0.014 and 5.79+-0.02, respectively. The saponins and tannins were found to be 0.17+-0.00 and 0.053+-0.00 respectively. Curcas seed had phytic acid (0.34+-0.008), phytate (0.31+-0.005), total phenolics (0.17+-0.008) and lectin (4.26+-0.024). The result of this analysis was in line with the previous studies conducted by Abou-Arab and Abu Salem (2010) in the kernel meal, with a little difference in crude protein contents (32.8%). However, contradictory results were reported by Azzaz et al., (2010) and Francis et al., (2013). Moreover, the fiber contents reported by Azzaz and Francis were 3.4 and 4.9 %, whereas in present study were 37% and 35.32% in defatted and detoxified seed samples. Dry matter percentage was found to be 96.7% and 96.2% respectively in defatted and detoxified seed samples of present study which were found to be similar as reported by Chivandi et al., (2004).

In the present study, the detoxification of J. curcas seeds were carried out with 4% NaOH, aqueous methanol and moist heat treatment. After detoxification the amount of anti-nutrients present in J. curcas seed were determined (Table 2). In samples of defatted J. curcas the amount of Phytic acid and Phytate was 0.34 mg/mL and 0.31 mg/mL and reduced to 0.15 mg/mL and 0.11 mg/mL respectively in detoxified samples. This reduction in the Phytic acid amount suggested that the combination of 4% NaOH and moist heat was effective to reduce the level. The total phenolics were determined using standard curve of phenolics. The amount was 0.17mg/mL in defatted and reduced to 0.04mg/mL in detoxified J. curcas seeds. Phenols interfere with the normal process of digestion either binding directly to digestive enzymes and indirectly to feed components like proteins and minerals and impaired the process of digestion.

The amount of saponin in defatted and detoxified J. curcas seed sample was found to be 0.17 ug/g and 0.14ug/g. Saponin is secondary metabolite of plants that cause the hemolysis of RBCs and release of hemoglobin. The results indicated that the saponin can be degraded by combination of NaOH and heat treatment as reported by Martinez et al. (2006) and Abou-Arab and Abu Salem (2010).Trypsin inhibitory activity of defatted and detoxified curcas seed samples were found to be 0.18mg/mL and 0.05mg/mL, respectively.

Lectin hemagglutination Assay was performed for both the defatted (A) and detoxified (B) samples the results showed no agglutination (Fig. 1) which confirmed the absence of lectins. In similar study by Aregheore et al. (1998) using cattle blood have shown the presence of lectin activity but the results of present study were in contrast. Lectin is heat labile and might be destroyed by the moist heat (Aregheore et al., 1998).

Biological evaluation: The detoxified J. curcas seed meal was fed to rats and biochemical parameters determined are presented (table 3).

In present study, biological evaluation of rats fed with detoxified J. curcas seed meal showed no significant (P<0.05) differences with control (fed with casein protein). There was non-significant difference in the growth rate of detoxified J. curcas seed meal (DJM) fed rats. The average weight gain of DJM (25%) and DJM (50%) was increased compared to the control group (Table 3). The protein efficiency ratio (PER), true digestibility (TD), net protein utilization (NPU) and biological evaluation (BV) of detoxified J. curcas seed meal (DJM) 25%(B) and DJM50% was also increased. In detoxified J. curcas seed meal DJM (75%) and DJM (100%) these value were decreased, which may be due to poor utilization of protein. Zero weight gain in protein free group indicated that protein enables rats to grow properly as it's a necessary component of the diet and is the building block of the life and no protein intake in this group showed no PER, NPU and BV ratios.

The results are in agreement with the results of Azzaz et al., (2011) and Rahma et al., (2013). The studies of Herrera et al., (2012) and Martinez Ayala. (2013) observed a decrease in PER and NPU value compared to control group. Friedman and Brandon, (2001) and Rozan et al., (1997) reported little increase in weight gain using soya bean meal.

The biochemical parameters showed no significant change (P<0.05) between rats fed with DJM and control group (Table 4). The amount of glucose and total protein was decreased in six groups. While there was an increase in the concentration of creatinine, urea, ALT, AST, ALP of 75% and 100% DJM fed rats. Similar results were interpreted by Awasthy et al., (2010); Wang et al., (2011); and Kumar et al., (2010a). Although a slight reduction in blood sugar and protein level was observed in the Nubian goats fed with DJM (Adam and Magzoub, 1975). The reduced blood sugar and protein levels of rats may be due to impaired digestion of carbohydrate because of the presence of anti-nutrient amylase inhibitor (tannins/polyphenols) in the seeds (El-Sayed, 1997; Glick and Joslyn, 1970). The trypsin inhibitor interferes with digestion of dietary protein and the tannins complex with it and inactivates proteins in general, thus reducing the total protein of rats as also described by (Awasthy et al., 2010).

The increase in plasma urea, ALT, AST and ALP of group DJM (75%) and DJM (100%) was an indication of impairment of liver function while normal level of these parameters in groups DJM (25%) and DJM (50%) showed that the percentage of detoxified J. curcas can't be exceeded from certain limits.

In present study, non-significant difference (P<0.05) for the hematological parameters (Table 5) among all the six groups including control was observed. Although an increase in the RBCs count of DJM25% and DJM50% was observed when compared to the control group fed with casein protein. Similar results were observed by (Kumar et al., 2010). An increase in the RBCs count of DJM fed groups may suggest that the plant ingredients or DJM may have caused an immature release of erythrocytes that increased the RBC count (Hemre et al., 2005). The Hb levels of different groups (Table 5) did not differ significantly and were within the normal range. Although there was an increase in the Hb level of DJM (25%) and DJM (50%) groups suggested the normal health of the rats. These results were in agreement with Kumar et al., (2010) and Akinleye et al., (2012) whereas contradicted with Azzaz et al., (2011) and Katole et al., (2011).

Table-3: Biochemical Parameters of rats fed with detoxified Jatropha curcas meal.

Biochemical parameters###Control###25% DJM###50% DJM###75% DJM###100% DJM###Protein free

Alanine Transaminase###29.2+- 0.42 abd###24.13+-0.37 acd###21.133+-0.48 abcd###32.006+-1.39 abd###75.93+- 2.16abcd###24.75+-0.377 abcd

Aspartate transaminase###3.20+-1.01 abcd###2.08+-1.03 bcd###1.25+- 1.4 acd###1.33+-0.71 abd###1.82+-0.72 abc###1.81+-1.47 abc

Alkaline phosphatase###4.96+-1.09 abcd###4.14+-0.71 bcd###4.79+-1.47 acd###5.30 +-1.5 abd###5.6+-2.3 abc###2.08+-2.3 abcd

Serum Total protein###5.33+-0.09 bcd###5.17+-0.86 bcd###4.6+- 0.096abc###4.41+- 0.109 abc###4.1+- .096 abc###3.82+-0.13 abcd

Serum Glucose###115+-0.38 abcd###112+-0.60 bcd###97.41+- 0.46 ad###95.51+-0.71 ad###75.866+-1.15 abc###44.60+-0.66 abcd

Urea###32.25+-0.82bcd###34.83+-0.32 bcd###54.83+-0.84 acd###61.75+-0.68 ab###62.36+-0.71 ab###35.26+- 0.34bcd

Creatinine###0.841+- 0.017**###.801+- .018**###0.841+- 0.027**###0.833+- 0.032**###0.801+- 0.14**###1.44+-0.049 abcd

Table-4: Analysis of the rats fed with detoxified Jatropha curcas seed meal.

Parameters###Control###25% DJM###50% DJM###75% DJM###100% DJM###Protein free

Weight gain###10.33+-1.3 abcd###13.66+-0.33 bcd###12.33+-0.66 bcd###6.8+-1.4abc###5.0+-0.70 abc###0+-0 abcd

Protein efficiency ratio###0.36+-0.004 abcd###0.323+-0.009 abcd###0.42+-0.007 acd###0.283+-0.009 ab###0.27+- 0.02ab###0.036+-0.036abcd

True Digestibility###90.5+-0.22 abcd###77.66+-0.21 bcd###88.08+-0.26 acd###31.108+-0.44 abd###18.56+-0.33 abc###.00+-.00 abcd

Net protein Utilization###75.25+-0.17###65.33+-0.17 bcd###75.5+-0.182 abcd###17.35+-0.16 abd###10.16+-0.08 abd###.00+-.00abcd

Biological Value###83+-0.14 abcd###83.42+-0.16 bcd###85.10+-0.073 acd###57.00+-0.22 abd###53.37+-0.07 abc###.00+-.00abcd

Table-5: Hematological Analysis of the rats fed with detoxified Jatropha curcas seed meal.

Parameters###Control###25% DJM###50% DJM###75% DJM###100% DJM###Protein free

Red blood cells###6.73+-0.109 abcd###7.61+-0.18 bcd###7.00+-0.056 bcd###6.68+-0.96 abc###6.28+-0.0 abc###5.83+-0.18 abcd

White blood cells###7.23+-0.29 abcd###10.56+-0.16 bcd###8.29+-0.30 acd###5.94+-0.17 ab###5.6+-0.177 ab###7.07+-0.27 abcd

Haemoglobin###11.94+-0.11 abcd###12.29+-0.19 abcd###12.66+-0.27 acd###11.78+-0.14 abcd###10.97+-0.177 abcd###10.97+-0.177 abcd

Conclusion: The results of present study indicated that the detoxified J. curcas meal using combination of heat and chemical treatment can efficiently replace 50% of the ssoybean meal in the diets of poultry and small ruminants without negative effects on health or production.

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