Evaluation of the Antidiabetic Potential of an Isolated Hydroalcoholic Fraction from the Fruit of Withania coagulans.

The hydro-alcoholic extract of Withania coagulans fruits was investigated for preliminary phytochemical screening and characterized by high-performance thin-layer chromatography. Column chromatography of the hydro-alcoholic extract of W. coagulans eluted with four different combinations of ethyl acetate and methanol yielded four fractions (WCF01, WCF02, WCF03, and WCF04). One of these fractions, WCF02, significantly (P < 0.05) inhibited in vitro α-amylase and α-glucosidase activity with IC50 values of 104.71 μg/mL and 70.79 μg/mL, respectively. WCF02 further reduced blood glucose levels in comparison to control in the starch tolerance test. The extract showed a relative dose-dependent effect. It was observed that none of the extracts could delay the peak blood glucose that was achieved after 60 min of carbohydrate challenge, but these blunted the glycemic peak. Copyright:

INTRODUCTION

Diabetes mellitus (DM) is a chronic metabolic disorder characterized by hyperglycemia resulting from insufficient insulin secretion, ineffective insulin action, or both.[1] It has been referred to as pandemic as more than 400 million cases of DM prevailed across the globe by 2014, accounting for 1.6 million deaths in 2016.[2] Type 2 DM covers the major diabetic populations around the world and mostly attributed to excess body weight (body mass index >30) and decreased physical activities.[3] It has been always related to long-term complications such as retinopathy, neuropathy, nephropathy, atherosclerosis, and cataracts, which make it one of the leading causes of blindness, acute and chronic renal failure, lower-extremity amputations, vision loss, and neuronal pathologies.[4] Diabetic complications lead to the risk of myocardial infarction and stroke and are thus largely responsible for the mortality observed in these patients.[56789] Diet and exercises are crucial in healthy maintenance of the blood glucose levels and thus preventing and treating Type 2 diabetes. The majority of diets contain carbohydrates which are the greatest source of calories.[10] Dietary polysaccharides are broken down by digestive enzymes such as α-amylase which is chiefly present in pancreatic juice and saliva that randomly cleaves α (1 → 4) glycosidic linkages and intestinal α-glucosidase that hydrolyzes the terminal nonreducing (1 → 4)-linked alpha-glucose residues.[121112] Thus, α-amylase and α-glucosidase are regarded as good targets for antidiabetic agents and their inhibition is one of the important ways to control postprandial hyperglycemia in Type 2 diabetics by delaying carbohydrate digestion to absorbable monosaccharide. Acarbose and voglibose are clinically used α-glucosidase inhibitors which potentially reduce the DM complications.[1112] Though different α-glucosidase inhibitors are available as oral hypoglycemic agents for the management of Type 2 diabetes, there is an increased patient demand of natural products with blood glucose-lowering activity.[13] Ethnobotanical use has a long history in Type 2 DM management. A large number of plants and their products have been reported to possess α-amylase and α-glucosidase inhibitory activities.[1314151617181920] W. coagulans fruits, commonly known as Indian rennet, are distributed in drier parts of India. The fruits are superior indehiscent, many-seeded berries, round to globous in shape, yellow to brown in color enclosed in the leathery persistent calyx, and the fruit decoction has been recommended to be taken in the morning or before meals for glycemic control in diabetic patients.[21] Withanolides from different parts of the plant has been reported for diverse biological activities such as antimicrobial, anthelmintic, oxidative stress and anti-inflammatory,[2] antitumor, hepatoprotective, antispasmodic, antihyperlipidemic,[21] antidiabetic activity[11321] and wound healing in diabetic rats.[22] There is no safe and effective therapy in modern medicine to cure DM. Insulin therapy has many adverse effects such as insulin resistance, fatty liver, brain atrophy and anorexia nervosa. To the best of our knowledge, this is the first work on the hydroalcoholic extract of W. coagulans fruits and methanol-ethyl acetate fraction. Although W. coagulans fruit extracts have been described to possess anti-diabetic activity, the compounds responsible for this hypoglycemic activity remain unknown. Therefore, the present work was planned to carry out phytochemical screening of W. coagulans fruits and investigate in vitro and in vivo antidiabetic potentials of W. coagulans fruits.

EXPERIMENTAL METHODS

Plant material

W. coagulans fruits were procured from the Hansraj and Sons, Khari Baoli, local crude drug market of Delhi, and authenticated by Dr. Sunita Garg, Emeritus Scientist and Taxonomist, CSIR-NISCAIR, New Delhi-12, and the sample specimen (authentication no. NISCAIR/RHMD/Consult/2019/3423-24) was deposited in the Raw Material Herbarium and Museum, Delhi (RHMD), for future references. The samples were stored at room temperature.

Chemical and reagents

All the supplies, chemicals, and reagents of analytical grade were received from SD Fine chemicals & glass ware in Mumbai, Maharashtra (Retailer of Merck Chemicals). Column drying was carried out by using anhydrous sodium sulfate. Pancreatic α-amylase (porcine; Type VI-B; A3176), α-glucosidase (Type I from baker's yeast) G5003, 3,5-dinitrosalicylic acid (DNS), starch (85642), and acarbose (C25H43NO18, molecular weight 645; A8980) were supplied from Sigma Aldrich (St. Louis, USA).

Extraction

Air-dried W. coagulans fruit (3 kg) was crushed to get the coarse powder and defatted with hexane by refluxing it for approximately 48 h to remove waxy material. After defatting, the sample was completely dried to remove any traces of hexane. The sample was then packed in a Soxhlet apparatus with hydroalcoholic solvent (methanol: water, 90:10), and extraction was continued until the color of the siphoning tube disappeared. The extract was then collected and concentrated under reduced pressure to yield a dark brown viscous syrupy mass (23.5% yield).

Hydrolysis of extract and its preliminary analysis

The hydroalcoholic extract (10 g) was mixed with a small amount of 2 N hydrochloric acid to get the final pH of 3.5 and the mixture was refluxed for 3–4 h to completely hydrolyze the extract. The mixture was put on crushed ice with stirring and allowed to attain room temperature. The preliminary phytochemical screening was carried out to check the presence of primary/secondary metabolites in the biologically active extract of the crude drug.[232425]

High-performance thin-layer chromatography procedure

A precoated silica gel (high-performance thin-layer chromatography [HPTLC] 60 F254) was used to carry out HPTLC and chromatography in a presaturated developing chamber (20 mL of 80:20:12, v/v/v ethyl acetate–methanol–water) for 30 min at 50% ± 5% relative humidity and room temperature.

The sample was prepared by diluting hydroalcoholic extract (100 mg) with methanol (10 mL) and sonicating at 50°C ± 2°C for 15 min. It was filtered through 0.2-μ filter paper, diluted further to prepare test solutions, and applied by a thin-layer chromatography (TLC) auto sampler on 6-mm wide bands on plate. There was a 9-cm long chromatogram from the base. TLC plate was dried under hot air in oven and the bands were visualized under Camag Scanner video documentation system (Camag Muttenz, Switzerland).

Isolation of fractions from extract

To prepare the slurry, the solution was made by dissolving hydroalcoholic extract of dark brown syrupy mass (100 g) in sufficient amount of methanol and absorbed on 60–120 mesh size silica gel. The development and elution were carried out with sequential solvents in different proportions of ethyl acetate: methanol (95:5, 90:10, 85:15, and 80:20 v/v). Homogeneity of various fractions collected was checked through TLC. Identical fractions of the extract were combined and pooled and kept for crystallization.

Biological activity

In vitro α-amylase inhibition study

A volume of 1 mL of each fraction of hydroalcoholic extract and 1.2 mL of porcine α-amylase of 0.5% concentration in 20 mM phosphate buffer were mixed and put for 10 min of incubation at 25°C. Afterward, 1.2 mL of starch solution (0.5% w/v) in 20 mM phosphate buffer (pH 7) was added to the reaction mixture, and then further incubated for another 10 min. The reaction was then stopped with 2 mL of 96 mM DNS.[26] Then, the test tubes were heated for 5 min in a boiling water bath and left to cool to room temperature. The absorbance (A) of samples was taken at 540 nm using Amersham Biosciences spectrophotometer (Buckinghamshire, United Kingdom). The inhibitory enzyme activity was calculated in percentage as

where A540Cntrol is the absorbance of control sample without extract at 540 nm, and A540Extract is the absorbance of treatment with extract at 540 nm.

In vitro α-glucosidase inhibition study

A volume of 100 μl of each fraction of hydroalcoholic extract was diluted with phosphate buffer (pH 7) solution and incubated for 5 min with 50 μL (1 U/mL) of α-glucosidase. After the incubation, 50 μL of the substrate was added and incubated further for 5 min at room temperature. The absorbance on a microplate reader after substrate addition was taken at 405 nm. Increased absorbance after a substrate addition was observed and percent α-glucosidase inhibition was calculated as:

where A540Cntrol is the absorbance of control sample without extract at 405 nm and A540Extract is the absorbance of treatment with extract at 405 nm.[26]

In vivo oral starch tolerance test

Eight- to ten-week-old Swiss albino mice (Mus musculus) weighing in the range of 25 ± 2 g were used in the present study. The animals were brought from the animal house facility of Rajendra Memorial Research Institute of Medical Sciences, Patna. They were segregated into five groups each consisting of six mice (n = 6). The animals were subjected to free access of water and fasted overnight. Group 1 was given 4 mL/kg (normal control) distilled water orally using oral gavage, Group 2 was given acarbose 10 mg/kg body weight (positive control/standard), treatment Group 3 was subjected to 500 mg/kg b/w of 20% (v/v) hydroalcoholic extract of W. coagulans (D1), Group 4 received 750 mg/kg of 20% (v/v) hydroalcoholic extract of W. coagulans (D2), and treatment Group 5 was treated orally 1000 mg/kg of 20% (v/v) hydroalcoholic extract of W. coagulans (D3). Ten minutes later, all the mice were given starch solution (3 g/kg body weight) orally and the blood glucose was estimated before and after starch administration at 0 min and at 30, 60, 90, and 120 min. All the experimental procedures were approved by the Institutional Animal Ethics Committee, S.S. Hospital and Research Institute, in collaboration with Department of Botany, Thakur Prasad Singh College, Patna (Magadh University), Bodh Gaya (India) vide letter no. 1840/PO/ReBi/S/15/CPCSEA.

Statistical analysis

Experiments were performed in triplicate. Data were presented as mean, standard deviation, and standard error of mean (SEM). The inhibitory concentration (IC50) of extracts was obtained by nonlinear regression using GraphPad Prism 5.0 (GraphPad Software, San Diego, CA, USA). IC50 values were expressed as mean. Oral starch test data were reported as mean ± SEM. One-way ANOVA followed by Dunnett's multiple comparison t-test determined statistical significance at P < 0.05.

RESULTS

Extraction and preliminary analysis

Extraction of W. coagulans fruit with hydroalcoholic solvent (methanol: water, 90:10) yielded a dark brown viscous syrupy mass 690 g (23.5% yield). A portion of this extract was analyzed for the presence of alkaloids, glycosides, phenolic compounds, tannins, flavonoids, saponins, and mucilage, etc., [Table 1].

Table 1

Phytochemical screening of hydroalcoholic extract of Withania coagulans Dunal fruits

Constituents	Hydroalcoholic extract
Alkaloids	+
Carbohydrates	+
Glycosides	+
Phenolic compounds and tannins	+
Flavonoids	+
Proteins and free amino acids	+
Saponins	+
Mucilage	+
Resins	+

+: Present, −: Absent

High-performance thin-layer chromatography study of hydroalcoholic extract

HPTLC of the hydroalcoholic extract of W. coagulans at different dilutions exhibited a number of bands when visualized with CAMAG HPTLC scanner IV. The developed plate was scanned at a wavelength of 372 nm for the densitometric measurements. A close examination of peaks revealed that nearly 11 fractions were present in the hydroalcoholic extract of W. coagulans fruit [Figure 1]. Chromatograms showed a number of peaks with different Rf values [Figure 2].

Figure 1

Chromatogram (two dimensional), densitometric measurements of hydroalcoholic extract scanned at wavelength 372 nm. Peaks shows nearly 11 fractions of the hydroalcoholic extract of Withania coagulans fruit

Figure 2

Chromatogram (three-dimensional), densitometric measurements of hydroalcoholic extract scanned at wavelength 372 nm

Four different combinations of ethyl acetate: methanol (95:5, 90:10, 85:15, and 80:20 v/v) were used as eluent, and development of chromatogram gave four fractions (WCF01, WCF02, WCF03, and WCF04). The concentration of these fractions under reduced pressure yielded crystals of different color and melting points. These fractions were evaluated for their antidiabetic potential.

Biological evaluation

The antidiabetic activities were assessed in vitro and in vivo for four different fractions (WCF01, WCF02, WCF03, and WCF04) of hydroalcoholic extract of W. coagulans.

Different fractions (WCF01, WCF02, WCF03, and WCF04) of hydroalcoholic extract of W. coagulans fruit were used for the α-amylase inhibition study. Each of these fractions was studied for in vitro α-amylase inhibition study for four different twofold concentrations (100 μg/mL, 200 μg/mL, 400 μg/mL, and 800 μg/mL) as per the standard procedure.[17] One fraction (WCF02) of hydro-alcoholic extract of W. coagulans showed statistically significant (P < 0.05) enzyme inhibition compared to other fractions. The inhibition varied from 37.81% to 68.36% in the case of WCF01, 47.30%–97.48% for WCF02, 18.24%–38.72% for WCF03, and 23.03%–53.94% for WCF04 [Table 2]. α-amylase inhibition was dose dependent for all the fractions. The IC50 values for α-amylase inhibition were 269.15 μg/ml for WCF01, 104.71 μg/ml for WCF02, 1528.64 μg/ml for WCF03, and 616.50 μg/ml for WCF04 [Table 2].

Table 2

α-amylase inhibition studies of various fractions of hydroalcoholic extract of Withania coagulans Dunal fruits

Hydroalcoholic extract fractions	Concentration (μg/ml)	Log [C]	Percentage Inhibition	IC50 (μg/mL)
WCF01	100	2	37.81	269.15
	200	2.301	44.46
	400	2.601	51.23
	800	2.90	68.36
WCF02	100	2	47.30	104.71
	200	2.301	63.84
	400	2.601	83.76
	800	2.90	97.48
WCF03	100	2	18.24	1528.64
	200	2.301	23.78
	400	2.601	35.88
	800	2.90	41.72
WCF04	100	2	23.03	616.50
	200	2.301	36.53
	400	2.601	42.11
	800	2.90	53.94

Log [C]: Log Concentration; IC50: The half-maximal inhibitory concentration

Different fractions (WCF01, WCF02, WCF03, and WCF04) of hydroalcoholic extract of W. coagulans fruit were used for the α-glucosidase inhibition study. Each of these fractions was studied for in vitro α-glucosidase inhibition study for four different twofold concentrations (100 μg/mL, 200 μg/mL, 400 μg/mL, and 800 μg/mL) as per the standard procedure.[17]

One of the fractions (WCF02) of hydroalcoholic extract of W. coagulans showed statistically significant (P < 0.05) inhibition compared to other fractions. The inhibition varied from 39.81% to 68.36% for WCF01, 57.30%–98.48% for WCF02, 27.32%–58.18% for WCF03, and 33.13%–53.94% for WCF04. Moreover, all the fractions showed a relatively dose-dependent inhibition of α-glucosidase activity [Table 3]. The IC50 values for α-glucosidase inhibition were 177.82 μg/mL for WCF01, 70.79 μg/mL for WCF02, 447.66 μg/mL for WCF03, and 645.69 μg/mL for WCF04 [Table 3].

Table 3

α-glucosidase inhibition studies of various fractions of hydroalcoholic extract of Withania coagulans Dunal fruits

Hydroalcoholic extract fractions	Concentration (μg/mL)	Log [C]	Percentage Inhibition	IC50 (μg/mL)
WCF01	100	2	39.81	177.82
	200	2.301	54.46
	400	2.601	61.23
	800	2.90	68.36
WCF02	100	2	57.30	70.79
	200	2.301	69.08
	400	2.601	88.41
	800	2.90	98.48
WCF03	100	2	27.32	447.66
	200	2.301	39.08
	400	2.601	48.41
	800	2.90	58.18
WCF04	100	2	33.13	645.69
	200	2.301	39.53
	400	2.601	42.11
	800	2.90	53.94

Log [C]: Log concentration; IC50: The half-maximal inhibitory concentration

The fractions obtained from W. coagulans fruit extract showed a significant enzymatic inhibition in vitro experiment was thus subjected to in vivo studies. The oral carbohydrate challenge in Swiss albino mice was carried out with starch (3 g/kg) after pretreatment with different concentrations of WCF02. The WCF02 fraction was tested at three different doses of 500 mg/kg (D1), 750 mg/kg (D2), and 1000 mg/kg (D3) compared with standard (acarbose 10 mg/kg) and normal control. In all the animals, the blood glucose concentration was recorded. The group treated at lower doses D1 and D2 [Table 4] did not show significant effect till 90 min. However, D3 extract of W. coagulans showed significant reduction against starch control mice at 90 and 120 min.

Table 4

Oral starch tolerance test in Swiss albino mice with different concentrations of WCF02

Group	Dose and route of administration (per oral)	Blood glucose level in (mg/dL)
		0 (min)	30 (min)	60 (min)	90 (min)	120 (min)
Control	4 mL/kg	77.19±2.74	89.41±2.3	98.37±1.99	104.26±2.04	101.04±1.99
Standard	10 mg/kg	78.06±2.62	81.08±2.54	87.24±2.09*	83.36±1.98**	76.38±2.02**
D1	500 mg/kg	79.21±2.35	89.25±2.16	96.19±2.21	99.35±1.92	98.48±1.86
D2	750 mg/kg	80.30±2.56	88.39±1.97	96.02±1.86	94.37±1.86	87.54±1.65*
D3	1000 mg/kg	76.55±2.40	82.86±2.47	93.86±4.3	88.37±2.10*	78.14±1.72**

Blood glucose level in mg/dL taken at 30 min of interval of time from 0 to 120 min. Data were expressed as mean±SEM. Evaluation by one-way ANOVA followed by Dunnett’s multiple comparison t-tests. *P<0.05 and **P<0.01 as compared to control. SEM: Standard error of mean

DISCUSSION

DM is a long-term serious complication with the major impact on individuals, families, and the societies and their lives. It is one of the top ten causes of adult death globally. This study was intended to examine the antidiabetic potential of W. coagulans fruit extract using in vitro and in vivo tests. The extract tested positive for the presence of alkaloids, glycosides, phenolic compounds, tannins, flavonoids, saponins, resins, and mucilage. Many studies of W. coagulans extract from different parts of the plant reported the similar active compounds.[1] Because DM is a complex disease involving multiple factors, different models were used. Commonly, studies of natural products for prevention of DM primarily focused on to investigate the inhibition of α-glucosidase that hydrolyzes starch/disaccharides to glucose. The partial inhibition of α-amylase is rate limiting and thus α-amylase inhibition decreases the glucose release from starch.[11] Inhibition of α-amylase activity by W. coagulans leaf extract is reported in literature[18] that suggests a possible causal relationship between the bioactive component and enzyme inhibition. In the present study, all the fractions showed a relatively dose-dependent decrease in α-amylase activity. The IC50 values for α-amylase inhibition were 269.15 μg/mL for WCF01, 104.71 μg/mL for WCF02, 1528.64 μg/mL for WCF03, and 616.50 μg/ml for WCF04. Thus, the results of the present study of α-amylase inhibition are in agreement with the results of Heo et al.[18] Polyphenolic components from plants[181921] have been recently reported to be efficient α-amylase inhibitors.[11925] α-glucosidase is the source of hydrogen in catalytic hydrolysis of α-(1,4)-glucosidic link, and hydrogen scavenging is the primary mechanism of α-glucosidase inhibition by polyphenols.[27] Therefore, it is expected that polyphenols and withanolides are responsible for α-glucosidase inhibition. Complexation is supposed to be the principal mechanism of α-amylase inhibition[28] that delays the carbohydrate digestion and decrease glucose absorption.[121] Most of the pharmacological studies have shown that the biological properties of Withania are due to withanolides and polyphenols. The present study showed α-amylase and α-glucosidase inhibition by hydroalcoholic extract of W. coagulans.

CONCLUSION

Hydroalcoholic extract of W. coagulans fruits showed significant α-amylase and α-glucosidase inhibition in vitro. The oral starch tolerance test also showed a significant reduction in blood glucose level. On the basis of the result obtained, it is established that W. coagulans fruit extract possess antidiabetic properties. The underlying inhibitory action of W. coagulans fruit extract on α-amylase and α-glucosidase is the delayed digestion and decreased gut carbohydrate absorption. It is further postulated that a prolonged pretreatment for 20–30 min before carbohydrate overload may result in better glycemic control. Hence, further investigations shall be carried out to explore the principal bioactive components of the extract and their antidiabetic effect.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.