Int J Curr Pharm Res, Vol 9, Issue 2, 42-47Original Article

IN VITRO ANTI-DIABETIC AND ANTI-OXIDANT ACTIVITIES OF ETHANOL EXTRACT OF TINOSPORA SINENSIS

ANINDITA BANERJEE¹, BITHIN MAJI¹, SANDIP MUKHERJEE¹, KAUSIK CHAUDHURI², TAPAN SEAL^2*

¹Department of Physiology, Serampore College, Hooghly, West Bengal, India, ²Plant Chemistry Department, Botanical Survey of India, Howrah, India
Email: kaktapan65@yahoo.co.in

Received: 27 Nov 2016, Revised and Accepted: 23 Jan 2017

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ABSTRACT

Objective: The aim of the present study was to evaluate the alpha (α)-amylase and alpha (α)-glucosidase inhibitory activities and in vitro antioxidant activities of the 80 % aqueous ethanol extracts of Tinosporasinensis Lour (Merr.).

Methods: The 80% aq. ethanol extract of the plant was prepared. The plant extract was examined for its antioxidant activity by using free radical 1,1-diphenyl-2-picryl hydrazyl (DPPH) scavenging method, ABTS radical scavenging ability, reducing power capacity, estimation of total phenolic content, flavonoid content and flavonol content. Different concentrations (2, 4, 8,10and 15 μg/ml) of the extract was subjected to α-amylase inhibitory and α-glucosidase inhibitory activities and IC₅₀were calculated.

Results: The study revealed that the different concentrations of the plant extract possessed a very good amount of total phenolics, flavonoid and flavonol and exhibited potent radical scavenging activity using DPPH and ABTS as a substrate. The ethanol extracts exhibited significant α-amylase and α-glucosidase inhibitory activities with an IC₅₀ value1.093µg and 1.04µg dry extract respectively and well compared with standard acarbose drug.

Conclusion: Thus, it could be concluded that due to the presence of antioxidant components the plant extracts could be used for the treatment of hyperglycemia, diabetes and the related condition of oxidative stress. This knowledge will be useful in finding more potent components from the natural resources for the clinical development of antidiabetic therapeutics.

Keywords: Tinosporasinensis, 80% aq. ethanol extract, Antioxidant activities, α-amylase inhibitory activity, α-glucosidase inhibitory activity

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© 2017 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)
DOI: http://dx.doi.org/10.22159/ijcpr.2017v9i2.17379

INTRODUCTION

Diabetes mellitus is an important chronic metabolic disorder that affects the digestion of carbohydrate, fat and protein. It includes a group of metabolic diseases characterised by hyperglycemia, in which blood sugar levels are elevated either because the pancreas does not produce enough insulin or cells throughout the body do not respond properly to the insulin produced. The effects of diabetes mellitus include long-term complications include heart disease, stroke, dysfunction and failure of various organs [1].

There are three forms of diabetes. The three main kinds of diabetes are type 1, type 2, and gestational diabetes. Both women and men can develop diabetes at any age. The type 1 diabetes includes the cases which can be attributed to an autoimmune process and/or those with β-cell destruction for which unknown pathogenesis. Type 2 includes the common major form of diabetes, which results from defects in insulin secretion or rather insulin resistance. Gestational diabetes can develop to a pregnant woman when a pancreas does not make enough insulin during gestation.

The only therapy of type 1 diabetes is the substitution of insulin. Many and diverse therapeutic strategies for the treatment of type 2 diabetes are known. The conventional treatments for 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 [2-3].

One group of drugs introduced into the management of type 2 diabetes is represented by the inhibitors of α-glucosidase. The enzymes summarised as α-glucosidase is responsible for the breakdown of oligo and/or disaccharides to monosaccharides. The inhibitory action of these enzymes leads to a decrease of the blood glucose level because the monosaccharides are the form of carbohydrates which is absorbed through the mucosal border in the small intestine.

Another effective method to control diabetes is to inhibit the activity of a α-amylase enzyme which is responsible for the breakdown of starch to simpler sugars (dextrin, maltotriose, maltose and glucose) [4]. This is contributed by α-amylase inhibitors, which delays the glucose absorption rate thereby maintaining the serum blood glucose in hyperglycemic individuals [5].

Some inhibitors currently in clinical use are acarbose and miglitol which inhibit glycosidases such as α-glucosidase and α-amylase while others such as and voglibose inhibit α-glucosidase. However, many of these synthetic hypoglycemic agents have their limitations, are non-specific, produce serious side effects and fail to elevate diabetic complications. The main side effects of these inhibitors are gastrointestinal viz., bloating, abdominal discomfort, diarrhea and flatulence [6]. Recently herbal medicines have acquired more importance in the treatment of diabetes as they are free from side effects and less expensive when compared to synthetic hypoglycemic agents [7-8].

Various plants such as Proteus vulgaris, Euphorbia hirta, Cassia glauca containing active constituents like saponin, phenols, flavonoids, etc showed potential α-amylase inhibitors [9]. The role of medicinal plants in disease prevention is attributed to its antioxidant properties due to their bioactive constituents [10].

Tinosporasinensis (Lour.) Merrbelongs to the family Menispermaceae and the stem of the plant are used as medicine. The plant grows wild in most parts of India, both in forests and plains. The plant is reported to be used for fumigation in piles and ulcerated wounds, and for the preparation of medicated baths for liver complaints. The boiled roots are given in fever. Fresh leaves and stems are used in chronic rheumatism and also a muscle relaxant [11].

Thus, in this study, the antioxidant and antidiabetic activities of the 80 % aq. ethanol extract from the stem of T. sinensis was carried out and a relationship of these activities was established. To evaluate the potential of T. sinensis stem extract as antidiabetic agents, we investigated the effect of extracts on the α-glucosidase and α-amylase inhibitory activities.

MATERIALS AND METHODS

Plant materials

The stem of Tinospora sinensis was collected from Hooghly district West Bengal, India and authenticated from Botanical Survey of India, Howrah. The voucher specimens were preserved in our department under registry no PHYSIOL/BM/AB 001. The plant parts were shed-dried, pulverised and stored in an airtight container for further extraction.

Chemicals

Alpha (α)-Glucosidase, porcine pancreas alpha (α)-amylase, p-nitrophenyl-α-D-glucopyranose (p-NPG), 3,5-dinitrosalicylic acid (DNS),1,1-Diphenyl-2-picrylhydrazyl (DPPH), 2,2’-azino-bis(3-ethyl benzothiazoline-6-sulfonic acid) (ABTS), butylated hydroxytoluene (BHT), gallic acid, rutin, quercetin, ascorbic acid and acarbose were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Soluble starch, sodium potassium tartarate, sodium dihydrogen phosphate (NaH₂PO₄), Di-sodium hydrogen phosphate (Na₂HPO₄) sodium chloride, sodium hydroxide, butylated hydroxytoluene (BHT), potassium persulfate, sodium carbonate, Folin-Ciocalteu (FC) reagent, potassium ferricyanide, potassium per sulphate, aluminum chloride, ferric chloride (FeCl₃) were from Merck Chemical Supplies (Damstadt, Germany). All the chemicals used including the solvents were of analytical grade.

Extraction of plant material

The air-dried and coarse powdered plant sample of T. sinensis (10 g) was extracted with 100 ml 80% aq. ethanol by maceration on an orbital shaker with agitation for five days at room temperature. The extract was filtered and the residue was again extracted with the same solvent for another five days and filtered. The filtered extracts were combined and concentrated using a rotary evaporator, under reduced pressure at approximately 40 °C and lyophilized to obtain the powdered extract. The powdered extracts were analysed for their α-glucosidase and α-amylase inhibition assays. The total phenolic, flavonoid and flavonol content, reducing power and their free radical scavenging capacity of the ethanol extracts from the plant were also investigated.

Estimation of total phenolic content

The amount of total phenolic content of crude extracts was determined according to Folin-Ciocalteu procedure [12]. The tested extracts (100 ml) were introduced into test tubes. 1.0 ml of Folin-Ciocalteu reagent and 0.8 ml of sodium carbonate (7.5%) were added. The tubes were mixed and allowed to stand for 30 min. Absorption at 765 nm was measured (UV-visible spectrophotometer Shimadzu UV 1800).

Estimation of total flavonoids

Total flavonoids were estimated using the method of Ordonez et al., 2006[13]. To 0.5 ml of extracts, 0.5 ml of 2% AlCl3 ethanol solution was added. After one hour, at room temperature, a yellow color developed, indicated the presence of flavonoids and the absorbance was measured at 420 nm (UV-visible spectrophotometer Shimadzu UV 1800).

Estimation of total flavonols

Total flavonols in the plant extracts were estimated using the method of Kumaran and Karunakaran, 2006 [14]. To 1.0 ml of extracts1.0 ml of 2% AlCl₃ ethanol and 3.0 ml (50 g/l) sodium acetate solutions were added. The absorption at 440 nm (UV-visible spectrophotometer Shimadzu UV 1800) was read after 2.5 h at 20 °C for the estimation of total flavonol content in the plant extract.

Measurement of reducing power

The reducing power of the extracts was determined according to the method of Oyaizu, 1986 [15]. The plant extracts (100 µl) were mixed with phosphate buffer (2.5 ml, 0.2 M, pH 6.6) and 1% potassium ferricyanide (2.5 ml). The mixture was incubated at 50 °C for 20 min. Aliquots of 10% trichloroacetic acid (2.5 ml) were added to the mixture, which was then centrifuged at 3000 rpm for 10 min. The upper layer of the solution (2.5 ml) was mixed with distilled water (2.5 ml) and a freshly prepared ferric chloride solution (0.5 ml, 0.1%). The absorbance was measured at 700 nm and reducing power is determined.

Determination of DPPH free radical scavenging activity

The free radical scavenging activity of the plant extract and butylated hydroxyl toluene (BHT) as a positive control was determined using the stable radical DPPH (1,1-diphenyl-2-picrylhydrazyl) [16]. Aliquots (20, 40 and 80 ml) of the tested extracts were placed in test tubes and 3.9 ml of freshly prepared DPPH solution (25 mg L^-1) in methanol was added in each test tube and mixed. 30 min later, the absorbance was measured at 517 nm (UV-visible spectrophotometer Shimadzu UV 1800). The capability to scavenge the DPPH radical was calculated, using the following equation:

DPPH scavenged (%) = {(Ac–At)/Ac} x 100

Where Ac is the absorbance of the control reaction and At is the absorbance in the presence of the sample of the extracts. The antioxidant activity of the extract was expressed as IC₅₀. The IC₅₀ value was defined as the concentration in mg of dry material per ml (mg/ml) that inhibits the formation of DPPH radicals by 50%. Each value was determined from the regression equation.

Determination of scavenging activity of ABTS radical cation

The 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical cation (ABTS.+)-scavenging activity was measured according to the method described by Re et al.[17]. ABTS was dissolved in water to a 7 mmol concentration. The ABTS radicals were produced by adding 2.45 mmol potassium persulphate(final concentration). The completion of radical generation was obtained in the dark at room temperature for 12–16 h. This solution was then diluted with ethanol to adjust its absorbance at 734 nm to 0.70 ± 0.02. To determine the scavenging activity, 1 ml of diluted ABTS.+solution was added to 20,40 and 80 µl of plant extracts and the absorbance at 734 nm was measured 6 min after the initial mixing, using ethanol as the blank. The percentage of inhibition was calculated by the equation:

ABTS scavenged(%) = (A_cont-A_test)/A_cont× 100

where A_c and A_s are the absorbencies of the control and of the test sample, respectively. From a plot of concentration against % inhibition, a linear regression analysis was performed to determine the IC₅₀ value of the plant extract.

In vitro 𝛼-amylase inhibitory assay

The α-amylase enzyme solution was prepared by mixing 27.5 mg of porcine pancreatic amylase (PPA) in 100 ml 20 mmol of phosphate buffer (pH 6.9) containing 6.7 mmol of sodium chloride. To 100 μl of (2,4,8,10,15μg/ml) plant extracts, 200 μl PPA solution was added and the mixture was incubated at 37 °c for 20 min. To the reaction mixture 100 μl (1%) starch solution (A starch solution (1% w/v) was prepared by stirring 1g starch in 100 ml of 20 mmol of phosphate buffer (pH 6.9) containing 6.7 mmol of sodium chloride) was added and incubated at 37 °C for 10 min. The reaction was stopped by adding 200 μl DNSA (1g of 3,5 di-nitro salicylic acid, 30g of sodium potassium tartarate and 20 ml of 2N sodium hydroxide was added and made up to a final volume of 100 ml with distilled water) and kept it in a boiling water bath for 5 min. The reaction mixture diluted with 2.2 ml of water and absorbance was read at 540 nm.

For each concentration, blank tubes were prepared by replacing the enzyme solution with 200 μL in distilled water. Control, representing 100% enzyme activity was prepared in a similar manner, without extract. The experiments were repeated thrice using the same protocol [18].

In vitro 𝛼-glucosidase inhibition assay

The inhibition of 𝛼-glucosidase activity was determined using the modified published method [19]. One mg of 𝛼-glucosidase was dissolved in 100 ml20 mmolof phosphate buffer (pH 6.8). To 100 μl of (2, 4, 8, 10, 15μg/ml) plant extracts, 200 μl 𝛼-glucosidase were added and the mixture was incubated at 37 °C for 20 min. To the reaction mixture 100 μl 3 mmol 𝑝-nitrophenyl𝛼-D-glucopyranoside (p-NPG) was added and incubated at 37 °C for 10 min. The reaction was terminated by the addition of 2 ml Na₂CO₃ (0.1M) and the 𝛼-glucosidase inhibitory activity was determined spectrophotometrically at 405 nm on spectrophotometer UV-VIS (Shimadzu UV-1800) by measuring the quantity of 𝑝-nitrophenol released from p-NPG.

Acarbose was used as positive control of 𝛼-amylase and 𝛼-glucosidase inhibitor. The concentration of the extract required to inhibit 50% of 𝛼-amylase and 𝛼-glucosidase activity under the assay conditions was defined as the IC₅₀ value.

Method for calculation of α-amylase and α-glucosidase inhibitory concentration (IC50)

The concentration of the plant extracts required to scavenge 50% of the radicals (IC50) was calculated by using the percentage scavenging activities at five different concentrations of the extract. Percentage inhibition (I %) was calculated by

I % = (Ac-As)/Ac X 100,

Where Ac is the absorbance of the control and As is the absorbance of the sample [20].

Values are presented as mean±standard error mean of three replicates. The total phenolic content, flavonoid content, flavonol content, reducing power and IC₅₀ value of each plant material was calculated by using Linear Regression analysis.

RESULTS AND DISCUSSION

The results showed that the ethanol extract of the plant exhibited good antioxidant property and dose-dependent α-amylase and α-glucosidase inhibitory activities by in vitro assay using starch and p-NPG respectively as substrate.

Total phenol, flavonoid and flavonol content of the extracts

Polyphenols have been said to be important phytochemicals with significant antioxidant capacities and other potent medicinal characteristics. Total phenolic content in the plant extract was determined by the FC method and the calibration curve developed using Gallic acid. A regression equation was obtained from the standard curve and the amount of Gallic acid in the ethanol extract of T. sinensis was calculated from the regression equation: y = 0.0013x+0.0498, R² = 0.999 where y was the absorbance and x was the Gallic acid equivalent (mg/g).

The investigation showed that the plant under study contained a very good amount of total phenolics (18.18±0.09 mg GAE/mg plant extracts) which is well compared with the extract of Terminalia arjuna (20.862 mg GAE/g plant extracts)[21].

Total flavonoid content in the plant extract was calculated as rutin (mg/g) equivalent using the equation based on the calibration curve: y = 0.0182x-0.0222, R² = 0.9962, where y was the absorbance and x was the rutin equivalent (mg/g) and the flavonol content in the plant extract was estimated as quercetin (mg/g) equivalent using the equation based on the calibration curve: y = 0.0049x+0.0047, R²= 0.9935, where y was the absorbance and x was the quercetin equivalent (mg/g). The experimental result showed the presence of an appreciable amount of flavonoid 0.35±0.005 mg/gm) and flavonol (0.31±0.012 mg/gm) in the ethanol extract of T. sinensis.

The result of investigation also showed a very good reducing power (0.52±0.006 mg/gm dry extract) with the ethanol extract of the plant. The reducing power of the extract was evaluated in ascorbic acid equivalent (AAE) in milligram per gram (mg/g) of dry extract using the following equation based on the calibration curve: y = 0.0023x-0.0063, R²= 0.9955 where y was the absorbance and x was the ascorbic acid equivalent (mg/g).

It has been established that phenolic compounds are the major plant compounds with antioxidant activity and this activity is due to their redox properties. Phenolic compounds are a class of antioxidant agents which can adsorb and neutralise the free radicals [22].

Flavonoids and flavonols are considered as one of the most widespread groups of natural constituents found in the plants. It has been recognised that both flavonoids and flavonols show antioxidant activity through scavenging or chelating process [23].

Table 1: Antioxidant activities of the 80 % aq. ethanol extract of T. sinensis

Name of the plant	Total phenolic content (GAE mg/g dry extract)	Total flavonoid content (Rutin equivalent mg/g dry extract)	Total flavonol content (Quercetin equivalent mg/g dry extract)	Reducing power (Ascorbic acid equivalent mg/g dry extract)	Free radical scavenging ability (DPPH) (IC50 mg/g dry extract)	Free radical scavenging ability (ABTS) (IC50 mg/g dry extract)
T. sinensis	18.18±0.09	0.17±0.002	0.24±0.004	0.30±0.005	0.72±0.005	0.32±0.001

Each value in the table was obtained by calculating the average of three experiments and data are presented as mean±SEM

The results strongly suggest that phenolics are important components of these plants. The other phenolic compounds such as flavonoids, flavonols, which contain hydroxyls are also responsible for the radical scavenging effect in the plants.

Thus after determining the total phenol content of the plants, the antioxidant activities of the plants were estimated for their free radical scavenging activity.

The evaluation of anti-radical properties of the ethanol extract of T. sinensis was executed by DPPH radical scavenging assay. The 50% inhibition of DPPH radical (IC₅₀) by the plant extract was determined (table 1), a lower value would reflect the greater antioxidant activity of the sample. DPPH stable free radical method is an easy, rapid and sensitive way to survey the antioxidant activity of a specific compound or plant extracts [24]. The antioxidant effect is proportional to the disappearance of the purple colour of DPPH in test samples. Thus antioxidant molecules can quench DPPH free radicals by providing hydrogen atom or by electron donation and a colourless stable molecule 2,2-diphenyl-1-hydrazine is formed and as a result of which the absorbance (at 517 nm) of the solution is decreased.

Hence the more potent antioxidant, more decrease in absorbance is seen and consequently, the IC₅₀ value will be minimum. In the present study, the potent radical scavenging activity (IC₅₀ = 0.72±0.005 mg/gm dry ext) was shown by the 80% aq. ethanol extract of T. sinensis.

The antioxidant activity of the ethanol extract of T. sinensis using ABTS assay was also carried out. The antioxidant effect is proportional to the disappearance of the colour of ABTS in test samples. The concentration of sample that could scavenge 50 % free radical (IC₅₀) was used to determine antioxidant capacity of the sample compared to standard. The sample that had IC₅₀<50 ppm, it was a very strong antioxidant, 50-100 ppm strong antioxidant, 101-150 ppm medium antioxidant, while weak antioxidant with IC₅₀>150 ppm. A strong inhibition was observed (IC₅₀ =0.32±0.001 mg/gm dry ext) with the 80% aq. ethanol extract of the plant under investigation.

In vitro 𝛼-amylase inhibitory assay

𝛼-amylaseis one of the key enzymes that play a role in digestion of starch and glycogen and carbohydrate metabolism. Its inhibition is one of the strategies for the treatment of disorders in carbohydrate uptake, such as diabetes and obesity. It is involved in carbohydrate metabolism and thus inhibiting it would lead to reduced post prandial blood sugar [25].

A number of medicinal plants and their formulations are used for treating diabetes in the traditional Indian Ayurvedic system as well as in ethnomedicinal practices as their principal bioactive components showed good 𝛼-amylase inhibitory and antioxidant properties [26].

The result of the present experiment showed that there was a dose-dependent increase in percentage inhibitory activity against the 𝛼-amylase enzyme. The 80% aq. ethanol extract (2-15μg/ml) of T. sinensis exhibited potent 𝛼-amylase inhibitory activity in a dose dependent manner. The 80 % aq. ethanol extract of the plant showed inhibitory activity from 2.07±0.06 to 13.87±0.05%with an IC₅₀ value of 1.093μg dry extract (table 2).

Acarbose is a standard drug for the 𝛼-amylase inhibitor. Acarbose at a concentration of (2-15 μg/ml) showed 𝛼-amylase inhibitory activity from 6.99±0.03 to 56.17±0.05% with an IC₅₀ value 0.32μg dry extract. A comparison of 𝛼–amylase inhibitory activity between the standard drug and plant extracts has been depicted in fig. 1. So the ethanol extract of T. sinensis might be used as starch blockers since it prevents or slows the absorption of starch in to the body mainly by blocking the hydrolysis of 1,4-glycosidic linkages of starch and other oligosaccharides into maltose, maltriose and other simple sugars.

Table 2: In vitro antidiabetic activity of the 80 % aq. ethanol extract of T. sinensis using alpha amylase method and comparison with standard drug acarbose

S. No.	Plant extract/standard drug	Concentration µg/ml	% of Inhibition	IC 50 µg dry extract
1	80 % aq. ethanol extract of T. sinensis	2 4 8 10 15	2.07±0.06 4.80±0.02 8.85±0.04 10.27±0.08 13.87±0.05	1.093
2	Acarbose	2 4 8 10 15	6.99±0.03 11.58±0.05 26.45±0.02 41.64±0.06 56.17±0.05	0.32

Each value in the table was obtained by calculating the average of three experiments and data are presented as mean±SEM

Fig. 1: 𝛼-amylase inhibitory activity of acarbose vs 80% aq. ethanol extract of T. Sinensis

In our study, the ethanol extract of the plant showed maximum α-amylase inhibitory activity (IC₅₀=1.093μg dry extract) which could be attributed to the presence of polyphenols (18.18±0.09 mg/g) and flavonoids (0.17±0.002) because polyphenols are not only capable of reducing oxidative stress but also of inhibiting carbohydrate hydrolyzing enzymes because of their ability to bind with proteins [1].

Our results are in accordance with the previous study wherein; there is a positive relationship between the total polyphenol and flavonoid content and the ability to inhibit intestinal α-glucosidase and pancreatic α-amylase [27-28].

In vitro 𝛼-glucosidase inhibitory assay

𝛼-glucosidase enzyme located in the brush border of the small intestine. It breaks down starch and disaccharides to glucose. 𝛼-glucosidase inhibitors are oral anti-diabetic drugs used for diabetes mellitus type 2 that work by inhibiting the digestion of carbohydrates (such as starch and table sugar). Carbohydrates are normally converted into simple sugars (monosaccharides), which can be absorbed through the intestine. Hence, 𝛼-glucosidase inhibitors reduce the impact of carbohydrates on blood sugar. α-glucosidase inhibitors from natural sources have received tremendous attention because of the highly abundant compounds in nature and their promising biological activities. On the basis of literatures published worldwide, it has been established that natural products isolated from medicinal plants showed potent α-glucosidase inhibitory activity.

Structurally these natural product inhibitors includes terpene, alkaloid, quinine, flavonoid, phenol, phenylpropanoid, and steride frameworks rich in organic acid, ester, alcohol, and allyl functional groups. A majority of the compounds reported contain flavonoid, terpene, and phenylpropanoid ring structures [29].

The results of antidiabetic activity using 𝛼-glucosidase inhibitory assay of the 80% aq. ethanol extracts of T. sinensisstem are shown in table 3. The extract revealed a significant inhibitory action of 𝛼-glucosidase enzyme. The percentage inhibition at 2-15 μg/ml concentrations of T. sinensis extract showed a dose dependent increase in percentage inhibition. The percentage inhibition varied from 17.89±0.11%-6.50±0.09 % for highest concentration to the lowest concentration.

Thus the inhibition of the activity of 𝛼-glucosidase by T. sinensis would delay the degradation of carbohydrate, which would in turn cause a decrease in the absorption of glucose, as a result the reduction of postprandial blood glucose level elevation [30].

Table 3: In vitro antidiabetic activity of the 80 % aq. ethanol extract of T. sinensis using alpha-glucosidase method and comparison with standard drug acarbose

S. No.	Plant extract/standard drug	Concentration µg/ml	% of Inhibition	IC 50 µg dry extract
1	80 % aq. ethanol extract of T. sinensis	2 4 8 10 15	6.50±0.09 10.43±0.07 13.11±0.12 15.31±0.09 17.89±0.11	1.04
2	Acarbose	2 4 8 10 15	29.57±0.14 31.58±0.17 34.93±0.12 38.28±0.09 41.82±0.08	0.46

Each value in the table was obtained by calculating the average of three experiments and data are presented as mean±SEM

A comparison of 𝛼–glucosidase inhibitory activity between the standard drug and plant extracts has been depicted in fig. 2.

Fig. 2: 𝛼-Glucosidase inhibitory activity of Acarbose vs 80% aq. ethanol extract of T. Sinensis

In this study, acarbose was also used as a standard drug for the α-glucosidase inhibitor. Acarbose at a concentration of (2-15 μg/ml) showed α-glucosidase inhibitory activity from 29.57±0.14 to 41.82±0.08 % with an IC₅₀ value 0.46μg dry extract.

This indicates that the ethanolic extract of T. sinensis is very potent α-amylase and α-glucosidase inhibitor in comparison with acarbose. This could be justified that the nature of some extract constituents (phenols, flavonoids saponins, steroids, alkaloids, terpenoids) present in the extract could be responsible as being effective inhibitors of α-amylase and α-glucosidase.

CONCLUSION

To investigate the biological activities of T. sinensis tuberous stem, the antioxidant and antidiabetic activities of the 80 % ethanol extract of the plant has been analysed. As a result, we found that the extract of T. sinensis have free radical scavenging activity and inhibitory activity against α-amylase and α-glucosidase and this therapeutic potentiality could be exploited in the management of postprandial hyperglycemia in the treatment of type 2 diabetes mellitus. Although the effects of T. sinensis extract have been established in vitro, these results indicate that T. sinensis has potential as a crude drug and a dietary health supplement.

The plant showed significant enzyme inhibitory activity, so the compound isolation, purification and characterization which is responsible for inhibiting activity, has to be done for the usage of antidiabetic agent. Further studies are also required to elucidate whether the plant have antidiabetic potential by in vivo for corroborating the traditional claim of the plant.

ACKNOWLEDGEMENT

Authors of this paper are highly grateful to Dr. P. Singh, Director, Botanical Survey of India, Kolkata, for providing all facilities. We are also thankful to Dr. R. Gogoi, Scientist D, Botanical Survey of India, Howrah for identifying the plant specimens.

CONFLICT OF INTERESTS

None declared conflict of interest

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How to cite this article

• Anindita Banerjee, Bithin Maji, Sandip Mukherjee, Kausik Chaudhuri, Tapan Seal. In vitro anti-diabetic and antioxidant activities of ethanol extract of Tinospora sinensis. Int J Curr Pharm Res 2017;9(2):42-47.