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International Journal of Recent Academic Research (ISSN: 2582-158X)

Vol. 01, Issue 08, pp.404-418, November, 2019 Available online at http://www.journalijrar.com

RESEARCH ARTICLE

UTILIZATION OF MULBERRY METHANOLIC LEAF EXTRACTIVE (MMLE) FOR TREATING HYPERCHOLESTEROLEMIA IN RAT, RATTUS NORVEGICUS (L).

1Sonali Mahadev Jambhale, 2Avram Hershko and 3, *Vitthalrao Bhimasha Khyade

1, 3Science Association, Shardabai Pawar Mahila Mahavidyalaya, Shardanagar Tal. Baramati Dist. Pune – 413115, India 3Unit of Biochemistry, The B. Rappaport Faculty of Medicine, and the Rappaport Institute for Research in the Medical

Sciences, Technion-Israel Institute of Technology, Haifa 31096, Israel

ARTICLE INFO ABSTRACT

Healthy diet is the foundation for a healthier workforce and lower healthcare costs. The diet therapy for regulation cholesterol is exerting influence to reduce mortality and morbidity. The methanolic mulberry leaf extractives (MMLE) (at the rate of 100 and 200 mg/kg body weight) and the standard hypolipidemic medicine Questran were administered through the diet to the rats of hypercholesterolemia (maintained on high cholesterol diet). Treatment was five times per week for the period of nine weeks. Analysis of blood plasma and it’s postmitochondrial fraction (PMF) was carried out through the bioassay of total cholesterol; triglyceride; lipid peroxidation (LPO) and reduced glutathione. Treating the rats of hypercholesterolemia with MMLE caused the dose dependent reduction in cholesterol level in plasma and it’s PMF. The second dosage of Methanolic Mulberry Leaf Extractives (MMLE) (200 mg/kg) was significantly attenuated the cholesterol-induced increase in cholesterol plasma. Similarly, the cholesterol-induced decrease in PMF reduced glutathione levels of hypercholesterolemic rats were significantly ameliorated in experimental animals treated with Methanolic Mulberry Leaf Extractives (MMLE). The consumption of leaves of mulberry, Morus alba (L) may act as a potent hypocholesterolemic nutrient. It may open a new avenue in the field of regulation cholesterol through diet.

Key Words: Hypercholesterolemia, Lipid-lowering effect, Morus alba, Cholesterol.

Copyright © 2019, Sonali Mahadev Jambhale et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

INTRODUCTION

Elevated cholesterol, or hypercholesterolemia, is the presence of high levels of cholesterol in the blood. It is not a disease but a metabolic imbalance, caused by abnormalities in the levels of lipoproteins, the particles that carry cholesterol in the bloodstream. This may be related to diet, genetic factors and the presence of other diseases such as diabetes and an underactive thyroid. The high level of cholesterol in the blood leads to the condition recognized as hypercholesterolemia. In this condition, the lipoproteins in the blood are elevated. Therefore, it is also called as hyperlipoproteinemia (Durrington, 2003). The reasons for the condition of hypercholesterolemia may a consequence of diet, obesity, inherited (genetic) diseases and the other diseases (like: type 2 diabetes and an underactive thyroid). The three major classes of lipids include: Storage lipids; Structural lipids and other lipids. Cholesterol is one of three major classes of lipids. In the animal cell, cholesterol is utilized for the formation of membranes. Therefore, cholesterol is manufactured in all animal cells. It is also the precursor of the hormones of steroid category and acids of the bile. The cholesterol is insoluble in water. Therefore, it is transported in the blood plasma within protein particles (lipoproteins). The lipoproteins are categorized on the basis of their density. Accordingly, the lipoproteins include: VLDL (Very Low Density Lipoprotein); IDL (Intermediate Density Lipoprotein); LDL ( Low Density Lipoprotein); and HDL (High Density Lipoprotein) (Biggerstaff and Wooten, 2004).

According to Carmena, et al, (2004), all the lipoproteins carry cholesterol, but elevated levels of the lipoproteins other than HDL (termed non-HDL cholesterol), particularly LDL-cholesterol, are associated with an increased risk of atherosclerosis and coronary heart disease. In contrast, higher levels of HDL cholesterol are protective (Kontush and Chapman, 2006). Avoiding trans fats and replacing saturated fats in adult diets with polyunsaturated fats are recommended dietary measures to reduce total blood cholesterol and LDL in adults (de Souza, et al., 2015). In people with very high cholesterol (e.g., familial hypercholesterolemia), diet is often not sufficient to achieve the desired lowering of LDL, and lipid-lowering medications are usually required. If necessary, other treatments such as LDL apheresis or even surgery (for particularly severe subtypes of familial hypercholesterolemia) are performed. About 34 million adults in the United States have high blood cholesterol (Ito, et al., 2011). According to Wald and Law (1995) and Krieger (1998), hypercholesterolemia is a risk factor for cardiovascular diseases (CVD) such as atherosclerosis and myocardial infarction. It is a common cause of mortality and morbidity. The factors, like life style, a diet rich in cholesterol, age and hypertension, have been reported to cause heart failure (Schaefer et al., 1995). The high levels of cholesterol, particularly LDL cholesterol, are mainly responsible for hypercholesterolemia (Krieger 1998). Adaramoye et al (2005) reported relation between “hypercholesterolemia and enhanced oxidative stress” for increase in the lipid peroxidation. Increase in the generation of oxidized LDL is a major factor in the vascular damage associated with high cholesterol levels

Article History:

Received 24th August 2019, Received in revised form 27th September 2019,

Accepted 18th October 2019, Published online 25th November 2019.

*Corresponding Author: Vitthalrao Bhimasha Khyade

(Pritchard et al 1995). Therefore, the inhibition of oxidative stress through hypercholesterolemic conditions is considered to be an important approach for treatment. The efforts have been made to identify the antioxidative functions of various medicinal plants (Hu et al 2006; Tomotake et al 2006; Visavadiya and Narasimhacharya, 2007). The fibrates are the medicines of a class of amphipathic carboxylic acids. The fibrates are used for a range of metabolic disorders, mainly hypercholesterolemia (high cholesterol), and are therefore hypolipidemic agents. The acid sequestrants of bile are next to the fibrates used for several decades. The high prevalence of adverse effects of fibrates and bile acid sequestrants led to the introduction of statins (HMG CoA inhibitors) (Miller 2001). Adverse effects of the statins are relatively low. Miller (2001) reported, rhabdomyolysis as a serious effect of statin. The mild muscle pain is a relatively common side effect of statins, some people who take statin medications to lower their cholesterol may have severe muscle pain. This intense pain may be a symptom of rhabdomyolysis (rab-doe-my-OL-ih-sis), a rare condition that causes muscle cells to break down. Herbal source of food and medicine have been the companions of human being since time immemorial. Herbal source formed the basis of useful drugs since they are less toxic than synthetic drugs. Medicinal plants screening for suitable medicine for the betterment of health of human being constitute a new avenue for the discovery of safe drugs. Man’s dependence on plants for his existence dates back to the beginning of the human race. In the early days, he had only limited needs like food, shelter and clothing but with the advancement of civilization, his requirements also grew. The present day man depends heavily on a very large numbers of plants and animal produced to meet his daily needs. Traditional medicines are used by about 60% of the world population theses are not only used for primary health cure not just in rural areas in developing countries, but also in developed countries as well as where modern medicines are predominantly used. Chattisgarh is a remarkable place, not only because of the tribal population and dense forest, but also having a lot of rare and useful natural resources including some rare species of medicinal plants which are used for curing different kinds of diseases. Mulberry is a deep-rooted perennial plant with highly branching root and shoot systems Mulberry belongs to the family moraceae,. In Japan, more than 1000varieties are stored and in India at the CSTRI Mysore, there are about 400 mulberry varieties of which 85 are indigenous, 114 are exotic origin. Some of the important medicinal values of mulberry plants are it contains different chemical of medicinal value in leaf fruit stem and root portions. Mulberry can grow both in the tropics and in the temperate regions. It can be cultivated in different soil types. It can be raised both in rain-fed and irrigated conditions. It is comparatively resistant to environmental fluctuations and is relatively free from pests and diseases. The quality and quantity of leaves harvested from mulberry plantation for silkworm feeding depends upon the availability of soil water. The nutritive value of leaf changes according to the photosynthetic and respiratory activities of the leaf. Mulberry cultivation and sericulture have been practiced for a very long time. But in the past mulberry was cultivated more for its medicinal use than for rearing of silkworms alone. The leaves of mulberry are simple alternate, stipule and petiolate. Leaf may be glossy or scabrous in texture leaf apex nay be long tailed acute or double acute in some countries, mulberry leaves

are directly used in preparing the tea or these together with the mulberry roots and stem are boiled to prepare a both which is effective in lowering down the blood pressure. In Cambodia, mulberry leaves are used in the treatment of conjunctivitis. Mulberry leaves contain variety of chemical such as phenylalanine, leucine, valine, tyrosine, proline, alanine, glutamic acid , glycine, serine, arginine, aspartic acid, ascorbic acid, carotene, vitaminB, folic acid, folinic acid, vitaminD, Cystine, threonine, sarcosine, raminobutyric acid, n-butanol ,B -y-hexenol, methyl ethyl acetaldehyde, n-butyl aldehyde ,iso-butyl, aldehyde,iso-butylaldehyde, valeraldehyde, hexaldehyde, pipecolicaid, acetone, methyle ethyle ketone, methyl-hexel ketone, propionic acid, isobutyric acid, besides tartaric acid, succinic acid and calcium malate. Accordingly, leaves of mulberry have great medicinal value. Mulberry leaves have been found effective in lowering the blood -sugar level and arterial pressure. High performance liquid chromatography reveals the presence of aromatic amino acids in the leaves of mulberry, Morus alba (L). Various biogenic amines are detected (at picogram level), which include dopamine dyhydroxyphenyl acetic acid, hydroxyphenyl acetic acid L-DOPA, Norepinephrine, Tyramine and Netanephrine. Dopamine is found at high concentrations [54.37 mg./gm. dry powder ]. These are also used in gargles for curing the throat inflammation. Moreover, the aqueous and alkali extract of mulberry leaves are active against gram-positive bacteria and yeasts. Some rural people as food because of being highly nutritious also use mulberry leaves. Several compounds of calcium, phosphorus, slicon, manganese, magnesium, iron, copper, zinc and vitamin are found in abundance in mulberry leaves. In view of this, it has been seen that mulberry leaves provide excellent material for preparing paratha, pakoda, baji as compared to spinach. As like pea, cucumber and drumstick etc. man also now consumes mulberry leaves as vegetable. The green leaves of various plants are more nutritive, mulberry leaves, satisfy the dietary requirement of human beings and contain all the elements required for the body. The mulberry is widely developed to feed the silkworms employed in the commercial production of silk. It is also notable for the rapid release of its pollen, which is launched at over half the speed of sound. Its berries are poisonous when unripe, but are otherwise edible. For the eternal health, longevity and remedy, to remove pain and discomfort, fragrance, flavor and food, all over the world mankind depend upon the plants to meet their demands. Medicinal plants still play important role in emerging and developing countries. Mulberry plant is one of the important traditional herbs widely used in medicine from centuries ago. It is a rich source of flavonoids and other compounds that showed antimicrobial potential and free radical scavenging activity. Due to its pharmacological properties, all parts of plant are used as medicine. Mulberry is proved in protecting liver, improving eyesight, facilitating discharge of urine, lowering of blood pressure, anti-diabetic and weight controlling in human beings. Mulberry leaves are rich in protein and widely used as silkworm food, in food formulations and also have neuroprotective functions can be used against neurodegenerative disorders such as Alzheimer and Parkinson. Other useful effects such as immune-modulation and chemo-protective properties need further exploration by researchers. It is the need of the hour to explain medicinal values of mulberry by scientists. (Vitthalrao Bhimasha Khyade and Avram Hershko, 2018). The fruit of mulberry is a multiple one, as all the flower of the inflorescence give rise to a single fruit. The

405 International Journal of Recent Academic Research, Vol. 01, Issue 07, pp.404-418, November, 2019

fruit is green but changes to purplish through various shades of pink, red, brown, etc. due to certain water contained in the juice. It is also good for brain heart, spleen and helps curing diarrhea and intestinal ulcers. It is considered as a laxative and oral juice administration good drink during convalescence after a febrile illness. Mulberry fruit is well known as esteemed dessert fruit and having high source of vitamin C which is commonly used for the jams, Jellys cooling beverages and wines. The fruit has special taste and attracts women who are on their family way. This is due to vitamin C, which gives a sour taste and makes them chew the fruit repeatedly. Contains moisture [85percent] proteins [0.7percent], fats [0.4percent], carbohydrates [12.2percen], calcium [80mg, phosphorous [20mg, iron [2.6mg, carotene [vitamin A, 60IU], thiamin [58g], nicotinic acid [0.2ug], riboflavin [2ug] and ascorbic acid [10ug] per 100 g. Besides, these, it contains malic acid, citric acid, pectin, mucilage and coloring matter . Extracts of fruits of Morus laevigata contains high concentration of hydroxyproline. Medicinal value of Stem: Among various substances extracted from mulberry shoots, polyphenols or its derivatives have been seen to increase the fur growth in rabbits and sheep. Some of the polysaccharides including pectin and glucose are used in cosmetics for making hair and skin supple. While diverse uses of mulberry shoots have been demonstrated by various studies, details on the use of various substances present in the shoots are yet to come out. Wood contains tannin, extracts prepared from wood [tannin content 0.32%] that is suitable for tanning and coloring purpose and bark is used as purgative and vermifuge. Mulberry being a member of moracae, which is specialized for their latex producing capacity, has the property of healing the wounds and injuries. It can be used as dermal ointment. Medicinal value of Root-Mulberry is a deeprooted perennial plant its root system is very well developed through the growth and spread of the root depend upon the texture of the soil the root shows the same structure as in other dicotyledonous. Root the bark of root is bitter and possesses cathartic, antihelmintic and stringent properties. It is also used as restorative tonic and as remedy for nervous disorders in China. An alkaloid deoxyjirimycin [DNJ] has been extracted from the root bark of the black mulberry, M.nigra, which resembles glucose and interferes with the synthesis of sugar chains. The chemical hinders the addition of sugar molecules to the outer coat of the human immunodeficiency virus. It has been observed that the surface glycoprotein of HIV is rich in sugar chain and is involved in the budding activity of the virus on and from a cell. Although the alkaloid’s [DNJ’S] activity at the cellular level is yet to be analyzed, it is crystal clear that DNJ inhibits the enzymes glycosidase, responsible for trimming the sugar chains on glycoproteins. In a sense, DNJ hinders the docking of HIV on the human cell and hence makes the attack futile thus DNJ, easy to synthesize and seems to be a promise as a potential medicine against AIDS. [RAY, 1989].The GD searle Pharmaceuticals Company has taken keen interest in DNJ and is working closely with chemists at Oxford and Cambridge . Butyl-DNJ is reported more active in treating AIDS. [Ray 1989, Tewary and Rao 1990] Mulberry root juice has the capacity of agglutinating the blood. The medicine prepared from the root is called Glucosidae which can be administered to the patients suffering from high blood pressure. Japanese have already isolated a hormone called Moranoline which contains 5 carbon atoms and one nitrogen atom. Its use as medicine is yet to be confirmed. Conclusion: mulberry contains different chemicals of medicinal value in

leaf, fruit, stem, seed and root portions. The chemical substances are normally extracted in to aqueous media in the form decoctions and concoctions .The knowledge of medicinal plants is species specific and restricted to a few individuals which differs from ancient system and due to lack of written documentation, many prime remedies are vanishing with generation. The mulberry cultivation would be helpful or encouraging for making index of medicinal plants of the region. The leaf decoction of mulberry, M. alba (L), had significant nephroprotective effect against the hyperglycemia-induced oxidative stress instreptozotocin-induced diabetes brown rat, R. norvegicus (L) (Vitthalrao Bhimasha Khyade and Avram Hershko, 2018). Aqueous Mulberry Leaf Decoction (AMLD) was reported to reduce the oxidative stress by inducing mechanism for antioxidant, thereby showing protective effect against DMBA induced liver damage (Vitthalrao B. Khyade and James P. Allison, 2018). On this much background, the attempt on Lipid-lowering influence of methanolic extractives of leaves of mulberry, Morus alba(L) leaves in rats fed on high cholesterol diet has been planned.

MATERIALS AND METHODS The attempt was completed through the steps, which include: Preparation of Methanolic Mulberry Leaf Extractives (MMLE); Rearing of brown rat, Rattus norwegicus (L), the experimental animals; Design of Experiment through Grouping the experimental animals; Processing for the assay sample preparation (Serum Assay Sample and Liver tissue homogenate); Biochemical Analysis; Histochemical studies and Statistical analysis. Preparation of Methanolic Mulberry Leaf Extractives (MMLE): Fresh leaves of mulberry, Morus alba (L) were obtained from the mulberry garden of Sericulture Unit at Malegaon Sheti Farm of Agriculture Development Trust, Baramati (Malegaon Khurd) Post Box No - 35, Baramati, Pune, Maharashtra, India. The botanical identification and authentication was confirmed at the Shardabai Pawar Mahila Mahavidyalaya, Shardanagar, where voucher specimen was kept at the herbarium (Voucher No. VBK/Dr.APIS/786).

Figure: M.1: The Soxhlet Extraction


The leaves of mulberry, Morus alba (L) were allowed for shade drying at room temperature. Leaves were then powdered through the use of domestic mixture. The mulberry leaf powder was defatted with n-hexane. The mulberry leaf powder was kept in n-hexane (1 Kg leaf powder in 2.5 n-hexane) for overnight period. Defatted mulberry leaf powder then processed for extraction with 75% methanol (2.5 litres) overnight in a soxhlet extractor. Methanolic Mulberry Leaf Extractives (MMLE) was concentrated and evaporated to dryness at 50oC with a rotary evaporator under reduced pressure, and the yield of the preparation was 6.8%. The Methanolic Mulberry Leaf Extractive (MMLE) was dissolved in water at a concentration of 4 g/100 ml, and aliquots of different concentrations were given orally to the animals with a gavage needle. Rearing of brown rat, Rattus norwegicus (L), the experimental animals: For the present attempt on utilization of the Methanolic Mulberry Leaf Extractives (MMLE) for treating the hypercholesterolemia in Norwegian rat, Rattus norvegicus (L), seventy adult male (12 week-old) Brown rats (Rattus norvegicus L) (Dr. APIS Laboratory), weighing 120 - 130 g were procured from the Department of Zoology, Savitribai Phule Pune University. The adult male rats were housed in quiet cages (20 - 25°C; 50 - 60% relative humidity). They were kept in laboratory with a condition of “12 hour light/dark cycle (7 a.m. - 7 p.m.)”. They were fed with a commercial standard rat diet (Abaliogu YemSanayi, Denizli, Turkey) and water ad libitum. All animal procedures were approved by the Animal Care and Use Protocol (Department of Zoology, Shardabai Pawar Mahila Mahavidyalaya, Shardanagar Baramati). The method for rearing the brown rat, Rattus norwegicus (L), the experimental animals belong to International Journal of Scientific Research in Chemistry (IJSRCH) | IJSRCH | Volume 3 | Issue 5 ISSN: Page 106 – 123. Experimental Design (Table-1): The experimental animals, Norwegian rat, Rattus norvegicus (L) were divided into seven groups, each with ten individuals. Remaining individuals of experimental animals were maintained as reservoir. The individuals of group: [(A) water treated control group] were served as Water treated Control group and they received drug-vehicle, water. The individuals of group: [(B) Positive Control (MMLE)] were served as positive control group and they were received Methanolic Mulberry Leaf Extractives (MMLE) at dose 100 mg/kg body weight. The individuals of group: [(C) Q. Only] were served as Questran treated group and they were received Questran. The Questran was given at a therapeutic dose of 0.26 g/kg body weight (Adaramoye et al., 2005). The individuals of group: [(D) CH. Only] were served as Cholesterol Treated and they were intubated with cholesterol-only. Animals in this group: [(D) CH. Only] was considered as hypercholesterolemic animals. The Cholesterol was orally administered at a dose 30 mg/0.3 ml/ animal. The individuals of group: [(E) MMLE (I) + CH] were served as Methanolic Mulberry Leaf Extractives (MMLE) Treated group and they were received Methanolic Mulberry Leaf Extractives (MMLE) at dose 100 mg/kg body weight in addition to oral administration of cholesterol. The Cholesterol was orally administered at a dose of 30 mg/0.3 ml/ animal. The individuals of group: [(F) MMLE (II) + CH] were served as Methanolic Mulberry Leaf Extractives (MMLE) Treated group and they were received Methanolic Mulberry Leaf Extractives (MMLE) at dose 200 mg/kg body weight in addition to oral

administration of cholesterol. The Cholesterol was orally administered at a dose of 30 mg/0.3 ml/ animal. The individuals of group: [ (G) CH + Q] were served as Questran Treated group and they were received Questran at a therapeutic dose of 0.26 g/kg body weight (Adaramoye et al., 2005) in addition to oral administration of cholesterol. The Cholesterol was orally administered at a dose 30 mg/0.3 ml/ animal. The dosage for oral cholesterol administration was 30 mg/0.3 ml/ individual in the experimental group. The dosage for oral questran administration was 0.26 g/kg body weight (Adaramoye et al. 2005). The dosage for oral Methanolic Mullberry Leaf Extractives (MMLE) administration was 100 and 200 mg/kg body weight. The Methanolic Mullberry Leaf Extractives (MMLE); Questran, and cholesterol were administered five times in a week for a period of nine weeks. Processing for the assay sample preparation: After the last dose of drugs, rats were fasted overnight and sacrificed by cervical dislocation. Blood was collected from the heart into EDTA tubes. Plasma was prepared by centrifugation at 3,000 g for 15 minutes in an MSC bench centrifuge (Beckman and Hirsch, Burlington, IO, USA). The plasma was used in the estimation of lipid profile. Liver from the animals were rinsed in ice- cold 1.15% KCl, dried and weighed. It was homogenized in 4 volumes of ice-cold 5 mM phosphate buffer, pH 7.4, and centrifuged at 10,000 g for 20 min to obtain post mitochondrial fraction (PMF). Organs such as kidneys, lungs, liver and heart were removed from the animals and weighed. Bioassay of cholesterol: The cholesterol is a sterol and lipid present in the cell membranes, and is transported in the bloodstream of all animals. It is used to form cell membranes and hormones, and plays important roles in cell signalling processes. Elevated levels (hypercholesterolemia) have been associated with cardiovascular diseases such as atherosclerosis; whereas, low levels (hypocholesterolemia) may be linked to depression, cancer and cerebral hemorrhage. Simple, direct and automation-ready procedures for measuring cholesterol are very desirable. The “Enzyme Chrome Cholesterol Assay” (ECCH004) method was utilized for the determination of levels of cholesterol in assay sample. This method is based on cholesterol esterase hydrolysis of cholesterol esters to form free cholesterol and cholesterol dehydrogenase catalyzed conversion of cholesterol to cholest-4-ene- 3-one, in which NAD is reduced to NADH. The optical density of the formed NADH at 340 nm is directly proportionate to the cholesterol concentration in the sample. The chemical reagents in the kit include: Assay Buffer (20 mL); Enzyme Mixture (120 microlitre); NAD Solution (2 x 1 mL) and Standard cholesterol (1 mL 300mg/dL). All the chemicals and reagents were brought to room temperature prior to assay. Standard Curve: Ten-fold diluted standard (STD) of cholesterol was prepared by mixing 40 μL 300 mg/dL Standard and 360 μL Assay Buffer. The dilutions are shown in Table 2. Fifty microlitres of diluted standard solution of cholesterol was transferred into wells of the 96-well plate. Assay samples were diluted 10-fold (e.g. 10 μL sample with 90 μL Assay Buffer). 50 μL diluted sample was transferred in separate wells. For each reaction-well, the NAD solution in assay Buffer was prepared. For each reaction-well, 40 μL assay buffer with 18 μL the provided NAD solution were mixed. Addition of 50 μL of diluted NAD to standards and sample wells was carried out. For the purpose of well mixing, the well-plate was taped. The reaction mixture was kept for five minutes at room temperature. The optical density of the contents in each well plate was read at 340nm on


spectrophotometer. The optical density readings and respective concentrations of standard solution were used for plotting the standard graph. Through the use of standard graph, cholesterol concentration in the assay sample was determined. The results were expressed in the unit: mmol/L. Preparation of Enzyme Mixture: Enough enzyme mixture was prepared. For each reaction-well, the content for mixing include: 10 μL Assay Buffer and 1 μL provided Enzyme Mixture. Addition of 10 μL diluted enzyme mixture was carried out. For the purpose to mix well, the well-plate was taped thoroughly. The reaction mixture was processed for incubation for thirty at room temperature. The optical density of the contents in each well was read at 340nm on spectrophotometer. Through the use of optical readings and standard graph, cholesterol concentration in the assay sample was determined. sample cholesterol. Bioassay of Triglyceride: The triglyceride is the main constituent in vegetable oil and animal fats. There is important role of triglycerides as energy sources and transport of dietary fat. In the body of human being, the diseases like atherosclerosis; thrombosis and pancreatitis are linked with high levels of triglycerides in the bloodstream. Significant application of drug discovery lies in the assay method which deserve simplicity and automation in ready procedures for measuring the desired moiety. Bioassay Systems' triglyceride assay uses a single Working Reagent that combines triglyceride hydrolysis and glycerol determination in one step, in which a dye reagent is oxidized to form a colored product. The color intensity at 570nm is directly proportional to triglyceride concentration in the sample. The “Enzy-Chrom-TM Triglyceride Assay Kit” ETGA006 was used for the determination of triglyceride level in assay sample. The kit was stored in refrigerator at minus twenty degree Celsius. The chemical reagents in the kit include: assay buffer (24 mL); ATP ( 250 μL); dye Reagent (220 μL); Enzyme Mixture (500 μL Lipase: 1000 μL) and Standard triglyceride solution [100 μL (equivalent to 100 mmol/L Triglyceride)]. All components were equilibrated to room temperature. The dilutions for standard triglyceride are shown in Table –3. With the help of distilled water, the plasma sample was diluted 5-fold in distilled. It was then used for the assay. The ten microlitres (μL) of assay sample was transferred into each of wells of the 96-well plate. Working Reagent was prepared for each well, by mixing 100 μL Assay Buffer, 2 μL Enzyme Mix, 5 μL Lipase, 1 μL ATP and 1 μL Dye Reagent in a clean tube. Hundred μL of working reagent was transferred into standards and sample wells. The plate was taped well to mix. Incubation of the contents was carried out for thirty minutes at room temperature. The optical density was read at 570nm (550-585nm) on spectrophotometer. The optical density readings and respective concentrations of standard solution were used for plotting the standard graph. Through the use of standard graph, triglyceride concentration in the assay sample was determined. The results were expressed in the unit: mmol/L (1 mmol/L triglyceride equals 38.67 mg/dL or 10 ppm). Bioassy of lipid peroxidation (LPO): Lipid peroxidation products such as malionaldehyde (MDA) are considered useful and reliable indicators of oxidative damage, due to the susceptibility of membranes to attack by reactive oxygen species (Wise, 1995; Hodges et al., 1999). Malionaldehyde is a secondary end product of the oxidation of polyunsaturated fatty acids, and reacts with thiobarbituric acid (TBA) to yield a

pinkish-red chromagen with maximal absorbance at 532nm (Hodges et al., 1999). Measurements of these thiobarbituric acid reactive substances (TBARS) have been made since the 1950’s, often using the method of Heath and Packer (1968). However, many plant tissues contain interfering compounds, including sugars, anthocyanins and other phenolics, that absorb at 532 nm, leading to overestimations of MDA by up to 96.5% (Du and Bramlage, 1992; Merzlyak et al., 1993; Hodges et al., 1999). The assay sample was transferred to a 2 mL Eppendorf tube and centrifuged at 5000 rpm for 10 min. Solutions with or without thiobarbituric acid (+TBA or –TBA, respectively) were added to 1 mL of supernatent in a glass centrifuge tube. The –TBA solution comprised 20% trichloroacetic acid (TCA; Sigma, USA) and 0.01% butylated hydroxytoluene (2, 6-Di-tertbutyl-4-methylphenol; Sigma Aldrich, WI, USA). The +TBA solution contained the same chemicals as –TBA solution plus 0.65% thiobarbituric acid (TBA; Sigma, MO, USA). The test mixtures were shaken, then heated in a water bath at 95°C for 25 min and then cooled on ice water. Solutions were transferred to Eppendorf tubes containing 1 mL of 80% ethanol (for dilution). The solution was centrifuged at 5000 rpm for 10 min. Absorbance was scanned from 400-600 nm on a Cary 50 Bio UV-Visible scanning spectrophotometer (Varian, Mulgrave, Victoria) with Cary Win-UV scanning kinetics software version 2.0 for data collection. Absorbance at 440 nm were used for calculation of MDA (H). Bioassay of Glutathione: Glutathione consists of a linkage called as unusual peptide linkage that is between carboxyl group of glutamate side chain and amine group of cysteine. (Kretzschmar, 1990). Oxidation of reduced glutathione converts it into its oxidized form (Irwin et al. 1975). The glutathione bioassay was carried out through the use of Ellman’s method explained by Hashmat Ullah, et al. (2011). Various chemicals and reagents required for the bioassay of reduced glutathione inclue: Potassium dihydrogen phosphate (Merck), Glutathione (GSH) (Fluka), Sodium Hydroxide (Fluka), Ellman’s Reagent, (Sigma), HCl 35% (Kolchlight), Cadmium nitrate tetrahydrate (Aldrech), Distilled water Double refined distilled water, UV Visible spectrophotometer (UV-1601) (Schimadzu, Japan), pH meter: Nov:2010 model (Scientific Nova Company Ltd Korea), Balance Analytical model AX200 (Schimadzu, Japan), Magnetic Stirrer, Memmert oven model U-30854 (Schwabach, Germany), 50ml beakers (Pyrex Iwaki Glass, Japan) 200 µl, 500 µl, 1000 µl micro- pipette digital (Scorex Swiss Finland) were used in this research work. Preparation of Stock Solutions: 0.2M Phosphate buffer was prepared by taking 42.4 ml of 0.2M sodium hydroxide solution into a 250 ml volume metric flask, to this 50 ml of 0.2M Potassium dihydrogen phosphate solution was mixed and total volume was made 200 ml with quantity sufficient of distil water. 1mM of Glutathione (GSH) solution was prepared by taking/dissolving 15.375 milligram of L. Glutathione (GSH) in 50ml of 0.1 N hydrochloric acid solution. DTNB or 5,5-dithobes-2-nitrbenzoic acid 1mM solution was prepared by dissolving 19.8 mg into 50 ml of phosphate buffer pH 7.6. 2mM stock solution of cadmium nitrate tetra hydrate was prepared by dissolving 61.8 mg in 50 ml of distilled water. Processing For the Standard Graph of Reduced Glutathione: After preparing 1mM stock solution of GSH (Glutathione), its different concentrations (0.20 mM; 0.40 mM; 0.60 mM; 0.80 mM and 1.00 mM) were prepared and from each of the GSH


dilution 200µl was taken and added into 2300µl of 0.2 M phosphate buffer pH 7.6 then 500µl of 1mM 5,5-Dithiobis,2-nitro benzoic acid (DTNB) was added, these five mixtures were well shaken and incubated for five minutes. After incubations period, absorbance of each mixture was recorded at fixed wave length λmax: 412 nm. DTNB blank was prepared by adding 500µl DTNB (5,5-dithiobis-2-nitrobenzoic acid) to 2500µl phosphate buffer pH 7.6. Absorbance of DTNB was also taken at same fixed wavelength λmax: 412nm. By subtracting absorbance of DTBN blank from absorbance of each of the mixture, a real absorbance of each mixture was obtained as shown in table- 4. The readings of used concentration of glutathione in incubated mixture and the absorbance were accounted for plotting the graph and further calculations. The mathematical equation considered in the attempt was: y = 0.157 X + 0.006 and R2 = 0.999. Statistical analysis: For consistency in the results, each step in the study attempt was repeated for three times. The statistical parameters like mean; standard deviation (±) and percent change were calculated through the use of collected data. A one way analysis of variance (ANOVA) was used for the data analysis, using SPSS (Statistical Package for Social Sciences) (10.0) software. Significant differences between groups were detected in the ANOVA using Duncan’s Multiple Range Test at p values less than 0.05 and 0.001.

RESULTS AND DISCUSSION The results on the influence of methanolic extractives of leaves of mulberry, Morus alba (L) on the hypercholesterolemia in rat, Rattus norvegicus (L) are summarized in presented in table – 5; 6; 7; 8 and presented in Fig. RD.1; RD.2; RD.3 and RD.4. The results are explained away through the points like: Cholesterol level in blood plasma and it’s Post Mitochondrial Fraction (PMF); Triglyceride level in blood plasma and it’s Post Mitochondrial Fraction (PMF); Lipid Peroxidation (LPO) level in blood plasma and it’s Post Mitochondrial Fraction (PMF) and Glutathione level in blood plasma and it’s Post Mitochondrial Fraction (PMF). Cholesterol level in blood plasma and it’s Post Mitochondrial Fraction (PMF) (Table- 5 and Fig. RD.1): The contents of cholesterol in blood plasma and it’s Post Mitochondrial Fraction (PMF) in the experimental animals (rat, Rattus norvegicus L.) belong to group: [(A) water treated control group] was found measured 81.846 (±6.315) and 17.231 (±2.198) units (mmol/L) respectively. The contents of cholesterol in blood plasma and it’s Post Mitochondrial Fraction (PMF) in the experimental animals (rat, Rattus norvegicus L.) belong to group: [(B) Positive Control Group (MMLE) group] [treated with Methanolic Mulberry Leaf Extractives (MMLE)@100 mg/kg body weight] was found

Table 1. Experimental design for treating the extractives of leaves of mulberry, Morus alba (L) on the hypercholesterolemia in rat, Rattus norvegicus (L).

Serial No. Group Treatment

1. (A). Water Treated Control Water 2. (B). [MMLE] Positive Control Methanolic Mulberry Leaf Extractives (MMLE)@100 mg/kg body weight 3. (C).[Q Only] [email protected] g/kg body weight 4. (D). [CH Only] Cholesterol 5. (E). [MMLE (I) + CH] Methanolic Mulberry Leaf Extractives (MMLE)@100 mg/kg body weight and cholesterol. 6. (F). [MMLE (II) + CH] Methanolic Mulberry Leaf Extractives (MMLE)@200 mg/kg body weight and cholesterol. 7. (G). [CH + Q] [email protected] g/kg body weight and cholesterol.

Table 2. Dilutions of standard cholesterol solution for standard curve

Serial No. STD + Assay Buffer Total Volume (Microlitre) 10 x Conc. (mg/dL)

1. 100 Microlitre + 000 Microlitre 100 300 2. 080 Microlitre + 020 Microlitre 100 240 3. 060 Microlitre + 040 Microlitre 100 180 4. 040 Microlitre + 060 Microlitre 100 120 5. 030 Microlitre + 079 Microlitre 100 090 6. 020 Microlitre + 080 Microlitre 100 060 7. 010 Microlitre + 090 Microlitre 100 030 8. 000 Microlitre + 100 Microlitre 100 000

Table 3. Dilutions of standard Triglyceride solution for standard curve

Serial No. Standard + Distilled Water Total Volume Strength of Standard Triglyceride Solution (mmol/L)

1. 10 Microlitre + 990 Microlitre 1000 Microlitre 1.0 2. 06 Microlitre + 994 Microlitre 1000 Microlitre 0.6 3. 03 Microlitre + 997 Microlitre 1000 Microlitre 0.3 4. 00 Microlitre + 1000 Microlitre 1000 Microlitre 0.0

Table 4. Different concentrations of Glutathione plus 5, 5-dithiobis-2 -nitrobenzoic acid (DNTB) and Absorbance Readings

Serial No.

Used Concentration of Glutathione (GSH)

Final Concentration of Glutathione (GSH) in incubating mixture

First Absorbance

Second Absorbance

Third Absorbance

Mean Absorbance

Corrected Absorbance

1. 0.20 mM 13.33 µM 00.215 00.213 00.210 00.213 00.156 2. 0.40 mM 26.66 µM 00.365 00.362 00.360 00.362 00.365 3. 0.60 mM 40.00 µM 00.525 00.523 00.519 00.522 00.465 4 0.80 mM 53.32 µM 00.688 00.685 00.680 00.684 00.627 5. 1.00 mM 66.65 µM 00.845 00.841 00.838 00.841 00.784

Absorbance of 5,5-dithiobis- 2-nitrobenzoic acid (DTNB) blank solution was 0.057


measured 80.769 (±7.962) and 17.058 (±3.171)respectively. In comparison with the water treated control group, there was 01.316 percent reduction in cholesterol level in plasma and 1.005 percent reduction in cholesterol level in Post Mitochondrial Fraction (PMF) of plasma through treating the experimental animals (rat, Rattus norvegicusMethanolic Mulberry Leaf Extractives (MMLE)@100body weight. The contents of cholesterol in blood plasma and it’s Post Mitochondrial Fraction (PMF) in the experimental animals (rat, Rattus norvegicus L.) belong to group: [(C) Q. Only] [treated only with [email protected] g/kg body weight] was found measured 81.817 (±5.578) and units (mmol/L) respectively. Questran is the standard medicine used to reduce the lipid level in human body.

Table 5. Influence of Extractives of Leaves of Mulberry, Mitochondrial Fraction (PMF) In Hypercholesterolemic Rat,

Serial No. Group Blood Plasma Cholesterol level (mmol/L)

1. (A). Water Treated Control 81.846(±6.315)76.771

2. (B). Positive Control (MMLE)

80.769(±7.962)75.761

3. (C). Q. Only

81.817(±5.578)76.744

4. (D). CH.Only

106.61(±6.485)100.00

5. (E). MMLE [I] + CH

80.977(±7.819)75.956

6. (F). MMLE [II] + CH

77.786(±4.927)72.963

7. (G). CH + Q

75.624(±4.843)70.935

- Each figure is the mean of three replications. - Figures with ± sign in parentheses are the standard deviations.– Figures below standard deviations represent percent.-MMLE: Methanolic Mulberry Leaf Extractive; CH: Cholesterol; Q: Questran.

MMLE: Methanolic Mulberry Leaf Extractive; CH: Cholesterol; Q: Questran.

Fig. RD.1. Influence of Extractives of Leaves of Mulberry,

Fraction (PMF) In Hypercholesterolemic Rat,

0

20

40

60

80

100

120

W MMLE

81.846 80.769 81.817

17.231 17.058

410 International Journal of Recent Academic Research

(±3.171) units (mmol/L) respectively. In comparison with the water treated control group, there was 01.316 percent reduction in cholesterol level in plasma and 1.005 percent reduction in cholesterol level in Post Mitochondrial Fraction (PMF) of plasma through treating

Rattus norvegicus L.) of with Methanolic Mulberry Leaf Extractives (MMLE)@100 mg/kg

The contents of cholesterol in blood plasma and it’s Post Mitochondrial Fraction (PMF) in the experimental

L.) belong to group: [(C) Q. Only] [treated only with [email protected] g/kg body weight]

and 17.228 (±2.289) mmol/L) respectively. Questran is the standard medicine

ody.

In comparison with the individuals of water treated control group, administration of Quuestran was found resulted into 00.035 percent reduction and 00.099 percent reduction in cholesterol in plasma and in Post Mitochondrial Fraction (PMF) of plasma respectively.blood plasma and it’s Post Mitochondrial Fraction (PMF) in the experimental animals (rat, group: [(D) CH. Only] [treated with Chfound measured 106.61 (±6.485)(mmol/L) respectively. It is obvious to appear elevation in the level of cholesterol in both plasma and Post Mitochondrial Fraction (PMF) of plasma through treating the experimentanimals (rat, Rattus norvegicuscontents of cholesterol in blood plasma and it’s Post

Influence of Extractives of Leaves of Mulberry, Morus alba (L) on the levels of cholesterol in blood plasma and it’s Post Mitochondrial Fraction (PMF) In Hypercholesterolemic Rat, Rattus norvegicus

Blood Plasma Cholesterol level (mmol/L) Post Mitochondrial Fraction (PMF) Cholesterol (mmol/L) in Blood Plasma

81.846 (±6.315) 76.771

17.231 (±2.198) 57.145

80.769 (±7.962) 75.761

17.058 (±3.171) 56.571

81.817 (±5.578) 76.744

17.228 (±2.289) 57.135

106.61 (±6.485) 100.00

30.153 (±4.384) 100.00

80.977 (±7.819) 75.956

17.538 (±2.345) 57.343

77.786 (±4.927) 72.963

17.584 (±2.111) 58.315

75.624 (±4.843) 70.935

16.451 (±3.263) 54.558

sign in parentheses are the standard deviations. below standard deviations represent percent.



Influence of Extractives of Leaves of Mulberry, Morus alba (L) on the levels of cholesterol in blood plasma and it’s Post Mitochondrial Fraction (PMF) In Hypercholesterolemic Rat, Rattus norvegicus (L)

Q. Only CH. Only MMLE (I) + CH

MMLE (II) + CH

CH + Q

81.817

106.61

80.977 77.786 75.624

17.228

30.153

17.538 17.584 16.451

International Journal of Recent Academic Research, Vol. 01, Issue 07, pp.404-418, November, 2019

In comparison with the individuals of water treated control group, administration of Quuestran was found resulted into

percent reduction and 00.099 percent reduction in and in Post Mitochondrial Fraction

(PMF) of plasma respectively. The contents of cholesterol in blood plasma and it’s Post Mitochondrial Fraction (PMF) in the experimental animals (rat, Rattus norvegicus L.) belong to group: [(D) CH. Only] [treated with Cholesterol only] was

(±6.485) and 30.153 (±4.384) units mmol/L) respectively. It is obvious to appear elevation in the

level of cholesterol in both plasma and Post Mitochondrial Fraction (PMF) of plasma through treating the experimental

Rattus norvegicus L.) with cholesterol. The contents of cholesterol in blood plasma and it’s Post

(L) on the levels of cholesterol in blood plasma and it’s Post tus norvegicus (L)

Post Mitochondrial Fraction (PMF) Cholesterol (mmol/L)

.

(L) on the levels of cholesterol in blood plasma and it’s Post Mitochondrial

16.451

Plasma

PMF

, November, 2019

Mitochondrial Fraction (PMF) in the experimental animals (rat, Rattus norvegicus L.) belong to group: [(E) MMLE (I) + CH] [treated with Methanolic Mulberry Leaf Extractives (MMLE)@100 mg/kg body weight and cholesterol] was found measured 80.977 (±7.819) and 17.538 (±2.345) units (mmol/L) respectively. In comparison with the individuals of the group treated with Cholesterol only, there was 24.044 percent reduction in cholesterol level in plasma and 42.657 percent reduction in cholesterol level in Post Mitochondrial Fraction (PMF) of plasma through treating the experimental animals (rat, Rattus norvegicus L.) with methanolic mulberry leaf extractives (MMLE)@100 mg/kg body weight and cholesterol. The contents of cholesterol in blood plasma and it’s Post Mitochondrial Fraction (PMF) in the experimental animals (rat, Rattus norvegicus L.) belong to group: [(F) MMLE (II) + CH] [treated with Methanolic Mulberry Leaf Extractives (MMLE)@200 mg/kg body weight and cholesterol] was found measured 77.786 (±4.927) and 17.584 (±2.111) units (mmol/L) respectively. In comparison with the individuals in the group treated with Cholesterol only, there was 27.037 percent reduction in cholesterol level in plasma and 41.685 percent reduction in cholesterol level in Post Mitochondrial Fraction (PMF) of plasma through treating the experimental animals (rat, Rattus norvegicus L.) with methanolic mulberry leaf extractives (MMLE)@200 mg/kg body weight and cholesterol. The contents of cholesterol in blood plasma and it’s Post Mitochondrial Fraction (PMF) in the experimental animals (rat, Rattus norvegicus L.) belong to group: (G) [CH + Q] [treated with [email protected] g/kg body weight and cholesterol] was found measured 75.624 (±4.843) and 16.451 (±3.263) units (mmol/L) respectively. In comparison with the individuals in the group treated with Cholesterol only, there was 29.065 percent reduction in cholesterol level in plasma and 42.865 percent reduction in cholesterol level in Post Mitochondrial Fraction (PMF) of plasma through treating the experimental animals (rat, Rattus norvegicus L.) of with [email protected] g/kg body weight and cholesterol. Triglyceride level in blood plasma and it’s Post Mitochondrial Fraction (PMF) (Table 6 and Fig. RD.2): The contents of Triglyceride in blood plasma and it’s Post Mitochondrial Fraction (PMF) in the experimental animals (rat, Rattus norvegicus L.) belong to : group [(A) water treated control group] was found measured 22.089 (±2.465) and 05.632 (±0.371) units (mmol/L) respectively. The contents of Triglyceride in blood plasma and it’s Post Mitochondrial Fraction (PMF) in the experimental animals (rat, Rattus norvegicus L.) belong to group: [(B) Positive Control (MLE)] [treated with Methanolic Mulberry Leaf Extractives (MMLE)@100 mg/kg body weight] was found measured 21.827 (±4.962) and 05.629 (±1.213) units (mmol/L) respectively. In comparison with the individuals of water treated control group, there was 01.186 percent decrease in Triglyceride level in plasma and 00.053 percent decrease in Triglyceride level in Post Mitochondrial Fraction (PMF) of plasma through treating the experimental animals (rat, Rattus norvegicus L.) of with Methanolic Mulberry Leaf Extractives (MMLE)@100 mg/kg body weight. The contents of Triglyceride in blood plasma and it’s Post Mitochondrial Fraction (PMF) in the experimental animals (rat, Rattus norvegicus L.) belong to group: [(C) Q. Only] [treated with [email protected] g/kg body weight] was found measured 18.718 (±3.617) and 05.435 (±1.641) units (mmol/L) respectively. In comparison with the individuals of water treated control group, there was 15.260 percent reduction in Triglyceride level in

plasma and 03.497 percent reduction in Triglyceride level in Post Mitochondrial Fraction (PMF) of plasma through treating the experimental animals (rat, Rattus norvegicus L.) of with [email protected] g/kg body weight and cholesterol. The contents of Triglyceride in blood plasma and it’s Post Mitochondrial Fraction (PMF) in the experimental animals (rat, Rattus norvegicus L.) belong to group: [(D) CH. Only] [treated with Cholesterol] was found measured 36.786 (±5.536) and 08.456 (±1.613) units (mmol/L) respectively. In comparison with the individuals of water treated group, there was 66.535 percent elevation in Triglyceride level in plasma and 50.142 percent elevation in Triglyceride level in Post Mitochondrial Fraction (PMF) of plasma through treating the experimental animals (rat, Rattus norvegicus L.) with cholesterol. The contents of Triglyceride in blood plasma and it’s Post Mitochondrial Fraction (PMF) in the experimental animals (rat, Rattus norvegicus L.) belong to group: [(E) MLE (I) + CH] [treated with Methanolic Mulberry Leaf Extractives (MMLE)@100 mg/kg body weight and cholesterol] was found measured 31.124 (±5.113) and 07.922 (±0.867) units (mmol/L) respectively. In comparison with the individuals of the group treated with cholesterol only, there was 15.391 percent reduction in Triglyceride level in plasma and 6.315 percent reduction in triglyceride level in Post Mitochondrial Fraction (PMF) of plasma through treating the experimental animals (rat, Rattus norvegicus L.) with methanolic mulberry leaf extractives (MMLE)@100 mg/kg body weight and cholesterol. The contents of Triglyceride in blood plasma and it’s Post Mitochondrial Fraction (PMF) in the experimental animals (rat, Rattus norvegicus L.) belong to group: [(F) MLE (II) + CH] [treated with Methanolic Mulberry Leaf Extractives (MMLE)@200 mg/kg body weight and cholesterol] was found measured 24.387 (±3.349) and 07.519 (±1.143) units (mmol/L) respectively. In comparison with the individuals of the group treated with cholesterol only, there was 33.605 percent decrease in cholesterol level in plasma and 11.080 percent decrease in Triglyceride level in Post Mitochondrial Fraction (PMF) of plasma through treating the experimental animals (rat, Rattus norvegicus L.) with methanolic mulberry leaf extractives (MMLE)@200 mg/kg body weight and cholesterol. The contents of Triglyceride in blood plasma and it’s Post Mitochondrial Fraction (PMF) in the experimental animals (rat, Rattus norvegicus L.) belong to group: [(G) CH + Q] [treated with [email protected] g/kg body weight and cholesterol] was found measured 22.684 (±4.631) and 07.624 (±1.897) 16.451 (±3.263) units (mmol/L) respectively. In comparison with the individuals of the group treated with cholesterol only, there was 38.335 percent reduction in Triglyceride level in plasma and 9.839 percent reduction in Triglyceride level in Post Mitochondrial Fraction (PMF) of plasma through treating the experimental animals (rat, Rattus norvegicus L.) of with [email protected] g/kg body weight and cholesterol. Lipid Peroxidation (LPO) level in blood plasma and it’s Post Mitochondrial Fraction (PMF) (Table-7 and Fig. RD. 3): Lipid peroxidation is the degeneracy of the lipid through oxidation. The free radicals steal the electrons from the lipids of cell membrane and results into damage of cell membrane. Up to some extent, Vitamin C and Vitamin E stop the lipid peroxidation in cell. The toxicity of lipid hydroperoxides to animals is best illustrated by the lethal phenotype of glutathione peroxidase 4 (GPX4) knockout mice.


These animals do not survive past embryonic day 8, indicating that the removal of lipid hydroperoxides is essentialmammalian life (Muller, et al., 2007). The levels of Lipid Peroxidation (LPO) in blood plasma and it’s Post Mitochondrial Fraction (PMF) in the experimental animals (rat, Rattus norvegicus L.) belong to the group: [(A)] [water treated control group] was found measured 09.615 and 04.232 (±0.894) units (mmol/L) respectively.Lipid Peroxidation (LPO) in blood plasma and it’s Post Mitochondrial Fraction (PMF) in the experimental an(rat, Rattus norvegicus L.) belong to the group: [(B) Positive Control (MMLE)] [treated with Methanolic Mulberry Leaf Extractives (MMLE)@100 mg/kg body weightmeasured 09.518 (±1.742) and 04.189 (±0.786)respectively. In comparison with the individuals of the water treated group, there was 01.008 percent decrease in Lipid Peroxidation (LPO) level in plasma and 01.016

Table 6. Influence of Extractives of Leaves of Mulberry, Mitochondrial Fraction (PMF) In Hypercholesterolemic Rat,

Serial No. Group Blood Plasma Triglyceride level (m

1. (A). Water Treated Control

22.089(±2.465)60.158

2. (B). Positive Control (MMLE)

21.827(±4.962)59.335

3. (C). Q. Only

18.718(±3.617)50.883

4. (D). CH.Only

36.786(±5.536)100

5. (E). MMLE [I] + CH

31.124(±5.113)84.608


24.387(±3.349)66.294

7. (G). CH + Q

22.684(±4.631)61.664

- Each figure is the mean of three replications. - Figures with ± sign in parentheses are the standard deviations.– Figures below standard deviations represent percent.-MMLE: Methanolic Mulberry Leaf Extractive; CH: Cholesterol; Q: Questran.


Fig. RD.2: Influence of Extractives of Leaves of Mulberry, Mitochondrial Fraction (PMF) In Hypercholesterolemic Rat,

0

5

10

15

20

25

30

35

40

W MMLE. Only


These animals do not survive past embryonic day 8, indicating that the removal of lipid hydroperoxides is essential for

The levels of Lipid Peroxidation (LPO) in blood plasma and it’s Post Mitochondrial Fraction (PMF) in the experimental animals

g to the group: [(A)] [water treated control group] was found measured 09.615 (±1.537)

mmol/L) respectively. The levels of Lipid Peroxidation (LPO) in blood plasma and it’s Post Mitochondrial Fraction (PMF) in the experimental animals

L.) belong to the group: [(B) Positive Control (MMLE)] [treated with Methanolic Mulberry Leaf

mg/kg body weight] was found (±0.786) units (mmol/L)

comparison with the individuals of the water percent decrease in Lipid

01.016 percent decrease

in Lipid Peroxidation (LPO) level in Post Mitochondrial Fraction (PMF) of plasma through treating the experimental animals (rat, Rattus norvegicusMulberry Leaf Extractives (MMLE)@100The levels of Lipid Peroxidationit’s Post Mitochondrial Fraction (PMF) in the experimental animals (rat, Rattus norvegicusOnly] [treated with [email protected] g/kg body weight] was found measured 09.231 (±1.384)(mmol/L) respectively. In comparison with the individuals of the water treated group, there was 03.993 percent reduction in Lipid Peroxidation (LPO) level in plasma and 04,017 percent decrease in Lipid Peroxidation (LPO) level in Post Mitochondrial Fraction (PMF) of plasma through treating the experimental animals (rat, Rattus [email protected] g/kg body weight and cholesterol.of Lipid Peroxidation (LPO) in blood plasma and it’s Post

Influence of Extractives of Leaves of Mulberry, Morus alba (L) on the levels of Triglyceride in blood plasma and it’s Post Mitochondrial Fraction (PMF) In Hypercholesterolemic Rat, Rattus norvegicus

Blood Plasma Triglyceride level (mmol/L) Post Mitochondrial Fraction (PMF) Triglyceride (mmol/L) in Blood Plasma

22.089 (±2.465) 60.158

05.632 (±0.371) 66.603

21.827 (±4.962) 59.335

05.629 (±1.213) 66.568

18.718 (±3.617) 50.883

05.435 (±1.641) 60.273

36.786 (±5.536) 100

08.456 (±1.613) 100

31.124 (±5.113) 84.608

07.922 (±0.867) 93.684

24.387 (±3.349) 66.294

07.519 (±1.143) 88.919

22.684 (±4.631) 61.664

07.324 (±1.897) 86.613

sign in parentheses are the standard deviations. Figures below standard deviations represent percent.

Extractive; CH: Cholesterol; Q: Questran.


Fig. RD.2: Influence of Extractives of Leaves of Mulberry, Morus alba (L) on the levels of Triglyceride in blood plasma and it’s Post Mitochondrial Fraction (PMF) In Hypercholesterolemic Rat, Rattus norvegicus

MMLE. Q Only CH Only

MMLE (I) + CH

MMLE (II) + CH

CH + Q


Lipid Peroxidation (LPO) level in Post Mitochondrial Fraction (PMF) of plasma through treating the experimental

Rattus norvegicus L.) of with Methanolic Mulberry Leaf Extractives (MMLE)@100 mg/kg body weight. The levels of Lipid Peroxidation (LPO) in blood plasma and it’s Post Mitochondrial Fraction (PMF) in the experimental

Rattus norvegicus L.) belong to group: [(C) Q. Only] [treated with [email protected] g/kg body weight] was

(±1.384) and 04.916 (±1.217) units mmol/L) respectively. In comparison with the individuals of

the water treated group, there was 03.993 percent reduction in Lipid Peroxidation (LPO) level in plasma and 04,017 percent decrease in Lipid Peroxidation (LPO) level in Post

ction (PMF) of plasma through treating the Rattus norvegicus L.) of with

[email protected] g/kg body weight and cholesterol. The levels of Lipid Peroxidation (LPO) in blood plasma and it’s Post

(L) on the levels of Triglyceride in blood plasma and it’s Post Rattus norvegicus (L)

Post Mitochondrial Fraction (PMF) Triglyceride (mmol/L) in Blood Plasma

.

(L) on the levels of Triglyceride in blood plasma and it’s Post Rattus norvegicus (L)

Plasma

PMF

, November, 2019

Mitochondrial Fraction (PMF) in the experimental animals (rat, Rattus norvegicus L.) belong to (D) CH. Only [treated with Cholesterol] was found measured 18.076 (±2.641) and 06.923 (±1.577) units (mmol/L) respectively. In comparison with the individuals of the water treated group, there was 87.99 percent elevation in Lipid Peroxidation (LPO) level in plasma and 63.58 percent elevation in Lipid Peroxidation (LPO) level in Post Mitochondrial Fraction (PMF) of plasma through treating the experimental animals (rat, Rattus norvegicus L.) with cholesterol. The levels of Lipid Peroxidation (LPO) in blood plasma and it’s Post Mitochondrial Fraction (PMF) in the experimental animals (rat, Rattus norvegicus L.) belong to the group: [(E) MLE (I) + CH] [treated with Methanolic Mulberry Leaf Extractives (MMLE)@100 mg/kg body weight and cholesterol] was found measured 11.923 (±1.254) and 04.246 (±0.931) units (mmol/L) respectively. In comparison with the individuals of the group treated with Cholesterol (only), there was 34.039 percent reduction in levels of Lipid Peroxidation (LPO) in plasma and 38.668 percent reduction in Lipid Peroxidation (LPO) level in Post Mitochondrial Fraction (PMF) of plasma through treating the experimental animals (rat, Rattus norvegicus L.) with methanolic mulberry leaf extractives (MMLE)@100 mg/kg body weight and cholesterol. The levels of Lipid Peroxidation (LPO) in blood plasma and it’s Post Mitochondrial Fraction (PMF) in the experimental animals (rat, Rattus norvegicus L.) belong to (F) MLE [II] + CH [treated with Methanolic Mulberry Leaf Extractives (MMLE)@200 mg/kg body weight and cholesterol] was found measured 10.786 (±1.639) and 03.846 (±0.857) units (mmol/L) respectively. In comparison with the individuals of the group treated with Cholesterol (only), there was 40.329 percent decrease in Lipid Peroxidation (LPO) level in plasma and 44.446 percent decrease in Lipid Peroxidation (LPO) level in Post Mitochondrial Fraction (PMF) of plasma through treating the experimental animals (rat, Rattus norvegicus L.) with methanolic mulberry leaf extractives (MMLE)@200 mg/kg body weight and cholesterol. The levels of Lipid Peroxidation (LPO) in blood plasma and it’s Post Mitochondrial Fraction (PMF) in the experimental animals (rat, Rattus norvegicus L.) belong to (G) CH + Q [treated with [email protected] g/kg body weight and cholesterol] was found measured 10.201 (±2.416) and 05.415 (±1.613) units (mmol/L) respectively. In comparison with the individuals of the group treated with Cholesterol (only), there was 43.567 percent decrease in Lipid Peroxidation (LPO) level in plasma and 21.782 percent decrease in Lipid Peroxidation (LPO) level in Post Mitochondrial Fraction (PMF) of plasma through treating the experimental animals (rat, Rattus norvegicus L.) of with [email protected] g/kg body weight and cholesterol. Glutathione level in blood plasma and it’s Post Mitochondrial Fraction (PMF) (Table- 8 and Fig. RD. 4): The glutathione (GSH) is recognized as antioxidant. It is present in most of the plants, animals, fungi, bacteria and archaea. It deserve capability of preventing the cellular damage caused through the reactive oxygen species (ROS) (such as free radicals, peroxides, lipid peroxides, and heavy metals). The levels of reduced Glutathione in blood plasma and it’s Post Mitochondrial Fraction (PMF) in the experimental animals (rat, Rattus norvegicus L.) belong to group: [(A) water treated control group] was found measured 76.086 (±6.764) and 39.132 (±4.456) units (mmol/L) respectively. Both the readings were considered for comparison with other groups in the attempt. The levels of reduced Glutathione in blood plasma and it’s Post Mitochondrial Fraction (PMF) in the

experimental animals (rat, Rattus norvegicus L.) belong to group: [(B) Positive Control (MMLE)] [treated with Methanolic Mulberry Leaf Extractives (MMMLE)@100 mg/kg body weight ] was found measured 109.81 (±11.095) and 42.283 (±4.768) units (mmol/L) respectively. In comparison with the individuals of water treated group, there was 44.32 percent increase in reduced Glutathione level in plasma and 08.05 percent increase in reduced Glutathione level in Post Mitochondrial Fraction (PMF) of plasma through treating the experimental animals (Positive Control Group) (rat, Rattus norvegicus L.) of with Methanolic Mulberry Leaf Extractives (MMLE)@100 mg/kg body weight. The levels of reduced Glutathione in blood plasma and it’s Post Mitochondrial Fraction (PMF) in the experimental animals (rat, Rattus norvegicus L.) belong to group: [(C) Q. Only] [treated with [email protected] g/kg body weight] was found measured 126.23 (±16.467) and 45.835 (±7.119) units (mmol/L) respectively. In comparison with the individuals of water treated group, there was 64.34 percent increase in reduced Glutathione level in plasma and 17.170 percent increase in reduced Glutathione level in Post Mitochondrial Fraction (PMF) of plasma through treating the experimental animals (rat, Rattus norvegicus L.) of with [email protected] g/kg body weight and cholesterol. The levels of Glutathione in blood plasma and it’s Post Mitochondrial Fraction (PMF) in the experimental animals (rat, Rattus norvegicus L.) belong to group: [(D) CH. Only] [treated with Cholesterol] was found measured 59.826 (±13.024) and 21.369 (±2.451) units (mmol/L) respectively. In comparison with the individuals of water treated group, there was 21.380 percent reduction in reduced glutathione level in plasma and 45.393 percent reduction in reduced glutathione level in Post Mitochondrial Fraction (PMF) of plasma through treating the experimental animals (rat, Rattus norvegicus L.) with cholesterol. The dietary cholesterol induce decrease in the level of glutathione in rat, Rattus norvegicus ( L.). The levels of reduced Glutathione in blood plasma and it’s Post Mitochondrial Fraction (PMF) in the experimental animals (rat, Rattus norvegicus L.) belong to group: [(E) MLE (I) + CH] [treated with Methanolic Mulberry Leaf Extractives (MMLE)@100 mg/kg body weight and cholesterol] was found measured 97.591(±23.508) and 48.621 (±11.163) units (mmol/L) respectively. In comparison with the individuals of water treated group, there was 28.240 percent elevation in levels of reduced Glutathione in plasma and 24.240 percent elevation in reduced Glutathione level in Post Mitochondrial Fraction (PMF) of plasma through treating the experimental animals (rat, Rattus norvegicus L.) with methanolic mulberry leaf extractives (MMLE)@100 mg/kg body weight and cholesterol. In comparison with the individuals of the group: [(D) CH. Only] [treated with Cholesterol], there was 63.124 percent increase in levels of reduced Glutathione in plasma and 127.53 percent increase in reduced Glutathione level in Post Mitochondrial Fraction (PMF) of plasma through treating the experimental animals (rat, Rattus norvegicus L.) with methanolic mulberry leaf extractives (MMLE)@100 mg/kg body weight and cholesterol. The levels of reduced Glutathione in blood plasma and it’s Post Mitochondrial Fraction (PMF) in the experimental animals (rat, Rattus norvegicus L.) belong to (F) MLE (II) + CH [treated with Methanolic Mulberry Leaf Extractives (MMLE)@200 mg/kg body weight and cholesterol] was found measured 129.58 (±36.115) and 52.554 (±13.024) units (mmol/L) respectively.


In comparison with the individuals of water treated group, there was 70.300 percent increase in reduced Glutathione level in plasma and 34.290 percent increase in reduced Glutathione level in Post Mitochondrial Fraction (PMF) of plasma through treating the experimental animals (rat, Rattus norvegicuswith methanolic mulberry leaf extractives (MMLE)@200mg/kg body weight and cholesterol. In comparison with the individuals of the group: [(D) CH. Only] [treated with Cholesterol], there was 116.59 percent increase in levels of reduced Glutathione in plasma and 145.93 percent increareduced Glutathione level in Post Mitochondrial Fraction (PMF) of plasma through treating the experimental animals (rat, Rattus norvegicus L.) with methanolic mulberry leaf extractives (MMLE)@200 mg/kg body weightThe cholesterol-induced decrease in reduced glutathioneof hypercholesterolemic rats were significantly (p less than

Table 7. Influence of Extractives of Leaves of Mulberry, and it’s Post Mitochondrial Fraction (PMF) In Hypercholesterolemic Rat,

Serial No.

Group Lipid Peroxidation (LPO) (mmol/L) in Blood Plasma


09.615 (±1.537) 53.192

2. (B). Positive Control (MLE)

09.518 (±1.254) 52.655

3. (C). Q. Only

09.231 (±1.384) 51.067

4. (D). CH.Only

18.076 (±2.641) 100

5. (E). MMLE [I] + CH

11.923 (±1.254) 59.702


10.786 (±1.639) 65.960

7 (G). CH + Q

10.201 (±2.416) 56.433

- Each figure is the mean of three replications. - Figures with ± sign in parentheses are the standard deviations.– Figures below standard deviations represent percent.-MMLE: Methanolic Mulberry Leaf Extractive; CH: Cholesterol;


Fig. RD.3. Influence of Extractives of Leaves of Mulberry, and it’s Post Mitochondrial Fract

0

2

4

6

8

10

12

14

16

18

20

W MMLE


In comparison with the individuals of water treated group, percent increase in reduced Glutathione level

in plasma and 34.290 percent increase in reduced Glutathione level in Post Mitochondrial Fraction (PMF) of plasma through

Rattus norvegicus L.) eaf extractives (MMLE)@200

In comparison with the individuals of the group: [(D) CH. Only] [treated with

was 116.59 percent increase in levels of percent increase in

reduced Glutathione level in Post Mitochondrial Fraction (PMF) of plasma through treating the experimental animals

L.) with methanolic mulberry leaf mg/kg body weight and cholesterol.

uced decrease in reduced glutathione levels of hypercholesterolemic rats were significantly (p less than

0.05) ameliorated in animals treated with both the dosages [(MMLE)@100 mg/kg body weightleaf extractives (MMLE) and [(MMLE)@200weight of methanolic mulberry leaf extractives (MMLE)].levels of reduced Glutathione in blood plasma and it’s Post Mitochondrial Fraction (PMF) in the experimental animals (rat, Rattus norvegicus L.) belong to group: [(G) CH + Q] [treated with [email protected] g/kg body weight and cholesterol] was found measured (±12.694) units (mmole/L) respectively.the individuals of water treated percent increase in reduced Glutathione level in plasma and 34.590 percent increase in reduced Glutathione level in Post Mitochondrial Fraction (PMF) of plasma through treating the experimental animals (rat, [email protected] g/kg body weight and cholesterol.

Influence of Extractives of Leaves of Mulberry, Morus alba (L) on the levels of Lipid Peroxidation (LPO) in blood plasma and it’s Post Mitochondrial Fraction (PMF) In Hypercholesterolemic Rat, Rattus norvegicus

Lipid Peroxidation (LPO) (mmol/L) in Lipid Peroxidation (LPO) (mmol/L) in Post Mitochondrial Fraction (PMF) of Blood Plasma

04.232 (±0.894) 61.129 04.189 (±0.931) 60.508 04.062 (±1.217) 58.673 06.923 (±1.577) 100 04.246 (±0.931) 61.331 03.846 (±0.857) 55.553 05.415 (±1.613) 78.217

sign in parentheses are the standard deviations. Figures below standard deviations represent percent.



Fig. RD.3. Influence of Extractives of Leaves of Mulberry, Morus alba (L) on the levels of Lipid Peroxidation (LPO) in blood plasma and it’s Post Mitochondrial Fraction (PMF) In Hypercholesterolemic Rat, Rattus norvegicus

Q Only CH Only MMLE (I) + CH

MMLE (II) + CH

CH + Q


0.05) ameliorated in animals treated with both the dosages mg/kg body weight of methanolic mulberry

leaf extractives (MMLE) and [(MMLE)@200 mg/kg body of methanolic mulberry leaf extractives (MMLE)]. The

levels of reduced Glutathione in blood plasma and it’s Post Mitochondrial Fraction (PMF) in the experimental animals

L.) belong to group: [(G) CH + Q] [treated with [email protected] g/kg body weight and cholesterol] was found measured 129.63 (±47.786) and 53.843

mmole/L) respectively. In comparison with the individuals of water treated group, there was 70.370 percent increase in reduced Glutathione level in plasma and 34.590 percent increase in reduced Glutathione level in Post Mitochondrial Fraction (PMF) of plasma through treating the experimental animals (rat, Rattus norvegicus L.) with [email protected] g/kg body weight and cholesterol.

(L) on the levels of Lipid Peroxidation (LPO) in blood plasma Rattus norvegicus (L)

L) in Post Mitochondrial Fraction

.

(L) on the levels of Lipid Peroxidation (LPO) in blood plasma Rattus norvegicus (L)

Plasma

PMF

, November, 2019

In comparison with the individuals of the group: [(D) CH. Only] [treated with Cholesterol], there was 116.67 percent increase in levels of reduced Glutathione in plasma and 151.96 percent increase in reduced Glutathione level in Post Mitochondrial Fraction (PMF) of plasma through treating the experimental animals (rat, Rattus [email protected] g/kg body weight and cholesterolto Lipid Research Clinics Program (LRCP) (1984), the presence of a high level of cholesterol in the diet has been demonstrated to elevate plasma cholesterol. This condition is responsible to increase the possibility of aortic atherosclerosis. Kwiterovich (1997) mentioned that, the diet therapy for regulation cholesterol is exerting influence to reduce subsequent CVD- associated mortality and morbidity. The therapeutic advantages of herbal food materials hafocus of many extensive dietary attempts (Yokozawa 2006; Zhang et al 2007).

Table 8. Influence of Extractives of Leaves of Mulberry, it’s Post Mitochondrial Fraction (PMF) In Hypercholesterolemic Rat,

Serial No.

Group Blood Plasma Triglyceride level (mmol/L)


76.086 (±6.764) 100.000

2. (B). Positive Control (MLE)

109.81 (±11.095) 144.32

3. (C). Q. Only

126.23 (±16.467) 164.34

4. (D). CH. Only

59.826 (±13.024) 078.62

5. (E). MLE [I] + CH

97.591 (±23.508) 128.24

6. (F). MLE [II] + CH

129.58 (±36.115) 170.30

7. (G). CH + Q

129.63 (±47.786) 170.37

- Each figure is the mean of three replications. - Figures with ± sign in parentheses are the standard deviations.– Figures below standard deviations represent percent. -MMLE: Methanolic Mulberry Leaf Extractive; CH: Cholesterol; Q: Questran.


Fig . RD.4. Influence of Extractives of Leaves of Mulberry, it’s Post Mitochondrial Fraction (PMF) In Hypercholesterolemic Rat,

0

20

40

60

80

100

120

140

W MMLE

76.086

109.81

39.132 42.283


In comparison with the individuals of the group: [(D) CH. was 116.67 percent

increase in levels of reduced Glutathione in plasma and 151.96 percent increase in reduced Glutathione level in Post Mitochondrial Fraction (PMF) of plasma through treating the

Rattus norvegicus L.) with [email protected] g/kg body weight and cholesterol. According

to Lipid Research Clinics Program (LRCP) (1984), the presence of a high level of cholesterol in the diet has been demonstrated to elevate plasma cholesterol. This condition is

possibility of aortic atherosclerosis. Kwiterovich (1997) mentioned that, the diet therapy for regulation cholesterol is exerting influence to reduce

mortality and morbidity. The therapeutic advantages of herbal food materials have been the

of many extensive dietary attempts (Yokozawa et al

In the present attempt reporting the lipidmethanolic leaf extractives of mulberry, rats fed on high cholesterol diet for a period of nine weeks. The experimental animals intubated with dietary cholesterol reported higher level of plasmacholesterol and triglyceride thanmulberry leaf extractives (MMLE). The reduction in plasma cholesterol level through the mulberry leaf extractives (MMLE) treatment in the present attempt found dose dependent. According to Fungwe,cholesterol reduce fatty acid oxidation, which, inincreases the levels of hepatic and plasma triglycerol.findings of Adaramoye and colleagues (2005) andand colleagues (2006) appear to be parallel with present attempt. The results of present attempt sugMulberry Leaf Extractives (MMLE) at the rate of two hundred milligram per kilogram body weight is responsible for

Leaves of Mulberry, Morus alba (L) on the levels of Reduced Glutathione in blood plasma and it’s Post Mitochondrial Fraction (PMF) In Hypercholesterolemic Rat, Rattus norvegicus

Blood Plasma Triglyceride level Post Mitochondrial Fraction (PMF) Triglyceride (mmol/L) in Blood Plasma

39.132 (±4.456) 100.000 42.283 (±4.768) 108.05 45.835 (±7.119) 117.17 21.369 (±2.451) 054.607 48.621 (±11.163) 124.24 52.554 (±13.024) 134.29 53.843 (±12.694) 137.59

sign in parentheses are the standard deviations.



Influence of Extractives of Leaves of Mulberry, Morus alba (L) on the levels of Reduced Glutathione in blood plasma and it’s Post Mitochondrial Fraction (PMF) In Hypercholesterolemic Rat, Rattus norvegicus

MMLE Q only CH only MMLE (I) + CH

MMLE (II) + CH

CH + Q

126.23

59.826

97.591

129.58 129.63

42.283 45.835

21.369

48.621 52.554 53.843


In the present attempt reporting the lipid-lowering influence of methanolic leaf extractives of mulberry, Morus alba (L) in the rats fed on high cholesterol diet for a period of nine weeks. The experimental animals intubated with dietary cholesterol

higher level of plasma and hepatic levels of total cholesterol and triglyceride than the animals treated with mulberry leaf extractives (MMLE). The reduction in plasma cholesterol level through the mulberry leaf extractives (MMLE) treatment in the present attempt found dose dependent. According to Fungwe, et al. (1993), the dietary

fatty acid oxidation, which, in turn, increases the levels of hepatic and plasma triglycerol. The findings of Adaramoye and colleagues (2005) and Yokozawa and colleagues (2006) appear to be parallel with present

The results of present attempt suggest that Methanolic Mulberry Leaf Extractives (MMLE) at the rate of two hundred milligram per kilogram body weight is responsible for

(L) on the levels of Reduced Glutathione in blood plasma and us norvegicus (L)

Post Mitochondrial Fraction (PMF) Triglyceride (mmol/L) in Blood

.

(L) on the levels of Reduced Glutathione in blood plasma and Rattus norvegicus (L)

Plasma

PMF

, November, 2019

reduction in biosynthesis of hepatic triglyceride and the redistribution of cholesterol among the molecules of lipoproteins. The oxidative stress is distinguished by disruption of the balance between oxidative and antioxidative processes. According to Yokozawa, et al. (2006), the oxidative status of the body plays an important role in the pathogenesis of atherosclerosis. The diet with rich cholesterol is responsible for accelerative rate of peroxidation of the lipids. It is followed by hypercholesterolemia (Adaramoye, et al., 2005). The relationship between oxidative stress and cholesterol level should be confirmed through the level reduced glutathione. The reduced glutathione is an indicator of antioxidant defense, exhibit a negative correlation with the hepatic total cholesterol levels of hypercholesterolemic rats. The positive correlation with the cholesterol levels of extract pre-treated rats. Therefore, methanolic mulberry leaf extractives (MMLF) may elicit some health benefits through the modulation of physiologic functions including the atherogenic lipid profile. Evidence from the present attempt confirms the lipid-lowering effects of mulberry, Morus alba (L) in rats fed on high cholesterol diet. The methanolic mulberry leaf extractives (MMLF) at 200 mg/kg was effective in reducing the levels of plasma and PMF total cholesterol. as well as low-density lipoprotein Further studies are warranted to determine the exact component in methanolic mulberry leaf extractives (MMLF) responsible for the observed effect and, such component may be a mulberry derived herbal medicine for use as a prophylactic agent against hypercholesterolemia. Conclusion The contents of cholesterol in blood plasma and it’s Post Mitochondrial Fraction (PMF) in the experimental animals (rat, Rattus norvegicus L.) belong to group: [(E) MMLE (I) + CH] [treated with Methanolic Mulberry Leaf Extractives (MMLE)@100 mg/kg body weight and cholesterol] was found measured 80.977 (±7.819) and 17.538 (±2.345) units (mmol/L) respectively. In comparison with the individuals of the group treated with Cholesterol only, there was 24.044 percent reduction in cholesterol level in plasma and 42.657 percent reduction in cholesterol level in Post Mitochondrial Fraction (PMF) of plasma through treating the experimental animals (rat, Rattus norvegicus L.) with methanolic mulberry leaf extractives (MMLE)@100 mg/kg body weight and cholesterol. Mulberry plant is one of the traditional herbs which are used in medicine from centuries before. Due to its pharmacological properties mulberry is used as medicine currently in many countries. Mulberry is proved in protecting liver, improving eyesight, facilitating discharge of urine, lowering of blood pressure, anti-diabetic and controlling weight in humans as well as animal model. Acknowledgement: 12 December is the birthday of Hon. Sharad Govind Pawar (Indian leader from Maharashtra with many years of public service). With the best compliments from International Journal of Recent Academic Research (IJRAR), the present attempt is wishing Hon. Sharad Govind Pawar Happy Birthday. The major research project on “Influence of Methanolic Extractives of Leaves of Mulberry, Morus alba (L) on the Hypercholesterolemia in Rat, Rattus norvegicus (L)” would not have been possible without the financial support of the Minister, Food Processing and Industries, Government of India. The authors indebted to International Science Community Association and Science Association, Shardabai Pawar Mahila Mahavidyalaya, Shardanagar Tal. Baramati

Dist. Pune – 413115 (India). Supportive inspiration from Hon. Editor, International Journal of Recent Academic Research (IJRAR) deserve appreciations. … and … and exert a grand salutary influence.

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