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2015|Volume 1 | Issue 1 | Page 1 - 5

Ewemen Journal of Herbal Chemistry & Pharmacology Research

Available online at http://ewemen.com/category/ejhcpr/

Original Research

IN VITRO STUDY ON ALPHA-AMYLASE INHIBITORY ACTIVITY OF ETHANOLIC EXTRACT OF MANGROVE SPECIES BRUGUIERA CYLINDRICA (L.)

*PITCHAIPILLAI, Revathi1; PONNIAH, Thirumalaikolundu subramanian2; THALAIMALAI, Jeyaseelan Senthinath3; SUBRAMANIAPILLAI, Manickavasagam1

1Postgraduate and Research Department of Pharmacology, Chennai Medical College Hospital and Research Centre

(SRM Group), Tiruchirapalli, INDIA 2Department of Medicine, Chennai Medical College Hospital and Research Centre (SRM Group), Tiruchirapalli, INDIA

3Department of Microbiology, Ponniah Ramajayam Institute of Medical Sciences, Kancheepuram, INDIA

ABSTRACT

Received 25 June, 2015 Revised on the 27 June, 2015 Accepted 30 June, 2015 *Corresponding Author’s Email: reva1923@gmail.com

Diabetes mellitus is a common endocrine system disease that mainly causes metabolic disorders and leads to multiple organ dysfunction. Clinically the cases of diabetes are divided in to type-1 and type-2, but type-2 diabetes is common in INDIA. With more side effects and increase in insulin resistance in using antidiabetic agents, the need of alternative medicines using phyto-constituents may be helpful. The use of pancreatic α-amylase inhibitors tenders an efficient approach to minimize the levels of post prandial hyperglycemia. The main objective of this current investigation is to analyze the inhibitory action of ethanolic extract of Bruguiera cylindrica against α-amylase by in vitro conditions compared to acarbose. The in vitro inhibitory action of the ethanolic extract of B. cylindrica against α-amylase was performed using spectrophotometry with acarbose as positive control. The results revealed a significant reduction (nearly 50%) of α-amylase activity. Thus the present study suggests a new source of herbal drug for managing diabetes using the leaves of B. cylindrica. Keywords: Bruguiera cylindrica, ethanolic extract, α-amylase, type-2 diabetes.

INTRODUCTION 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 (Okonji et al., 2014). Bruguiera cylindrica is rare mangrove tree in the southern tropical Indian Ocean

especially in Tamilnadu coastal areas. It has been used as traditional medicine for years with variety of biological activity such as antioxidant, antimicrobial, antiviral, antiulcer and anticancer properties (Lakshmi et al., 2012). Due to rich phytochemicals including steroids, triterpenes, saponins, flavonoids, alkaloids and tannins present in mangrove plants, they may be useful as best alternative for antidiabetic activity.

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Tamilnadu has a coastline of about 950 km along the coastline major mangrove wetlands are present in two areas – Pichavaram on Cuddalore district and Muthupet region in Thiruvarur-Thanjavur districts (Revathi et al., 2014). Small patches of mangroves are also present along the Palk bay, particularly in the Devipattinam region and also in some of the islands of the Gulf of Mannar in Ramanathapuram District. B. cylindrica is a rare, endangered and very important medicinal mangrove tree found along the western coast of INDIA. Traditionally B. cylindrica used to treat hepatitis, stop hemorrhage and applied to malignant ulcers. Only the stem part is proved scientifically as antioxidant effect. Further finger prints of antidiabetic activities had not been determined. The leaves are used to treat blood pressure and as fodder for livestock (Krishnamoorthy et al., 2011). Due to dysfunction of β cells of islets of langerhans in pancreas, the condition of insulin deficiency and/or resistance observed in diabetic patients. Multi-organ dysfunction is a major issue in this case by which micro and macro vascular complications are observed. The medicinal plants or natural products involved retard the absorption of glucose by inhibiting the carbohydrate hydrolyzing enzymes, such as pancreatic amylase. The inhibition of this enzyme delay carbohydrate digestion and protract overall carbohydrate digestion time, resulting in the reduction in glucose absorption rate and as a result dulling the postprandial plasma glucose rise (Iniyan, 2010). Several indigenous medicinal plants have a high potential in inhibiting α-amylase enzyme activity (Prashanth, 2001). The pancreatic α-amylase and alpha glucosidase are key enzymes responsible for the breakdown of long chain carbohydrates and degradation of starch to a mixture of oligosaccharides and oligoglucans, which further proceeds rapidly and leads to elevated post prandial hyperglycemia (PPHG) (Kim, 2005; Matsui, 1996; Sindhu, 2013). Thus, this α-amylase inhibition is considered as one of the mechanism to reduce blood glucose levels. The retardation of starch digestion confirms the control of diabetes (Tucci et al 2010). Alpha amylases and alpha glucosidases have been looked at as potential targets to control diabetes since 1960s (Imad and Fatima, 2010). However it was not before the early 1990s when the first alpha

glucosidase (alpha amylase as well) inhibitor, known as acarbose, came into clinical use (Jung et al., 1996; Lebovitz, 1997; Scheen, 2003). However, a major side effect associated with the use of acarbose is gastrointestinal disorders including bloating, abdominal discomfort, diarrhea and flatulence (Scheen, 2003; Chiasson, 2002). Therefore, the search of more safer, specific and effective hypoglycemic agents has continued to be an important area of investigation with natural plant extracts offering great potential for discovery of new antidiabetic drugs. The phytochemical composition of B. cylindrica of various extracts including n-hexane, chloroform, ethanol and aqueous extracts indicated the presence of various primary and secondary compounds (Revathi et al., 2015). On comparing with other solvent’s extract, the ethanolic extract showed significant quantity of phyto-constituents, thus the ethanolic extract was chosen for determination of α-amylase inhibitory assay. This biochemical method of qualitative determination of phyto-constituents of B. cylindrica compared further with the GC-MS analysis by the same author (Revathi et al., 2015) and data was correlated. Acarbose, the positive control used in this study has clinically high side effects (Scott and Spencer, 2000; Kim et al., 2000; Gomathi et al., 2012). Thus the need of screening the α-amylase inhibitors from medicinal herbs that are non-toxic, minimal side effects and cost effective is mandatory now to manage the diabetic patients. In this study, the effect of α-amylase inhibitory action of ethanolic extract of B. cylindrica compared with acarbose was performed and identified better. The aim of the present study is to examine the in vitro α-amylase inhibitory activity of ethanolic extract of Bruguiera cylindrica. MATERIAL AND METHODS Chemicals Alpha-amylase, ethanol, 3,5-dinitrosalicylic acid (DNSA) and soluble starch were products of Himedia, INDIA. Other chemicals and reagents used were of analytical grade.

Collection of plants The leaves of the mangrove plant Bruguiera cylindrica (L.) included in this study was collected from Pichavaram mangrove forest on 21st March 2014, after obtaining permission from District Forest Officer’s Office, Cuddalore, Tamilnadu, INDIA. The leaves were

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further authenticated by Botanist for the confirmation of the genus and species. Further, the leaves were preliminarily cleaned using running tap water and dried in shadow. The dried leaves were powdered coarsely using kitchen blender and stored in air tight polyethene bags until use. Sample extraction The dried leaves of B. cylindrica were weighed (100grams) and successive solvent extraction procedure was followed using soxhlet apparatus. Initially n-hexane was used as solvent followed by chloroform, ethanol and water (aqueous) each of 500ml. The extracted crude phyto-constituents was then evaporated using a rotary vacuum evaporator at 50 oC and further stored at low temperature (-4 oC) in a sterile air tight container. In vitro α-amylase inhibitory study This assay was carried out using a modified procedure of McCue and Shetty to determine the α-amylase inhibition. 250 μl of varying concentrations of the ethanolic extract of B. cylindrica (5-100 mg/ml), was placed in a tube and 250 μl of 0.02 M sodium phosphate buffer (pH 6.9) containing alpha-amylase solution (0.5 mg/ml) was added. This solution was pre-incubated at 25 °C for 10 min, after which 250 μl of 1% starch solution in 0.02 M sodium phosphate buffer (pH 6.9) was added at timed intervals and then further incubated at 25 °C for 10 min. The reaction was terminated by adding 500 μl of 3,5-dinitrosalicylic acid (DNSA) reagent. The tubes were then incubated in boiling water for 5min and cooled to room temperature. The reaction mixture was diluted with 5 ml distilled water and the absorbance was measured at 540 nm using spectrophotometer. The control sample was prepared using the same procedure replacing the extract with distilled water. The alpha-amylase inhibitory activity was calculated as percentage inhibition: concentrations of extracts resulting in 50% inhibition of enzyme activity (IC50) were determined graphically. The concentration of control and plant extracts required to inhibit 50% of α-amylase activity under the conditions was defined as the IC50 value. Evaluation of inhibitory action The results were expressed as % inhibition calculated using the formula:

Where Ac and Ae = Absorbance of control and extract respectively. The absorbance of control was determined as 1.530 in this study.

The IC50 values were determined from plots of percent inhibition against log inhibitor concentration and calculated by non-linear regression analysis from the mean inhibitory values. Acarbose (an α-glucosidase inhibitor) was used as positive control, (Jyothi et al., 2013). RESULT AND DISCUSSION The results of in vitro inhibitory study of alpha amylase by B. cylindrica is shown in Table 1. The results showed that B. cylindrica inhibited alpha amylase in a concentration-dependent manner. A plot of % inhibition against test samples concentrations (Figure 1) reveals that the IC50 of for B. cylindrica was 89.50±0.25. Acarbose (positive control) at a concentration of 0.075 mg/ml showed 49.60 % inhibitory effects on the α-amylase.

Table 1: Percentage inhibition of α-amylase by ethanolic extract of B. cylindrica (L.) Samples Conc.

(mg/ml) Absorbance (540 nm)

% Inhibition

IC50 (mg/ml)

Plant extract

5 1.306 14.64 89.5 ± 0.25 10 1.188 22.35 20 1.041 31.96 40 0.879 42.54 80 0.790 48.36 100 0.741 51.56

Figure 1: Percentage inhibition of α-amylase by ethanolic extract of B. cylindrica (L.)

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The α-amylase inhibitory activity of ethanolic leaves extract of B. cylindrica showed over 48% inhibition rate at 89.50 mg/ml. In previous studies, buffered extracts of several plant species namely Balanites aegyptiaca L., Camellia sinensis L., Galega officinalis L., Holarrhena floribuda (Don), Khaya senegalensis (Desr.), Melissa officinalis L., Mitragyna inermis (Willd), Rosmarinus officinalis L., Securidaca longepedunculata., Tamarindus indica L., Taraxacum officinale and Vaccinium myrtillus L were screened for α-amylase activity and they all showed remarkable inhibitory activity against α-amylase at 200 mg/ml (above 45% inhibition) (Paloma et al., 2012, Funke and Melzing, 2006). Hence, the percentage inhibition of α-amylase by the extracts of B. cylindrica indicates that it is a better inhibitor of the enzyme (48% inhibition at 89.50 mg/ml) than plants reported by Paloma et al. (2012). The management of postprandial hyperglycemia is a critical strategy in the control of diabetes and its complications and the determination of α-amylase and α-glucosidase inhibitors has been useful as oral hypoglycemic agents for the control of type 2 diabetes mellitus (Kimura et al, 2006). However the existing pharmaceutical hypoglycemic agents like acarbose, miglitol and voglibose have been found to possess more undesirable gastro intestinal side effects like abdominal discomfort, flatulence, bloating and diarrhea which limits their use (Kazeem et al 2013, Dayananda KS and Gopinath SM 2013). B. cylindrica is already approved as a food and feed for humans and animal consumption, respectively, or botanical supplements in the form of juice that have moderate α-amylase inhibition to control diabetes in INDIA (Sathyavani et al., 2012; Revathi et al., 2015). The result of this study showed that B. cylindrica is a mild α-amylase inhibitor which is more desirable as previous reports had indicated that excessive inhibition of pancreatic 𝛼-amylase could result in the abnormal bacterial fermentation of undigested carbohydrates in the colon (Apostolidis et al., 2007). The phyto-constituents including phenolic compounds like flavonoids, saponins, terpenoids and tannins present in the plants are believed to be responsible for the wider inhibitory action of α-

amylase (Paloma et al., 2012; Kazeem et al., 2013; Funke and Melzing, 2006). Another study had demonstrated, that both ethanolic and hexane extracts of Phyllanthus amarus exhibited appreciable α-amylase inhibitory effects when compared with acarbose and it was suggested that the inhibitory activity may be due to the presence of more chemical constituents such as lignans (phyllanthin and hypophyllanthin), terpenes, tripenes, flavonoids (quercetin, quercetrin, rutin), and alkaloids in the ethanol and hexane extracts (Inian, 2010). Another study had suggested flavonols or phenolic acids as the bioactive components responsible for the amylase inhibitory activity as various studies reported in literature had shown a clear link between polyphenols and antidiabetic activity of herbal extracts (Sindhu, 2013, Egharevba et al, 2015). The ethanolic extracts of B. cylindrica had been found to contain flavonoids, polyphenols, alkaloids, saponins, tannins and triterpenes, and this might be the reason for the α-amylase inhibitory action. Thus, this suggests that these active components in the extract compete with the substrate for binding to the active site of the enzyme thereby preventing the breaking down of oligosaccharides to disaccharides and monosaccharides (Matsuda et al., 2002; Shai et al., 2010). CONCLUSION It can be concluded that the ethanolic extract of B. cylindrica effectively inhibited the activity of α-amylase and this is one of the mechanism to reduce the hyperglycemia. This inhibitory property of the extract may be attributed to the presence of phytochemicals such as flavonoids, tannins, triterpenes and saponins but further study is required to prove this, and the extension of this study may lead to the isolation of the specific compound(s) responsible for the observed activity from the plant. Further in vivo analysis of the plant against type 2 diabetes mellitus needs to be done with a view toward identifying the responsible molecules and understanding its mode of action in the management of diabetes.

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Article Citation: Pitchaipillai R, Ponniah TS, Thalaimalai JS, Subramaniapillai M (2015). In vitro study on alpha-amylase inhibitory activity of ethanolic extract of Mangrove species Bruguiera cylindrica (L.). Ew J Herb Chem & Pharmacol Res 1(1): 1-5.