DETERMINATION OF INHIBITION ACTIVITY OF α- AMYLASE ENZYME, ANTIOXIDANT ACTIVITY, ANTIBACTERIAL Activity AND PHENOLIC COMPOUNDS BY USING SOME MEDICAL PLANTS

The inhibition of carbohydrate hydrolyzing enzymes such as α-amylase can be an important strategy in the postprandial blood glucose level in patients with type II diabetes, plants contains different chemical constituents with potential for inhibition of α-amylase and hence may be used as therapeutic. Nine types of plants include (Clove, Lemon, Chamomile, Radish, Ginger, Black seed, pomegranate, Beetroot and Garlic) were used in this study to select the optimum plant material that inhibited αamylase enzyme. Raphanus raphanistrum was chosen, it had the highest inhibition activity of the enzyme (95.5%). Also sodium phosphate buffer (0.2 M, pH 7.5) was selected as a best extraction buffer of plants inhibitor with inhibition activity 83%. The optimum extraction ratio represented by 1:20 (w:v) after 90 min, it was given 96.8% enzyme inhibition activity. Antioxidant activities of plants were performed using DPPH free radical scavenging assay, and lemon had a highest by 94.4%. This confirms that the plant might protect cells from oxidative damage, resulting in certain diseases. The antimicrobial properties of lemon, garlic, clove have been proven the highest effective against selected human pathogens such as Staphylococcus aureus, E. coli and Enterococcus sp. Also total phenolic content (TPC) of lemon and garlic were estimated spectrophotometrically according to the FolinCiocalteu procedure and calculated as gallic acid equivalents (GAE), lemon revealed the highest value compared with garlic.


INTRODUCTION
Diabetes is defined as a state in which homeostasis of carbohydrate and lipid metabolism is improperly regulated by the pancreatic hormone, insulin; resulting in an increased blood glucose level. Diabetes is a progressive disease and is one of the major killers in recent times. Most prevalent form of diabetes is non-insulin dependent diabetes mellitus (NIDDM/type II). The treatment of type II diabetes is complicated by several factors inherent to the disease and elevated post prandial hyperglycemia (PPHG) is one of the risk factors (14). PPHG is elevated by the action of glucosidases, a class of enzymes that helps in the breakdown of complex carbohydrates into simple sugars such as maltose and glucose. Glucosidase inhibitors such as α-amylase inhibitors plays a major role in managing PPHG in diabetic patients. These α-amylase inhibitors inhibit the action of αamylase enzyme leading to a reduction in starch hydrolysis which shows beneficial effects on glycemic index control in diabetic patients (2002). Oxidative stress is an important risk factor in the pathogenesis of numerous chronic diseases. Free radicals and other reactive oxygen species are recognized as agents involved in the pathogenesis of sicknesses such as asthma, inflammatory arthropathies, diabetes, Parkinson's and Alzheimer's diseases, cancers as well as atherosclerosis. Reactive oxygen species are also said to be responsible for the human aging (17,35). An antioxidant can be broadly defined as any substance that delays or inhibits oxidative damage to a target molecule (34). The main characteristic of an antioxidant is its ability to trap free radicals. Antioxidant compounds like phenolic acids, polyphenols and flavonoids scavenge free radicals such as peroxide, hydroperoxide or lipid peroxyl and thus inhibit the oxidative mechanisms that lead to degenerative diseases (9). Herbal plants considered as good antioxidant since ancient times. Rising antibiotic resistance and the scarcity of new antimicrobials has long been acknowledged (24). Some Staphylococcus spp. and Streptococcus spp. involved in the pathogenesis of respiratory and skin infections, along with Pseudomonads and members of the Enterobacteriaceae causing gastrointestinal, urogenital diseases and wound contamination are resistant to virtually all of the older antibiotics (19). Clinical isolates of Staphylococcus aureus, the leading cause of nosocomial infections, are increasingly resistant to an array of antimicrobial agents like penicillin, gentamicin, tobramycin, amikacin, ciprofloxacin, clindamycin, erythromycin, chloramphenicol, trimethoprimsulfamethoxazole and vancomycin (4). The problem of antibiotic resistance in humans and animals will continue for a long time (10). Against this backdrop, the development of alternative drug classes to treat such infectious diseases is urgently required (9). Plants have an amazing ability to produce a wide variety of secondary metabolites, like alkaloids, glycosides, terpenoids, saponins, steroids, flavonoids, tannins, quinones and coumarins (10). These biomolecules are the source of plant-derived antimicrobial substances (PDAms) (9). Some natural products are highly efficient in the treatment of bacterial infections (13). The aim of this project is trying to use some medical plants common in ways to reduce the effectiveness of amylase and reduce the proportion of glucose blood for diabetics. And to evaluate the antioxidant activity of several agents proposed for reversion of problems caused by bleaching procedures using the DPPH free radical assay, also to estimate the antibacterial activity and phenolic compounds of these plants extracts that might have support and help the research scientific community to study and encourage the use of medicinal plants for the treatment of diseases.

Determination Amylase assay
The activity of α-amylase enzyme was estimated according to the method describe by Bernfeld (6) which depend on standard curve of maltose throughout decomposition reducing sugars liberated by the enzyme. A standard calibration curve was prepared for the maltose taking 1ml of 360-1800 μg/ml dilution of maltose, the percentage (w:v) of maltose in the reaction wells was calculated from the corrected absorbance of each test and using the equation of the calibration curve. Mixing 0.8 ml of starch solution with 0.2 ml of enzyme solution (saliva) for 20 min in water bath at 37 °C, then add 1 ml of 3,5-Dinitrosalicylic acid (DNSA) with boiling for 5 min then cooling and add 10 ml of D.W with vortex. Amylase activity was detected by measuring the absorbance increase at 540 nm. Enzymatic activity unit known as the amount of enzyme liberated 1 μmole from reducing sugars (glucose and maltose) in one minute under optimum conditions. Estimation the standard curve of maltose Put the following volume of standard maltose solution (2 mg/ml) that prepared previously in test tubes then add the appropriate volume of D.W ( Table 1). After that add 1 ml of DNSA for each tube, then put the tubes in water bath at boiling temperature for 5 min. Thereafter the tubes were cooled by tap water and add 10 ml of distilled water for each tube. The solution was mixed by vortex for each tube and read the absorbance at 540 nm (Fig. 1).

Extraction and recovery of α-amylase inhibitor
One g of each plant was homogenizing with 10 ml of 0.2M of phosphate buffer, the mixing was crushed by mortar for 15 min at room temperature. The mixture was centrifuge at 10000 rpm for 15 min (17). The clear supernatant obtained represented the crude extract and used to assay the αamylase inhibitor activity. Determination of α-amylase inhibitor activity A crude extract of each plant was incubated separately with a known volume of α-amylase enzyme (saliva) by 1:1 ratio for 30 min at room temperature, thereafter the α-amylase activity was estimated, the control representing 100% enzyme activity were conducted in the same manner replacing the plant extract with distilled water. Mixing 0.8 ml of starch solution with 0.2 ml of a mixture (enzymeinhibitor) for 20 min in water bath at 37 °C, then add 1 ml of 3,5-Dinitrosalicylic acid (DNSA) with boiling for 5 min then cooling and add 10 ml of D.W with vortex. Amylase activity was detected by measuring the absorbance increase at 540 nm. The remaining activity is the present inhibitory activity of the enzyme with respect to the present enzyme activity without inhibitor according to the following equation:  The free radical scavenging activity of the nine medical plants was evaluated by 1,1-diphenyl-2-picryl-hydrazyl (DPPH) according to the previously reported method by (32). Briefly, 0.1mM solution of DPPH in methanol was prepared and 1mL of this solution was added to 3 ml of the solution of all extracts separately. The mixtures were shaken vigorously and allowed to stand in dark at room temperature for 30 minutes, then the absorbance was measured at 517 nm using a UV-VIS spectrophotometer. The ability of sample extract to scavenge DPPH radical was calculated by the following equation: Where: Ab of control = is the absorbance of DPPH radical only, Ab of sample = is the absorbance of DPPH radical with sample extract.

Determination of Antimicrobial Activity
Nutrient agar prepared and poured in the sterile petri dishes and allowed for solidify. Twenty-four hour overnight bacterial cultures of Staphylococcus aureus, E. coli and Enterococcus sp. were swabbed separately on the media using sterile cotton buds, then two wells (8 mm diameter) were made by using a sterile cork borer. One hundred microliter of each extract from chosen medical plants were loaded in the wells. The plates then incubated at 37°C for 24h. After incubation, the diameter of inhibition zone was measured. Tetracyclin, Pipracillin, Gentamycin, Sulfametazoltrimethoprim, Azthereonam and Ciprofloxacin were used as positive control.

Determination of the Total Phenolic Content (TPC)
The total phenolic contents of the lemon and garlic extracts were estimated using the Folin Ciocalteau reagent as described by Singleton and Rossi (33) with some modifications. The calibration curve fig. (2) was plotted by mixing 1 ml aliquots of 50, 100, 150, 200, 250, 300, 350 and 400 mg/ml of gallic acid solutions with 0.5 ml of Folin Ciocalteu reagent (diluted tenfold) and 4.0 ml of sodium carbonate solution (75 g/l). The absorbance was measured after 10 min at 765 nm. For both plants extracts, 1 ml was mixed separately with the same reagents, as performed for constructing the calibration curve. After 10 min, the absorbance was measured to determine the total phenolic contents in both extracts separately using the formula, = × / Where: C = total phenolic content in mg/g, in GAE (Gallic acid equivalent), C1= concentration of Gallic acid established from the calibration curve in mg/ml, V= volume of extract in ml, and m= the weight of the plant extract in g.

Result and Discussion Optimum Conditions of α-Amylase Inhibitor
Different bioprocess conditions that effect on αamylase inhibitor were optimized for maximum inhibition activity, a large number of factors affect the extraction of inhibitor such as types of plants, types of buffer, extraction ratio and extraction time etc. Hence, optimization of these conditions helps to reduce extraction cost and help to obtain a high yield of αamylase inhibitor.

Types of Plants
Nine types of plants, commonly used in this study, namely Clove (Syzigium aromaticum), Lemon (Citrus lemon), Chamomile (Matricaria chamomilla), Radish (Raphanus raphanistrum), Ginger (Zingiber officinale), Black seed (Nigella staiva), pomegranate (Punica granatum), Beetroot (Beta vulgaris) and Garlic (Allium sativum) were obtained and screened for their enzymatic inhibition activity. As can see from fig. (3) radish was gave the highest inhibition activity of 95.5%, followed by lemon 87.3% whereas the inhibition activity of Matricaria, Clove, Ginger, Black seed, Garlic, Pomegranate and Beetroot were reached to (83.5, 66.6, 60.6, 9, 72, 81 and 59.4) % respectively. Plants αamylase inhibitors are drug-design targets for treatment of diabetes and digestion disorderes (5). Radish has been identified as having antidiabetic effects, making it favorable for those with diabetic conditions. This may be due to its ability to enhance the antioxidant defense mechanism and reduce the accumulation of free radicals, affect hormonal-induced glucose hemostasis, promote glucose uptake and energy metabolism, and reduce glucose absorption in the intestine. Furthermore, the aqueous extract of radish inhibited both αamylase and α-glucosidase enzymes in vitro, it is well-known that these enzymes are required for the degradation of poly-and oligosaccharides in the intestine before absorption. Therefore, such enzymatic inhibition may reduce the amount of glucose absorbed, which could be effective for the management and prevention of diabetes (5).
Bibek with coworkers (7) found that radish seed and rapeseed have antioxidant activity, alpha-amylase inhibitory activity and antibacterial activity.

Type of Extraction Buffer
The inhibition activity of α-amylase was estimated after extraction, using different buffer, and the results were illustrated in (Fig  4). These results show that sodium phosphate buffer (0.2 M, pH 7.5) was a best extraction buffer with inhibition activity 83%. While other buffer with different pH were given low inhibition activity. α-amylase inhibitors was extracted from Pouteria sapota by using phosphate buffer in pH 7.5 (30). In addition, Hind with coworker (16) were extracted αamylase inhibitor from Ononis angustissima by using distilled water in pH 7. Wang with coworkers (34) were extracted α-amylase inhibitors from seeds of common bean mutants by water pH was adjusted to 6.5.

Extraction ratio
Three extraction ratio were chosen 1:10, 1:20 and 1:30 (w:v) to determine the best extraction ratio on extraction the inhibitor material from radish by using 0.2M phosphate buffer pH 7.5. The best result was obtained at extract ratio 1:20 in rate of 75.45%, While the other gave 66.48% and 30.39% for 1:10 and 1:30 respectively (Fig.5). Kondo and Ida (19) found that an α-Amylase inhibitors from wheat (Triticum aestivum) was extracted with water in a 1:10 (w:v). Also Wang with coworkers (1) were extracted α-amylase inhibitors from seeds of common bean mutants by using tertbutanol. A preliminary trial for optimization of ratio of crude extract to tert-butanol volume required for Three-phase partitioning (TPP) was determined by comparing the 1:0.5, 1:1, and 1:1.5 ratios of crude extract totert-butanol. Salivary αamylase inhibitor was extracted from barley by using water at a ratio (1:11) .

Extraction time
Four extraction periods were chosen (15,30,60 and 90) min to determine the best extraction time on extraction the α-amylase inhibitor by using sodium phosphate buffer (0.2 M, pH 7.5). The highest inhibition activity of radish was measured in 90 min; it was reached to 96.8 %, compared to the lower of inhibition activity in 15, 30 and 60 min (87, 89.3 and 91.5) % respectively (Fig. 6). Abood and Hakeem (1) were extracted salivary αamylase inhibitor from barley by using water at a ratio (1:11) at 60 min. Alpha-amylase inhibitor in white kidney beans (Phaseolus vulgaris) was extracted by stirred 1, 2, 3 hour with 0.05M, 0.10M, 0.15M (1:6 w/v) of solvent at room temperature (25 o C). The homogenate was centrifuged at 10,000 g for 30, 45, 60 min at 4 o C (21).

Antioxidant Assay: Free Radical Scavenging Activity
The nine types of medical plants were used to examine αamylase inhibition, also selected to measure the antioxidant activity by using DPPH. The results shown that lemon has a highest activity of radical scavenger (94.4%) between the other plants (Fig. 7). The DPPH radical scavenging assay is a commonly used tool for accessing the antioxidant capacity of plant materials because of the relative cheap cost and speed of completion. The model of scavenging the stable of DPPH radical was commonly used to evaluate the free radical scavenging ability of various samples. DPPH is a commercial oxidizing radical that can be reduced by antioxidants. In this assay, the violet color of DPPH changed to pale yellow fig (8), due to the abstraction of hydrogen atoms from the antioxidant compound. When there are more antioxidants in an extract, more DPPH was reduced (11). Natalie (24) was found that citrus fruits like lemons are high in vitamin C, a primary antioxidant that helps protect cells from damaging free radicals. Lemon fruit extract act as an antioxidant agent against histo-pathological changes induced by cyclophosphamide in the testes of albino mice (29).

Antibacterial Activity
The selected medical plants that used to determined αamylase inhibition and antioxidant activity were used to measure the antibacterial activity by selected three different pathogenic bacteria included Staphylococcus aureus, Escherichia coli and Enterococcus sp. after growing on nutrient agar at 37°C for 24 h. The results shown in table (2) that lemon has a highest antibacterial activity against E.coli comparison with other plants fig. (9), wheras Staph aureus was inhibited by lemon and Garlic separately fig (10), also garlic and ginger were used to inhibit the growth of Enterococcus sp. (Fig.11). Lemon is an important medicinal plant of the family Rutaceae. It is a rich source of vitamin C and it is cultivated mainly for its alkaloids, which are having anticancer activities and the antibacterial potential in crude extracts of different parts (leaves, stem, root and flower) of lemon against clinically significant bacterial strains has been reported also its consists of about 5% citric acid that gives a sour (tarty) taste to the lemon (31,26). (11) found that lemon juice has the antibacterial susceptibility against some gram-positive and -negative bacterial strains. Also garlic is a potential inhibitor for food pathogens. Foods contaminated with pathogens pose a potential danger to the consumer's health. The use of garlic can increase the shelf life and decrease the possibilities of food poisoning and spoilage in processed foods. Garlic aqueous extract has antibacterial properties against S. aureus. In addition, garlic has antibacterial properties against other Gram-positive and Gramnegative bacteria, which must be investigated in further studies (3).

Determination of the Total Phenolic Content
Two types of medical plants (Lemon and garlic) that gave the highest antibacterial activity against the selected pathogens were used to screening for TPC in aqueous extracts, and both of extracts showed the presence of phenolics. The total phenolic contents were determined using the Folin Ciocalteu methodin terms of the Gallic acid equivalent (GAE) in mg/g of the extract. The total phenolic content was calculated with the help of the graph shown in fig (2), and the standard curve equation was y = 0.001x+0.1719 where R 2 = 0.6339. The total phenolic contents (Gallic acid equivalents, mg/g) in the extracts were calculated to be 47.5 and 35 mg/g, respectively (Fig 12). Phenolic compounds are commonly reported in plants and they are known to exert various biological activities, including antioxidant activity (2,27) as well as possess antibacterial properties (23,28). Phenolic compounds are also known to possess αamylase inhibitory potential (22). Thus, the antioxidant, α-amylase inhibition and antibacterial activities of medical plants extracts of this study may be attributed to their high phenolic contents. Elkhatim with coworkers (15) were estimated the TPC of whole fruit, peel, and pulp containing seeds citrus (orange, lemon, and grapefruit) and founds that the peels of grapefruit had the highest total phenolic content followed by lemon and orange, which was reached to 77.3, 49.8, and 35.6 mg of gallic acid equivalent/g of peels, respectively. While (8) mentioned that garlic had a various levels of phenolics (0.05-0.98 mg gallic acid equivalents/g of dry extract).