Phytochemistry-i PGC 504

Cactus. Phytochemistry-i PGC 504.

Chemical composition and evaluation of possible alpha glucosidase inhibitory activity of eight Aloe species.

Abstract. The polysaccharides found in the gel of the leaves of Aloe species have been associated to many of the health benefits associated with the plant. There are eight different Aloe species, A. vera (A1), A. arborescens (A2), A. eru (A3), A. grandidentata (A4), A. perfoliata (A5), A. brevifolia (A6), A. saponaria (A7) and A. ferox (A8), these polysaccharides were extracted by hot extraction method then hydrolyzed. By using high-performance liquid chromatographic method, we identify The polysaccharide hydrolysates where, A7 (12.04%), A1 (8.51%), A8 (8.03%), A2 (5.32%) and A6 (2.18%) respectively had the highest yields of total polysaccharides detected. In alloxan-induced diabetic rats, the isolated polysaccharides were tested for antihyperglycemic activity and alpha glucosidase inhibitory activity..

In the eight Aloe species studied, chromatographic analysis revealed the presence of 18 saccharides, glucuronic acid, stachyose, galacturonic acid, sucrose, glucose, xylose, galactose, rhamnose, mannose, arabinose, fructose, but the main saccharides found were glucuronic acid, stachyose, and galacturonic acid. The biological activities demonstrated significant antihyperglycemic activity with varying degrees of effectiveness. Polysaccharides isolated from A. vera (A1) and A. arborescens (A2) were the most active after four weeks of daily treatment, lowering blood glucose levels by 40 and 44 percent, respectively. The alpha glucosidase inhibitory activity of all polysaccharides examined was considerable, with IC50 (g/ml) values of 11.70, 14.60, and 15.80 for A7, A6, and A1, respectively. Finally, the polysaccharides studied contribute to the anti-diabetic properties of these Aloe species..

Introduction. The International Diabetes Federation (IDF) estimated that there are 34.6 million people with diabetes in the Middle East and North Africa, a number that will almost double to 67.9 million by 2035 if concerted action is not taken to tackle the risk factors fuelling the epidemic of diabetes. Egypt is now ranked eighth highest in the world in terms of the disease..

Diabetes mellitus can cause complications where body cells cannot uptake and utilize glucose; therefore breakdown of fats increase with production of fatty acids and ketone bodies, this disorder is accompanied by decrease in protein synthesis..

In recent years, several synthetic drugs have been developed to combat against diabetes, but they aren’t able to combat with all the pathological complications. Some of medicinal plants including Allium sativa, Gymnema sylvestre, Ocimum sanctum were clinically tested; the results were recommendation that physicians can depend on herbs in alleviating diabetes and its complications. Therefore, these herbal plants could be an alternative therapy for diabetes and its complications..

A. vera is the most widely known species of the genus Aloe and one of the most important pharmaceutical herbs. Different Aloe species have been used in the treatment of a variety of disorders including infections, dermatologic conditions and also used as a laxative since ancient times..

It was claimed that the polysaccharides in Aloe vera gel had therapeutic properties such as anti-inflammatory, wound healing, promotion of radiation damage repair, antidiabetic and anti-neoplastic activities. During the past 20 years, reports have shown that Aloe preparations have beneficial therapeutic effects on diabetes. Aloe spp. is documented as one of the potential anti-diabetic plants. D ried sap, of the Aloe plant was administrated in five patients with noninsulin-dependent diabetes and in mice made diabetic using alloxan. It’s ability to lower the blood glucose was studied. A. vera gel was administered to diabetic rats. The results were decreased fasting blood glucose levels and improved the levels of the antioxidant enzyme. A polyphenol-rich A. vera extract with known concentrations of aloin and aloe-emodin was administered to insulin resistant mice for 4 weeks leading to improved insulin tolerance and fasting blood glucose levels..

The leaves of Aloe vera were collected from El-Orman Botanical Garden, Giza, Egypt. and were kindly authenticated by Dr. Mohamed El-Gebaly, Botany Specialist. kept in the herbarium of the Pharmacognosy Department, Faculty of Pharmacy, Cairo University. Extractions of polysaccharides were conducted in Pharmacognosy Department..

AIæveraL. Burm.f.(A1) AloearborescensMiII. (A1) Aloe xrfoIiataL.(As) Aloebrevifolia Mill. AIR. eru (A1) AIR "nana LIA,) identata Dyck (A4) Alæferox Mill.lAi).

10 milligram of each of the mucilage of each plant were separately heated in 2 ml of 0.5 M (H 2 SO 4 ) in a sealed test tube for 20 h in (BWB). At the end any precipitate was filtered off. The filtrate was freed of sulphate ions by precipitation with (BaCO3). The hydrolysates were separately concentrated under vacuum at a temperature >= 40°C to a syrupy consistency. It was diluted with 10% isopropanol in water..

Separation and determination were performed on column sugar (Shodex SUGAR Series) using deionized water as mobile phase with 1ml/min flow rate. 10 mg of each residue of isolated polysaccharides, and the aforementioned authentic sugars were separately dissolved in 1 ml of deionized water. 10 µl of each sample was injected into HPLC using an SGE syringe. Quantitative determination was based on peak area measurement, while qualitative identification was carried out by comparison of the retention times of the peaks with those of the authentic sugars (Figures 2A to D)..

Figure 2B. Figure 2C. Figure 2D.

Animals Adult male rats of Sprague- Dawley strain were obtained from the laboratory animal facility of the National Research Center , Dokki , Giza. Animals were housed in steel cages under standard conditions. Induction of diabetes mellitus in rats Diabetes was induced with a single dose of alloxan (150 mg/kg body weight). Alloxan was first weighed individually for each animal according to the body weight and then solubilized with 0.2 ml saline (154 mM NaCl) just prior to injection. Two days after alloxan injection, rats with plasma glucose levels of 140 mg/dl were included in the study. Treatment with polysaccharides samples was started 48 h after alloxan injection. Hyperglycaemia was assessed after 72 h by measuring blood glucose and after 2 and 4 weeks intervals..

Assessment of anti-hyperglycaemic effect The control diabetic group in each experiment received a single daily dose of 1% Carboxymethylcellulose sodium (CMC-Na), as a vehicle for the tested sample. The vehicle, metformin and polysaccharides were given orally by gavage as single daily treatments for 4 weeks. At the end of each study period, blood samples were collected from the retro-orbital venous plexus through the eye canthus of anesthetized rats after an overnight fast. Serum was isolated by centrifugation, and the blood glucose level was measured by enzymatic colorimetric method at zero time, at days 14 and 28 from the treatment (Trinder, 1969). Assay for alpha-glucosidase inhibitory activity The assay was performed to measure the alpha-glucosidase inhibitory activity of the polysaccharide samples isolated from the eight Aloe species (A1-A8). The enzyme inhibition study was carried out spectrophotometrically in a 96-well microplate reader using a procedure. Enzyme assay of 220 µl were prepared using phosphate buffer..

The inhibitor was replaced by water in case of incubations performed to determine 100% activity. Blank incubations were performed to cancel the color of the polysaccharide. Polysaccharide was dissolved in 600 µl hot water (90°C). The assays were incubated at 37°C for 7 min and color was measured at λ max 410 nm using a Spectra Max 340 spectrometer. Controls contained the same reaction mixture except the same volume of phosphate buffer was added instead of the inhibitor solution. Acarbose was dissolved in water and used as a positive control. Inhibition %= [(AB - AA)/AB] × 100%. Statistical analysis The statistical comparison of difference between the control group and the treated groups was carried out using two-way ANOVA followed by Duncan's multiple range test..

Results. From figures 1 and 2 it shown that: All investigated Aloe species were rich in polysaccharide , specially Aloe saponaria (A 7 :12%), Aloe vera (A 1 : 8.5%) and Aloe Ferox (A 8 : 8%) had the highest polysaccharide content . While Aloe perfoliata (A 5 : 0.5%) and Aloe grandidentata (A 4 : 1.2%) had the lowest polysaccharide content. High concentration of glucuronic acid, was detected in A 7 (2.1%), A 8 (1.5%) and A 2 (1.3%) . Stachyose is a major sugar in A 8 (5.4%), A 7 (2.6%) and A 1 (1.7%). Galacturonic acid is a major sugar identified in A 1 (4.5%), A 7 (1.45%) and A 5 (0.3%). The lowest concentration of most of the sugars were found in A2, A3, A4 and A6..

Chemical composition differs among the species of Aloe. For example, A. barbadensis Miller may contain 2.5 times the aloe-emodin of A. ferox Miller ,the reason the time of harvest may affect the chemical composition . Despite Aloe ferox is similar to Aloe vera but has many times more nutritional and medicinal value than it. HPLC analysis HPLC analysis of the polysaccharides revealed that their composition is more or less similar qualitatively. From quantitative HPLC analysis it shown that: 1) A. saponaria (A7) mucilage hydrolysate contained mainly glucuronic acid, stachyose, galacturonic acid, maltose and glucose in ratio 2.0, 2.4, 1.6, 2.5 and 0.5 % respectively. 2. A. vera (A1) mucilage hydrolysate contained mainly glucuronic acid, stachyose , galactouronic acid, xylose and galactose in the ratio 1.0,1.5, 4.4, 0.1 and 0.2% respectively..

From the previous we notice that: Glucuronic acid was the dominant sugar in all Aloe species. Usage of aloe species as antidiabetic : By experimental on diabetic rats it shown that: Aloe species specially A. vera and A. arborescens cause hypoglycemic effect as they induced 40% and 44% reduction in glucose levels, respectively. Polysaccharide samples isolated form the eight studied Aloe species had moderate inhibitory activities on the alpha- glucosidase enzyme thus they can be used as useful functional foods according to their association with reduced risk of diabetes ..

discussion. Type 2 diabetes mellitus is a complex disease, characterized by abnormal hepatic glucose output, insulin resistance and impaired insulin production. Some metabolic pathways can be affected leading to dysfunction. Non treated diabetic patients tend to have reduced glucuronic acid excretion; and a reduced glucuronidation capacity after a menthol load due to lowered glycogen content of the liver. Glucuronidation is the addition of glucuronic acid to a xenobiotic as drugs to make them more water soluble and ease their excretion from the body through urine or feces. But intestinal flora contains β-glucouronidase that hydrolyzes glucuronides to their respective aglycone and absorbed to the liver. Glucuronic acid is a precursor of ascorbic acid which binds toxins entering the liver and gets rids rid of them via kidneys..

Mucilages are soluble fibers that absorb water and swell, forming gel which increases the viscosity of intestinal content and adds bulk to the stool. It also lowers sugar absorption. Plant polysaccharides can be a glucuronic acid source through partial hydrolysis by intestinal bacteria. They have shown to be beneficial in reducing serum lipid in hyperglycemic rats (antioxidant) and partially recovering the secretory function of islet cells, causing increased blood insulin levels and improved glucose metabolism. Postprandial hyperglycemia is the major problem of diabetes mellitus. Increased blood glucose level is caused by a carbohydrate-rich diet which breaks down to monosaccharides by glycoside hydrolysis. Therefore, alpha-glucosidase inhibitors are effective in inhibiting disaccharide digestions. The polysaccharide samples isolated from the eight studied Aloe species have shown an inhibitory action on alpha-glucosidase enzyme as they prevent substrate pairing with the enzyme which in turn lowers disaccharides hydrolysis and the amount of free sugars absorbed; therefore reducing postprandial hyperglycemia..

conclusion. Glucuronic acid, and galacturonic acid the major identified sugar acids in all polysaccharide samples of Aloe species could be attributed to the observed anti hyperglycemic effect. Polysaccharide’s constituents of eight Aloe species have synergistic in vivo anti hyperglycemic activity in alloxan-induced diabetic rats. They have moderate alpha-glucosidase inhibitory activity. So, these plant polysaccharides can be used as useful functional foods to control elevated blood glucose levels in diabetic patients. The traditional antidiabetic plants can provide new compounds which is cheaper than current medicines, this will be useful for developing countries..

References. American Diabetes Association (ADA) (2009). Diagnosis and Classification of Diabetes Mellitus. Diabet . Care 32:S62-S67. Bailey LJ, Day C (1989). Traditional plant medicine as treatment for diabetes. Diabet . Care 12:553-564. Bhaludra CSS, Bethapudi R R , Murugulla AC, Pullagummi C, Latha T, Venkatesh K, Bheemagani AJ, Pudutha A, and Rani AR (2013). Cultivation, phytochemical studies, biological activities and medicinal uses of Aloe ferox , grandfather of Aloes an important amazing medicinal plant. Int. J. Pharmacol . 9:405-415. Córdova V, Barriguete M, Lara E, Barquera S, Rosas P, Hernández A, León M, Aguilar S (2008). Las enfermedadescrónicas no trasmisibles en México: Synopsis epidemiológicay prevención integral. Salud Pública México 5:419-427. Davis RH (1997). Aloe vera : A scientific approach. New York: Vantage Press. Gbolade AA (2009). Inventry of antidiabetic plants in selected districts of Lagos State, Nigeria. J. Ethnopharmacol . 121:135-139. Gertz CH (1990). HPLC Tips and Tricks. Great Britain, Oxford, P 608. Ghannam N, Kingston M, Al- Meshaal IA, Tariq M, Parman NS, Woodhouse N (1986). The antidiabetic activity of Aloes. Preliminary clinical and experimental observations. Horm . Res. 24:288-294. Ghorbani A (2013). Best herbs for managing diabetes: A review of clinical studies. Braz. J. Pharm. Sci. 49(3):413-22..

Golay A, Felber JP, Jequier E, DeFronzo RA, Ferrannini E (1988). Metabolic basis of obesity and noninsulin-dependent diabetes mellitus. Diabet . Metab . Rev. 4:727-747. Grindlay D, Reynolds T (1986). The Aloe vera phenomenon: A review of the properties and modern uses of the leaf parenchyma gel. J. Ethnopharmacol . 16:117-151. Khan V, Najmi AK, Akhtar M, Aqil M, Mujeeb M, Pillai KK (2012). A pharmacological appraisal of medicinal plants with antidiabetic potential. J. Pharm. Bioallied Sci. 4(1):27-42. King C, Rios G, Green M, Tephly T (2000). Current Drug Metabolism. "UDP- glucuronosyltransferases ". Curr . Drug Metab . 1(2):143-61. Li X, Yu Z, Long S, Guo Y, Duan D (2012). Hypoglycemic effect of Laminaria japonica polysaccharide in diabetes mellitus mouse model. ISRN Endocrinol . 4p. Muller- Oerlinghausen B, Hasselblatt A, Jahns R (1967). Impaired hepatic synthesis of glucuronic acid conjugates in diabetic rats. LifeSci . 6(14):1529-1533. Ni Y, Turner D, Yates KM, Tizard I (2004). Isolation and characterization of structural components of Aloe vera L. leaf pulp. Int.Immunopharmacol . 4(14):1745-55. Winchester B, Fleet GW (1992). Amino-sugar glycosidase inhibitors: versatile tools for glycobiologists . Glycobiology 2:199-210..