CHAPTER I INTRODUCTION In the present time, the rate of proliferation of diabetic people is continuously and rapidly increasing. Further studies show that the number of diabetes cases worldwide has doubled since 1980 and is now at startling 350 million diabetics. (Laino, 2011). Diabetes is a lifelong disease in which the level of glucose in the blood is abnormally high. Connected to this is a condition called hyperglycemia. Hyperglycemia is the technical term for high blood glucose (sugar).
High blood glucose occurs when the body can’t produce sufficient amount of insulin. And also, it can happen when the body can’t use insulin properly. (American Diabetes Association, 1995). In addition, Hyperglycemia is the major cause of complications with diabetes. If left untreated, hyperglycemia can become severe and lead to serious complications requiring emergency care, such as diabetic coma. In the long term, persistent hyperglycemia, even if not severe can lead to complications affecting your eyes, kidneys, nerves and heart. Mayo Foundation for Medical Education and Research, 2012) Due to the alarming incidences of hyperglycemia and adverse side effects of synthetic medicines, researchers sought for natural, indigenous and inexpensive herbal alternatives and botanical sources (Venkatesh et al. , 2003). In Britain,10 percent of the 150,000 diabetics who had been switched from the traditional animal-derived insulin to synthetic human insulin have suffered hyperglycemic episodes, coma and injuries. Brown, 1999) In archaic journals, 800 plants were reported to have anti-diabetes properties while survey says that more than 1200 plants were used for hypoglycemic activity. (Kesari et al. , 2007) Moreover, studies suggest that Banaba and Ampalaya can be alternatives to these synthetic medicines. (Klein et al. , 2007) Like the said herbal alternatives, there are many plants that have the potential to decrease the production of blood glucose such as Guava, Macopa and Camias. Guava (Psidium guajava) is a small tree with a height of approximately 33 feet (10 inches) with spreading branches.
Also it has smooth, thin, copper-colored bark that flakes off, showing the greenish layer beneath. The fruit exuding a strong, sweet, musky odor when ripe, may be round, ovoid, or pear-shaped, 2 to 4 in (5-10 cm) long, with 4 or 5 protruding floral remnants (sepals) at the apex; and thin, light-yellow skin, frequently blushed with pink. (Philippine Medical Plants, 2012) Furthermore, guava can be used for gastroenteritis, controlling blood pressure and cholesterol, can be source of Vitamin C, reduces the risk of acquiring cancer, an alternative pain reliever, can lower blood sugar, and other uses. Sarvani, 2007). According to a research conducted by Yoriko Deguchi and Kouji Miyazaki, guava leaves extract has the potential to reduce blood sugar. However, little is known regarding the therapeutic activity of the extract in human clinical trials as well as its underlying therapeutic mechanisms and safety. In Japan, Guava Leaf Tea (Bansoureicha®, Yakult Honsha, Tokyo, Japan) containing the aqueous leaf extract from guava has been approved as one of the Foods for Specified Health Uses and is now commercially available.
This review describes the active component of the aqueous guava leaf extract and its inhibition of alpha-glucosidase enzymes in vitro, safety of the extract and Guava Leaf Tea, reduction of postprandial blood glucose elevation, and improvement of hyperglycemia, hyperinsulinemia, hypoadiponectinemia, hypertriglycemia and hypercholesterolemia in murine models and several clinical trials. It is suggested that the chronic suppression of postprandial blood glucose elevation is important in preventing type 2 diabetes mellitus, and that Guava Leaf Tea is considered useful as an aliment therapy for chronic treatment. Philippine Medical Plants, 2012) Macopa (Syzygium samarangense) Makopa is a tree reaching a height of 10 meters. Leaves are pinkish when young. Older leaves are large, drooping, elliptic-oblong to broadly oblong-lanceolate, 15 to 30 centimeters long, 7 to 15 centimeters wide, narrowed and pointed at both ends. Flowers are large, showy, crimson, 5 to 6 centimeters in diameter, borne on the branches below the leaves, clustered on short, few-flowered racemes, 6 centimeters long or less. Fruit is shiny, oblong or pear-shaped, 5 to 7. centimeters long, either white splashed, striped with pink, or wholly crimson to purplish, and slightly shiny, seedless or one-seeded. Flesh is white, pithy, juicy. Although rather tasteless, some varieties have a pleasant flavor. (Philippine Medical Plants, 2012) Camias (Averrhoa bilimbi) is a tropical fruit abundant to Malaysia and Indonesia. It is widely known for its medicinal uses. Camias trees are described evergreen and have approximately 6 to 9 meters of height. The leaves tend to crowd towards the ends of branches, occurring in pairs of 7 to 19 leaflets which ovate, measuring estimately 5 to 12 cm long.
The branches are very few and upright while the flowers are small with reddish-purple or crimson free petals that measure 10 to 22 mm long. Flowers are auxiliary or cauliflorous, appearing directly on the branches and trunk. They are produced all year round. (National Library Board Singapore, 2012) In the Philippines, the leaves are applied as a paste or poultice on itches, swellings of mumps and rheumatism, and on skin eruptions. Elsewhere, they are applied on bites of poisonous animals. Malayans take the leaves fresh or fermented as a treatment for venereal disease.
A leaf infusion is a remedy for coughs and is taken after childbirth as a tonic. A leaf decoction is taken to relieve rectal inflammation. A flower infusion is said to be effective against coughs and thrush. In contrary to its uses, Camias contains oxalic acid, which can be imperiling when consumed improperly or without consulting a physician or an expert. Moreover, related studies were conducted that show Camias leaf extract has hypoglycemic, hypotriglyceridemic, anti-lipid peroxidative and anti-atherogenic properties in STZ-diabetic rats.
Moreover, a study showed the aqueous fraction was more potent than the butanol fraction in the amelioration of hyperglycemia and hyperlipidemia in high fat diet-fed STZ diabetic rats and suggests the AF as the potential source for isolation of the active principle for oral antidiabetic therapy. Norway Rat (Rattus norvegicus), also known as Brown Rat, is native to Texas. It has approximately 440 mm long; its tail is 205 mm and its hind legs measure 46mm. An average Norway rat can weigh 400-500 g. Also, it was reported that it has an average lifespan of 2-3 years.
This rat lives both as a commensal in close association with man and in the feral state, chiefly where vegetation is tall and rank and affords adequate protection. Although more at home on the ground, these rats are adept at climbing and have been observed traveling along telephone wires from one building to another, however they become exceedingly numerous and destructive. On contrary, Norway rat is used for laboratories because its blood can be easily obtained. Considering the mentioned information, the researchers decided to study the effectiveness of each said plant: Guava, Macopa, and Camias.
The plants will be compared according to their effectiveness in treating hyperglycemia or reducing blood sugar. Brown Rat will be used as the experimental sample. Protocols for animals will be followed strictly in conducting this study. The results of this research study will help many diabetic people and can contribute to the society. STATEMENT OF THE PROBLEM The researchers of this study aimed to find answers to the following questions: 1. Which among Camias (Averrhoa bilimbi) leaves, Guava (Psidium guajava) leaves and Macopa (Syzygium samarangense) leaf extracts is the most effective in treating hyperglycemia? 2.
Is there a significant difference between the hypoglycemic effect of the three leaf extracts in terms of reducing the glucose level of the rat (Rattus norvegicus)? SIGNIFICANCE OF THE STUDY The study would be of immense contribution in dealing with hyperglycemia, particularly those who suffer from it. Moreover, the exploration and comparison of the potential of Camias (Averrhoa bilimbi) leaves, Guava (Psidium guajava) leaves and Macopa (Syzygium samarangense) leaves will bring awareness of which among them is the most effective in treating or inhibiting the said disease, thus increasing the chances of survival and recuperation.
Looking at the large-scale impact of the study, it aims to slowly decrease the cases of hyperglycemic patients in the Philippines by the means of natural extracts found in some of the most accessible and abundant plants in the said country. Also the modernized process of treating hyperglycemia, though known to produce dependable results, is complicated and expensive. This is why many researchers are now focusing on finding the most effective plant yet in treating diabetes like banaba (Lagerstroemia speciosa ).
Folk medicine (or lay medicine) is "the ordinary person's concept of health, illness, and healing; it is the treatment of disease practiced traditionally among the common people stressing the use of herbs and other natural substances" (Webster). Because all the plants in the study are widely available and cheap, it can cater to everyone, specifically designed for the less fortunate. Aside from treating the condition itself, this study that uses readily available materials will also raise the economic value of Camias, macopa and guava. Our country is an agricultural one and more medicinal iscoveries validated may result to a beneficial use of our resources. Furthermore, the researchers aim to differentiate and analyze the effects of the different plants and the reaction of the specimen which is rat (Rattus norvegicus) towards the varying concentrations of the extracts to be sure that the extract did not jeopardize or compromise with the other aspects of the rat’s health. The researchers chose such plants for the study to be able to promote the three plants since they are not popular to possess anti-diabetic activity even if they are very effective.
The study would like to emphasize the wise and proper utilization of our natural resources and advancing the knowledge of people of how useful these plants are. The extracts are not composed of harsh chemicals thus promoting the conservation of the environment. SCOPE AND DELIMITATION The study is specifically limited to Camias (Averrhoa bilimbi) leaves, Guava (Psidium guajava) leaves and Macopa (Syzygium samarangense) leaves and thus the properties explored here are not generalized for all the species of the said plants..
The plants were not treated with pesticides and are free from culturing techniques to assure the purity and integrity of the extracts. The rats were induced with glucose orally (drinking) to put leverage in the glucose level, making the rats hyperglycemic. The effects to be studied are only the hyperglycemic potential on rat (Rattus norvegicus), with 100% concentration specification for each of the plants’ leaves extract. The conclusion in the study is limited to the comparison of camias, guava, macopa and their corresponding effects in hyperglycemia in rat and must not be deemed true for all plant comparisons.
Several tests must still be undergone to prove the effectiveness of the plants in other life forms so as to be sure of the safety. Variables other than the concentration of the three leaves and the glucose level of the rats will not be entertained in the study. DEFINITION OF TERMS 1. Hyperglycemia is the condition wherein the blood sugar level is very high. 2. Diabetes is the disease caused by hyperglycemia, or the lack of insulin in the body. 3. Glucose is a compound commonly known as sugar and the most common carbohydrate. . Exuding means to ooze out. 5. Aqueous means that water is contained. 6. Postprandial is an indication of time after a meal or dinner. 7. Cauliflorous refers to the production of flowers and fruits directly from the branches or trunks of tropical plants. 8. Oxalic Acid is an organic compound with the formula H2C2O4. It is a colorless crystalline solid that dissolves in water to give colorless solutions. Ingestion of oxalic acid through skin contact or orally is dangerous. CHAPTER II
Review of Related Literature Hyperglycemia is known to be a condition in which an excessive amount of glucose is present within the blood (WebMD, 2012). It is evident that it can serve as a principal health problem for those who are suffering from diabetes. To elaborate, there are two specific types of hyperglycemia that occur. One is the fasting hyperglycemia, in which blood glucose level exceeds 130 mg/dL (milligrams per deciliter), and the other one is called postprandial or after-meal hyperglycemia.
In the latter condition mentioned, the blood glucose level usually remains higher than 180 mg/dL. The guidelines provided by the American Diabetes Association state that a subject with a consistent range between 100 and 126 mg/dL is considered hyperglycemic, while above 126 mg/dl is generally held to have diabetes. Diabetes is defined by the World Health Organization (2011) as a chronic disease which occurs when the pancreas does not produce enough insulin, or when the body cannot effectively use the insulin it produces.
It is caused by insulin resistance, which is defined as defective insulin signaling and a decreased insulin efficiency to induce glucose transport from the blood into key target cells such as muscle and fat (adipocyte) cells. In general, obesity leads to hyperglycemia, which in turn, leads to, and exacerbates insulin resistance. Insulin resistance, if not treated, results in hyperinsulinemia. Reported to be the principal cause of blindness in adults and the most common cause of kidney failure (www. naturalways. om, 2011), the said disease, according to National Diabetes Statistics (2007), is known to be the seventh leading cause of death based on U. S. death certificates in 2007. Deleterious hyperglycemia is renowned to be exhibited by a patient with diabetes as it is recognized as one of the commonest chronic illnesses present in human beings (Jayanthi, 2010). In 1999, the World Health Organization (WHO) has estimated that by the year 2030, the number of people with diabetes will have reached 370 million.
At present, Hensley has published an article in npr. org, revealing that 346 million people have been affected by diabetes, globally. The said case remains a hot topic for research, with more than 194,000 papers published. All those research have lead to making it possible for all forms of diabetes to be treatable, since insulin became available in 1921; at the same time, type 2 diabetes may already be controlled with medications. Nevertheless, both types 1 and 2 are chronic conditions that cannot be cured.
From a study conducted by Moorthy, et al. in 2010, it is generally claimed that insulin (including human insulin) and many synthetic drugs currently in use for the treatment of diabetes are effective; however, prolonged use causes side-effects on patients, and this has been the reason why scientists all over the world have turned into working on medicinal plants which have potential in becoming anti-hyperglycemic agents, most of which were mentioned in Ayurveda, reviewed by Mukherjee et al. and Shukla et al.
Cures for both type 1 and type 2 diabetes remain elusive to medical science, but several studies on different plants show promising hypoglycemic effects. Browsing through books related to health as well as articles regarding alternative home remedies provide reliable information concerning health benefits of certain plants. Though not widely known as helpful in cases of hyperglycemia, there are a few studies that attest to the efficiency of guava, kamias, and macopa leaf extracts in reducing the blood glucose level of a certain individual.
One of these published studies includes the work of Pushparaj, et al. (2000), entitled Effects of Averrhoa bilimbi Leaf Extract on Blood Glucose and Lipids in Streptozotocin-Diabetic Rats. In this particular study, the hypoglycemic activities of an ethanolic extract of Averrhoa bilimbi Linn. leaves in streptozotocin (STZ)-diabetic rats were investigated. Through the oral glucose tolerance test in both normal and STZ-diabetic rats, the optimal hypoglycemic dose was determined.
It was found out that Averrhoa bilimbi significantly lowered blood glucose by 50% when compared with the vehicle (distilled water), which leads to a conclusion that the said plant has hypoglycemic properties. In 2010, Deguchi and Miyazaki published an article entitled anti-hyperglycemic and anti-hyperlipidemic effects of guava leaf extract. It was stressed that the leaf extract, particularly, of guava has already been traditionally used for the treatment of diabetes, but little is known regarding the therapeutic activity of the extract in human clinical trials as well as its underlying therapeutic mechanisms and safety.
Murine models were initially used for the testing, wherein normal mice were immediately given saline (control) following overnight fasting, then loaded with sucrose or maltose (2 g/kg). Afterwards, to evaluate the effects of a single ingestion of guava leaf tea on postprandial blood glucose elevation in normal and pre-diabetic subjects, a human study was designed. Eventually, results showed that long-term feeding of the extract significantly reduced blood glucose level, increased plasma insulin level in an oral glucose tolerance test, and stimulated activities of some glucose metabolic enzymes.
They stated that guava leaf tea, in Japan, has been approved as part of Foods for Specified Health Uses (FOSHU) in March 2000. It is recommended for individuals who are anxious about their high blood glucose and control of sugar uptake, as the consecutive ingestion of guava leaf tea with every meal is also expected to benefit pre-diabetic and diabetic patients as an alimentotherapy—the treatment of a certain disease by dietetic methods.
This is related to the present study since guava (Psidium guajava) is one of the components that will be utilized by the researchers for performing the comparative analysis, along with kamias (Averrhoa bilimbi) and macopa (Syzygium samarangense); it is important to assure that all of these medicinal plants are potential hypoglycemic agents. Another study conducted by Won et al. (2004) using an extract from guava leaves exhibited significant blood glucose lowering effects after intraperitoneal injection at a dose of 10 mg/kg in mice, thus, suggesting that extract from guava leaves possess anti-diabetic properties. Owen et al. 2008) also commended that habitual intake of guava is proposed to offer better protection against diabetes development as guava bud extract displayed significant insulin-mimetic and potentiating activity. Recently, Barbalho et al. (2012) has published a study entitled Psidium guajava (Guava): A Plant of Multipurpose Medicinal Applications, wherein the medicinal capacities of guava are identified. With bioactive compounds contained in several plants that make way for prevention of risk factors of many diseases, many fruits, vegetables, and seeds have been used to reduce risk factors associated with the occurrence of chronic disorders.
Guava, a medicinal plant traditionally utilized throughout the world for a number of ailments, has been found out to contain vitamins, tanins, phenolic compounds, flavonoids, essential oils, sesquiterpene alcohols and triterpenoid acids (Haida et al. , 2011). Farinazzi et al. (2012) showed that animals treated with guava pulp juice had significantly lower body weight, glycemia, cholesterol and triglycerides levels and significantly augmented the levels of HDL-c when compared to the animals from the control group.
Lyophilized pulp of the said fruit in diabetic rats induces significant hypoglycemic effects probably due to its antioxidant activity of compouns present in the pulp (Huang et al. , 2011). In the said article, it was stated that Deguchi and Miyazaki reported that guava leaves infusion not only reduces postprandial glycemia and improved hyperinsulinemia in murine models but also contributes to reduce hypercholesterolemia, hypertriglyceridemia and hypoadiponectinemia in the animals of their study.
On the other hand, Ojewole (2005) identified the presence of phenolic compounds in the leaves demonstrating their hypoglycemic and hypotensive effects on diabetic rats treated with aqueous leaf extract. His study, Hypoglycemic and hypotensive effects of Psidium guajava Linn. (Myrtaceae) leaf aqueous extract, is used by the researchers as a basis for using guava leaves and its leaf aqueous extract in the study.
Traditionally, guava leaves have been used in African folk medicine to manage, control, and/or treat a plethora of human ailments, including diabetes and hypertension. Ojewole's study was conducted to investigate the claim on the plant's anecdotal, folkloric, and ethnomedical uses scientifically. It has been noted also that there are numerous tannins, polyphenolic compounds, flavonoids, pentacylic triterpenoids, guiajaverin, quercetin, and other chemical compounds present in the plant which are believed to account for its observed hypoglycemic and hypotensive effects.
This research thus lends pharmacological credence to the suggested folkloric, ethnomedical uses of the plant in the management or control of adult-onset, type 2 diabetes mellitus and hypertension in some rural African communities, having the experimental animal study result to indications that the leaf aqueous extract of P. guajava possesses hypoglycemic and hypotensive properties. In a pharmacological review on camias, Roy et al. (2010) pointed out that, being medicinally used as a folk remedy for many symptoms, the plant as a whole serves for various purposes.
The leaves are said to have hypoglycemic and hypolipidemic activities; the claim was supported by Benny et al. (2005) through his work entitled anti-diabetic activity of the semi-purified fractions of camias in high fat diet fed-streptozotocin-induced diabetic rats, and Pushparaj et al. (2000), as he investigated the hypoglycemic activity of an ethanolic extract of camias leaves in STZ-diabetic rats by administering the diabetic rats with vehicle, camias leaves, and metformin twice a day for two weeks.
Both conclusions showed that camias significantly lowered blood glucose by 50%, thus considered as a good hypoglycemic agent in STZ-diabetic rats. As the prevalence of obesity and Diabetes mellitus are very common in our society, research on plants with anti-diabetic and anti-hyperlipidaemic action has great value in modern therapeutics. A study on the hypoglycemic, hypolipidemic and hepatic glycogen raising effect of macopa upon STZ-induced diabetic rats (Bairy et al. , 2005) was used as a related literature for another article done by Velmurugan et al. 2011) focused on the anti-diabetic and hypolipidaemic activity of bark of ethanolic extract of Ougeinia Oojeinensis using oral treatment on alloxan-induced diabetic rats. All the experiments were carried out using male, Swiss Albino mice (25-30 g) and Wister rats (150-200 g) given access to water and fed with standard comercial rat chaw pellets. A total of 24 overnight fasted rats were used, from which 18 were rendered diabetic by the intraperitoneal injection of alloxan. Mice which did not develop hyperglycemia after 48 hours were replaced with new mice.
Immediately after confirmation of diabetes, rats were classified into four groups of six rats each. Evaluation of anti-diabetic effect of test extracts was done by considering the first group as the normal control (saline), the second as the diabetic control (alloxan-induced), the third received ethanolic extract, and the fourth as reference standards. Treatment was continued for 14 consecutive days, with once a day dose. Before the treatment and at the end of the 5th, 10th, and 14th day, blood samples were collected from the retro-orbital vein of each rat.
Samples were subjected to glucose measurement using a semi-auto-analyzer. Employing one-way ANOVA followed by Dunnett's method of multiple comparisons rendered results that demonstrated the ability of the ethanolic extract of Ougenina Oojeinensis in managing diabetes associated with abormalities in lipid profiles. This work is related to the present study because the methodology of this research can serve as a point of reference for the researchers to decide which the best way of determining the hypoglycemic activity of specific plants will be.
After all, numerous mechanisms of actions have been proposed for these plant extracts. Some studies utilize the induction of streptozotocin, a naturally-occurring chemical that is particularly toxic to the insulin-producing beta cells of the pancreas in mammals, On the other hand, Velmuguran's work utilized alloxan-induced rats; alloxan is an oxidized product of uric acid that tends to produce diabetes. As both are now widely-used to induce experimental diabetes in animals, the mechanism of their action in B cells of the pancreas has been intensively investigated and now is quite well understood.
Somehow, the same study on the hypoglycemic, hypolipidemic and hepatic glycogen raising effect of macopa upon STZ-induced diabetic rats of Bairy et al. (2005) was also used as a reference by Salahuddin and Jalalpure (2010), in evaluating comparative hypoglycemic activity of the aqueous extract of Cassia glauca leaf and bark, which is related to the present study in such a way that it also is a comparative analysis regarding diabetes. The difference between this work and the previous studies cited is that, this experiment was carried out on normal, healthy rats.
No toxicity was observed with the extract in toxicity studies. Cassia glauca leaves and bark was air dried in the shade and cut into small pieces. Hundred grams each of leaves and bark was extracted with 1000 mL of water through a method called hot extraction. Regarding the evaluation of the hypoglycemic activity itself, rats were fasted for 16 hours prior to STZ-injection. Diabetes was induced in rats by intraperitoneal injection of STZ. Seven days after the injection, the blood glucose concentration level above 200 mg/dl was considered to be diabetic and used in the experiments.
Diabetic rats were then divided into three groups, wherein the first is the control, the second was given aqueous extract of leaves, and the third was given aqueous extract of the bark. The results then implied that the aqueous extract of Cassia glauca bark possessed a significant hypogylcemic effect in diabetic rats. However, the exact mechanism of action of the plant extract either alone or in combination cannot be stated. Even so, knowing that neuropathy, artery disease, and premature aging are common conditions associated with chronically elevated blood sugar levels, Dr.
Yamazaki (1998), professor of Pharmaceutical Science, Hiroshima University School of Medicine (Japan) studied the effects of corosolic acid in relation to its insulin-like properties. He found out that corosolic acid activates the transport of glucose across cell membranes, resulting in blood sugar reductions. Insulin is the hormone that naturally increases glucose transport activity across cell membranes. In the Type II diabetic, cell membranes are often less responsive to insulin-induced glucose uptake. The results of Dr.
Yamazaki’s study showed that oral administration of insulin does not reduce blood sugar, whereas orally administered corosolic acid can produce a drop in blood sugar levels. Large doses of injected insulin are capable of producing adverse reactions, while oral doses of corosolic acid have no known side effects. In rabbits, oral doses of corosolic acid have been shown to act similarly to subcutaneous injections of insulin. Hence, corosolic acid may be defined as a phyto-insulin or insulin-like plant extract.
According to Ray Sahelian, M. D. (2011), corosolic acid is a substance found in certain herbs such as almond hulls, Weigela subsessilis, Perilla frutescens, Campsis grandiflora, while it is a compound primarily extracted from a Southeast Asian tree commonly called as banaba (Lagerstroemia speciosa). Presently, corosolic acid has been the topic of many research studies and in spite of the conducted tests concluding that corosolic acid does have insulin-like activity, there are just as many studies that conclude that it does not.
Patients taking supplements containing corosolic acid do report positive results, anyway, as opposed to many diabetes drugs which carry warning labels about their dangerous side effects, including an increased risk of heart failure (De Livera, 2009). Corosolic acid has numerous biological properties including anti-diabetic, anti-inflammatory, anti-proliferative, and protein kinase C inhibition activity. (Wen et al. , 2005; Jung et al. , 2006). It is found in numerous plants species, particularly L. speciosa (Washino et al. 2004). Most medical research focuses on the compound's efficacy in diabetes, such as a published article that appeared in the April, 2008 issue of “Diabetes Research and Clinical Practice,” where Dr. Nobuya Inagaki and his colleagues at the Graduate School of Medicine of Kyoto University in Kyoto, Japan, used rat liver to study the effects of corosolic acid on glucose metabolism. Inagaki concludes that corosolic acid is able to lower blood glucose levels because it is an inhibitor of gluconeogenesis and a promoter of glycolysis.
Gluconeogenesis is the synthesis of glucose from substances that are not carbohydrates—for example, amino acids or fats—and it occurs primarily in the liver. Glycolysis is the metabolic breakdown of glucose. By inhibiting gluconeogenesis and promoting glycolysis, corosolic acid decreases the amount of glucose available to the body. Banaba leaves (Lagerstroemia speciosa L. Pers, Lythraceae) have been used in traditional medicine to treat diabetes mellitus in Southeast Asia for many years.
Banaba extracts are also known to have anti-obesity (Suzuki et al. , 1999), anti-oxidant (Unno et al. , 1997) and anti-gout (Unno et al. , 2004) effects. Corosolic acid, an active ingredient in these extracts, displays a potential anti-diabetic activity (Murakami et al. , 1993; Kakuda et al. , 1996; Lui et al. , 2001; Judy et al. , 2003; Miura et al. , 2004, 2006; Fukushima et al. , 2006; Shi et al. , 2008), as well as anti-oxidant, anti-inflammation, and anti-hypertension properties (Yamaguchi et al. 2006). Corosolic acid, equipped with numerous biological properties, including antidiabetic, anti-inflammatory, antiproliferative, and protein kinase C inhibition activity, is the component which guarantees a lowering effect on the glucose level of the blood, but none among camias, macopa, and does guava contain such. Nonetheless, each of the cited plants has its own anti-diabetic property that makes studies on its hypoglycemic activity imply results of glucose-reducing effects.
Herbal formulations are getting more importance in the treatment of diabetes, cancer and hepatic disorder because of the hazardous adverse effects of the current therapy, especially when diabetes can be controlled by allopathic medicine as well as herbal medicine. Medicinal plants continue to provide valuable therapeutic agents, in both modern medicine and traditional systems. According to Environ Health Insights (2010), over four hundred traditional plant treatments for diabetes have been reported, although a small number of these have received scientific and medical evaluation to assess their efficacy and safety.
Of these, ginseng species, bitter melon (Momordica Charantia), cloves, cinnamon, Fenugreek (Trigonella foenum graecum) and onion (Allium cepa) have been used for taste and flavor development in food preparations (Eldin et al. , 2010). It is widely known that several studies have already been performed on many other plants to test their anti-diabetic potential. Among these is the Allium cepa, which belongs to the family Liliaceae and is probably native of South West Asia and is widely cultivated throughout the world (Ikram, 1971).
It has a globose bulb that is an underground part of the stem and is so often treated as a single household vegetable. A. cepa has been used medicinally for hundreds of years (Ikram, 1971). Its most popular modern uses is to lower blood pressure (Ikram, 1971), antiseptic (Jain, 1976), hypoglycaemic and hypocholesterlemic properties (Mathew and Augusti, 1975). The active ingredient in A. cepa is allyl propyl disulfide (APDS), though other active sulphurous compounds are present (Kumari et al. , 1995).
The use of herbal products for medicinal benefits has played an important role in nearly every culture on earth and for many years, the search for anti-diabetic productswill continue to focus on plants and other natural resources (Osinubi et al. , 2006). The cost of administrating modern antidiabetic drugs is beyond the reach of most people in the low income group and those living in the rural areas, hence the use of plants for the treatment of common diseases such as diabetes are very common.
In line with the WHO (1980) expert committee on diabetes which recommends that traditional methods of management of diabetes should be further investigated. Also considering the economic resource constraints and cheapness of these herbal products, this present study was designed to determine the effects of increasing dosages of A. cepa (onions) on alloxan-induced diabetic Rattus novergicus and its possible mechanisms of action, for possible use in the control of hyperglycaemia and hyperlipidaemia characteristic of diabetes mellitus.
Two sets of compounds make up the majority of onion’s known active constituents—sulfur compounds, such as allyl propyl disulphide (APDS), and flavonoids, such as quercetin. Each of these groups of compounds has multiple medicinal actions (www. healthnotes. com, 2011). The higher the intake of onion, the lower the level of glucose found during oral or intravenous glucose tolerance tests. Experimental and clinical evidence suggests that allyl propyl disulfide is responsible for this effect and lowers blood sugar levels by increasing the amount of free insulin available (www. didea. com, 2011). Allyl propyl disulfide does this by competing with insulin, which is also a disulphide, to occupy the sites in the liver where insulin is inactivated (www. mdidea. com, 2011). This results is an increase in the amount of insulin available to usher glucose into cells causing a lowering of blood sugar. In addition, onions are a very good source of chromium, the mineral component in glucose tolerance factor, a molecule that helps cells respond appropriately to insulin.
Clinical studies of diabetics have shown that chromium can decrease fasting blood glucose levels, improve glucose tolerance, lower insulin levels, and decrease total cholesterol and triglyceride levels, while increasing good HDL-cholesterol levels (www. mdidea. com, 2011). Marginal chromium deficiency is common in the United States, not surprising since chromium levels are depleted by consuming refined sugars, white flour products, and lack of exercise. One cup of raw onion contains almost 20% of the Daily Value for this important trace mineral (www. didea. com, 2011). According to Nutraceuticals as therapeutic agents: A Review (2008), flavonoids are widely distributed in onion, endives, cruciferous vegetables, black grapes, red wine, grapefruits, apples, cherries and berries. Flavanoids in plants available as flavones (containing the flavonoid apigenin found in chamomile); flavanones (hesperidin—citrus fruits; silybin- milk thistle flavonols (tea: quercetin, kaempferol and rutin grapefruit; rutin-buckwheat; ginkgo flavonglycosides—ginkgo), play a major role in curing the cardiovascular diseases.
Flavonoids block the angiotensin-converting enzyme (ACE) that raises blood pressure; by blocking the "suicide" enzyme cyclooxygenase that breaks down prostaglandins, they prevent platelet stickiness and hence platelet aggregation. Flavonoids also protect the vascular system and strengthen the tiny capillaries that carry oxygen and essential nutrients to all cells. Flavonoids block the enzymes that produce estrogen, thus reducing the risk of estrogen-induced cancers (Rajasekaran, 2008). While onions have long been known to have several putative health benefits, onions are rich in flavinoids such as quercetin as well as sulfur compounds.
The health benefits of onions have been linked to everything from the common cold due to diabetes and osteoporosis (Morrow, 2010). Researchers from the department of pharmacology at the University of Gezira published the results of a preliminary study on the anti-diabetic properties of red onion (Allium cepa) in the journal, Environmental Health Insights. The purpose of their study was to investigate the hypoglycemic properties of red onion in patients with Type 1 and Type 2 diabetes. The flavonoids and sulphur compounds in the onions act as powerful agents in lowering blood sugar.
Researchers in the study concluded that onion it would be an effective aid in treating Type 1 and Type 2 diabetes, along with diet and exercise (www. wisegeek. com, 2011). In the study Preliminary Study of the Clinical Hypoglycemic Effects of Allium cepa (Red Onion) in Type 1 and Type 2 Diabetic Patients (2010) conducted by Imad M. Taj Eldin, Elhadi M. Ahmed and Abd Elwahab H. M, it was evident that, crude Allium cepa produced hypoglycemic effects, thus it could be used as a dietary supplement in management of type 1 and/or type 2 diabetes mellitus.
Makahiya (Mimosa pudica) plant is a creeping annual or perennial herb often grown for its curiosity value: the compound leaves fold inward and droop when touched, re-opening within minutes. This species is sometimes considered a troublesome weed in tropical crops. This plant is most often grown as an indoor annual, but is also grown for groundcover. Medicinally speaking, every part of this plant are important most especially the roots. It is said to have a bitter and astringent taste, and has a history of use for the treatment of various ailments.
Most commonly used is the root, but leaves, flowers, bark, and fruit can also be implemented. For treating kidney disorder, the plant is uprooted, the roots are wash and clean and put to boil in a pot of water. It is drink as a tea and as a substitute to water or liquid intake. Drinking boiled water of makahiya roots help eliminate kidney stones. (Bermosa, 2008) Mimosa pudica Linn is traditionally used in Indian system of medicine for the treatment of diabetes. The stem bark extract of Mimosa pudica Linn has been reported for the treatment of hyperglycemic patients, but other parts of plants such as leaves or pods have not been studied.
Hence it was thought worthwhile to screen different extracts of leaves of Mimosa pudica Linn for its anti-diabetic effects. (Sutar et al. , 2009) According to the study Anti-diabetic Activity of the Leaves of Mimosa pudica Linn in Albino Rats (2009), results show that the ethanolic extract showed hypoglycemic activity and the extract was subjected to TLC. Various solvent systems were tried. The most suitable solvent system was Chloroform: Methanol: Benzene (6:2:2). The spots were detected using iodine as a detecting reagent.
The ethanolic extract showed 3 spots, which indicated the number of constituent present in ethanolic extract. Also, because of its existence in many cultures, several medicinal uses have been developed. For example, Mimosa pudica has been taken as an antidepressant. Rodent studies have confirmed its efficacy. For example, in a study done by the University of Veracruz, Mexico, rats treated with tricyclic anti-depressants showed similar benefits as rats did with treatment with Mimosa pudica in specialized diagnostic tests.
Referring to www. ehow. com (2011), in the journal Acta Poloniae Pharmaceutica, ayurvedic concoctions containing Mimosa pudica have been shown to be helpful in the reduction of blood sugar in rodents with high blood sugar, suggesting that Mimosa pudica might be helpful as a diabetic treatment in humans as well. Corosolic acid may improve the insulin pathway. The action of insulin is mediated by tyrosine phosphorylation and initiated by the binding of insulin to the insulin receptor, promoting glycolysis.
However, although other plants are known to contain corosolic acid as well, Weigela (Weigela subsessilis) and Murtilla (Ugni molinae) are both unavailable in the Philippines and thus, cannot be utilized for the present study. The researchers, however, are aware that there are studies going on regarding plants which could contain corosolic acid, and guava, is considered a potential resource rich in corosolic acid revealed by high performance liquid chromatography, as investigated by Ying et al. (2011).
It stresses out the fact that the corosolic acid is mainly contained in the leaves, not the fruit. More researchers are getting interested in testing out the therapeutical potential of many herbal plants for convenience in medical purposes. Contrastingly, a study on alpha-glucosidase inhibitors, which are known to be oral anti-diabetic drugs used for Diabetes mellitus type 2 (American Accreditation Health Care Commission, 2012) show a different type of result as for the plant Averrhoa bilimbi. The study ? -Glucosidase inhibitory activity of selected Philippine plants (Lawag, et al. 2012) made use of six plants, specifically, Antidesmabunius (Linn. ) Spreng. (Phyllantaceae), Averrhoa bilimbi Linn. (Oxalidaceae), Biophytum sensitivum (Linn. ) DC. (Oxalidaceae), Ceriops tagal (Perr. ) C. B. Rob. (Rhizophoraceae), Kyllinga monocephala Rottb. (Cyperaceae), and Rhizophora mucronata Lam. (Rhizophoraceae) as remedies to control diabetes. The research methodology states that the 80% aqueous ethanolic extracts were screened for their ? -glucosidase enzyme inhibitory activity using yeast alpha glucosidase enzyme.
Summarizing the results, the researchers found out that except for Averroha bilimbi with IC50 at 519. 86±3. 07, all manifested a significant enzyme inhibitory activity. R. mucronata manifested the highest activity with IC50 at 0. 08±1. 82 ? g mL? 1, followed by C. tagal with IC50 at 0. 85±1. 46 ? g mL? 1 and B. sensitivum with IC50 at 2. 24±1. 58 ? g mL? 1. This is not an implication, however, that Averrhoa bilimbi is not effective as an alpha glucosidase enzyme; rather, it was pointed out in the study’s conclusion that the work serves as the first report on the ? glucosidase inhibitory effect of the six Philippine plants; thus, partly defining the mechanism on why these medicinal plants possess anti-diabetic properties. The aforementioned study is one of the most recently conducted work, and also a local one. Several factors may have affected the results of the study which was conducted in several locations, namely, Research Center for the Natural and Applied Sciences, Thomas Aquinas Research Complex, and University of Santo Tomas, an institution located at Espana, Manila, Philippines. Presently, Ranilla et al. 2010), Loarca-Pina et al. (2010), and Chee et al. (2007) highlighted that in response to the increasing economic crises and other predicaments individuals all over the world are eventually suffering from, herbs have become widely used as an agent in controlling diabetes mellitus. Because such disease is hereditary, there are unavoidable circumstances wherein one cannot do anything to remedy a pancreas that is not functioning well, thus, researches regarding antidiabetic herbs have become necessary subjects for many studies being conducted nowadays.
Some antidiabetic herbs have been reported to control diabetes mellitus by enhancing insulin secretion (Gray et al. , 1999), or by mimicking insulin activity (Gray et al. , 1998). An original article published last August 2011 showed successful results after testing the capability of agarwood leaf extracts to reduce blood glucose level. At the time, agarwood was being widely cultivated for its resin; its various parts were then reported to have several pharmacological activities. A reason that pressed Pranakhon, et al. 2011) in conducting the study entitled Antihyperglycemic activity of agarwood leaf extracts in STZ-induced diabetic rats and glucose uptake enhancement activity in rat adipocytes is basically a report that states that there has been one diabetic patient who drank water infusion of agarwoo leaf instead of water for six months. Consequently, the blood glucose of the said patient significantly decreased from 184 mg/dL to 128 mg/dL. Satisfied, the tea made from agarwood was then decided to be taken twice a day, once in the morning and once at bed time.
The next recorded blood glucose level decreased further to normal level (117 mg/dL), according to Akrarapholchote (2008). Nonetheless, no formal studies have yet been conducted by then to confirm this antihyperglycemic activty. Thus, the particular study conducted in Thailand made use of hexane, methanol, and water extracts of A. sinensis to be tested on STZ-induced diabetic rats. According to the journal itself, the glucose uptake enhancement activity of hexane, methanol and water extracts were tested in normal rat adipocytes to check the possible mean of the anti-diabetes activity.
Thin layer chromatographic (TLC) fingerprints of the extract were detected by UV absorption at 254 and 366 nm wavelengths and color reaction was detected by spraying with anisaldehyde-sulphuric acid reagent (Wagner et al. , 1996). Detection for antioxidant or radical scavenging activity of the crude extracts by spraying with 0. 1 M DPPH was also performed since oxidative stress is involved in diabetes mellitus in many ways. For their methodology, the process by which the study was done is divided into several parts. First is the extraction of the plant, wherein an amount of 2 kg of leaves of A. inensis was dried and powderized, then extracted sequentially with hexane, ethyl acetate and methanol. Each extraction was carried out three times with 6 L of solvent. Water extraction was carried out by infusing the dried powder leaves (150 g) in boiling water (2. 8 L) for 30 minutes, filtering through cotton cloth and then lyophilizing. Lyophilization, or freeze-drying, is a dehydration process typically used to preserve a perishable material or make the material more convenient for transport. All extracts were kept at -20°C until used. Thin layer chromatography fingerprints were performed to characterize the extracts.
Fingerprint analysis approach using chromatography has become the most potent tool for quality control of herbal medicines due to its simplicity and reliability, according to Chitlange (2008). Added to this, the said approach can serve as a tool for identification, authentication, and quality control of herbal drugs. As for inducing experimental diabetes in rats through the use of the chemical streptozotocin, male Sprague-Dawley rats (200-280 g) were maintained in an air conditioned room (25±1°C), with a 12 h light - 12 h dark cycle and fed with standard diet (C.
P. mouse feed, Bangkok, Thailand) and water ad libitum. All procedures have complied with the National Standards for the care and use of experimental animals and were approved by the Animal Ethics Committee of Khon Kaen University, Khon Kaen, Thailand (Rec. No: AEKKU28/2551). This is related to the study such that the same species of rats were used. The researchers, however, cannot afford to keep the experimental subjects in an air conditioned room because of certain circumstances. The rats were definitely provided standard diet, though, and water ad libitum also.
Going back to the study Antihyperglycemic activity of agarwood leaf extracts in STZ-induced diabetic rats and glucose uptake enhancement activity in rat adipocytes conducted in 2011 by Pranakhon et al. , the rats were acclimated to the Animal Transit Room where the experiment is to be performed for seven days before starting the experiment. Animals were diabetes-induced by a single intraperitoneal injection of 45 mg/kg body weight of STZ dissolved in 0. 1 M citrate buffer (pH 4. 5). After seven days of STZ injection, venous blood was collected from rat tail to determine fasting blood glucose level.
Only the rats with fasting blood glucose over 200 mg/dL were considered diabetic and were included in the experiments. Their experimental design was decided such that the rats were separated into five groups with six rats in each group as follows: Group I: diabetic control rats orally administered with distilled water; Group II: diabetic rats subcutaneously injected with 4 U/kg/day insulin (Mixedtard®) and Group III-V: diabetic rats orally administered with 1 g/kg body weight/day of methanol, water and hexane extracts, respectively. The treatments were continued for 1 week.
Fasting blood glucose levels of all rats were determined by a glucometer (Accu-Chek Advantage II, Roche Diagnostics, Mannheim, Germany). During fasting, rats were deprived of food overnight for 12 h but had free access to water. As for the statistical analysis they employed, all results are expressed as mean ± SEM. Comparisons of blood glucose levels between base line and after treatments were performed using Student’s paired t-test. For glucose uptake, the values among groups were compared and tested by analysis of variance (ANOVA) followed by Student Newman-Keuls test to show specific group differences.
A P value less than 0. 05 was considered statistically significant. In conclusion, their research claims that the results from the study showed that the methanol and the water extracts had anti-hyperglycemic activity in STZ-induced diabetic rats, increased glucose uptake by adipocytes from normal rats and contained antioxidant activities. However, hexane extract had no anti-hyperglycemic activity. The methanol and water crude extracts have anti-hyperglycemic activity in diabetic rats, most likely to be associated with glucose uptake increasing mechanism.
Lower doses of the extract should be tried in future study to establish the most appropriate dose for clinical trial. Another parallel study conducted in 2010 by Moorthy et al. , entitled Anti-hyperglycemic compound (GII) from fenugreek (Trigonella foenum-graecum Linn. ) seeds, its purification and effect in diabetes mellitus, shows an alternate process of inducing experimental diabetes to rats. As stated in the published Indian Journal of Experimental Biology, the researchers used alloxan in place of streptozotocin, in spite of the chemical being preferred for inducing diabetes.
Streptozotocin (Streptozocin, STZ, Zanosar? , CAS No. 18883-66-44) is a naturally occurring compound, produced by the bacterium Streptomyces achromogenes, that exhibits broad spectrum antibacterial properties (Vavra et al. , 1959). Antibacterial therapy, however, is not currently a widely used application for STZ (NTP, 2005). Primary contemporary uses for STZ include treatment of metastasizing pancreatic islet cell tumors, malignant carcinoid tumors, and as an investigational drug for diabetes research due to its specific toxicity associated with pancreatic ? cells (NTP, 2005). Streptozotocin is a mixture of ? - and ? -stereoisomers that appear as a pale yellow or off-white crystalline powder. STZ is very soluble in water, ketones, and lower alcohols and only slightly soluble in polar organic solvents (NTP, 2005). Streptozotocin functions as a DNA synthesis inhibitor in both bacterial and mammalian cells (Bolzan and Bianchi, 2002). In bacterial cells, a specific interaction with cytosine moieties leads to the degradation of the bacterial DNA (Reusser, 1971).
In mammalian cells, the mechanism of action that results in cell death has not been fully identified, but is thought to be a result of DNA and chromosomal damage brought forth by mechanisms involving free radical generation during STZ metabolism (Bolzan and Bianchi, 2002). In many animal species, STZ induces diabetes that resembles human hyperglycemic nonketotic diabetes mellitus (Weir et al. , 1981). This effect has been extensively studied and appears to be mediated through a lowering of beta cell nicotinamide adenine dinucleotide (NAD+) and results in histopathologic alteration of pancreatic islet beta cells (Karunanayake et al. 1974). Alloxan, on the other hand, is a toxic glucose analogue, which selectively destroys insulin-producing cells in the pancreas (that is beta cells) when administered to rodents and many other animal species. Nonetheless, alloxan (80 mg/kg body weight in citrate buffer, pH 4. 5) was injected into the tail vein of rabbits, the experimental subject in the study. These rabbits were initially normal and healthy; the test was carried out in such controls, untreated diabetic and treated diabetic rabbits.
The research aims to know the anti-diabetic activity of various fractions during purification and also for assessing the improvement in diabetic rabbits after treatment with the drug. By the end of the study, it has been stated that despite the numerous plants confirmed to be useful in aiding diabetes, it is a great advantage that fenugreek seeds need not be taken daily but can be taken intermittently. To synopsize, hyperglycemia is a condition wherein there is too much glucose circulating within the blood plasma of an individual, and this can principally lead to diabetes mellitus, a chronic ondition whose complications, according to the Australian Institute of Health and Welfare (2010) contribute significantly to ill health, disability, poor quality of life, and premature death. Searching for treatments for a deadly disease, isolation of insulin, a hormone produced in the pancreas, began in 1922 at the University of Toronto. However, prolonged use of insulin leads to certain side effects, such as severe allergic reactions that can pose a significant risk to health. Thus, researchers around the world started looking for alternatives in treating diabetes.
Though the cure remains undiscovered, many researches worked on have been providing information on the hypoglycemic effects of different plants. Corosolic acid, or glucosol, found in several herbal supplements, is a compound whose exact mechanism of activity and benefits are not yet fully determined. Even so, it is being anticipated as the component contained in several herbal plants which accounts for their capability to significantly lower blood glucose levels, as it did exhibit an insulin-like activity in a study conducted on banaba leaves.
Researchers found out later that there are other active compounds in the said plant which account for its hypoglycemic activity. Through the study of Ying et al. (2011), corosolic acid is found out to be contained in guava (Psidium guajava L. ). As for macopa (Syzygium samarangense Bl. ) and kamias (Averrhoa bilimbi L. ), they are not well-known as hypoglycemic agents but there are studies which show their capability to significantly lower blood glucose levels. The processes involved in the related studies are followed by the researchers of the present study in order to assure accuracy of the methodology and consequently, the results.
Chapter III Research Design, Methods and Procedures The framework design developed and employed, the locale of the study established, the sampling strategy and methods that were utilized, the information gathering process observed, and the data interpretation and analysis done is shown in this chapter of the research. Research Framework The study, entitled “Comparative Analysis of Hypoglycemic Activity of Camias (Averrhoa bilimbiL. ), Guava (Psidium guajavaL. ) and Macopa (Syzygium samarangense) Leaf Extracts in Glucose-treated Rats (Rattus norvegicus)”, ade use of the fundamental experimental design to identify the ranking of efficiency of the independent variables which are the Macopa, Camias, and Guava leaves in reducing the sugar level (dependent variable) of the blood of the glucose-treated albino rats. In accordance with the structured protocol, the researchers had their methodology submitted, reviewed and approved by Bureau of Animal Industry (BAI) in the Department of Agriculture, Animal Welfare Division. Afterwards, a permit was issued for them to conduct experiment involving the utilization of albino rats.
Twelve (12) male albino rats of approximately 6-8 weeks in age and bodyweight of 150-180 grams were purchased from Food and Drug Administration (FDA). Only 10 were used and the other 2 were used as spares in case of emergency that one of the experimental rats will die before the experimentation or become too weak to undergo blood extraction. There were 5 set-ups for the experiment- 1 rat in negative control, 1 rat in positive control, 1 rat fed with guava extracts, 1 rat fed with macopa extract, and 1 rat fed with camias extract.
There were two trials done, therefore justifying the 10 specimens acquired. Fortunately, no rats died before the start of experiment so the 2 spares were put in separate cages and isolated. The researchers housed each of the 10 albino rat specimens in separate steel cages of floor area 23 inches2 and cage height of 7 inches, adhering to the recommended space for rats by the Institute for Laboratory Animal Research (ILAR) in the Manual “Guide for the Care and Use of Laboratory Animals” (1996) for rats weighing 100 to 200 grams.
The sizes were the same but of different colors. For identification purposes, the specimens were marked using a red marker (nontoxic) in the following areas for identification: right ear, left ear, right leg (anterior), left leg (anterior), right leg (posterior), left leg (posterior), base of tail, tip of tail, lower middle of the tail and upper middle of the tail. Cages were cleaned and litter boxes were replaced every morning to ensure comfortable and sanitized environment of the rats.
As for the feeding routine for the whole experiment, 4 tablespoons of rice grain and 10 ml of water per day is given to each rat as stated in Guidelines for Proper Conduct of Animal Experiments of Science Council of Japan (2006). | | The researchers were supervised by a professional veterinarian, Dr. Wenceslao C. Ortiz Jr. , of De La Salle Araneta University for the acquisition of the baseline of the blood glucose level of the rats. The equipments (lancet needles, strips and glucometer) were bought from "Mary Immaculate Medical Supplies" and "Gemry Medical Supplies", both located in Bambang.
The blood was extracted from the tip tail using a lancet needle. The droplet of blood was then placed on the test strip. Finally, the strip was inserted onto the glucometer, which in turn, revealed the glucose reading of the blood measured in mg/dL. The lancets and strips were disposed in a glass waste bottle right after every usage to prevent contamination and erroneous results. In the process, the veterinarian and his assistant showed the researchers the proper handling of rats as well as the precise method of extracting blood.
Stress, factor which may negatively affect the results of the study, was prevented by giving the rats an ample time of three days of rest. A 200 ml bottle of Glucolin was bought from Intercon Diagnostic Laboratory, Inc. , Binondo, Manila. Each rat was given 15 mL of glucolin using a dropper (5 ml every day within 3 days). Within the period that glucolin was orally-administered to the specimens; provision of drinking water was strictly prohibited. Eventually, blood glucose level was obtained twice during the 3 days of treatment.
This was needed to ascertain that the raise in glucose level was made by the glucolin and not the natural insulin mechanism of the specimen as well as to check whether the rats reached the hyperglycemic state already (blood glucose above 100 mg/dL). Camias leaves were collected from Barangay Cembo, Makati City; the Guava and Macopa leaves were both gathered from Valenzuela City. The trees, from which the leaves were handpicked, were free from chemical fertilizers to ensure the purity of the extracts and to avoid undesired side effects on the animals as well.
The leaves were brought to National Museum for accurate and valid identification. A certificate was issued confirming and validating the identities of the leaves. Afterwards, the samples were immersed into the water for 24 hours. Subsequently, leaves were blended until the required 1. 5 liters of each extract was obtained. The blended leaves were brought to Adamson University for filtration and rotary evaporation. Rotary Evaporation was used since the researchers needed 100% concentration and this process eliminates the solvent from the extract.
Eight (8) hours was allotted for the filtration and distillation of the three samples. Approximately, 200 mL extract was retrieved from each concoction with viscosity identical to water’s consistency. Aside from the extracts, insulin was also required in conducting the experiment. Thus, injectable insulin was bought from Mercury Drugstore in Bambang Mercury Drugstore. The insulin functioned as the positive control and was expected to reduce the rats’ glucose level the most.
The gathered extracts and insulin were administered to the rats at the same time. The insulin was injected to the rats with a mark in their front left or right leg. Guava extract was provided to the rats with a mark in their left or right ear, Camias extract for the rats with a mark on their right or left leg, macopa extract for rats with a mark on the base or tipoff their tail. And water only was provided to the rats with a label on the middle or upper mid-tail; these rats functioned as the negative control in the experiment. mL of each extracts were administered using a dropper and the dosage of insulin is . 5 IU. The blood of each rat was extracted to determine the glucose level. The results obtained were recorded. After the experiment, the specimens were sent to De la Salle Araneta Veterinary Hospital for the euthanizing process via cervical dislocation in accordance with the “Current Protocols in Immunology” (2006) for laboratory specimens.
One Way ANOVA for Repeated Measures was used in determining if there was a significant difference between the hypoglycemic effects of the three leaf extracts namely guava, macopa and camias since each rat was repeatedly used to obtain blood glucose level and the level of measurement was ratio. It was the most appropriate statistical test when comparing whether the mean of any of the individual experimental conditions differ significantly from the aggregate mean across the experimental conditions.
In ranking the lead extracts from most effective to least effective, the decrease in glucose level before and after treatment of hyperglycemic rats caused by the extracts was averaged since there were two trials. The greatest average will be deemed the most effective. MONTH| AUGUST| SEPTEMBER | DAY| 28| 29| 30| 31| 1| 2| 3| 4| 5| 6| 7| 8| 9| 10| 11| 12| 13| 14| 15| 16| SpecimenGathering| | | | | | | | | | | | | | | | | | | | | MaterialGathering| | | | | | | | | | | | | | | | | | | | Baseline GlucoseLevel| | | | | | | | | | | | | | | | | | | | | Leaves Gathering| | | | | | | | | | | | | | | | | | | | | Extraction of leaves| | | | | | | | | | | | | | | | | | | | | Glucose Induction| | | | | | | | | | | | | | | | | | | | | Acquisition of Glucose level| | | | | | | | | | | | | | | | | | | | | Inducing of Extracts and Insulin| | | | | | | | | | | | | | | | | | | | | Final Acquisition of Glucose Level| | | | | | | | | | | | | | | | | | | | | Euthanasia| | | | | | | | | | | | | | | | | | | | | Figure 1.
Gantt Chart Showing the Time Schedule Allotted for the Experiment of the Study CHAPTER IV Results and Discussion The glucose levels results for two trials are presented in this chapter. The results are presented in table form. The results displayed were measured in mg/dL. The measured glucose levels were categorized into three conditions: baseline, glucolin, and after treatment. In the baseline, the glucose levels shown were obtained a day after the rats were brought. As for the drinking of glucolin, the rats were administered with ‘Glucolin 100’ each with 15 mL.
And in the after treatment group, the glucose levels were obtained after the rats were given with the extracts, insulin, and water. And the results were summarized and computed using One Way ANOVA for Repeated Measures to know if there is a significant difference among the hypoglycemic effects of the three leaf extracts. To rank the leaves according to the most effective to the least effective, the average of the inflicted reduction in glucose level for the two trials was obtained. The greater the average, the more effective the extract is. Statistical Treatment
One way ANOVA for repeated measures was used in this study. An ANOVA with repeated measures is for comparing three or more group means where the participants are the same in each group. This usually occurs in two situations - when participants are measured multiple times to see changes to an intervention or when participants are subjected to more than one condition or trial and the response to each of these conditions wants to be compared. In this case, the effects of the different conditions were analyzed. TABLE 1. 1 SUMMARY OF THE GLUCOSE LEVEL OF RATS After treatment
Table 1. 1 as portrayed above, shows the consolidated results obtained in the glucose level testing within the timeframe. These data were the ones used in the computations and analysis of results. Two trials were done with 3 experimental groups (guava,camias and macopa) , 1 negative control and 1 positive control per trial. The blood glucose level was acquired for baseline, after drinking glucolin and after giving the corresponding treatments. Glucolin was given until the rats were hyperglycemic. The hyperglycemic level for rats is greater than or equal to 100 mg/dL.
After which, the corresponding treatments were given to the specimens. LEVEL OF SIGNIFICANCE The level of the significance used is ? =0. 05 and the degrees of freedom (df) is a=3 N=15 s=5, where a is the number of sets of condition; N is the total number of data; s is the number of subjects (rats). The equations that were used for computing the degrees of freedom: dfbetween = a-1Eq. 1. 1 dfwithin =N-aEq. 1. 2 dfsubjects =s-1Eq. 1. 3 dferror = dfwithin - dfsubjectsEq. 1. 4 dftotal = N-1Eq. 1. 5 The computed results were dfbetween= 2, dfwithin = 12, dfsubjects = 4, dferror = 8, and dftotal = 14.
Using the F –table, the critical value is 4. 4590 and if the computed F value is greater than 4. 4590, the null hypothesis is rejected, then, the alternative hypothesis can be accepted. The null hypothesis states: There is NO significant difference between the hypoglycemic effects of the leaf extracts. The alternative hypothesis states: There IS significant difference between the hypoglycemic effects of the leaf extracts. TRIAL 1 TABLE 1. 2 GLUCOSE LEVEL OF RATS IN THE FIRST TRIAL Table 1. 2 shows the glucose level from the rats in the first trial.
The list of the subjects and their corresponding treatment was indicated in the first column. In the second column, the baseline or the first obtained glucose level was shown. There is a large increase in the glucose levels of the rats after glucolin was induced to them, as shown in the third column. And in the last column the glucose levels declined after the rats were given with the extracts, insulin and water. The total of the baseline is 351, for the glucolin is 842 and after the treatment is 407. These summations are needed for the computation for the F- value TABLE 1. TEST STATISTIC TRIAL 1 | SS| df| MS| F value| Between| 28896. 1| 2| 14448. 1| 5. 71| Within| 32189. 2| 12| 2529. 7| | -Subject| 11952| 4| | | -Error| 20237. 2| 8| | | Total| 32189. 2| 14| | | Table 1. 3 shows the results computed using the One Way ANOVA. The F-value obtained is 5. 71. Base on the decision rule, since F value which was computed to be 5. 71 is greater than critical value of 4. 4590, reject null hypothesis and accept the alternative hypothesis. Therefore, there is a significant difference between hypoglycemic effects of leaf extracts in Trial 1. TRIAL 2 TABLE 1. GLUCOSE LEVEL OF RATS IN THE SECOND TRIAL Table 1. 4 shows the glucose level from the rats in the second trial. The list of the subjects and their corresponding treatment was specified in the first column. The first glucose levels obtained in the second set of rats was shown in the second column. In the third column, it was shown that there is a surge in the sugar level of each rat. And in the last column the glucose levels reduced after the rats were given with the treatment. The total of the baseline is 344, for the glucolin is 799 and after the treatment is 461. TABLE 1. 5 TEST STATISTIC TRIAL 2 SS| df| MS| F value| Between| 22330. 5| 2| 11165. 3| 5. 87| Within| 24174. 4| 12| 1901. 2| | -Subject| 8964. 9| 4| | | -Error| 15209. 5| 8| | | Total| 68835. 4| 14| | | Table 1. 4 shows the results computed using the One Way ANOVA. The F-value obtained is 5. 87. Base on the decision rule, since F value which was computed to be 5. 87 is greater than critical value of 4. 4590, reject null hypothesis. Therefore, there is a significant difference between hypoglycemic effects of leaf extracts in Trial 2. Table 1. 6 Average Reduction Inflicted by Extracts in Blood Glucose Level of Rats
Extract| Difference of Glucose Level (Trial 1) in mg/dL| Difference of Glucose Level (Trial 2) in mg/dL| Average| Guava| 104| 136| 120| Camias| 62| 25| 43. 5| Macopa| 22| 18| 20| Water | -19 (increased)| 2| -8. 5 (increased)| Insulin| 228| 157| 192. 5| As seen in the table, the greatest average inflicted reduction was the commercially bought insulin with 192. 5 mg/dL. It was followed by the Guava leaf extract which caused a total of 120 mg/dL reduction in the glucose level of the rat. The third was the Camias leaf extract, earning 43. 5 mg/dL decrease in blood glucose level.
The macopa, was the least effective of the three plants, with only 20 mg/dL reduction in the glucose level. Meanwhile, in the water, there was an increase in the glucose level instead of lowering. As portrayed in the table, we can now rank the effectiveness of the three difference herbal hypoglycemic extracts from the strongest to the weakest. The extract having the greatest average inflicted reduction in the glucose level will be declared the strongest and followed accordingly by the next highest and the lowest. The strength can be now summarized as follows: Guava leaf extract > Camias leaf extract > Macopa leaf extract.
CHAPTER V Conclusion and Recommendations Conclusion There is a significant difference among the hypoglycemic effect of the three leaf extracts namely Guava, Macopa and Camias in the glucose- treated rats since the computed F-values for trial 1(F=5. 71) and trial 2 (F=5. 87) are greater than the critical value which is 4. 4590. This means that the reducing power in terms of blood glucose level of the extracts is varied at a significance level of ? = 0. 05. When the efficiency is ranked accordingly, Guava leaf extract > Camias leaf extract > Macopa leaf extract.
The guava has the highest average of inflicted reduction in glucose level which was 120 mg/dL, followed by the camias leaf which reduced the glucose level by 43. 5 mg/dL and last would be the macopa leaf which reduced blood glucose by 20 mg/dL. When compared to the inflicted reduction caused by the insulin, the extracts are less effective but it is still beneficial since it is safer to intake, with no synthetic products and harsh chemicals. Recommendation The researchers would recommend lengthening the duration of the whole experiment to about a month to fully monitor the blood glucose level after the extracts have been administered.
This way, more data would be yield, thus giving more possibility for exploration of deeper relationships. If there were more data, various statistical treatments can be further employed to test different aspects of the hypoglycemic effects like within groups. In addition, it would be best to check the glucose level of the rats when being fed with glucolin at an interval of at most 6 hours to be assured that the raise in glucose was really cause by the glucolin and not much affected yet by the insulin production of the rats.
Also it would help the researchers in determining what is the exact time that all the rats become hyperglycemic (reached 100 mg/dL or above). This way, the results will be more accurate. APPENDIX A Certificates and Receipts A. Animal Research Permit from the Bureau of Animal Industry B. Certification of Plant Species from the National Museum C. Rat Purchase and Delivery Receipt from the Food and Drug Administration D. Animal Research Permit Payment Receipt E. Glucolin Purchase Receipt from the Intercon Diagnostic Laboratory, Inc. F. Request Letter to the Director of the Adamson University G.
Extraction through Rotary Evaporation in Adamson University H. Purchase of Glucometer Strips and Lancets from Gemry Medical Supply I. Purchase of Droppers and Surgical Gloves from the Mercury Drugstore J. Purchase of Mixtard® 30 HM Flex Plen® Human Biphasic Isophane Insulin from the Mercury Drugstore K. Statement of Charges in De La Salle Araneta University Veterinary Hospital APPENDIX B Solutions TRIAL 1 Repeated Measures ANOVA Subjects| Baseline| Glucolin| After Treatment| Rat 1 (Guava)| 48| 161| 57| Rat 2 (Kamias)| 64| 108| 46| Rat 3 (Macopa)| 88| 104| 82| Rat 4 (Negative Control/Water)| 67| 185| 166|
Rat 5 (Positive Control/Insulin)| 84| 284| 56| ? ai| 351| 842| 407| ?(? a i ) = 1600 1. Hypotheses Ho: There is NO significant difference between the hypoglycemic effects of the leaf extracts. Hi: There IS significant difference between the hypoglycemic effects of the leaf extracts. 2. Level of Significance ? = 0. 05 3. Degrees of Freedom a = 3N = 15 s = 5 where: a is the number of sets of condition N is the total number of data s is the number of subjects (rats) dfbetween = a-1= 3-1=2 dfwithin =N-a=15-3=12 dfsubjects =s-1=5-1=4 dferror = dfwithin - dfsubjects =12-4 =8 dftotal = N-1 =15 -1 =14 . Decision Rule dfbetween = a-1= 3-1=2 dferror = dfwithin - dfsubjects =12-4 =8 (2,8) Looking at F-table ? =0. 05 CRITICAL VALUE = 4. 4590 If F value is greater than 4. 4590 then reject null hypothesis and accept the alternative hypothesis. 5. Test Statistic | SS| df| MS| F value| Between| 28896. 1| 2| 14448. 1| 5. 71| Within| 32189. 2| 12| 2529. 7| | -Subject| 11952| 4| | | -Error| 20237. 2| 8| | | Total| 32189. 2| 14| | | SS between = ? (? a i )2 -T2 = (3512 + 8422 + 4072) - 16002 = 28896. 1 s N 5 15 SS within = ?
Y2 - ? (? a i )2 = 482 + 642 + 882 + 672 + 842 + 1612 + 1082 + - (3512 + 8422 + 4072) s 1042+1852 + 2842 + 572 + 462+ 822+ 1662+ 562 15 = 32189. 2 SS subject = ? (? s i )2 - T2 = 266+ 218+ 274+ 418+ 424 - 16002 = 11952 a N 3 15 ?s I (sum across rows) Rat 1= 48 +161+ 57= 266 Rat 2= 64+ 108+ 46= 218 Rat 3= 88+ 104+ 82= 274 Rat 4= 67+ 185+ 166= 418 Rat 5= 84+ 284+56= 424 Error = within- subjects = 32189. 2- 11952= 20237. 2 Total = between + within= 28896+ 32189. 2 = 61085
MSbetween= SSbetween = 288961. 1 = 14448. 1 df between 2 MSerror= SSerror = 20237. 2 = 2529. 7 df error 8 F= MSbetween = 14448. 1 = 5. 71 MSerror 2529. 7 Since F value which was computed to be 5. 71 is greater than critical value of 4. 4590, reject null hypothesis. Therefore, there is a significant difference between hypoglycemic effects of leaf extracts in Trial 1. TRIAL 2 Repeated Measures ANOVA Subjects| Baseline| Glucolin| After Treatment| Rat 1 (Guava)| 64| 189| 53| Rat 2 (Kamias)| 74| 100| 75| Rat 3 (Macopa)| 67| 101| 83|
Rat 4 (Negative Control/Water)| 70| 189| 187| Rat 5 (Positive Control/Insulin)| 69| 220| 63| ? ai| 344| 799| 461| ?(? a i ) = 1604 1. Hypotheses Ho: There is NO significant difference between the hypoglycemic effects of the leaf extracts. Hi: There IS significant difference between the hypoglycemic effects of the leaf extracts. 2. Level of Significance ? = 0. 05 3. Degrees of Freedom a = 3N = 15 s = 5 where: a is the number of sets of condition N is the total number of data s is the number of subjects (rats) dfbetween = a-1= 3-1=2 dfwithin =N-a=15-3=12 dfsubjects =s-1=5-1=4 ferror = dfwithin - dfsubjects =12-4 =8 dftotal = N-1 =15 -1 =14 4. Decision Rule dfbetween = a-1= 3-1=2 dferror = dfwithin - dfsubjects =12-4 =8 (2,8) Looking at F-table ? =0. 05 CRITICAL VALUE= 4. 4590 If F value is greater than 4. 4590 then reject null hypothesis and accept the alternative hypothesis. 5. Test Statistic | SS| df| MS| F value| Between| 22330. 5| 2| 11165. 3| 5. 87| Within| 24174. 4| 12| 1901. 2| | -Subject| 8964. 9| 4| | | -Error| 15209. 5| 8| | | Total| 68835. 4| 14| | | SS between = ? (? a i )2 -T2 = (3442 + 7992 + 4612) - 16042 = 22330. 5 N 5 15 SS within=? Y2 -? (? a i )2 = 642 + 742 + 672 + 702 + 692 + 1892 + 1002 +1012 - (3442 + 7992 + 4612) s + 1892 + 2202 + 532+ 752+ 832+ 1872+632 15 = 24174. 4 SS subject = ? (? s i )2 - T2 = 3062 + 2492 +2512 + 4462 + 3522 - 16042 = 8964. 9 a N 3 15 ?s I (sum across rows) Rat 1= 64+189+53 = 306 Rat 2= 74+ 100+ 75 = 249 Rat 3= 67+101+83= 251 Rat 4= 70+189+ 187= 446 Rat 5= 69+220+63= 352
Error = within- subjects = 24174. - 8964. 9 =15209. 5 Total = between + within= 22330. 5-46509. 9= 68835. 4 MSbetween= SSbetween = 22330. 5= 11165. 3 df between 2 MSerror= SSerror = 15209. 5 = 1901. 2 df error 8 F= MSbetween = 11165. 3 = 5. 87 MSerror 1901. 2 Since F value which was computed to be 5. 87 is greater than critical value of 4. 4590, reject null hypothesis. Therefore, there is a significant difference between hypoglycemic effects of leaf extracts in Trial 2. APPENDIX C Documentation A. At DLSAU Veterinary Hospital for Glucose Level Baseline
B. Extraction of Guava, Macopa and Kamias C. Blood Glucose Testing After Glucolin Treatment D. Feeding the Extracts BIBLIOGRAPHY * American Diabetes Association (2012). Living With Diabetes: Hyperglycemia. http://www. diabetes. org/living-with-diabetes/treatment-and-care/blood-glucose-control/hyperglycemia. html * Guidelines for Proper Conduct of Animal Experiments (2006) Science Council of Japan http://www. scj. go. jp/ja/info/kohyo/pdf/kohyo-20-k16-2e. pdf * Guide for the Care and Use of Laboratory Animals (1996) Institute for Laboratory Animal Research (ILAR) http://www. aaalac. org/resources/Guide_2010. df * PubMed Health (2011). ADAM Medical Encyclopedia: Diabetes. http://www. ncbi. nlm. nih. gov/pubmedhealth/PMH0002194/ * Medicinal plants of India with anti-diabetic potential (2002). Grover JK et al. http://ukpmc. ac. uk/abstract/MED/12020931 * Morton, J. (1987). Fruits of Warm Climates: Guava, p. 356-363 * Morton, J. (1987). Fruits of Warm Climates: Bilimbi, p. 128-129 * NCBI (2010). Anti-hyperglycemic and anti-hyperlipidimic effects of guava leaf extract. Deguchi, Y. and Miyazaki, K. http://www. ncbi. nlm. nih. gov/pmc/articles/PMC2831039/ * Singapore Infopedia (2005). Camias: Belimbing. ttp://infopedia. nl. sg/articles/SIP_856_2005-01-28. html * Philippine Medicinal Plants (2012). Family Oxalidaceae: Kamias. http://stuartxchange. com/Kamias. html * The Mammals of Texas (2012). Norway Rat: Rattus Norvegicus. http://www. nsrl. ttu. edu/tmot1/rattnorv. htm * Quality of life associated with diabetes mellitus in an adult population (1999) Brown et al. http://www. jdcjournal. com/article/S1056-8727(00)00061-1/abstract * Alternative therapies useful in the management of diabetes: A systematic review (2011) Pandey, Awanish et al. http://www. ncbi. nlm. nih. gov/pmc/articles/PMC3249697/