Abstract

Background

Diabetes mellitus is generally characterized by hyperglycemia that leads to disturbances in the metabolism of carbohydrates, lipids and proteins [1]. Chronic inflammation leads to obesity and it may be prevented by avoiding sugar containing food items. Nowadays insulin therapy is encouraged for the prevention of diabetes mellitus, but the therapy has several side effects like insulin resistance [2], anorexia nervosa, brain atrophy etc. Recently, the use of medicinal plants in modern medicine has been increased in order to prevent or to cure diseases [3–6]. At present food safety is an important concern due to the presence of food borne and other clinical pathogens. The quest for new antimicrobials have been taken into account by researchers worldwide due to the emergence of antibiotic resistant organisms and toxicity of synthetic drugs. Herbal plants and their extracts have been investigated in last few years due to the toxicological concerns of synthetic drugs [7, 8]. The secondary metabolites obtained from medicinal plants have also been investigated for their radical scavenging property.

P. granatum Linn. (Pomegranate) is abundantly present in India and belongs to family Punicaceae. Pomegranate peel contains tannins, flavonoids, polyphenols and some anthocyanins such as cyanidins and delphinidins [9]. Extracts from the peels of pomegranate has been proposed to play vital role in various pharmacological activities [10, 11]. The natural antioxidant food supplement will give the anti- aging process of skin, cells, tissues and organs. Antioxidants are present in certain fruits and vegetables that can protect human cells from oxidative damage and prevent aging of cells and body [12]. It reduces the incidence of tumors and infections. The plant constitutes gallotannic acid and the alkaloid such as isopelletierine, pelletierine, methypelletierine, psuedopelletierine, gallic acid, tannic acie, sugar, cacium oxalate, etc. However, the phytochemical constituents of the plant and antimicrobial activity of this plant have been reported in literature [13, 14]. It is very important to explore the findings of the research by investigation it in vivo and understanding its interactive effect.

C. elegans is a model organism that can be grown cheaply and in large numbers on plates. The worm is preferred over other model organisms especially mouse because they have a short life cycle of only 2 weeks which reduces the experimental cycles and the behaviour of individual cells can be studied because of its transparent body. Additionally, C. elegans genome have functional counterparts in humans which makes it a convenient model for human diseases especially diabetes study.

The nematode Caenorhabditis elegans contains abundant fat droplets in intestinal and hypodermal tissue. Compared to droplets in mammalian adipose tissue, which can expand to sizes of 100 μm [15], C. elegans lipid droplets are small, typically in the size range of 1–1.5 μm [16]. C. elegans has a multistep developmental process due to multicellularilty property. Regulation of lipid droplets inside the nematode using potential herbal extracts will yield key insights into the understanding of obesity, diabetes, and other metabolic diseases [17, 18]. From thios point of view, the present study was evaluated to determine antioxidant activity, α-Glucosidase activity, antimicrobial and antidiabetic property of pomegranate fruit peel extracts.

Methods

Results and discussion

Medicinal plants and their extracts have exploited continuously by researchers in order to produce potential drugs of medicinal properties with reduced toxicity. In the line of this, we reported significant in vitro α-glucosidase inhibition activity of ethyl acetate extract of pomegranate peel. The α-glucosidase inhibiting potential of solvent extract such as hexane, ethyl acetate and methanol extracts from the fruit peel were tested and the results are summarized in Table 1. Ethyl acetate extract inhibited alpha glucosidase with the maximum value of 75.6 ± 2.03 % at 500 μg/ml with IC₅₀ value of 285 ± 1.98 μg/ml. Alpha glucosidase inhibiting activity of fruit peel extracts are in the order of ethyl acetate > hexane > methanol.

Pomegranate fruit peel extract potently scavenged DPPH radicals similar to catechin, it is likely that peel extract possessed proton-donating ability and in association with a number of hydroxyl groups to stabilize free radicals [25, 26]. The results of this study suggest that the extracts contain phytochemical constituents that are capable of donating hydrogen to a free radical. Ethyl acetate extract of pomegranate peel has the ability to reduce the stable radical DPPH to diphenylpicryl hydrazine. The different concentrations of solvent extract of pomegranate peel showed antioxidant activities in a concentration-dependent manner (26–71.2 %) in the DPPH scavenging assay. Ethyl acetate extract (100–500 μg/ml) showed the highest activity (30.5–71.2 %). Figure 1 shows the scavenging effects of various extracts of pomegranate fruit peel on DPPH˙ in the following order: ethyl acetate > methanol > hexane.

The total antioxidant capacity of various solvent extracts of pomegranate fruit peel at different concentrations (100–500 μg/ml) was found to be in the following order: ethyl acetate > methanol > hexane (Figs. 2, ,33 and and4).4). Ethyl acetate extract (500 μg/ml) showed the highest total antioxidant activity (69.5 %).

The antibacterial activity of P. granatum fruit peel may be because of the presence of metabolic toxins or broad spectrum antimicrobial compounds that act against both Gram + ve and Gram –ve bacteria. The results of antibacterial activity of ethyl acetate extract of P. granatum (Pomegranate) fruit peel (PGPEa) against various human pathogens are listed in the Table 2. The MIC was significantly lower in ethyl acetate extracts that inhibits T. rubrum and T. mentagrophytes (31.25 μg/ml) (Table 3).

Various investigations were carried out to determine antioxidant, anticarcinogenic, and anti-inflammatory properties of pomegranate constituents [27–29]. Hajoori et al. [30] evaluated the antibacterial activity of different solvent extracts of P. granatum peel against human pathogens including four gram positive bacteria and six gram negative bacteria. According to Rathi et al. [31] P. granatum fruit peel can be used as an easily accessible source of natural antioxidant. They clearly demonstrated broad spectrum antimicrobial activity of pomegranate against bacteria. Additionally they mentioned that the presence of phytocompounds in the extracts including phenols, tannins and flavonoids as major active constituents may be responsible for these activities. Thus, the present study provides a strong direction for proper investigation of pomegranate fruit peel to explore molecules having antimicrobial properties against human pathogens. The presence of active inhibitors in pomegranate fruit peels including phenolics and flavonoids were revealed by phytochemical analysis as potent constituents.

The observed lifespan of C. elegansas model organism (control) was about 17 ± 1 days and the normal worms about 25 ± 1 days sillar to the other reported study [32]. When the observed result of the control was compared with that of the tests, we found that the ethyl acetate extract of P. granatum fruit peel showed the increased lifespan of the worms than that of the control i.e., about 24 ± 1 days. The second highest activity was shown for methanol extract of P. granatum (Table (Table44).

C. elegans provides a reliable tool to understand the changes in lipid accumulation in the body by glucose concentrations that are within the range observed in poorly controlled diabetic patients [33]. In the present study ethyl acetate extract of pomegranate fruit peel has the ability to reduce the lipid accumulation in the worm body. The fluorescence microscopic analysis clearly shows that ethyl acetate peel extract treated worms have accumulated less lipid compared to the normal worms (Figs. 5 and and6).6). Reduction in glucose level and lipid content in C.elegans indicates that ethyl acetate extract of pomegranate fruit peel has potential antidiabetic compounds which need to be isolated and identified through chromatographic techniques. Undoubtly ethyl acetate extracts of the test plant showed the presence of diverse molecules when subjected to GC-MS. In PGPEa a total of 48 compounds were detected, out ofwhich the maximum area was found for 5-hydroxymethylfurfural and 4-fluorobenzyl alcohol (Table 5) with a value of 48.59 % (Fig. (Fig.77).

Conclusion

It is concluded that the ethyl acetate extract of pomegranate fruit peel contained considerable levels of phenols and flavonoids which are responsible for α-glucosidase inhibition and antioxidant activities. These in vitro assays also indicate that the PGPEa not only has potential antidiabetic and natural antioxidant compounds but also has the ability to increases the life span of C. elegans. Future studies are required to prove whether the process described in C. elegans can be translated to the situation in diabetic patients. 5-hydroxymethylfurfural and 4-fluorobenzyl alcohol compounds detetcted in GCMS might plaied the role in antioxidant and antimicrobial potentials of the extracts.

Abbreviations

GC-MS, gas chromatography–mass spectrometry

Acknowledgement

Contributor Information

Mariadhas Valan Arasu, Email: moc.liamg@usaranalavm.

Han Sung Choi, Email: ten.liamnah@8473gsh.

Young Ock Kim, Email: rk.aerok@8219oyk, Email: moc.liamg@8219oyk.

References