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Full Project – INVITRO ANTI-OXIDANT CAPACITY OF YOYO BITTERS AND ITS NUTRITIONAL EFFECTS ON MALE ALBINO WISTAR RATS

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ABSTRACT

In order to investigate invitro antioxidant capacity of yoyo bitters and its nutritional effect on the weight of male albino wistar rats. Sixteen(16) rats weighing (163±2.00g) were randomly picked into two equal group; (Group A and B). The rats were acclimatized for 7days, after which 1ml 0f yoyo bitters were administered once daily to group A respectively for the period of 28days while group B were received distilled water which serve as control. The feed was weighed throughout the period of the experiment. Each group were given 300g of mash grower feed daily and animals were also weighed weekly. At the end of 4week, a significant increase was observed in the weight of the test rat (208.08±9.37g) compared to control (192.69±10.20g). The relative weight of the following organ: Liver, Kidney, Heart, Lungs, Spleen, Testes and Pancreas were observed and its was statistically significant (P<0.05). The total antioxidant capacity was compare using T test, the value of the test and control are statistically significant (P<0.05), Hydroxyl free radical n al activity (P<0.05) and DPPH scavenging activity (P<0.05).

CHAPTER ONE

1.0INTRODUTION

1.1HERBAL BITTERS

Herbal medicine is “the knowledge, skills and practice based on the theories, beliefs and experiences indigenous to different cultures, used in maintenance of health and in the prevention, diagnosis, improvement or treatment of physical and mental illness” (W.H.O 2005). There are different systems of herbal medicine and the philosophy and practices of each are influenced by the prevailing conditions, environment, and geographic area within which it first evolved (W.H.O 2005), however, a common philosophy is a holistic approach to life, equilibrium of the mind, body and the environment and an emphasis on health rather than on diseases. Generally, the focus is on the overall condition of the individual rather than the particular ailment or disease from which the patient is suffering and the use of herbs is a core part of all system of traditional medicine. (Ergebreten 2002; Conboy et al., 2007; Rishon 2008, Schmidt et al., 2008).

Over the past 100, the development and mass production of chemical synthesized drugs have revolutionized healthcare in most part of the world. However, large section of the population in developing countries still rely on traditional practitioners and herbal medicine for primary care. In Africa, up to 90% and in India, 70% of their population depend on herbal medicine to help meet their healthcare needs. In China, herbal medicine account for around 40% of all healthcares delivered and more than 90% of general hospitals in China have unit for herbal medicine (W.H.O 2005). However, use of herbal medicine is not limited to developing countries, and during the past two decade public interest in natural therapies has increased greatly in industrialized countries, with expanding use of ethnobonicals. In the united state, in 2007, about 38% of adult and 12% of children were using some form of herbal medicine (Ernst, Schmint and Wider; Barnes, Bloom, and Nahim).

The most common reasons for using herbal medicine are that, it is more affordable; more closely corresponds to patients idealogy, allays concerns about the adverse effects of chemical (synthetic) medicine, satisfies a desire for more personalized healthcare, and allows greater public access to health information. The major use of herbal medicine is for health promotion and therapy for chronic ailment, as opposed to life threatening conditions. However, the usage of herbal remedies, increases when conventional medicine is ineffective in the treatment of diseases, such as in advance cancer and in the face of new infectious diseases. Furthermore, herbal medicines are widely perceived as natural and safe, that is not toxic. This is not necessarily true, especially when herbs are taken with prescription drugs, over-the-counter medications, or other herbs, as is very common (Canter and Ernst 2004; Qato et al, 2008; Loya, Gonzalez-Stuart, and Rivera 2009; Cohen and Ernst 2010).

Herbs and plants can be processed and can be taken in different ways and forms and they includes the whole herbs, teas, syrup, essential oils, ointments, salves, rubs, capsules, bitters and tablets that that contain a ground or powdered form of a raw herb or its dried extract.Plants and shrubs extract vary in the solvent used for extraction, temperature and extraction time and include alcoholic extract (tinctures), vinegars (acetic acids extracts), hot water extract (tisanes), long term boiled extract,usually roots or bark (decoction), and cold infusion of plants (macerates). There is no standardization and components of an herbal extract or a product are likely to vary significantly between batches and producers. Plants are rich in a variety of compounds. Many are secondary metabolites and include aromatic substances, most of which are phenol or their oxygen substitution derivatives such as tannis. (Hartmann 2007; Janke-KODAMA, Muller and Dittmann 2008). Many of these compound have anti oxidant properties.

1.2INTRODUCTION OF YOYO BITTERS AS HERBAL PLANTS

Yoyo bitters is a plant based medicine in the class of herbal bitters that was launched into the market in 2003 by Abllat company Nigeria Limited. Abllat Nigeria limited is an indigenous manufacturer nature green medicine. After it was introduced into Nigeria drug market. Yoyo bitters tonic has received wide acceptance and usage by general populace. The ingredients used for production of yoyo bitters as published by the manufacturers are: Acinos avenis, Chenopodium murale, Citrus aurantifolia and Cinamomum aromaticum. Each of these components has several medicinal properties.

1.3 COMPONENT OF YOYO BITTERS

Acinos avensis: it serves as antiseptic stimulant tonic. It is also used for shortness of breath ,improving digestion, treating bruises, toothaches, sciatica and neuralgia.

Chenopodium murale: it is commonly known as nettle leaf goosefoot and is an important annual weed. It is distributed throughout the temperate and tropical regions and found in Pakistan in almost every field in winter season.

Citrus auantifolia: citrus species are among the native plants of Iran and the history of their cultivation dates back to 4000years ago from which time, they have been widely used in the ethno medicine. These species with the wide range of bioactive ingredients have been found to exert anti-infection and anti- inflammatory properties. Citrus fruits have also been found beneficial for cancer prevention in an epidemiological survey. These fruits contain several classes of phytochemicals and micronutrients such as limonoids and flavonoids, which have been reported to have anti-tumor effects in vivo and vitro.

Cinamomum aromaticum: Aqueous cinnamon extract from bark of cinnamomum cassia (cinamomum aromaticum) cause apoptosis in human cervical cancer cell line through loss of mitochondrial membrane potential. Yoyo bitters is formulated in such a way that the ingredients have a synergetic effect on the management of the digestive circulatory, nervous, urinary and systems bydecreasing the stomach acidicity in case of ulcer, diminishing the irregular production of gastric juice stimulating the liver to ensure proper and complete digestion of heavy and fatty food, enhancing blood circulation, assists in the elimination of cholesterol, sugar triglycerides, creatine and ulric acid, enhances effective function of the secretive glands and is beneficial in the treatment of disorders such as insomnia, stress, depression, kidney stone and bladder infections.

It is also reported to dissolve any encased toxic materials in the body, enhance cell formation and growth, reduce excess body fat and boost healthy weight loss(Allbat company Nigeria Limited, 2009) “Yoyo bitters leaflet”

1.4LITERATURE REVIEW

1.4.1BENEFITS OF HERBAL BITTERS

When a bitters substance is recognized by bitters receptions on tongue a chain of neural and endocrine events begins, labeled as the bitter reflex (Mills and Bone K,2000).Mediated by the release gastric hormone gastrin, thisreflex results in an overall stimulation of digestive function, which overtime strengthens the structure and functions of all digestive organs (liver, stomach, gallbladder, pancreas, etc). (Yarnell et al. 2003).

Starting from the mouth, when you tasted bitter tasting substances, you’ll notice that your salvary glands have increased their output of enzyme-rich saliva, helping to breakdown complex starches into smaller and more easily digested oligosaccharides. (Mills S, Bone K)

In the stomach, the hormone gastric has stimulated the secretion of hydrochloric acid.(Hoffman, David.2003; Mills S, Bone K. 2000).

The acidity helps breakdown protein, enhance the bioavailability of many minerals (especially calcium) and destroys and harmful microbes present in your food. It’s interesting to note that more people have levels of gastric acid that are too low rather than the opposite due to stress. (Esplugues Jv, et al. 1996.) or simply ageing. Low level of gastric acid contribute to poor nutrition and increased susceptibility to gastrointestinal infections. Low stomach acidity is associated with a variety of allergic and immune –mediated disorders, including asthma, skin disorders such as eczema, psoriasis and rosacea, gallbladder disease and arthritis. (Mills S, Bone K. 2000); (Bone K, 2003).

Gastric also stimulates secretion of pepsin an enzyme necessary for breaking large protein molecules intosmaller parts and intrinsic factors necessary for the absorption of vitamin B12. The smooth muscle of the stomach is also stimulated by the bitters reflex, which increases the rate of gastric empting and contracts the esophageal sphincter to prevent the movement of acidic stomach contents upwards into the esophagus.(Mills S, Bone K. 2000); (Hoffmann, David, 2003).

Self-repair mechanism of the intestinal wall are stimulated enchancing division and growth.(Hoffmann, David, 2003). While many people with GERD are hesitate to partake of bitters due to the potential increase in stomach acidity, the combined effect of these actions actually can help this condition by ensuring that the stomach contents are moved downward rather than allow to reflux back up and out of the stomach . Bitters also act to heal any damage done to the gastric mucosa.

Down in the small intestinal, the stimulation caused by the bitter taste prompts your liver to increase its production of bile excretion. (Mills S, Bone K. 2000); (Hoffmann, David 2003). Ble is necessary of fat digestion and absorption of fat-soluble nutrients such as vitamins A, D and E.(Marieb E. 2004). Healthy bile flow helps rid the liver of waste products such as oxidized cholesterol and hormonal metabolic, prevents gall stone formation and provides lubrication of intestines, easing the passage of stool. (McDonald, Jim, 2010); (Marieb E.2004). Te enhancing movement of waste products out of the liver, bitter herbs have been found to exert a protective effect in liver conditions such as hepatitis and cirrhosis. (Mills S, Bone K. 2000).

1.4.2THE EFFECT OF BITTERS

The effect of bitters also extends to the pancreas. With bitters digestive enzyme secretions are increased, helping to promote the complete breakdown of nutrients into their absorbable units, preventing gas information. When large molecules are acting upon by bacteria further down the small intestine.

The complete breakdown of proteins is particularly important, as the cross cells plays an important role in the etiology of conditions such as celiac disease. (Bergner P.2005). Insulin and glucagon secretions are stimulated, helping to stabilizing blood sugar levels. ( Hoffmann, David 2003). Many bitters formulations have een traditional used in the treatment of type 2 diabetes. The naturally sweet substances are often paired with some bitterness in nature. (Jim McDONALD, 2010).

1.4.3TYPES AND USES OF HERBAL BITTERS

Bixa orellena

MEDICAL USES: Astingent, febrifuge, antidysenteric, aphrodisiac, detoxifer. The seed is thought to reduce acid,killer bacteria, fight free radicals, kills parasites and germs, stimulatd digestion, lower blood pressure, malaria, fever, kidney diseases, skin diseases.

Senna siamea

MEDICAL USES: Stimulates immune system by activating large number of anti RBC producing cells in the spleen that suggest its therapeutic usefulness. But further detailed studies of mechanism of immune-modulation and probable use in immunocomprised individual are still to be investigated and their possible use as an adjuvant during vaccination.

Tamarindus indica

MEDICAL USES: It is used as laxative, acarminative and a stomachric. It is an ingredient in cardiac and blood sugar reducing medicine. Acts as a cathartic, astringent, febrifuge, antseptic, refrigerant. The seed extract is a polyphenolic flavonoid that has been shown to have antioxidant properties. The seed extract possesss anti-snake nom properities.

Aranomum sceptrum A.melagueta

MEDICAL USES: An enhanced antimalarial which is exploited in traditional medicine. Vernifuge, smallpox and chicken pox, catarrh, congested chest.

Prosopis Africana

MEDICIAL USES: Cure male sterility and diarrhea.

Dichrostachys cinerea

MEDICAL USES: An ethnobotanical study revealed that it is used for treatment of sexually transmitted diseases as it possess antibacterial properties.

Monodora myristica

MEDICAL USES: It possesses anti hypertensive activities

Hura crepitans.

MEDICAL USES: These seed are emetic when green are purgative. Oil extracted from the dried seed I also used as a purgative. The leaves are used against eczema. (Idu and Gill, 1997, Idu and Gill, 1998; Gill and Idu, 2001a; Gill and Idu,2001b; Idu and Oyibe, 2007).

Garlic

MEDICAL USES: Prevent the oxidant of LDL cholesterol. Prevent the liver from producing excess fat and cholesterol. In one study, adding a little as thirty(30g) of garlic juice to a fatty, cholesterol laden meal was found to actually lower the cholesterol by up to 7percent. Another study found that 600mg of garlic powder a day could push the total cholesterol down by some 10percent. Other research has corroborated these finding reporting that garlic can lower both total and LDL cholesterol while raising the HDL (good) cholesterol.

A 10month study found that eating three cloves of garlic a day keeps the cholesterol down for extended periods. And because it contains ajoene and other substances, garlic also helps to keep the blood “thin” and free of potential deadly blood clots. Garlic cloves is use primarily for its cardiovascular effects, principally as a support to dietetic measures at elevated levels of lipids in blood and as a preventive measure for age-dependent vascular changes. Garlic is also used for atheroma, prophylaxis of atherosclerosis, hypertension, respiration infections, catarrh conditions as well as a natural antibiotic.

Ginger

MEDICAL USES: Ginger is an important herbs for a healthy heart, traditional physician suggest that eating a little bit of ginger everyday will help to prevent heart attack. It reduce cholesterol. It also reduce blood pressure and prevent blood clots.

Terminalia catappaL. (Indian almond)

MEDICAL USES: The leaves , bark, root, fruit and seeds of Indian almond have been used to effectively to reduce blood sugar, regulating functions in damaged pancreas of diabetics, prolong ejaculation, stop the spread of lung cancer and sickle cell anaemia. All part of the plant (leaves, bark, roots, fruits and seed) are used in herbal medicine, such as n dysentery, dressing rheumatic joints, treating coughs, asthma. The fruits may be helpful in treating of leprosy, headaches. In reducing travel nausea. Leaves have been used to get rid of intestinal parasites, treat eyes problems, rheumatism, wounds and stop bleeding during tooth extraction. Leaves have antioxidant as wee as anti- clasnogenic (preventing breakage of chromosomes) properties.

Vernonia amydalina L.(Bitter leaf)

MEDICAL USES: Bitter lesf can be the cure for the dreaded breast cancer and diabebetes? Chukuma Muanya on Monday, August18, 2008- Guardian Nigeria reported that Bitter leaf- based drud for diabetes and cancer have passed human clinical trials and received United stated patents. (Cunnane etal.,, 1993); (Impari Radosevich et al., 1998).

Peperomia pellucid L.

MEDICAL USES: Use as infusion, decoction or salad for gout and rheumatic pain, pounded plant warm poultice for boils and abscesses.

Momordica charantia L.

MEDICAL USES: Is useful for rheumatism. To treat diseases of the spleen and liver. It also aids in lowering blood pressure, relieve headaches. (Ataman et al., 2006b; Atama and Idu, 2007)

Curcuma longa L. (Tumeric)

MEDICAL USES: It is used to aid digestion and liver function, relieve arthritis pain, and regulate menstruation. Tumeric has also been applied directlt to the skin for eczema and wound healing. Tumeric is used for condition such as heartburn, stomach ulces and gallstone. It is also used to reduce inflammation as well as to prevent and treat cancer.

1.5CHEMICAL COMPONENTS OF HERBAL PLANTS

ALKALOIDS

These are natural occurring plant compound found in 15-30% of all flowering plants and are essentially commonly in some families. It occurs in plants parts but majorly in plant organs such as bark, roots, leaves and finally the fruits. Their accumulation serve as aprecursor to amino acids biosynthesis such as ornithin, tyrosine, tryptophan and even lysine. They differ chemically due to nitrogen component. Alkaloids are generally alkaline in nature and this derivea the name alkaloid contrary to their point are example; Berbarnine and Colchincine that are acidic and insoluble in water. They need organic solvent like alcohol to dissolve them. Alkaloid contain basic amino groups and have medicinal uses as drugs for malaria, colds, cough, hypertension, diabetes, cancer and other disease. They are of many type (Alkaloid). (Akpuaka, 2009). It acts on nervous system of the body (Beckelt et al, 1988).

GLYSOCIDES

These compounds are also the known called cardiotonic glycoside. Many of contain phenolic group and occur in most plants. The simple glycosides are colourless and soluble in water. Many plants steroids occur as glycosides and have properties of stimulating heart , muscles. (Pendleton 2010).

FLAVONOIDS

Flavonoids are phenolic compounds which are present in all plant . A number of medical diseases could be prevented or improved with the use of dietary flavonoid such as liver problem, diabetes,eyes problem (Sahaliam 2005). They are derived from flovones which occur in plant. They are mainly water soluble and can be extracted with 10% ethanol. Thet are phenolic and hence change in colour when treated with base or ammonia. Flaonoids are generally present in plants boun to suga as glycoside and any one flavonoids glycone may occur in a single plant. They are responsible for colours of fruits, seeds leaves and bud of plant (Harborne 1998).

SAPONINS (Harborne 1973)

This occur mainly in plant and are associated with cardio tonic glycoside. It is a group of steroidal glycoside which foam in water like soap solution. Saponins prevent increase in blood cholesterol and are beneficial to patients with arteriosclerosis and hypertension. It can be detected by its foaming action in water or aqueous alcohol. The leaves they contain can be used as natural soaps though may be toxic to fish. Saponins are also importantly in controlling cholesterol in the body by preventing its re-absorption into the body.(Akpuaka, 2009).

TANNIN

Tannin are commonly found in fruits and vegetable or leaves. They bind to both proteins and carbohydrate and provoke astringent reaction. Tannins precipitate protein in the gut, reduce digestibility or inhibit digestive enzyme like trypsin, amylase and lipase thus inhibit microbial; activities (Grifits 1979). They have been found to be insecticidal anti-inflammation, antifungal, antiseptic and wound healer. (Beckelt et al 1988).

TERPENOID

These are sweet smelling liquid extract which are souble in water. They contain molecular structure containing carbon backbone made of isoprene units which contain fiv cabon atoms derivatives if the isopentienyl pyrophosphate. Steroids have common tetracyclic carbon skeleton and are modified terpenoids that are synthesized from tetracyclic triterpene lanosterol. (Deven et al, 1974)

The role of terpenoid is as akey precursor for vitamin A or retinol for the pigment in eye which detect light, therefore it is responsible for the sense of light. Vitamin E or tocophenol is an important antioxidant which prevent damage to cell. It is also a key factor of photosynthesis through which atmospheric CO2 is converted to glucose. These functions as thefunctional role of terpenoid and steroid. They can also be used in the treatment of asthma and in crms for reducing inflammation (Birch 1983).

STEROIDS

Plant steroids (or steroid glycoside) also referred to as “cardiacglycosides” are one of the most naturally ocrring plant phytoconstituent that have found therapeutic application as arrow poision or cardiac drugs.(Firn, 2010). The cardiac glycosides are basically steroids with an inherent ability to afford a very specific and powerful actionmainly on the cardiac muscle when administered through injection into man or animal. Steroid (anabolic steroid) have been observed to promote nitrogen retention in osteoporosis and in animals with wasting illness. (Maurya et al, 2008; Madziga et al, 2010)

1.6ANTIOXIDANT

Antioxidant protect cells against the damaging effects of reactive oxygen species otherwise called free radicals such as singlet oxygen, super oxide perosyl radicals, hydroxyl radicals and peroxynite which results in oxidative stress leading to cellular damage (Mattson & Cheng, 2006). Natural antioxidant play a key role in health maintenance and prevention of the chronic and degenerative diseases, such as artherosclerosis, cardiac and cerebral ischema, carcinogenesis, neurodegenerative disorder, diabetics, pregnancy, rheumatic isorder, DNA damage and ageing.(Uddin et al,2008; Jayasri et al, 2009). Antioxidant exert their activitives by scavenging the free-oxygen radical thereby giving raise to a fairly stable radical. The free radicals are metastable chemical species which tend to trap electron from the molecules in the immediate surroundings. These radicals if not scavenged effectively in time, they may damage crucial biomolecules like lipids proteins including those presenting in all membrane mitochondria and, the DNA resulting in abnormalities leading to diseases conditions. ( Uddin et al, 2008). Thus, free radicals are involved in a number of diseases including; tumor inflammation, hemorrhagic shock, astherosclerosis, diabetes, infertility, gastrointestinal ulcerogenesis,asthma, rheumatoid arthritis, cardiovascular disorders, cystic fibrosis, neurodegenerative diseases (eg parkisonim, Alzhiemer’s disease) AIDS and even senescene.(Chen et al, 2006; Uddin et al., 2008). The human body produces insufficient amount of antioxidants which are essential for preventing oxidative stress. Free radicals generated in the body can be removed by the body’s own natura antioxidant defences such as glutathione or catalases. (Sen, 1995). Therefore this deficiency had to be compensated by makind use of natural exogenous antioxidants. Such as vitamin C, Vitamin E,flavones,beta carotene and natural products in plants.(Madsen & Bertelsen, 1995; Rice Evans et al, 1997; Diplock et al, 1998). Plants contains wide variety of free radical scavenging molecules including flavonoid, vitamins, terpenoids that are rich in antioxidant activity.( Madsen & Bertelsen, 1995; Cai & Sun, 2003).

1.6.1TYPES OF ANTIOXIDANT

Ascorbic acid or “vitamin C” is required for conversion of the procollagen to collagen by oxidizing proline residues to hydroxyproline. In other cells, it is maintained in its reduced form by reacting with glutathione, which can be catalysed by protein disulfide isomerase and glutaredoxine (Meister, Alton; 1994.; Wells, William W,et al.;1990). Ascorbic acid is a redox catalyst which can reduce, and thereby neutralize,reactive oxygen species such as hydrogen peroxide (Padayatty et al.; 2003).

Vitamin C offer a wide variety of health benefits, includingprotecting from infection and damage to body cells, helping produce collagen ( the connective tissue that holds bones and muscle together) protecting your body from bruising by keeping capillary walls and blood vessels firm; helping in the absorbing of iron and folate.

VITAMIN E

Research has demonstrated the broad role of vitamin E in promoting health. The main role of vitamin E is as an antioxidant, it help protect the body from cells damage that can lead to cancer, heart disease and cataract as we age. Vitamin E works with other antioxidants like vitamin C to offer protection from some chronic diseases.

GLUTATHIONE

Glutathione dose more than simply fight free radicals on its own. This antioxidant also has ability to regenerate other antioxidants such as vitamin C and E. After these antioxidants neutralize free radicals they recycle these unstable molecules, returning them to active duty.(Xiang c et al., 2001)

1.7NUTRITIONAL COMPONENT OF HERBAL BITTERS

Potassiionic (K)

Potassium is important as diuretic and it take part in the ionic balance of human body and maintains tissue excitability potassium is the principle intracellular cation and also considered as a very important for transmission of electrical impulse in the nerve. Venketaraman and Gopal kristian (2002) reported concentration of ca, k, fein plants traditionally used for treatment of disease (jaundice).

Magnesium (Mg)

Magnesium is the most abundant cation in the body. In muscle and other tissue it functions as activation of many enzymes involve d in metabolism and synthesis of nucleic acid (DNA and RNA). Magnessium causes irritability of nervous system, peripheral vasudialetion and cardiac arrhythmias (singh et al, 2006). Magnesium is also abundant in most plants at a given proportion.

Calcium (Ca)

Calcium impacts strength and rigidity to bones and teeth, calcium ions are needed in neuromuscular transmission in excitability of nervous for normal excitability of heart in clothing of blood and promoting muscular contraction. It is also an activator of most enzymes such as phospholipase, arginin kinase, aolenosin triphosphate and adenyl kinase. Excess quantity of calcium ion in the extracellular fluids such as metal acts as depressant though low level calcium causes spontaneous discharge of nerve fibres resulting in tetany. Calcium is reported in plant at different concentration in plant (venkataraman et al. ; 2002)

Iron (Fe)

Iron is present in most plant such Q-infectoria (omolo et al, 1997) and responsible for transport of oxygen to tissue in animals. It forms the integra part of the cytochrome, flaroprotein and certain enzymes.

Zinc (Zn)

Zinc is a enssential component of a number of enzymes presentin animals such as acohol dehydrogenase, alkaline phosphatase atc. It is important for reproduction and helps in the process of tissue repair and wound healing. It is mostly found in plant in which its deficiency causes growth retardation and skin lesion (chalterjec et al.; 1995) the sloen accumulation of zinc in the plant is due to its use absorption from the soil.

Phosphorous (P)

Phosphorous ions are major anion intracellular fluid, phospholipide and the co-enzyme NAD and NADP and especially of ATP and other high energy compound, it helps in the process of ossification of bones by getting deposite in the form of calcium phosphate (indrayan et al, ; 2005); (kar et al, : 1999) reported that the in-organic parts (k, zc, ca traces of cr) of some plants showed more pronounced action of glucose. Tolerance factor than their corresponding organic part.

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