NUTRACEUTICAL EFFECTS OF MORINGA OLEIFERA, LEAVES, SEED AND EXTRACT ON SIDE EFFECTS OF ISOPROTERENOL INDUCED MYOCARDIAL DAMAGE IN MALE RATS

INTRODUCTION

Despite the advances in the treatment of coronary artery disease, acute myocardial infarction is one of the leading causes of morbidity and mortality worldwide in both men and women Adams(2002);and Whellan (2005). When myocardial cells are injured or destroyed due to ischemia (deficient oxygen supply or glucose), the cardiac membrane becomes permeable or may rupture which results in leakage of macromolecules from damaged tissue Sasikumar and Shyamala Devi (2000). Among these macromolecules, enzymes are the best markers of tissue damage because of their tissue specificity and catalytic activity. These cellular enzymes are released as a response to β-adrenergic stimulation Ebenezar et al (2003) or due to the alterations in the integrity and permeability plasma membrane. These alterations may be due to the damage caused by the β-agonist (ISO) to the sarcolemma. Isoproterenol (ISO) is a synthetic catecholamine and [beta]-adrenergic agonist, which has been found to cause a severe stress in the myocardium, resulting in infarct-like necrosis of the heart muscle. Chagoya et al (1997) Catecholamines rapidly undergo autoxidation, and it has been suggested that the oxidative products of catecholamines are responsible for changes in the myocardium. Yates and Dhalla (1975). Lipid peroxide is a causative factor for irreversible damage to the myocardial membrane and thus favors the deposition of myocardial lipids. ISO-induced myocardial necrosis showed alterations in membrane permeability, which bring about the loss of function and integrity of myocardial membranes. Padmanabhan, (2007). Moringa oleifera Lam that belongs to Moringaceae family is commonly known as Drumstick tree that possesses various nutritional and medicinal values attributed to its roots, bark, leaves, flowers, fruits, and seeds are used in the management of obesity and heart diseseKumar et al (2010.).Recently cardiac stimulation and hypocholesterolemic activity from crude extract of Moringa oleifera crude extract was explored Ghasi et al (2000)Therapeutic potential of M. oleifera in chronic hyperglycemia and dyslipidemia. Chronic hyperglycemia is an indicator of diabetes mellitus (DM), and chronic dyslipidemia a risk factor for cardiovascular disease (CVD). These metabolic disorders are global epidemics (Yusuf et al.,2001; Wild et al., 2004).The dietary consumption of M. oleifera could be advocated in these and other countries as an inexpensive prophylactic strategy against DM and CVD. (Hossain et al., 2007; Aje and Miller, 2009 The safety of an aqueous leaf extract given orally to rats at doses of 400, 800, 1600, and 2000 mg/kg bodyweight was examined the consumption of Moringa. oleifera leaves at doses of up to 2000 mg/kg were safe. A dose-dependent decrease in body weights of the rats occurred over the 21 days (Adedapo et al., 2009).

MATERIALS AND METHODS

A – Materials

1-Isoproterenol hydrochloride was purchased from Sigma Chemical Company St.Louis, MO, USA

2- Moringa oleifera, leaves, seed and extract were purchased from Egyptian Society Moringa National Center for Research in Giza City, Egypt

 3-Experimental animals:

     Forty adult albino male rats Sprague –Dawley strain were purchased from the Agricultural Research Center, Giza, Egypt.The average weight was 145 ±6 g. The animals were kept under observation for five days before experiment and fed on standard diet according to NRC, (1995) and water ad-libitum. The standard diet comprised of casein (200g/kg), corn starch (497g/kg), sucrose (100g/kg), cellulose (30 g/kg), corn oil (50g/kg), mineral mixture (100g/kg), vitamins mixture (20g/kg) and DL-methionine (3g/kg).

B- Methods:

1-Preparation of Isoproterenol hydrochloride (ISO)

(ISO) was prepared at a concentration of 85mg/kg body weight in cold saline. Cardio toxicity was induced by subcutaneous administration of ISO (85mg/kg body weight) (Goyal et al. 2009).

2-Cardiotoxicity albino rats

Cardiotoxicity was induced in Wistar albino rats by two subcutaneous injection of freshly prepared solution of Isoproterenol hydrochloride (85 mg/kg ) body weight in cold saline after overnight fasting for18 h interval for two days 1 and 2 according to (Goyal et al. 2009)

3-Preparation of Moringa oleifera

- Moringa oleifera leaves powder

Fresh Moringa oleifera leaves were cut into small pieces, dried at 60°C in hot oven and crushed to a fine to obtain moringa oleifera leaves powder and given to rats at dose 5oo mg/kg daily by stomach tube

-Moringa oleifera extract

5 kg of Moringa oleifera plant powder was mixed five times with 5 L hot water the extract was filtered and the filtrate was evaporated to dryness with a rotatory vacuum evaporator at 50°C to give 150 g. The Moringa oleifera plant extract was given to rats at dose 300mg/kg daily by stomach tube

- Moringa oleifera seeds powder

Dried Moringa oleifera seeds were washed with tap water to remove possible potential dust and exposed to air-circulated oven at 40°C to complete dryness then grinded to fine powder and given to rats at dose 5oo mg/kg daily by stomach tube

4- Grouping of rats and experimental design:

The rats were randomly classified into five groups (8 rats each) and fed on the standard diet. The rats classified into control negative (–ve) group and four rat groups which injected ISO (85mg/kg body weight) in cold saline solution and reclassified into untreated control positive (+ve),and treated rat groups which were administration of Moringa oleifera leaves and seed powder (500 mg/kg b.w) Moringa oleifera water extract (300mg/kg b.w) in 10 ml of normal saline daily. All treatments were given in stock solution by stomach tube all over the period of the experiment. The study was assigned for 60 day. The rats were subjected daily to physical examination for observation of healthy condition such as external appearance, color of hair, body condition and activity of rats. The food intake was calculated daily and the body weight gain was recorded weekly Feed efficiency ratio and protein efficiency ratio were determined according to the method of Chapman et al., (1950)

 5- Collection of blood and heart samples:

At the end of the experiment period, the rats were anaesthetized by diethyl ether and sacrificed to obtain blood samples of each rat were withdrawn in test tubes The tubes of blood were left for coagulation then centrifuged at 3000 rpm for 15 minutes to obtain serum for each individual sample and then stored at -20 °C for some laboratory analyses. Heart of male rats was rapidly removed.

6-Determination of some of serum biochemical parameters:

Estimation of serum total cholesterol(CHO), triglyceride (TG), high density lipoprotein cholesterol (HDLc), and Low density lipoprotein cholesterol (LDL) were estimated by using the spin react enzymatic kits according to Young (2001), David and Buccolo (1973) ,Tietz (1976), Lee and Nieman (1996) Very low density lipoprotein (VLDL)was calculated VLDL-c = TG/ 5, according Friedewald et al., (1972)Atherogenic indices (CHO/HDL-c) was calculated according to Castelli and levitar, (1977). Serum alanine and aspartate amino transferase (ALT & AST) ,alkaline phosphatas (ALP) and Lactatet dehydrogenase (LDH) enzymes activity were performed according to the method of Bergmeyer andHorder (1980), Kind and King (1954), and Caband and Wroblewski (1958)respectively.The heart of each were immediately removed and homogenized in cold KCl solution (1.5%) to give a 10% homogenate and used for measuring Lactatet dehydrogenase (LDH) , Nitric oxide (NO) and Xanthine oxidase (XO) were estimated in the heart tissues according toCaband and Wroblewski (1958),Williams(1984) and Bergmever (1974)), respectively. Superoxide dismutase (SOD),catalase(CAT),glutathione-S-transferase(GST)enzymes and malodialdehyde (MDA) were estimated in the Serum according to Misra and Fridovich (1972)Cohen et al., (1970) Hissin and Hiff (1976) and Draper and Hadley (1990), respectively.

7-Histopathological examination of the heart

 The heart sample was fixed in 10% neutral buffered formaldehyde solution at pH 7.5 and cleared in xylol and embedded in paraffin. 4-5 µm thick section were prepared and stained with Hematoxylin and Eosin (H&E) for subsequent histopathological examination according to Bancroft et al, (1996).

C-Statistical analysis

All the obtained data were statistically analyzed by SPSS computer soft ware. The calculated occurred by analysis of variance ANOVA and follow up test LSD by SPSS ver.11 according to Armitage and Berry (1987).

RESULTS AND DISSCUSION

 Nutritional Results

 Feeding and growth performance in terms of food intake, body weight gain, food efficiency ratio and protein efficiency ratio of control (-ve), control (+ve) and myocardial damage rat groups treated with moringa oleifera leaves, seed powder and water extract were presented in table (1). In comparing with control (-ve) group, the myocardial damage control (+ve) group showed significant decrease in weight gain; food intake ,feed efficiency ratio(FER),protein efficiency ratio(PER), The myocardial damage rat groups treated with Moringa oleifera leaves, seed powder and water extract showed significant increase in weight gain; food intake ,feed efficiency ratio(FER) and protein efficiency ratio(PER) when compared with control (+ve) group. On the other hand, there were non-significant difference in weight gain; food intake ,feed efficiency ratio(FER) and protein efficiency ratio(PER) in all treated rat groups when compared with control (-ve) group. The improvement of weight gain, FER and PER in rats administration of moringa oleifera may be attributed to the biologically active components as moringa oleifera,. The leaves are highly essential nutritious when compared to other parts, being a source of protein, β-carotene, vitamins A, B, C and E, riboflavin, nicotinic acid, folic acid, pyridoxine, amino acids, minerals and various phenolic compounds (Khalafalla et.al.,2010).The chemical constituents in the dried, powdered leaves of moringa oleifera. contained 28.7% crude protein, 7.1% fat, 10.9%ash, 44.4% carbohydrates, 3.0mg calcium and 103.1mg of iron per 100 g, 161μg β-carotene/g, The dried moringa oleifera leaves extract contained 22.6% fiber, 2.73% ash, 3.78% protein, 9.53% total sugars, 0.00746% calcium, 0.0549% iron, and 0.0468% total catechins/flavonoids (0.0323% epicatechin). No carotenoids, vitamin C, or phytosterols were present in the extract (Teixeira et.al.,2014). The moringa oleifera was high concentrations of ascorbic acid; oestrogenic substances and β-sitosterol; iron; calcium; phosphorus; copper; vitamins A, B and C; α-tocopherol; riboflavin; nicotinic acid; folic acid; pyridoxine; β-carotene; protein; and in particular essential amino acids such as methionine, cysteine, tryptophan, and lysine present in Moringan leaves and make it ideal dietary supplement. (Westerterp 2004)

Table (1): Nutritional indicators of normal control and myocardial infarction rat groups treated with Moringa oleifera leaves powder ,seed powder and water extract at the end of the experimental period

Each value is the mean ±SD

Mean values in each column having different superscript (a, b, c & d) are significantly different at P <0.05 feed efficiency ratio(FER),protein efficiency ratio(PER)

Biochemical Results

Table (2) presented some serum lipid parameters. In comparing withcontrol negative rat groups, the levels of CHO, TG, LDL-c,VLDL-c, and atherogenic indices (CHO/HDLc).in control positive rat groups were significantly increased but HDLc was significant decreased . On the other hand, there were non-significant difference in serum levels of CHO, TG, HDLc , LDL-c, VLDL-c and atherogenic indices (CHO/HDLc) among Moringa oleifera leaves, seed powder and water extract rat groups In comparing with control negative rat groups The observed effects of Moringa oleifera leaves, seed powder and water extract on lipid profile and CHO/HDLc could be related to antioxidant activity which might attribute to those identified compounds such as, flavones, alkaloids, and so on Nandave et al.,( 2009). It is believed that the treatment with crude extract of Moringa. oleifera led to an increased serum HDL level and decreased levels of total cholesterol, LDL, and triglyceride. Thus, it can be concluded that leaves of Moringa oleifera possess cardioprotective potential Similar results were obtained by Mbikay,(2012) Illustrated that the atherogenic index is regarded as a marker for various cardiovascular disorders; the higher the value, the higher the risk of developing cardiovascular disease and vice versa High-fat diet exposure resulted in the increased atherogenic index. Treatment with 200 mg/kg and 400 mg/kg of Moringa oleifera showed a significantly decrease the atherogenic index and thus provides cardio protection. The decreased atherogenic index by Moringa oleifera thus supports the cardioprotectant Nambiar et al. (2010) examined the anti-dyslipidemic effects of Moringa. oleifera in 35 type 2 diabetic subjects. The treated group received 4.6 g of a leaf powder in a tablet form daily for 50 days. Compared with the control group, the treated subjects experienced a 1.6% decrease in total plasma cholesterol and a 6.3% increase in HDL.Comparing this study with the previous studies suggests that higher doses may be more effective.

Table (2): Effect of experimental Moringa oleifera leaves, seed powder and water extract on some serum lipid pattern and atherogenic index (CHO /HDL) of control and myocardial infarction rat groups at the end of study

Each value is the mean ±SD

Mean values in each column having different superscript (a, b, c & d) are significantly different at P <0.05 cholesterol, (CHO) ,triglyceride (TG), high density lipoprotein (HDLc),low density lipoprotein (LDLc),very low density lipoprotein (VLDLc),

Table (3) showed the effect of Moringa oleifera leaves, seed powder and water extract on cardiac markers in serum The levels of amino transferase, (AST,ALT), alkaline phosphates (ALP) and Lactatet dehydrogenase(LDH) were significantly increased in control positive rat groups, In comparing with Moringa oleifera treated rat groups and control negative rat groups. But the levels of cardiac markers in serum were significantly increased in Moringa oleifera treated rat groups when compared to control negative rat groups On the other hand, there were non-significant difference in serum levels of cardiac markers among treated rat groups with moringa oleifera leaves, seed powder and water extract These results agree with .Das et al., (2012) have shown that in mice fed with a high-fat diet, an aqueous extract of Moringa oleifera leaves and seeds protects against liver damage as demonstrated by reductions in tissue histopathology and serum activities of marker enzymes aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP) as well as reduced lipid peroxidation and increases in reduced glutathione Nandave et al.,(2009). Demonstrated that the cardioprotective effect of N,α-L-rhamnopyranosyl vincosamide, an indole alkaloid isolated from the leaves of Moringa oleifera Anwar,et al.,( 2007) Observed that the medicinal properties of the plant’s edible parts have been recognized by both the Ayurvedic and Unani systems of medicine in India The various plant parts have wide medicinal applicability for the treatment of cardiovascular diseases as the roots, leaves, gum, flowers, and seed infusion contain nitrile, mustard oil glycosides, and thiocarbamate glycosides as their important bioactive constituents, which are thought to be responsible for their diuretic, cholesterol lowering, and antiulcer properties Sidney et al ,(2015) studies have shown the aqueous, hydroalcohol, or alcohol extracts of Moringa. oleifera leaves possess a wide range of additional biological activities including antioxidant, tissue protective the liver, kidneys, heart, testes, and lungs, analgesic, antiulcer, antihypertensive, radioprotective, and immunomodulatory actions. because its cntains awide variety of polyphenols and phenolic acids as well as flavonoids, glucosinolates, and possibly alkaloids is believed to be responsible for the observed effects.

Table (3): Effect of experimental Moringa oleifera leaves, seed powder and water extract on some cardiac markers AST, ALT , ALP and LDH of control and myocardial infarction rat groups at the end of study

Each value is the mean ±SD

Mean values in each column having different superscript (a, b, c & d) are significantly aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP) Lactatet dehydrogenase(LDH)

Data in table (4) illustrated the cardiac markers in heart tissue functions in the five rat groups. Control positive rat groups showed significant increase in heart tissue Lactatet dehydrogenase(LDH),Nitric oxide(NO)and Xanthine oxidase (XO) compared to control negative rat groups Administration of Moringa oleifera leaves, seed powder and water extract to rat groups showed non significant changes compared with control negative rat groups exception Xanthine oxidase in heart tissue but showed significant decrease compared with +ve control rat groups. It is known that the increase of cardiac markers in heart tissue indicated abnormal heart functions and restoration of normal levels of these enzymes These results agree with Panda et al.,( 2012). Illustrated that the various derivatives of salicylic acid, gallic acid, coumarin acid, and caffeic acid also exist in extracts of M. oleifera. In addition, indole alkaloid N,α-L-rhamnopyranosyl vincosamide has been isolated from M. oleifera leaves and shown to exert a cardioprotective effect on rats Nandave et al.,(2009) Reported that the treatment of Moringa. oleifera resulted in significant favorable modulation of the biochemical enzymes superoxide dismutase, catalase, glutathione peroxidase, lactate dehydrogenase, and creatine kinase-MB compared to the ISP control group. Moringa treatment significantly prevented the rise in lipid peroxidation in myocardial tissue. Furthermore, M. oleifera also prevented the deleterious histopathological and ultrastructural perturbations caused by ISP. Based on the results of the present study, it can be concluded that M. oleifera extract possesses significant cardioprotective effect, which may be attributed to its antioxidant, antiperoxidative, and myocardial preservative properties. Randriamboavonjy et al., (2016 ) Moringa oleifera seed powder treatment did not modify blood pressure in spontaneous hypertensive rats but reduced nocturnal heart rate and improved cardiac diastolic function (reduction of isovolumetric relaxation time and deceleration time of the E wave, increase of ejection volume and cardiac output compared to nontreated spontaneous hypertensive rats Left ventricular anterior wall thickness, interseptal thickness on diastole, and relative wall thickness were reduced after Moringa oleifera seed powder treatment. Furthermore, we found a significant reduction of fibrosis in the left ventricle of Moringa oleifera seed powder treated spontaneous hypertensive rats this antihypertrophic and antifibrotic effect of Moringa oleifera seed powder was associated with increased expression of peroxisome proliferator-activated receptor and, reduced cardiac triglyceride level, and enhanced plasmatic prostacyclins.

Table (4): Effect of experimental Moringa oleifera leaves, seed powder and water extract on some cardiac markers in heart tissue LDH, N0 and XO of control and myocardial infarction rat groups at the end of study

Each value is the mean ±SD

Mean values in each column having different superscript (a, b, c & d) are significantly

Lactatet dehydrogenase(LDH),Nitric oxide(NO)and Xanthine oxidase (XO)

Data in table (5) illustrated the antioxidant system enzyme in the five rat groups. In comparing with -ve control rat groups, the myocardial damage +ve control rat groups showed significant decrease in serum values of superoxide dismutase( SOD) ,catalase (CAT) and glutathione-S-transferase (GST) but significant increase in serum Malondialdehyde (MDA. The moringa oleifera leaves, seed powder and water extract showed a significant increase compared to +ve control rat groups but showed non significant different in (SOD), (CAT) , (GST) and (MDA) compared to - ve control rat groups On the other hand, there were a significant increase in serum Malondialdehyde (MDA) in + ve control rat groups compared to all treated rats and – ve control rat groups these results agree with. Verma et al.,(2009) and Sreelatha and Padma,. (2011). showed that the Phenolic compounds are commonly found in both edible and other traditional medicinal plants, of Moringa oleifera and they have been reported to have multiple biological activities, including free radical scavenging activity. Antioxidants in food appear to play an essential role in the prevention of oxidative stress-related diseases/disorders and in the reduction of total mortality Typical phenolics that Possess antioxidant activity is mainly phenolic acids and flavanoids. Flavonoids commonly accumulate in the epidermal cells of plant organs such as flowers, leaves, stems, roots, seeds and fruits, being found in glycosidic form (glycosides) and non-glucosidic form Showed the strong in vitro antioxidant properties of the methanolic extract of Moringa oleifera edible parts. This antioxidant activity of Moringa oleifera extracts is due to the presence of various bioactive compounds such as chlorogenic acid, rutin, quercetin Glucoside, and kaempferol rhamnoglucoside furthermore, the extract of Moringa oleifera leaves and other parts have been shown to have potent antioxidant action in vivo Wojdylo et al (2007 ) Illistreted that the Moringa oleifera contains certain phenolic compounds also may induce production of glutathione-S-transferase and other antioxidant enzymes. The ability of certain phenolic compound to bind to minerals may be beneficial in some cases, since copper and iron can be initiators of hydroxyl radical production by the Fenton and Haber-Weiss reactions Jaiswal et al. (2013) have investigated the antioxidant activity of an aqueous extract of Moringa oleifera leaves in normal and diabetic rats. Oxidative free radical scavenging enzymes were measured in response to 200mg/kg of lyophilized powder. A significant increase in activities of superoxide dismutase, catalase, and glutathione S-transferase and a decrease in lipid per oxidation were observed. It was suggested that the high phenolic and flavonoid contents in the extract can protect against oxidative damage in normal diabetic and heart patients Vongsak et al. (2014) have conducted a quantitative analysis of an ethanol extract of Moringa oleifera leaves by HPLC, and have shown that the average values for crypto-cholorgenic acid, isoquercetin, and astragalin in the dried extract were 0.081%, 0.120%, and 0.153%, respectively. They have suggested that these compounds and this analysis may serve as a guideline for the standardization of Moringa oleifera extracts. However, these standards could only be applied to ethanol extracts and not to aqueous extracts

Table (5): Effect of experimental Moringa oleifera leaves, seed powder and water extract on SOD, GST , CAT and MDA in serum of control and myocardial infarction rat groups at the end of study

Each value is the mean ±SD

Mean values in each column having different superscript (a, b, c & d) are significantly

Histopathological Examination of Heart:-

Microscopically heart of–ve control rat groups revealed the normal myocardial muscle fibers (pict.1) Conversely, heart of rat from Isoproterenol non treated +ve control rat groups showed focal necrosis of myocardial muscle fibers associated with leucocytic cells infiltration, inflammatory cells and intermuscular edema (pict.2). However, heart of rats from treated with Moringa oleifera leaves, seed powder and water extract revealed no histopathological changes (pict 3,4,5). The histopathological results denoted some changes but less than that of the +ve control rat groups These histopathological results changes agree with Panda et al.,( 2012)This alkaloid in Moringa oleifera leaves, seed powder and water extract when administered at an oral dose of 40mg/kg per day for 7days markedly reduced isoproterenol-induced cardiotoxicity in rats. The cardioprotective effects were demonstrated by decreases in serum cardiac biomarkers, increases in cellular antioxidants and antioxidant enzymes, a reduction in cardiac necrosis, a decrease in cardiac lipid peroxidation, and a reduction in cardiac histopathology and electrocardiographic (ECG) changes Nandave et al.,( 2009).Showed that the rats were treated daily with saline, isoproterenol, or isoproterenol plus the leaf extract (200 mg/kg) orally for 1 month. The M. oleifera leaf extract prevented biochemical, histopathological, and ultrasound changes in the heart induced by isoproterenol. The extract prevented isoproterenol-induced hemodynamic changes in the heart including changes in heart rate, left ventricular end-diastolic pressure, left ventricular peak positive pressure, and left ventricular negative pressure .The myocardial infarction by isoproterenol (ISP) group showed the greatest aortic changes compared with the control groups.From obtained results It can be recommend that the increase consumption of Moringa oleifera leaves, seed powder and water extract of their effective impact in reducing the side effects of isoproterenol rats, which proved some chemical analyses of serum, and confirmed by the analysis of histopathological tissue of the heart, and nutritional status, and recommended research also need to enter the Moringa oleifera leaves, seed powder and water extract the food plan of myocardial infarction diet and general beverages in quantities which achieved results on rats, which have been turned into quantities to humans, dose.