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Inhibitory Effect of Curcuma longa and Zingiber officinale on Glucose Absorption in Rat Small Intestine

Andrea Carla M. AdajarNelle Rey Anne G. AsorShaira B. HernandezJanella Mariz N. JazarenoNos Amev V. Serrano

Group 3 Sec. AB-2L

May 4, 2015

INTRODUCTIONGlucose is the primary source of energy in order for the cells to function and its absorption is an essential metabolic process in the living body system. The small intestine which is responsible for the absorption of the majority of glucose taken in is also important for the regulation of glucose metabolism (Thazhath et al., 2014). Hence, foods that have properties that can alter the structural or functional capabilities of the small intestine may either improve or impede glucose uptake. Furthermore, the occurrence of these changes may lead to serious metabolic problems such as obesity, short- gut syndrome, Type 2 diabetes mellitus, and critical illnesses.Ginger has been an indispensable crop in Southeast Asia which is generally used as a culinary spice and an herbal medicine for different illnesses. Recent studies have shown that ginger has anti- microbial, anti- inflammatory, and possible anti- cancer properties (Habib et al., 2008). In the Philippines, common ginger (Zingiber officinale) and yellow ginger (Curcuma longa) are widely cultivated. Both of the said varieties have long been used in cooking and in medicinal purposes, specifically in treating wounds, diarrhea, rheumatism, and sore throat. Recently, extensive studies have been performed on the action of ginger on the inhibition of vitamin C and glucose intestinal transporters (Song et al., 2002). Ginger was shown to normalize blood glucose level by reducing glucose absorption in the gut. Ginger phenolics, specifically flavonoids, which are abundant in fruits and vegetables, were proposed to be the active compounds in delaying or inhibiting ascorbate and glucose transporters. Moreover, these compounds from ginger extract have been suggested to have broad antioxidant properties in humans. However, their effects in vivo remain unclear.If the extracts from two ginger varieties are established to inhibit glucose absorption in rat small intestine, then this study may serve as a reference in developing a drug that will assist in the treatment of illnesses such as hyperglycemia, mild glucose intolerance, and obesity, which are caused by high concentrations of glucose being absorbed in the small intestine.It can also be of great help for the medical industry in searching and developing a potential drug in treating diseases caused by elevated concentrations of glucose being absorbed in the small intestine, which can be an economically important alternative for expensive insulin-based medications. The results of this study can be used as a source for further studies on plants having glucose- enhancing or -inhibiting properties.The specific objectives of the study are:1)to evaluate the inhibitory effect of the extract of two ginger varieties, namely, Curcuma longa and Zingiber officinale, on glucose absorption in rat small intestine; and2)to compare the effectiveness in the inhibition of glucose absorption of the two ginger varieties.The study was conducted at the Animal House of the Institute of Biological Sciences from March 19 until April 19 and at room 405 of the New College of Arts and Sciences (NCAS) in the University of the Philippines Los Baos on April 20, 2015.

REVIEW OF RELATED LITERATUREMany past studies had been made in order to determine the effect of various substances on the glucose absorption in the small intestine of rats and most of the experiments were done in vitro. Mostly, studies on glucose absorption in the small intestine were done to understand it. However, since the widespread of obesity and diabetes, many substances are being tested as potential inhibitors of fat, cholesterol, and glucose absorption (Kwon et al., 2006). Also, flavonoids are well known inhibitors of glucose absorption. In spite of ginger being rich in flavonoids, no research had been made about its dietary effect in vivo on glucose absorption in the small intestine. Ginger was used in this experiment to test its potential inhibitory effect on small intestine glucose absorption, since it was found out that chronic intake of ginger decreases blood sugar (Nwachukwu and Ohiri, 2012).Nwachukwu and Ohiri(1) (2012) did a research on the effect of the chronic intake of common ginger on the gastrointestinal sections of rats. Four groups of rats were fed with powdered ginger mixed with normal rat food in varying concentrations. The ginger was prepared by drying the rhizomes under the sun and grinding them into powder by using an electronic milling machine. The researchers did this for eight weeks and they found out that blood glucose content increased in the first four weeks of the treatment and decreased significantly in the following weeks. In addition, they also observed the triacylglycerol and cholesterol profiles of the rats. However, the flavonoid content of ginger was not taken into consideration as a means to explain their results; they only explained that some compounds found in ginger caused a higher level of bile production in the liver that eventually lead to faster digestion. According to them, this caused the food to move faster along the digestive tract and therefore allowed little time for glucose to be absorbed in the intestines.The paper showed significant results but the method used in feeding the rats may have affected the results. This is because they did not control the amount of food that the rats can feed on. Although the concentration of the ginger was controlled, the fact that some rats may have eaten more food than others that may also have an effect on the blood sugar, cholesterol, and fatty acids cannot be completely ignored. Because of this, the gavage method will be employed in this experiment. In addition, the amount and concentration of ginger will be controlled by adjusting it according to the rats body weight.A similar study was conducted by Elshater, Salman, and Mussa (2009), except that they induced the rats with diabaetes through Alloxan. Also, instead of feeding ginger to rats, they obtained ginger extracts and had the rats intake them orally through the gavage method. The treatment was done for six weeks. Results showed that post- treatment of ginger has an overall anti- hyperglycaemic effect on diabetic rats. Unlike the formerly mentioned studies, blood glucose will not be measured in this experiment. Instead, the transport of glucose will be observed in vivo by conducting a modified experiment done by Rider, Schedl, Nokes, & Shining (1967). The experiment involved the isolation of the small intestine of rats. The insides were pumped with ionic glucose solution and the initial and final concentration of glucose was measured. The modification is that the method will be done in a more controlled environment.

MATERIALS AND METHODSGinger Extracts PreparationFresh ginger rhizomes of Zingiber officinale and Curcuma longa, weighing approximately 2.5 kilograms each, were purchased in a public market. These were washed several times with water until remnants of soil were removed. The gingers were thinly sliced, were placed in clean dry tray, and were sun- dried for 13 hours. After sun- drying, the rhizomes were grinded, separately, in a blender.

Treatment of ratsFifteen, two weeks old male rats of a certain weight range were housed in separate cages made of chicken wire. A week of acclimatization was provided; rats were fed with normal rat food that was 20% of their body weight. For the duration of three weeks, they will be given drinking water (ad libitum). This was based on Zvinorova, Lekhanya, Erlwanger, and Chivandis experimental methods in 2014.The rats were divided into three groups; five rats were randomly assigned to each experimental setup. All the rats, during the course of the experiment, were given food that was 10% of their body weight. Three dietary treatments were given to each group: the normal rat food, wherein no ginger extracts were introduced this served as the control setup; the Zingiber officinale- based diet, where common ginger extract were introduced (mL/kg); and, the Curcuma longa- based diet, introduced with yellow ginger extract (mL/kg). The amount of ginger extract fed to each rat was computed by dividing 4 mL to the respective weights (kg) of the subjects as presented on the study conducted by Elshater, Salmann, and Moussa in 2009.The weight of each rat was recorded before the experiment started (Day 0). During the experimentation proper, the rats were weighed every other day in order for the researchers to accordingly adjust the amount of food to be given to a particular rat. This was done for three weeks. The rats were then fasted overnight; only water was provided. Prior to dissection, the rats were weighed.

Determination of the effect of Ginger root powder on glucose absorptionUpon sacrificing a rat by applying a strong force to its hind head, the abdominal cavity was dissected by carefully cutting through the abdominal wall. The surrounding mesenteries and fats were removed. Measuring from the duodenum, 18 cm of the small intestine was obtained by severing it from the stomach and the colon. The inside of the intestine was flushed using Krebs PO4- Ringers solution until clear fluid flowed at the other end. The intestine was then inverted inside out using a thread and a steel rod immersed in Krebs PO4- Ringers solution. Afterwards, it was immersed in a Petri dish containing Ringers solution. Four centimeters of the small intestine was cut then tied at one end. The open end of the gut was tightly tied after slipping the tip of a cannula inside it. In a 15 mL test tube, an amount of 10 mL of Krebs PO4- Ringers solution + glucose (500 mg glucose/ 100 mL Krebs PO4- Ringers solution) was placed. A stopper containing the cannula, gut sac, and hypodermic needles were inserted into the tube upon submerging the gut into the before mentioned fluid. The inside of the sac was then filled with the same fluid amounting to about two centimeters above the tube fluid level. A fine stream of oxygen gas bubbles was then introduced into the fluid via the rubber tubing from the cylinder reducing valve that was connected to the no. 21 hypodermic needle. The apparatus (Fig. 1) was then immersed in a water bath at 37 C for 30 minutes.

Figure 1. Setup for the determination of amount of glucose absorbed in rat small intestine

Image source: Tuttle, W.W., Schottelius, B., Thomson, J. (1963). Physiology Laboratory Manual p. 162 Fig. 18.

The initial glucose concentration was then determined in the setup. After mixing five drops of Krebs PO4- Ringers solution and five drops of distilled water in a 15 mL test tube, a Clinitest tablet was added. The resulting mixture was only mixed by shaking for 15 seconds after the boiling stopped. The color of the mixture was then compared against a Clinitest color chart to determine the initial concentration of the glucose.The fluid from the sac then displaced to the broader part of the cannula by the no. 15 hypodermic needle. Afterwards, displaced fluid was placed in a 15 mL test tube and was subjected to the previous glucose test to determine the final glucose concentration.

REFERENCESElshater, A., M. Salman Arn, & M. Moussa. (2009). Effect of ginger extract consumption on levels of blood glucose, lipid profile and kidney functions in Alloxan- induced diabetic rats. Egypt. Acad. J. Biolog. Sci., 2 (1), 153-162.Habib, S.H., Makpol, S., Hamid, N.A.A., Das, S., Ngah, W.Z.W., & Yusof, Y.A.M. (2008). Ginger extract (Zingiber officinale) has anti-cancer and anti-inflammatory effects on ethionine-induced hepatoma rats. Clinics (Sao Paulo), 63(6), 807-13.Kwon, O., Eck, P., Chen, S., Corpe, C., Lee, J., Kruhlak, M., & Levine, M. (2007). The inhibition of intestinal glucose transporter GLUT2 by flavonoids. The FASEB Journal, 21 (2), 366-77. Retrieved from US National Library of Medicine National Institute of Health. Retrieved from http://www.ncbi.nlm.nih.gov/Nwuachukwu, N. & Ohiri, R. C., (2012). Effect of chronic intake of Zingiber officinale (ginger) enriched diet on the gastrointestinal sections of albino rats. African Journal of Food and Science, 6 (12), 330-334. DOI: 10.5897/AJFS11.105Rider, A., Schedl, H., Nokes, G., & Shining, H. (1967). Small intestinal glucose transports: Proximal- distal kinetic gradients. The Journal of General Physiology, 50, 1173-1182.Song, J., Kwon, O., Chen, S., Daruwala, R., Eck, P., Parks, J.B., & Levine M. (2002). Flavonoid inhibition of Sodium- Dependent Vitamin C Transporter 1 (SVCT1) and Glucose Transporter Isoform 2 (GLUT2), intestinal transporters for Vitamin C and glucose. The Journal of Biol. Chem., 277, 15252-260.Thazhath, S.S., Wu, T., Young, R.L., Horowitz, M., & Rayner, C.K. (2014). Glucose absorption in small intestinal diseases. Expert Rev Gastroenterol Hepatol, 8(3), 301-12.Umayal Sundari, A.R., Neelamegam, P., & Subramanian, C.V. (2013). Study and analysis of drying characteristics of ginger using solar drier with evacuated tube collectors. Research Journal of Pharmaceutical, Biological and Chemical Sciences, 4 (3), 1264-1265.