Inhibition of alpha-amylase by “ insulin plant ” ( Myrcia sphaerocarpa DC ) extracts : an alternative for the treatment of diabetes mellitus ?

Article history: Received on: 20/02/2015 Revised on: 17/03/2015 Accepted on: 09/04/2015 Available online: 27/05/2015 Diabetes mellitus is an important medical-economical-social problem requiring up search for less costly alternatives for treatment, such as use of medicinal plants. Of the species cited in ethnobotanical studies, the "insulin plant" (Myrcia sphaerocarpa DC) appears among those used by diabetics. The objective of this work was to evaluate the inhibitory activity of alpha-amylase by aqueous extracts of "insulin plant", prepared by different methods at a proportion of 1:200 (w:v), for information about the alleged hypoglycemic effect. For this, five samples of commercially teas "insulin plant" were obtained; aqueous extracts were prepared; and thereafter, they were tested for inhibition of alpha-amylase, before and after exposure to simulated gastric fluid. Results demonstrated before exposure to gastric fluid, a significant inhibition of enzyme activity (greater than 50%) in the samples A and C, using the decoction and sample E in all methods of extraction. Comparing methods of extraction, the decoction was more effective. After exposure to gastric fluid, there was a reduction in the activity of enzyme inhibition, suggesting negative influence of low pH on the properties of the inhibitor. These results do not elucidate the mechanism of the hypoglycemic action reported in folk medicine and literature, but indicate a promising path.


INTRODUCTION
The Chronic Noncommunicable Diseases (CNCD) are currently the main causes of death in the world.The increased prevalence is accompanied by a high number of premature deaths, loss in life quality due high limitation for work and leisure activities and economic impacts for families, communities and society at large (Alwan et al., 2010;Malta et al.,2011;Ministério da Saúde, 2012;Schmidt et al.,2011;Sociedade Brasileira de Diabetes, 2007).Among the CNCD diabetes mellitus has been highlighted as a problem of medical and socioeconomic importance in the context of public health.Once, there was a rapid increase in the risk group and the long-term metabolic control and the treatment of complications, result in high costs for individuals, families and health systems (Defani et al., 2011;Malta et al., 2011;Ministério da Saúde, 2007;Sociedade Brasileira de Diabetes, 2003).In the search for cheaper and more accessible alternative treatment for diabetes, there is a growing interest in the use of medicinal plants and herbal medicines (Borges et al, 2008;Defani et al., 2011;Rosa et al., 2012;Santos et al., 2012).Of the species cited in ethnobotanical studies in different regions of Brazil, the "insulin plant" appears among those used by diabetics.Under this popular nomenclature, several species are reported in the literature, especially Myrcia sphaerocarpa DC. (Rosa et al., 2012;Santos, 2005), Cissus sicyoides L. (Defani et al., 2011;Pepato et al.,2003;Santos et al., 2008;Santos et al., 2012;Viana et al., 2004), Cissus verticillata L.(Pereira da Silva and Proença, 2008), Myrcia guianensis, Myrcia uniflora, Myrcia salicifolia e Myrcia speciosa (Santos, 2005) e Myrcia multiflora (Matsuda et al., 2002;Santos, 2005).Despite the reported hypoglycemic effect, these species have not been validated in the mechanism of action, toxicity and usage, being rejected as ethical drug free prescription.Thereby, this study proposes: a) obtaining of "insulin plant" tea commercial samples, as well as the identification of the used scientific name, b) preparing of aqueous extracts by different methods (decoction, infusion, maceration and tisane), and c) conducting tests of αamylase inhibition, aiming to obtain information about the supposed hypoglycemic effect.

Sample Collection and Preparation
Five tea samples (A, B, C, D and E) of "insulin plant" were acquired in the form of dried leaves in drugstores and specialized stores of phytoterapy, at the cities of Lavras, Uberaba and Juiz de Fora, all of them at Minas Gerais state -through convenience sampling.The samples were taken to the Laboratory of Biochemistry ICBN, archived and identified with data on the species at the label and/or technical report, manufacturing dates and expiration dates, and informations about the provider.
The reduction of brute samples for to laboratory samples was performed manually, randomly, with the weighing carried out in a semi-analytical balance.The preparation of aqueous extracts was performed at the time leading up to the inhibition assays, using the different methods described below.

Decoction
The sample and water at a proportion of 1:200 (w:v) were put in heating in a closed vessel, after getting to ebullition point it was kept for 10 minutes.Following, the decoction was put in room temperature for 30 minutes and was filtered on tissue (organza), until the obtaining of a clear liquid used in the inhibition tests.

Infusion
Boiled water was added to the sample (in a vessel) at the proportion of 1:200 (w:v).The infusion was kept covered and put in room temperature for 10 minutes, after was filtered on tissue (organza), until the obtaining of a clear liquid used in the inhibition tests.

Maceration
The sample and water at a proportion of 1:200 (w:v) were put in a closed vessel and left resting for 10 days.After that, the macerated was filtered on tissue (organza), until the obtaining of a clear liquid used in the inhibition tests.

Tisane
The sample was immersed in boiling water at a proportion of 1:200 (w:v) and remained boiling for 5 minutes in a closed vessel.Removed from the heating, it was kept under room temperature for 10 minutes and filtered on tissue (organza), until the obtaining of a clear liquid used in the inhibition tests.

Enzyme Inhibition Test
The pancreatic enzyme α-amylase type VIB porcine (Sigma ®) was used for the inhibition test.The identification of the α-amylase activity was realized according to the methodology proposed by Noelting and Bernfeld (1948) using as substrate 1% starch prepared in Tris buffer 0,05 mol L -1 (pH 7.0) plus 38 mmol NaCl L -1 and CaCl 2 0.1 mmol L -1 .The plant extract and α-amylase were pre incubated for 20 minutes in a water bath at 37°C.Thereafter, added substrate and the mixture was incubated for four time periods (10, 20, 30 and 40 minutes).The reaction was stopped with adding 3,5 dinitro salicylic acid and the product was read in a spectrophotometer at 540 nm.
Additionally, to simulate the digestion process, were also conducted tests on enzyme activities in the presence of simulated gastric fluid prepared according to The United States Pharmacopeia -USP (1995).The extract was incubated in the presence of fluid for 1 hour in a water bath at 37°C, neutralized with sodium bicarbonate (pH 6.8 -7.0) and then carried out activity tests.
The enzyme inhibition was obtained by determining the slopes of the lines (absorbance vs. time) -testing the activity control (without extract) and sample (with extract).The slope of the line is due to the rate of product formation per minute of reaction and the presence of inhibitor causes a decrease in this gradient, expressed in percentage terms.All analyzes were performed in triplicate.

Experimental Design and Statistical Analysis
The slopes of the lines and the inhibition rates were obtained from the insertion of data into Excel spreadsheet.The results of the three replicates were analyzed by ANOVA -Scott-Knott test using the software SISVAR® (Ferreira, 2000).

RESULTS AND DISCUSSION
In literature, the popular name "insulin plant" is cited for different species, however in our study all samples corresponded to Myrcia sphaerocarpa DC.The results of enzyme inhibition tests, expressed as average percentage before and after exposure to simulated gastric fluid, are shown in Table 1.
Statistical analysis revealed significant differences between the samples and/or extraction methods.Despite the fact that the five samples are marketed as "insulin plant ", is important to consider that such variations in inhibition percentage can be attributed to factors edaphoclimatic, preparation methods and possible contamination with other species.Gobbo-Neto and Lopes (2007) report that the harvest is one of the most important factors, since the concentration and nature of the active constituents are not constant throughout the year.Souza (2011) cites that significant inhibition of the alphaamylase is greater than 50%; value shown by samples A and C, using the decoction, and sample E in all extraction methods.Souza (2011) also mentions that values exceeding the 80% -values presented for samples A and E of this study -represent high enzymatic inhibition.Comparing the extraction methods used in this study, the decoction was more effective in inhibiting alphaamylase in relation to others, with values significantly higher for samples A, C and D. There were exceptions the sample B in which was not observed inhibition and E where the value of inhibition was statistically similar to tisane.Also in relation to sample E, high percentages of inhibition were observed in all methods, suggesting an influence on the presentation, unlike the others, had the dried leaves, fragmented for commercialization.
The results were similar to those obtained by Martins et al.,(2014) in his work with Origanum vulgare L. leaves, in which the decoction was more effective to have higher antioxidant activity compared with infusion and hydroalcoholic extract.Funke and Melzig ( 2006) working with alpha-amylase inhibitors in 16 species, including Galega officinalis, also observed influence of the extraction method.The extract obtained at room temperature showed little effect on the enzyme, while that obtained with boiling buffer showed a 35% inhibition of enzyme activity.
Our results demonstrate a reduction in the activity of enzyme inhibition after exposure to simulated gastric fluid, except for sample D. These results corroborate Pereira et al.,(2010) who worked with green and black teas, observed inhibition of alphaamylase 42.19 and 73.44%, respectively, and absence of inhibition after simulation with gastric fluid.Freitas et al.,(2014) assessed the activity of enzyme inhibition of extracts of commercial teas Syzygium sp front of the alpha-amylase by different extraction methods (infusion, decoction and maceration) and different proportions (1:20, 1:50 and 1 : 100) obtained significant inhibition of the enzyme before exposure to fluid, with an average of 92.84%.However, the authors reported that the samples showed a reduction in the percentage of inhibition after exposure to gastric fluid.And this reduction may be related to the negative influence of low pH on the properties of the inhibitor.Unlike, Pereira et al.,(2011) assessed the crude extract of white bean flour found that the percentages of inhibition of α-amylase was 79.1% and 81.8% with and without gastric fluid, respectively, concluding that the inhibitor was stable.By comparing the results of this study to the literature, it can be inferred the presence of different inhibitors between species, requiring determination of these as a way to provide more data on the use of the same.
According Salunkhe et al., (1990) the inhibition of alphaamylase by teas can be associated with the presence of phenolic compound (non-protein inhibitors), because there interaction between the aromatic ring hydroxylated and aminoacids enzyme.Ali et al., (2006), Kandra et al., (2004) and Nickavar and Abolhasani (2013) discuss the association between inhibition by plant extracts and the presence of phenolic compounds, tannins and triterpenoids.Ali et al., (2006) worked with six species Malaysia's plants, including Phyllanthus amarus hexane extract, identified and isolated a triterpenoid mixture (oleanolic acid and ursolic acid -2: 1) that showed potent inhibition of alpha-amylase.It is noteworthy that, although authors as Sivakumar et al., (2006) and Wang et al.,(2006) cited Antunes ( 2008) affirm that the amylase inhibitors are produced by plants especially in seeds and tubers, this work shows the presence of inhibitors in the leaves showing the importance of further studies.
Among the species receiving designation "insulin plant", some studies have assessed their ability to enzyme inhibition.Matsuda et al., (2002) in their studies, found that the methanol extracts of Myrcia multiflora leaves show potent aldose reductase inhibitory activity-which catalyzes the reduction of glucose to sorbitol in rats, associating the use of the beneficial effects in diabetic patients, because the accumulation sorbitol has been implicated in chronic complications of diabetes, such as neuropathy, retinopathy and cataracts.Khalil et al.,(2008) worked with aqueous extract (decoction) of Cissus sicyoides and Bauhinia forficata to identify antioxidant activity related to prevention of diabetes complications found for the first, 48 % inhibition (100 μg/ml) of myeloperoxidase, an enzyme present in all stages of development of atherosclerosis and other diseases.According to the authors, the results support the use of the species in the prevention of diabetes.Ferreira et al., (2006) evaluated the effects of aqueous extract of Myrcia uniflora on thyroid function, isolating two flavonoids (mearnsitrin and myricitrin) of the species.The authors observed that a solution of these inhibited 50% of the peroxidase activity, which participates in the synthesis of thyroxine and triiodothyronine hormones, concluding that the indiscriminate use with nutritional iodine deficiency may contribute to the development of hypothyroidism.Ferreira (2010) conducted tests on samples of 2,4,6trihidroxiacetifenona (THA) isolated from Myrcia multiflora, in order to assess hypolipidemic potential of the compound in rats.There was a reduction in cholesterol, triglyceride and lipase levels of treated animals with THA higher than treated with available drugs in the market (lovastatin and orlistat), possibly by blocking the absorption of triglycerides due to an inhibitory effect on lipase, and induction of cholesterol 7α-hydroxylase.

CONCLUSION
All samples obtained commercially, with designation "insulin plant" correspond Myrcia sphaerocarpa DC.Aqueous extracts of "insulin plant" at a proportion of 1:200 (w:v), exhibit a high inhibition of α-amylase before to exposure to simulated gastric fluid, especially when used the decoction as extraction method.However, after exposure to the fluid, there is a significant reduction of the inhibitory activity, possibly related to the effect of low pH on the inhibitor.
These results do not elucidate the mechanism (hypoglycemic action) reported in folk medicine and literature, but indicate a promising path.There is an evident need for further studies to determine the bioactive compounds present as well as any toxic effects, and other possible mechanisms of action.

Table 1 :
Percent inhibition of alpha-amylase by aqueous extracts of Myrcia sphaerocarpa DC in proportion 1:200 (w:v) obtained by different extraction methods (infusion, decoction, maceration and tisane).Average percentage obtained from three replicates ± standard deviation.*a -i: Statistical variation between the results of the sample according to Scott-Knott (p <0.05).Same letter represent statistically identical results.