α -Amylase Inhibitors: A Review of Raw Material and Isolated Compounds from Plant Source

- Inhibition of α -amylase, enzyme that plays a role in digestion of starch and glycogen, is considered a strategy for the treatment of disorders in carbohydrate uptake, such as diabetes and obesity, as well as, dental caries and periodontal diseases. Plants are an important source of chemical constituents with potential for inhibition of α -amylase and can be used as therapeutic or functional food sources. A review about crude extracts and isolated compounds from plant source that have been tested for α -amylase inhibitory activity has been done. The analysis of the results shows a variety of crude extracts that present α amylase inhibitory activity and some of them had relevant activity when compared with controls used in the studies. Amongst the phyto-constituents that have been investigated, flavonoids are one of them that demonstrated the highest inhibitory activities with the potential of inhibition related to number of hydroxyl groups in the molecule of the compound. Several phyto-constituents and plant species as α -amylase inhibitors are being reported in this article. Majority of studies have focused on the anti-amylase phenolic compounds. _______________________________________________________________________________________


INTRODUCTION
Disorders of carbohydrate uptake may cause severe health problems such as diabetes (1), obesity (2), and oral diseases (3), all of which threaten an increasing worldwide population. Diabetes mellitus (DM) is a metabolic disorder resulting from deficiency in insulin secretion, insulin action, or both, promoting disturbance of carbohydrate, fat and protein metabolism. Longterm complications of diabetes mellitus include retinopathy, nephropathy, neuropathy, microangiopathy and increased risk of cardiovascular disease (1,4,5).
The therapeutic strategies for the treatment of type 2 diabetes include the reduction of the demand for insulin, stimulation of endogenous insulin secretion, enhancement of the action of insulin at the target tissues and the inhibition of degradation of oligo and disaccharides (6). The drugs commonly used in clinic to handle or control diabetes are insulin, sulfonylureas, biguanide, glucosidase inhibitors, aldose reductase inhibitor, thiazolidinediones, carbamoylmethyl benzoic acid, insulin-like growth factor. The effect of these drugs is aimed to lower the level of blood glucose (4,7,8). One therapeutic approach for treating type 2 diabetes mellitus is to decrease the post-prandial glucose levels. This could be done by retarding the absorption of glucose through the inhibition of the carbohydrates-hydrolysing enzymes, glucosidase and -amylase, present in the small intestinal brush border that are responsible for the breakdown of oligosaccharides and disaccharides into monosaccharides suitable for absorption (1,7,9,10). Inhibitors of these enzymes, like acarbose, delay carbohydrate digestion and prolong overall carbohydrate digestion time, causing a reduction in the rate of glucose absorption and consequently blunting the postprandial plasma glucose rise (1,4).
Dental caries and periodontal diseases are the most prevalent oral infectious diseases that cause significantly impact a person's overall health, having considerable economic impact, if not adequate treated (3,11). ________________________________________ Take part in etiopathology of dental caries, the most abundant enzyme in human saliva, αamylase salivary, possess at least three distinct biological functions in the oral cavity (12). First, its hydrolytic activity is responsible for the initial break down of starch to oligosaccharides. Second, several lines of evidence indicate that salivary αamylase bound to tooth enamel or hydroxyapatite may play a role in dental plaque formation. Third, α-amylase in solution binds with high affinity to viridans oral streptococci and bacteria-bound αamylase is capable of hydrolyzing starch to glucose, which can be used as a food source and then further metabolized to lactic acid. Localized acid production by bacteria can lead to the dissolution of tooth enamel, a critical step in dental caries progression (12,13). Because of its central role in the oral cavity, α-amylase salivary has been exploited as a target for the structureassisted design of compounds that might prevent unwanted dental plaque formation and the subsequent process of dental caries formation and progression.
Ethnopharmacological approach and bioassay-guided isolation have provided a lead in identifying potential α-amylase inhibitors from plant sources. Currently, methods to determine the levels of α-amylase inhibitor are based on the measurement of α-amylase activity resulting by the different iodine staining power in the presence or absence of an inhibitor during the action of the enzyme on soluble starch or by using an alkaline reactive whose brown reduction products are determined photometrically as reported by Bernfeld (14,15). This review highlights on the plants and their active constituents so far reported to have α-amylase inhibitory activity.
The human α-amylase is classical calciumcontaining enzyme composed of 512 amino acids in a single oligosaccharide chain with a molecular weight of 57.6 kDa (16). There are five α-amylase genes clustered in chromosome 1, at location 1q21, in humans. Three of them code for salivary R-amylase, AMY1A, AMY1B, and AMY1C, and the other two genes AMY2A and AMY2B are expressed in the pancreas (22,23). Human salivary and pancreatic α-amylases share a high degree of amino acid sequence similarity with 97% identical residues overall and 92% in the catalytic domains (12,18).
The amylase presents a three-dimensional structure capable of binding to substrate and, by the action of highly specific catalytic groups, promote the breakage of the glycoside links (20). The protein contains 3 domains: A, B, and C. Domain A, which has a (β/α) 8 barrel fold, constitutes the catalytic core domain. It contains about 280-300 residues. The catalytic triad (Asp, Asp, Glu) is present in domain A (24,25). The B domain is inserted between A and C domains and is attached to the A domain by disulphide bond. The C domain presents a β sheet structure linked to the A domain by a simple polypeptide chain and seems to be an independent domain with unknown function. The active site (substratebinding) of the α-amylase is situated in a long cleft located between the carboxyl end of both A and B domains. The calcium (Ca 2+ ) is situated between A and B domains and may act in stabilizing the three-dimensional structure and as an allosteric activator. The substrate-binding site contains 5 subsites (-3 -2 -1 +1 +2) (26).
α-Amylase catalyze the hydrolysis of starch via a double displacement mechanism involving the formation and hydrolysis of a covalent βglycosyl enzyme intermediate by using active site carboxylic acids for it (27). The residues, in particular, Asp 197 , Glu 233 , and Asp 300 were described to function as catalytic residues (26,27). Probably, Asp 197 acts as nucleophil that attacks the substrate at the sugar anomeric center, forming a covalently bound reaction intermediate. In this step, the reducing end of the substrate is cleaved off the sugar skeleton. In a second step a water molecule attacks the anomeric center to break the covalent bond between Asp 197 and the substrate, attaching a hydroxyl group to the anomeric center. In both steps Glu 233 and Asp 300 either individually or collectively act as acid/base catalysts. As a consequence, the active site of human α-amylase consists of several major binding subsites identified through kinetic studies (26). The same studies show that the "-1", "-2", and "-3" pocket is the core of the catalytic reaction (26).

INHIBITORS OF -AMYLASE FROM PLANTS
The potential role of the medicinal plants as inhibitors of α-amylase has been reviewed by several authors. A variety of plants has been reported to show α-amylase inhibitory activity and so may be relevant to the treatment of type 2 diabetes. About 800 plant species have been reported to possess antidiabetic properties. A wide range of plant-derived principles belonging to compounds, mainly alkaloids, glycosides, galactomannan gum, polysaccharides, hypoglycans, peptidoglycans, guanidine, steroids, glycopeptides and terpenoids, have demonstrated bioactivity against hyperglycaemia (28). A list of plants reported to have significant α-amylase inhibitory activity is shown in Table 1.
Syzygium cumini L. (syn: Eugenia jambolana Lam.) and Psidium guajava L. are widely used traditional system of medicine to treat diabetes in India (29). The aqueous extracts from S. cumini seeds and P. guajava leaves both showed a dosedependent inhibitory effect on α-amylase activity (29). The extract from seeds of S. cumini also significantly decreased the levels of blood glucose on diabetic rats (28,30). Conforti  Ktze., Rosmarinus officinalis L., Securidaca longepedunculata Fresen., Tamarindus indica L., Taraxacum officinale Web. ex Wigg., and Vaccinium myrtillus L. were screened for αamylase activity and showed remarkable inhibitory activity (above 45% inhibition rate at 0.2g/mL) (6). Methanol extracts of 41 plants, used in traditional Mongolian medicine have been tested for α-amylase inhibitory properties and significant inhibition of the enzyme was shown by Rhodiola rosea L., Ribes pullchelum Turcz, and Vaccinium uliginosum L; extracts from Geranium pretense L, Leontopodium ochroleucum Beauv., Paeonia anomala L., and Pentaphylloides fruticosa L. Schwarz showed αamylase inhibitory activity greater than 30% (32). Loizzo and cols (2008) screened the methanol, hexane and chloroform extracts from nine Lebanon traditional medicinal plants recommended in Lebanon for diabetes and found that the methanol extracts of Salvia acetabulosa L. and Marrubium radiatum Devile ex Benth exerted the highest inhibitory activity against αamylase (33).
Ayurveda, the traditional Indian herbal medicinal system practiced for over thousands of years have reports of antidiabetic plants with no apparent known side effects (34,35). Chloroform extracts of six plants namely Azadirachta indica A. Juss, S. cumini, Ocimum tenuflorum L., Murraya koenigii (L.) Spreng., and Linum usitatissimum L., traditionally used in Ayurveda along with Bougainvillea spectabilis Willd. used as a hypoglycemic plant in West Indies, and some parts of Asia were screened for inhibitory activity on α-amylase (34). A significant inhibition was observed with extracts of O. tenuflorum (34). Other six Indian medicinal plants were tested for their effect on α-amylase activity. Among them, Mangifera indica L., Embelia ribes Burm., Phyllanthus maderaspatensis Linn. and Punica granatum L. showed interesting α-amylase inhibitory activity (36).
The proteinaceous inhibitor of α-amylase (αAI), which inhibits animal salivary and pancreatic a-amylase, has been identified and isolated from various plant species (37). Amongst this plants, seeds of Phaseolus vulgaris L. contain proteinaceous inhibitors of the α-amylase and the isoform inhibitor αAI-1 have been isolated and characterized (38,39). The common bean αAI-1 has been reported to have relatively great potential as an extensive anti-obesity and antidiabetes remedy (37).

PHYTOCONSTITUENTS WITH -AMYLASE INHIBITORY ACTIVITY
A wide array of plant has derived numerous chemical compounds that have demonstrated activity consistent with their possible use in the treatment of diabetes. Research on new bioactive compounds from medicinal plants has led to isolation and structure elucidation of a number of exciting new pharmacophores. A list of phytoconstituents having significant α-amylase inhibitory activity is provided in Table 2.
Oligosaccharide inhibitors of the trestatin family that contain the acarviosine moiety (e.g., acarbose 1), proteinaceous inhibitors isolated from microbial sources and plant tissues (40) and molecules present in plants comprise the natural inhibitors of α-amylase (41). Acarbose [1], a well know drug widely used for clinical treatment of diabetes mellitus, is a pseudotetrasaccharide, produced by Actinoplanes sp. fermentation, consisting of a polyhydroxylated aminocyclohexene derivative (valienamine) linked via its nitrogen atom to a 6-deoxyglucose, which is itself α-1,4-linked to a maltose moiety. It is a competitive inhibitor of -amylase and the mechanism of inhibition seems to be due to the unsaturated cyclohexene ring and the glycosidic nitrogen linkage that mimics the transition state for the cleavage enzymatic of glycosidic linkages (42,43).
In the structural study of the human pancreatic α-amylase /acarbose complex, acarbose inhibitor was described to bind subsites "-3" through "+2" (26). In acarbose the valienamine moiety is found in binding subsite -1 and its strong inhibition is believed to result from enhanced binding of this moiety with the side chain of Asp 197 , Glu 233 , and Asp 300 . Kinetic studies also highlighted the importance in catalysis of the presence of hydroxyl groups in the ligand together with the Asp 197 , Glu 233 , and Asp 300 residues in the binding site (substitution of these residues leading to a considerable drop in catalytic activity) (26).
Acarbose [1] is metabolised by small and large intestinal carbohydrases to give acarviosineglucose and glucose (43). The main adverse effects observed with acarbose are gastrointestinal, including abdominal discomfort, flatulence, meteorism and diarrhea (8,43,44). These adverse effects might be caused by the increase of degradation products in the intestine resulting in the abnormal bacterial fermentation of undigested carbohydrates (43,44). Indeed, these main side effects are common to -amylase inhibitors. Specifically, bloating, abdominal discomfort, diarrhea and flatulence occur in about 20% of patients (45). Frequently such effects lead to therapy discontinuation (7). -Glucosidase inhibitors are contraindicated in patients with irritable bowel syndrome or severe kidney or liver dysfunction. Inflammatory bowel disease is a relative contraindication (4). There are also reports of an increased of renal tumors occurrence and serious hepatic injury and acute hepatitis (46).
Studies with healthy and type 2 diabetes subjects showed that natural α-amylases inhibitors isolated from wheat (47) and white bean (48) significantly reduced the peak of postprandial glucose. Inhibitory profiles were investigated in green, oolong and black teas and the results suggested that catechins may be responsible for its activity in human salivary α-amylase (49 Therefore, the present article reviews and shows in table 1 a list of compounds with human αamylase inhibitory capacity. Phenolic compounds are a large group of structurally diverse naturally occurring compounds that possess at least a phenolic moiety in their structures. Most of these compounds possess various degrees of antioxidant or free radical scavenging properties as well as medicinal properties and have long been used as drugs. Flavonoids are abundant class of natural phenolic compounds with several biological activities. They share a common structural skeleton consisting of two aromatic rings (A and B) linked through three carbons attached to the Aring, forming an oxygenated heterocycle (ring C) and are divided in groups ( Figure 1 and human αamylase in order to understand the molecular requirement for enzyme inhibition. They showed that the potency of inhibition is correlated with the number of hydroxyl groups on the B ring of the flavonoid skeleton. The interaction occurs with the formation of hydrogen bonds between the hydroxyl groups in position R6 or R7 of the ring A and position R4' or R5' of the ring B of the polyphenol ligands and the catalytic residues of the binding site and formation of a conjugated -system that stabilizes the interaction with the active site (41). These results are in general agreement with the mechanism of action proposed for acarbose (50). Tannins are another heterogenous polyphenol group widely distributed in the plant kingdom that are often present in unripe fruits, but can disappear during ripening. They have a relatively high molecular weight and can be classified into two major classes: hydrolysable tannins and condensed tannins. Hydrolysable tannins are subdivided into gallotannins, derived from gallic acid [2] units linked to a sugar moiety), while condensed tannins are complex polymers, where the building blocks are usually catechins and flavonoids (51). COOH HO OH OH 2 gallic acid Several polyphenolic compounds presenting α-amylase inhibitory activity are shown at Figures 2, 3, and 4. Tannins could cause several effects on the biological system because they are potential metal ion chelators and protein precipitation agents forming insoluble complexes with proteins, as well as biological oxidants (52). Tannic acid and tannin-rich nonalcoholic components of red wine have been shown to reduce serum glucose levels after starch-rich meals in a study of patients with non-insulin dependent diabetes mellitus (53). As the mechanism involved in this anti-hyperglycemic effect is unknown, it is possible that tannins can inhibit α-amylase activity in situ. The ability to strongly bind to proteins forming insoluble and indigestible complex is the basis of their extensive use in the leather industry (tanning process), and for the treatment of diarrhea, bleeding, skin injuries (54) and probably it is the action mechanism to cause inhibition of the enzyme amylase.
Terpenoids are compounds that comprise various structures commonly found in nature with a several function in plants and animals. They usually arise from head-to-tail joining of isoprene units and a combination of two or more isoprene units divide the terpenoids in monoterpene (C 10 ), sesquiterpene (C 15 ), diterpene (C 20 ), sesterterpene (C 25 ), triterpene (C 30 ) and tetraterpene (C 40 ) (55).
Triterpenoids are a large and structurally diverse group of natural products derived from squalene [33] or related acyclic 30-carbon precursors (56) with several potential uses in medicine. Some triterpenoids with wellcharacterized biological activities include sterols, steroids, and saponins (57).
A range of real and potential usable biological effects are being studied for triterpenoids. Antiinflammatory, analgesic, antimicrobial, antimycotic, antiviral, antiplasmodial, antiulcerogenic, anticariogenic, immunomodulatory, vascular protective, antiobese, anticancer and tonic effects are ones the use related uses for this class of compound (58,59). Hepato and cardioprotective activity were also related for triterpenoids (59)(60)(61). Triterpenoids represent a promising and expanding source for biologically active natural compounds whose potential for research and development of new substances with pharmacologic activity. However, despite the fact that triterpenoids are widely distribute in plants, inhibitory α-amylase activity was related only for oleanane, ursane and lupane types and the mechanism by which this activity occur still unknown. Some terpenes presenting inhibitory activity on α-amylase are shown at Figure 5.

CONCLUSION
α-Amylase, a salivary or pancreatic enzyme plays an important role in early breakdown of complex carbohydrates into simple molecules. Modulation of α-amylase activity affects the utilization of carbohydrates as an energy source and stronger is this modulation; more significant is the reduction is the breakdown of complex carbohydrates. Majority of studies have focused on the antiamylase phenolic compounds.
The action mechanism proposed for inhibitory capacity of flavonoids correlated the potency of inhibition of these compounds with the number of hydroxyl groups on the B ring of the flavonoid skeleton with the formation of hydrogen bounds between the hydroxyl groups of the polyphenol ligands and the catalytic residues of the binding site of the enzyme. The high inhibitory capacity is observed in flavonols and flavones groups.
The main inhibitory effects of the tannins is related with its the ability to strongly bind to carbohydrates and proteins. However, Kandra et al. (2004) suggested that the interaction between tannins, such galloylated quinic acid, and human α-amylase is also correlated with free OH groups in the tannin, that are able to participate in hydrogen bonding (51). However, in this review is possible to note that tannins are not always an effective inhibitor of α-amylase.
The significant differences in inhibitory activity for α-amylase were shown in luteolin-7-O-glucoside [9b] from different studies. This compound showed 100% of inhibition in one study and 50% of inhibition in another. The same methodology was carried out to evaluate this activity in both studies, however the concentration of tested compound and incubation time of enzyme were different for both (6,62).
Inhibitory activities ranging from 100% to 50% were also observed for fisetin [4c] and luteolin [9a]. The analyzed studies showed differences in the concentration of tested compound, incubation time of enzyme and substrate solution used (6,41,62), and the impact that changes can be noted in the obtained results.
The comparison of inhibitory activity to αamylase showing in the studies allows to observe significant differences in percentage of inhibition for the same compound. This is due a several number of valuable assay methods for available the amylase activity. Between them, two types of assays are largely used to determine the action of α-amylase. One is based on increase in reducing power of the substrate by the dinitrosalicylic acid (DNS) reagent (64), whereas the other is based on the change of the iodine-staining properties of the substrate (65). Thus, some modifications in this assays reported by researchers could express different results for α-amylase inhibitory activity.
As the intake of phenolic compounds is associated with many beneficial effects, it is also necessary to consider the dose for humans, because it is possible to reduce α-amylase activity by consuming food or medicinal herbs rich in polyphenols with strong α-amylase activity, if it takes in consideration that this source of polyphenols possess different kinds of this compounds in variable concentration. Therefore, more available evidences are necessary about the safety of using natural α-amylase inhibitor.
Also, there is need for novel agents, therapeutic strategies or designing functional foods that could act on the physiological regulation of sugar uptake, blood sugar levels, and prevention of oral diseases.
For the future, a standardized protocol to search potential inhibitors maybe should be designed in order to minimize the differences among obtained results.