Tridax Procumbens Inhibitory Properties of Alpha Amylase

Diabetes mellitus is a chronic endocrine disorder caused by an absolute or relative lack of insulin, which affects the metabolism of carbohydrates, proteins, fat, electrolytes and water. It includes a group of metabolic diseases characterized by hyperglycemia in post prandial and/or fasting state, in which blood sugar levels are elevated either because the pancreas does not produce enough insulin or cells do not respond to the produced insulin ⁶. In its severe form, it is accompanied by ketosis and protein wasting. Diabetes leads to a state in which the homeostasis of carbohydrates and lipid metabolism is improperly regulated by the pancreatic hormone, insulin ultimately resulting in increased blood glucose level. ¹⁴. Diabetes mellitus is one of the challenging diseases in the field of the medicinal chemistry.

Diabetes is the world’s largest endocrine disorder ¹⁴. Therefore, there is the need for a therapeutic approach to treat diabetes. The most common method is to decrease postprandial hyperglycemia ⁹. This can be achieved by the inhibition of carbohydrate hydrolyzing enzymes like alpha amylase and alpha glucosidase ¹⁴. Alpha glucosidase and alpha amylase are the important enzymes involved in the digestion of carbohydrates. Alpha Amylase is involved in the breakdown of long chain carbohydrates and alpha glucosidase breaks down starch and disaccharides to glucose. They serve as the major digestive enzymes and help in intestinal absorption. Alpha amylase and glucosidase inhibitors are the potential targets in the development of lead compounds for the treatment of diabetes ⁸.

Natural products from plants have been used for the treatment of diabetes, mainly in developing countries like Ghana where the resources are limited and affordability and access to modern treatment is a problem. Use of natural products has existed for a long time without any proper research conducted in the country into their efficacies and side effects. Extensive research has been carried out elsewhere to screen the bioactivity of these natural products because of their significant importance in health care and medicine. Plant food rich in secondary metabolites have been reported to cause effects similar to insulin in the utilization of glucose and act as good inhibitors of key enzymes like alpha amylase and alpha glucosidase associated with type 2 diabetes and lipid peroxidation in tissues. Studies have also shown that the bioactivity of secondary metabolites in plants is linked to their antioxidant activity and many of these plants also possess hypoglycemic properties ²³. To prevent the effects of free radicals, antioxidants donate electrons to rampaging free radicals and neutralize them, thereby reducing their capacity to damage tissue ²⁴.

Higher plants, animals and microorganisms are found to produce a large number of different protein inhibitors of alpha amylases and alpha glucosidases in order to regulate the activity of these enzymes ³. Some of these enzyme inhibitors act by directly blocking the active center of the enzyme at various local sites³. In animals, alpha amylase inhibitors decrease the high glucose levels that can occur after a meal by slowing the speed with which alpha amylase can convert starch to simple sugars ²⁰. This is of importance in diabetic individuals since low insulin levels prevent the fast clearing of extracellular glucose from the blood ²⁰. Hence, diabetics tend to have low alpha amylase levels in order to keep their glucose levels under control. These inhibitors alter the digestive action of alpha amylases and proteinases in the gut of insects and inhibit their normal feeding behavior. Thus, alpha amylase inhibitors have potential roles in controlling blood sugar levels and crop protection ²⁰. Alpha-amylase can be found in various organisms and show diverse substrate specificities while possessing a common topology formed from three domain, one of which being a typical alpha-beta barrel. Inhibition of insect’s alpha amylase is a proposed method of crop protection. On the other hand, inhibition of mammalian alpha-amylase is a proven therapeutic approach in diabetes and related disorders ²². Therefore, this research seeks to determine the ability of Tridax procumbens to inhibit the activity of alpha amylase and help in treatment of diabetes mellitus.

Tridax procumbens is a plant belonging to the daisy family, and found in various tropical and subtropical regions as well as mildly temperate regions worldwide ²⁰. It habitats waste places, road sides and hedges throughout Ghana ². The herb is used locally in Ghana for the treatment of wounds which lead to bleeding and also used to cause wounds to heal faster. It is boiled by some indigenes and drank as tonic for treatment of several ailments such as diabetes, typhoid fever and malaria. The plant is administered mainly by boiled extracts, macerated extract or raw squeezed extract.

Diabetes Mellitus is the world’s largest endocrine disease affecting millions of people. Individuals in developing countries are unable to afford conventional drugs to manage the condition. Hence, this work is aimed at studying the ability of Tridaxprocumbensto inhibit alpha amylase.

Alpha-amylase ( EC 3.2.1.1, PDB ID: 5U3A) is an enzyme that acts upon large linear carbohydrate (Starch) polymers at internal bonds. The hydrolytic products have alpha- configuration. In nature, starch (Carbohydrate) is the most abundant polysaccharide food store after cellulose and the primary accessible source of carbon and energy on the Earth. It is synthesized by plants and utilized in food, textile, paper, alcohol, pharmaceutical industries. Starch is deposited in plant cells in the form of granules as reserve material. ⁷

The enzyme is a glycoprotein ⁷. Its single polypeptide chain of about 475 residues has two free thiol groups, four disulfide bridges, and contains a tightly bound Ca²⁺, ²⁷ (see Appendices A, B and C). It exists in two forms (PPAI and PPAII), which have identical enzymatic properties and molecular mass of 55.4 kDa, differing only in electrophoretic mobility and Pi ⁵, ⁷

The structures of both forms of PPA consist of a major domain A (residues 1-99 and 170- 404) organized as an (alpha/beta)8-barrel that contains a loop (domain B). Following the C- terminal domain C (residues 405-496) is a ten-beta-stranded Greek key motif. ¹⁰

Pancreatic alpha-amylase hydrolyzes complex starches to oligosaccharides in the lumen of the small intestine. α -amylase and α-glucosidase are key enzymes involved in carbohydrates breakdown and intestinal absorption, respectively. It has an optimum pH of 7.0. ²⁹ Inhibition of these enzymes hamper blood glucose level increase after a carbohydrate diet and can be an important strategy in the management of non-insulin-dependent diabetes mellitus (NIDDM). In general, there is very little biological knowledge on the specific modes of action in the treatment of diabetes but most of the plants have been found to contain secondary plant metabolites like glycosides, alkaloids, terpenoids, flavonoids etc., that are frequently implicated as having antidiabetic effects by inhibiting the activity of alpha amylase.

Due to the role alpha amylases play in the metabolism of carbohydrates, research has been conducted to analyze the benefits of modulating and regulating the activities of this enzyme. Most of these researches have been done through inhibition and activation properties of various compounds. According to research carried out, inhibitors for alpha amylase were determined to be:

Phenolic compounds ¹¹

Plant extracts ⁴, ¹⁰

Urea and other amide reagents ¹⁴.

Diabetes Mellitus

Diabetes mellitus is one of the challenging diseases in the field of the medicinal chemistry.

It is classified in to two types ¹⁹:

Type 1- Insulin dependent diabetes mellitus. Type 2- Non-insulin dependent diabetes mellitus.

Type 1 diabetes (T1D) is a chronic disease caused by immune-mediated destruction of insulin producing beta cells in the pancreas. The destruction of beta cells results in insulin insufficiency, and patients develop life-threatening hyperglycemia that clinically manifests with weight loss, polyuria, and polydipsia²⁸. These are due to defective transport of glucose from the bloodstream into tissues, resulting in increased glucose levels in the blood, elevated glucose in the urine, and concomitant calorie and fluid losses in the urine. When insulin levels fall to such low levels that lipolysis cannot be suppressed, products of fat metabolism called ketone bodies (primarily acetoacetate and β- hydroxybutyrate) accumulate in the blood, leading to metabolic acidosis and compensatory respiratory alkalosis due to hyperventilation. If untreated, compensatory mechanisms eventually fail and ketoacidosis results in cerebral edema, mental confusion, unconsciousness, coma, and death¹⁵. Data from various researchers indicates that the disease process may be delayed by administering oral insulin to induce insulin specific regulatory T-cells in the gut, resulting in decreased inflammation in the pancreas.

Research has shown that Type 2 DM (T2DM) accounts for majority (90-95%) of diabetes and poses a huge burden on healthcare systems especially in developing countries ²⁰. Type 2 Diabetes occurs when the pancreatic beta cells cannot produce enough insulin or the cells acquire some degree of resistance to insulin. Normal regulation of glucose metabolism is determined by a feedback loop involving the islet β-cell and insulin-sensitive tissues in which tissue sensitivity to insulin determines the magnitude of the β-cell response. When insulin resistance is present, the β-cell maintains normal glucose tolerance by increasing insulin output. It is only when the β-cell is incapable of releasing sufficient insulin in the presence of insulin resistance that glucose levels rise ¹⁶.

Conventional therapies, as of yet, have been unable to achieve a cure for diabetes mellitus. Hence, systematic and intensive search in medicinal plants for new drugs to treat diabetes mellitus especially type 2 seem to be of great utility. Acarbose, currently marketed as a medicine in the treatment of diabetes, lowers post-prandial peaks of glucose. However, acarbose is principally known as an alpha-glucosidase inhibitor and causes side effects such as, abdominal distension, flatulence, meteorism and possibly diarrhea. Therefore it is attractive to find a substance that has strong inhibitory activity against α-glucosidase but minor effect on α-amylase activity ¹⁹.

Acarbose inhibits enzymes (glycoside hydrolases) needed to digest carbohydrates, specifically, alpha-glucosidase enzymes in the brush border of the small intestines and pancreatic alpha-amylase. Pancreatic alpha-amylase hydrolyzes complex starches to oligosaccharides in the lumen of the small intestine, whereas the membrane-bound intestinal alpha-glucosidases hydrolyze oligosaccharides, trisaccharides, and disaccharides to glucose and other monosaccharides in the small intestine. Inhibition of these enzyme systems reduces the rate of digestion of complex carbohydrates. Less glucose is absorbed because the carbohydrates are not broken down into glucose molecule ¹⁹.

Many common herbs and spices are claimed to have blood sugar lowering properties that make them useful for people with or at high risk of type 2 diabetes.

A number of clinical studies have been carried out in recent years that show potential links between herbal therapies and improved blood glucose control, which has led to an increase in people with diabetes using these more 'natural' ingredients to help manage their condition. ¹²

Herbal medicine in the scientific community is questioned on the basis of their modes of action and the ability of some active ingredients contained in them to interact with diabetic drugs and produce undesirable effects. Many researchers have therefore committed to demystifying herbal medicine. In the article “Antidiabetic effect of oral borapetol B compound, isolated from the plant Tinosporacrispa, by stimulating insulin release,” F. E. Lokman et al., have presented an evaluation of the antidiabetic property of a biologically active small compound borapetol B (C1) isolated from T. crispain normoglycemic control using Wistar (W) and spontaneously type 2 diabetic Goto-Kakizaki (GK) rats. They found that an acute oral administration of the compound significantly improves blood glucose levels in the treated group in comparison to the placebo group. They observed that plasma insulin levels were significantly enhanced by 2-fold in treated W and GK rats compared to the placebo group at 30 minutes. Their study provides evidence that borapetol B (C1)’s antidiabetic property is mainly due to stimulation of insulin release. ⁶

Another source of contention involves the mode of extraction of plant Extracts for use in diabetes management. It should be noted that plant extraction with a particular solvent is dependent on the type of plant and plant part. This is seen in work done by ². where efficacy of extract depended on the type of plant and solvent used for extraction.

The plant researched in this study, is Listed as a weed and a pest plant, it has been known by several names including ‘Tridax daisy’ in English, and ‘Herbecaille’ in French (see Appendix D). Many reports have focused on the immense potential of this plant which has antimicrobial, wound healing, anti-inflammatory and immunomodulatory properties (P et al., 2011). Antioxidant properties have also been demonstrated in various researches ²¹. Antioxidants can scavenge free radicals and play important role in prevention of diabetes. The role of oxidative stress in diabetes and diabetic complications has been reported by many researchers. ²⁵

Work by ³¹ determined by in-vitro assay that the Tridax procumbens plant extracts had alpha amylase inhibitory activities and these activities were dependent on the type of solvent used in extraction of Tridaxprocumbens. Among the extracts, methanol exhibited highest α-amylase activity. The methanol extract of Tridaxprocumbens exhibited highest α-amylase with an IC50 value of >10 µg/mL. The petroleum ether extract of Tridaxprocumbensextract showed α- amylase inhibitory activity with IC50 value of 70 µg/mL. ²⁰, also showed that the active components found in the extract include alkaloids, flavonoids, glycosides, saponins, polyphenols, tannins. Their research determined that inhibitory activities could be as a result of presence of these bioactive compounds and their interaction with proteins. The mode of inhibition of the aqueous extract of Tridaxprocumbensleaf on alpha-amylase was determined using the Lineweaver-Burk plot in this study, which displayed competitive inhibition of the enzyme.

This was done according to Bendelow’s method with some modifications. Maize obtained from the laboratory was subjected to the malting process to allow for a short duration of germination where alpha amylase is activated to break down stored carbohydrates in the seeds in order to nourish and support the growing young plant. After a considerable malting of the maize it was subjected to milling into a uniform powder. A mass of 3 g of milled malt sample was weighed into centrifuge tubes and 12 mL of phosphate buffer at pH of 6.9 (pKa 7.21). The enzymes were allowed to extract into the extraction medium for 30 minutes after which the enzyme suspension was centrifuged at a speed of 4500 rpm for 15 minutes using a swinging bucket rotor type centrifuge. The supernatant obtained was decanted and according to research by ³². contained alpha amylase of unknown activity. The pH of the supernatant obtained was reduced to 5.5 by the addition of 0.2 M hydrochloric acid. After this, there was a confirmatory test to determine the presence of the alpha amylase. To achieve this, 1mL of the enzyme extract was added to 5 mL of 1% starch solution and then 1 mL of iodine solution was added. The fading of the blue-black color with time is an indication of the presence of alpha amylase.

This was done according to Duffus’s method with some slight modifications. Different concentrations of crude alpha amylase were prepared. (1:10,1:100 and undiluted). Biuret reagent of volume 2 mL was then added to each of the concentrations and absorbance was taking at 562 nm wavelength using a UV/VIS Spectrophotometer.

This was done according to Sindhu’s method with some modifications. Alpha amylase was incubated with 250 ul alpha amylase for 10 minutes at 25˚C. To the incubated amylase, 500 ul of distilled water was added and 250 ul of starch solution of concentration 1% was added. The resulting solution was incubated for 3 minutes at 25˚C. Acidified Iodine solution of volume 500 ul was added to stop the reaction. Absorbance was taken at 620 nm wavelength using a UV/VIS spectrophotometer.

Tridax procumbens leaves were obtained from the lawn in front of the department of biochemistry after assessment was made on impact of human and other mechanical activities on the normal growth of the plant. Upon harvesting of the leaves, they were sent to the Teaching Assistant for verification of authenticity.

Fresh green leaves of the Tridax procumbens were washed with distilled water 3 times and air dried for 2 days. The dried leaves were subjected to blending using a warring blender (Speed 4, 10 Watts) for 15 minutes until a fine powder was obtained. A mass of 15 grams of the powdered Tridax procumbens according to Adrian’s method with slight modifications, was weighed into a beaker and 250 mL of Ethanol was added. Another 15 grams mass of powdered Tridaxprocumbenswas weighed into another beaker and 250 mL of petroleum ether was added. The mixtures were then transferred into 2 clean clearly labeled volumetric flasks. The mixtures were left standing with intermittent shaking for 3 days. Extract for each solvent was then obtained by filtration and evaporation of the sample. The slurry obtained was then stored for further analysis to be carried on.

This was a qualitative test performed to ascertain the kinds of phytochemicals present in Tridaxprocumbens(seeAppendixE). Phytochemical compositions of the leaves were determined using the methods variously described by Gramer and Walker with slight modification (Gramer & Walker, 2007).

Wagner’s reagent was prepared by dissolving 0.04 g of iodine crystals in 2 mL of water. A volume of 1 mL of the extract was aliquoted and 3 drops of the Wagner’s reagent was added to it. Observation for reddish brown was expected to indicate the presence of alkaloids.

From the extract, 2 mL was picked and 0.5 mL of biuret reagent was added. It was then allowed to stand for 5 minutes and the color change was observed and recorded.

To 1 mL of the extract ,1 mL of chloroform was added and 5 drops of concentrated hydrochloric acid (HCl) was added and kept for 60 seconds. Appearance of red colour in the lower layer was expected as an indication of the presence of steroids.

To 1 mL of the extract ,1 mL of chloroform was added and 5 drops of concentrated hydrochloric acid (HCl) was added and kept for 60 seconds. Appearance of red colour in the lower layer was expected as an indication of the presence of steroids.

The mode of inhibition of α -amylase by the leaf extract was conducted using the extract with the lowest IC50 according to the modified method described by ³². Briefly, 250 µL of the extract (5 mg/mL) was preincubated with 250 µL of α -amylase solution for 10 min at 25°C in one set of tubes. In another set of tubes α -amylase was preincubated with 250 µL of phosphate buffer (pH 6.9). 250 µL of starch solution at increasing concentrations (0.30–5.0 mg/mL) was added to both sets of reaction mixtures to start the reaction. The mixture was then incubated for 10 min at 25°C and 500 µl of acidified iodine was added to stop the reaction and absorbance was taken at 620 nm. The amount of starch remaining was determined spectrophotometrically using a Starch standard curve and converted to reaction velocities. A double reciprocal plot (1/V versus 1/(S)) where V is reaction velocity and (S) is substrate concentration was plotted.The type (mode) of inhibition of the crude extract on α-amylase activity was determined by analysis of the double reciprocal (Lineweaver-Burk) plot using Michaelis- Menten kinetics.

Data obtained from the experiment were analyzed statistically using Excel 2016. Analysis tool pack was used in Data projections and graphing.

Appendix 1.Stick model of alpha amylase. 29

Appendix 2.Model of alpha amylase. 1

Appendix 3.Surface model of alpha amylase with Ligand attached. 13

Appendix 4.Tridax procumbens plant and leaves

Appendix 5.Phytochemical analysis

Appendix 6.Alpha amylase inhibitory assay

Appendix 7.Alpha amylase activity and Inhibitory assay by Tridax procumbens extract