Biochemical and Antioxidant effects in crossbred calves fed with Morinda citrifolia

ABSTRACT Morinda citrifolia (Noni) is a medicinal plant widely distributed in the tropical regions of India, Indonesia and Malaysia and has a long history of treating a wide variety of diseases such as cancer, atherosclerosis and diabetes. The present investigation was designed to evaluate blood biochemical parameters and antioxidant effects in calves fed with M. citrifolia. A total of eight calves were divided into two groups as control (n = 4) and treatment (n = 4). The calves of treatment group were fed with fresh minced raw fruit (100 g/calf/day) and the calves in control group were fed with placebo. Blood samples were collected at weekly intervals for four weeks for estimation of biochemical parameters and to determine antioxidant activity. The crude extract of noni fruits significantly (P < .01) decreased the concentrations of serum total cholesterol, triglycerides, serum glucose and also decreased (P < .05) serum creatinine and urea. There was a reduction in lipid peroxidation (LPO) than control; however, superoxide dismutase (SOD) and catalase levels were dramatically increased (P < .01) in morinda-fed calves. The results of present preliminary study demonstrated hypolipidemic, hypoglycaemic and antioxidant effect of M. citrifolia in calves. The findings of this study could be exploited for stress amelioration and management of metabolic diseases in calves and cattle without adverse effects.


Introduction
Morinda citrifolia or noni (family: Rubiaceae) is a medicinal plant called as Indian mulberry in India, nunaakai in Tamil, mengkudu in Indonesia and Malaysia (Morton 1992). It has been used in folk remedies by Polynesians for over 2000 years, and is reported to have a broad range of therapeutic effects. Generally, the medicinal plants have secondary metabolites which are very effective to cure various diseases (Ghasemzadeh & Jaafar 2011). Almost all the parts of the plant are being used for its medicinal and nutraceutical properties. The noni fruit contains 90% of water and the main components of the dry matter appear to be soluble solids, dietary fibres and proteins (Singh 2012). Biological compounds such as glycosides, polysaccharides, iridoids, alkaloids, lignans, trisaccharide fatty acid esters, anthraquinones, scopoletin, morindin, vitamins and minerals have been isolated from noni fruits, roots and leaves (Yang et al. 2007). These isolated biological compounds exerts various health benefits, including anticancer, antibacterial, antiviral, antifungal, hypotensive, anti-inflammatory, cholesterol regulation, menstrual cycle regulator and immune enhancing effects (Wang et al. 2002;Saminathan et al. 2013). The pharmacologically active compounds derived from noni fruits, leaves and roots are available as readymade capsules, teas and juices, which are most popular (Saminathan et al. 2014). Plants produce various antioxidative compounds to counteract the reactive oxygen species in order to survive. Recently, peroxidation of unsaturated lipids in living organisms is receiving increasing attention in relation to the possible association between lipid oxidation and a wide range of degenerative diseases, including ageing, cancer, diabetes and cardiovascular diseases. Thus, antioxidants are important inhibitors of lipid peroxidation (LPO), not only for food protection but also as a defence mechanism of living cells against oxidative damage (Mohd Zin et al. 2002). Antioxidant plays an important role in cancer treatment by scavenging free radicals produced by cells. Palu et al. (2008) suggested that the fruit extract from M. citrifolia modulated the immune system in mice through activation of cannabinoid receptors (CB2), suppression of interleukin-4 production, and by stimulating the production of interferon-γ cytokines. Recently, Brooks et al. (2009) reported that supplementation of juice from M. citrifolia enhanced the activation of immune cells in neonatal calves. The immunomodulatory effects of supplementing Morinda-Max® showed an increase in total leucocytes and greater oxidative burst intensity by the primary neutrophil population. This larger pool of leucocytes and greater oxidative burst potential could conceivably improve resistance to disease in a high-risk population of cattle (Ponce et al. 2011). The aim of this preliminary study was to investigate the effect of M. citrifolia fruit extract on blood biochemical parameters and antioxidant properties in crossbred calves.

Experimental design
A total of eight crossbred (Jersey X Desi) calves (age: 4-6 months; average body weight: 60-70 kg) maintained under standard management condition were selected for the study. The calves were divided into two groups, such as control (n = 4) and treatment (n = 4). M. citrifolia raw fruit was cut into small pieces and minced using an electric mixer. Fresh, minced fruit (100 g/calf/day) was fed orally to the calves of treatment group, and the calves in control group were fed with placebo (distilled water @100 ml/calf/day). Blood samples were collected in tubes with and without anticoagulant ethylenediaminetetraacetic acid (EDTA) by jugular vein puncture prior to the start of feeding (zero week) and thereafter at weekly intervals for four weeks. Blood serum was separated from coagulated blood for biochemical parameter and antioxidant analysis.

Estimation of blood biochemical parameters
Total cholesterol (TC), triglycerides (TGL), LDL-Cholesterol, HDL-Cholesterol, serum glucose, total protein, TBARS (thiobarbituric acid reactive substances) assay for LPO, blood urea nitrogen and creatinine were estimated in blood serum spectrophotometrically using commercially available kits (ERBA BIO-SCIENCES). All other chemicals and reagents used were of analytical grade and are commercially available.

Measurement of antioxidant activity
The superoxide dismutase (SOD) activity was measured by the method of Marklund and Marklund (1974). Briefly, to 0.5 ml of serum, 0.25 ml of ice-cold ethanol and 0.1 ml of ice-cold chloroform (chilled in ice) were added. Then the contents were mixed well and centrifuged at 13,000 rpm for 15 min. Then 0.5 ml of supernatant was taken and 2 ml of tris EDTA buffer was added and the volume was made up to 4.0 ml with distilled water. The reaction was initiated with the addition of 0.5 ml of pyrogallol and the absorbance at 1 min interval for 3 min was measured at 470 nm. One unit of SOD activity was the enzyme concentration required for 50% inhibition of pyrogallol auto-oxidation per minute. The SOD activity was expressed as units/mg protein.
Catalase activity was measured using the method of Sinha (1972). Briefly, 3.5 ml of reaction mixture containing 1.0 ml of phosphate buffer solution, 0.4 ml of hydrogen peroxide, 0.1 ml of serum and 2.0 ml of dichromate acetic acid reagent. The activity was measured by keeping the reaction mixture in boiling water bath for 10 min and the absorbance was then read at 570 nm. One unit of Catalase activity was expressed as microgram of hydrogen peroxide consumed per minute per milligram of protein.

Statistical analysis
Data were analysed using standard statistical methods as per Snedecor and Cochran (1994) and expressed as mean ± SE.

Effect of crude extract of M. citrifolia on Lipid profile
The effects of crude extracts of M. citrifolia fruit (100 g/calf/day) on biochemical parameters are shown in Table 1. There was a continuous decline of the serum TC, TGL, LDL-cholesterol in the treatment group than in the control group and the levels significantly (P < .01) declined gradually till the fourth week of the experiment. However, in case of HDL cholesterol, no significant difference was found throughout the study at specific intervals. The mean concentration of serum TC, TGL, LDL-C and HDL-C in treatment group at 0th week was 129.1 ± 3.50, 123.8 ± 6.87, 68.75 ± 2.87 and 48.93 ± 4.01 and at 4th week was 92.55 ± 1.74, 92.00 ± 6.40, 48.93 ± 4.01 and 58.48 ± 3.39, respectively.

Effect on glucose and total protein
The serum glucose level was decreased significantly on 4th week of experiment in adult crossbred calves (93.17 ± 7.16) and statistically significant (P < .05) in the treatment group. It did not show any considerable differences till the third week of the experiment; however, the glucose level declined drastically on 4th week. There was a sharp increase in the serum total protein concentration (9.28 ± 0.55) at a specific interval in the treatment group compared to the control.

Effect on non-protein nitrogen compounds
Feeding of the calves with M. citrifolia fruit significantly reduced the levels of non-protein nitrogen compounds such as BUN and creatinine. At the 4th week, BUN level was found to decrease (23.66 ± 1.13) markedly and statistically significant (P < .01), whereas the creatinine level was reduced to about 0.82 ± 0.03 and statistically significant (P < .05) at 4th week. At the 4th week, BUN and creatinine levels were reduced significantly in treatment group than in the control.

Effect on antioxidant system
The mean value of LPO in the serum revealed a little decrease during the crude fruit extract feeding experiment and the decrease was statistically significantly higher (P < .05) in the treatment group. There was a marked increase in enzymatic antioxidants SOD (1.97 ± 0.12) and Catalase (18.58 ± 0.29) in fruit-extract-fed group compared to the control group. The results showed a significant increase (P < .01) in the serum levels of SOD and Catalase.

Discussion
The blood is the vital fluid that transports gases and nutrients to the tissues of the body. The biochemistry and functional capacity of the blood is directly linked to the status of the blood components. The M. citrifolia plant and especially its fruit have been used for centuries in folk medicine. Many researchers showed that this fruit contains several nutritional and functional compounds, but most of them have not been quantified. About 160 phytochemical compounds have already been identified in noni plant, and its major micronutrients are phenolic compounds, organic acids and alkaloids (Chan-Blanco et al. 2006). The main proven functional properties of noni fruit are related to the control of several diseases. In-vitro research and limited experiments with lab animals have shown that noni has antimicrobial, anticancer, antioxidant, anti-inflammatory, analgesic and cardiovascular activity (Yang et al. 2007). The aim of this present study was to observe the changes in blood biochemical constituents and antioxidant levels due to feeding of M. citrifolia fruit in crossbred calves. TC, TGL and LDL-Cholesterol was significantly reduced (P < .01) in calves fed with M. citrifolia fruit as compared to normal calves. On other hand, HDL-cholesterol was slightly increased but not statistically significant. The findings indicate that the crude extracts of noni fruit are effective in altering blood lipids' profile. The mechanism of hypolipidemic effect of fruit extract is unclear. However, hypolipidemic component present in most of the medicinal plants having a high fibrous content is responsible for reducing the lipid levels (Singh et al. 2002;Luo et al. 2004). Goh et al. (1995) reported that M. citrifolia significantly reduced lipid concentration in humans. And, it has some beneficial chemical constituents as already mentioned, such as alkaloids, flavanoids and some glycosides such as citrifolin. The hypolipidemic activity of flavanoids and alkaloids has been reported by several studies (Hadijah et al. 2008). Thus, these active compounds is responsible for the hypolipidemic activities observed in the present study. The decrease in serum glucose levels in Morinda-fed calves provided evidence in favour of the view that noni fruit could play an important role in treating hyperlipidemic and hyperglycaemic conditions. Our study showed that noni fruit could be used for both hypolipidemic and hypoglycaemic effects in calves. Mandukhail et al. (2010) also showed three extracts fruit, leaves and roots of noni caused reduction in TGL, TC, LDLcholesterol, atherogenic index and TC/HDL ratio. Serum total protein concentration were significantly higher (P < .01) in treated animals than in the control calves, which could be due to higher intake of available protein content in noni fruit. Chan-Blanco et al. (2006) reported that the protein content of M. citrifolia fruit was surprisingly high, representing 11.3% of the dry matter and main amino acids present in the fruit were aspartic acid, glutamic acid and isoleucine. On the contrary, the level of BUN and creatinine significantly reduced in Morinda-fed animals than in control. An elevated level of BUN and creatinine may be a result of increased protein catabolism from greater production of proteins (Huber et al. 1976). In the present study, surprisingly BUN and creatinine levels were decreased markedly even with increased level of protein. This could be due to the utilisation of protein for muscle growth. Presence of increased polyphenols in M. citrifolia plant extracts could be responsible for anabolic effect on protein metabolism (Yamaguchi et al. 2002).
LPO has been defined as the biological damage caused by free radicals that are formed under oxidative stress (Zin et al. 2006). The antioxidants' levels increase to compensate this oxidation reaction and prevent the tissue from the oxidative damage. The antioxidant property of natural sources is due to the presence of antioxidant compounds in the plants. Many natural antioxidants are found in fruit, root and leaves of various plants. Most of these compounds are normally phenolic or polyphenolic compounds in nature. These compounds as an antioxidant may act by scavenging the radicals directly or sustaining the activity of antioxidant enzymes or inhibiting the activity of oxidising enzymes. In present study, there is an increased level of enzymatic antioxidants such as SOD and catalase, which could be due to alkaloids, flavanoids and some glycosides such as citrifolin. Serafini et al. (2011) investigated the antioxidant activity of aqueous extract from M. citrifolia leaves against LPO, hydroxyl and nitric-oxide-induced radicals. Dussossoy et al. (2011) showed that Noni's antioxidant activities are possibly due to phenolic compounds, iridoids and ascorbic acid. The seed extract exhibited significant antioxidant potential against various types of free-radical-induced damage (West et al. 2011). M. citrifolia is well documented that noni fruit showed effective antioxidant activity (Mohd Zin et al. 2002). Besides, LPO activity, was measured by the amount of malondialdehyde (MDA) found in serum which is formed from decomposition of peroxides by oxidative damage. The levels of LPO gradually decreased at specific intervals, which could be due to the elevated levels of enzymatic serum antioxidants in calves fed with M. citrifolia fruit.

Conclusion
Feeding of M. citrifolia fruit extract to crossbred calves showed altered biochemical constituents (viz. hypolipidemic, hypoglycaemic) and enhanced antioxidant activity in calves. The findings of this study could be exploited for the stress amelioration and management of metabolic diseases in calves without adverse effects.

Disclosure statement
No potential conflict of interest was reported by the authors.