INTRODUCTION:

Diabetes mellitus continues to be a rising public health concern^1,2 with studies reporting a frequency ranging from 19% to 33% in general population^3,4 globally. The International Diabetes Federation estimated that there were 415 million adults with diabetes worldwide in 2015^5,6and currently reckoned as 500 million cases^7,8. If this growing trend is not arrested, it is estimated that there will be 642 million people with diabetes by 2040⁵. One strategy to avert diabetes mellitus or hyperglycemia is to control the glucose level in blood^9,10.

Glucose in blood originates from the hydrolysis of carbohydrates in the intestine by digestive enzymes such as α- amylase. Hence inhibition of alpha amylase can retard carbohydrate digestion and entrapment of the digested glucose within the intestine for a longer duration in diabetic patients would cause a reduction in the rate of glucose absorption into the blood. Therefore this approach is considered to be a therapeutic factor for managing diabetes.

Amaranthus viridis (Tamil: Kuppai keerai) is a cosmopolitan annual herb. Due its great traditional medicinal importance in the ailment of diabetes and also several diseases like gonorrhoea, haemorrhoids, kidney stones, dysentery, inflammations, cancer, blood purification, inflammation during urination, constipation, filaria, as an emmenagogue, emollient and vermifuge etc., and lack of literature to prove its pharmacological potential in vitro, the current research work was designed. In this study extracts from various parts of A. viridis have been examined for their α-amylase inhibitory action and their ability to entrap glucose within the dialysis tube which mimics human intestine.

MATERIALS AND METHODS:

1. Collection of plants and Solvent extraction:

Amaranthus viridis plant parts (leaves, stems, seeds and roots) were collected from a garden (Latitude 8.3381691^o N, Longitude 77.164017^o E) in Kanyakumari District, Tamilnadu, India during August 2018. The identity was confirmed by Dr. P. Ravichandran, Professor and Head, Department of Plant Sciences, Manonmaniam Sundaranar University, Tirunelveli, Tamilnadu, India. The plant parts were shade dried and ground into fine powder. The powders of each part were taken in labelled bottles at concentrations of three grams of powder per fifty millilitres of solvent (3g/50 ml). Six different solvents namely methanol, ethanol, ethyl acetate, petroleum ether, hexane and water were added to the powder. The bottles were tightly closed and kept for 2–3 days at room temperature to extract maximum metabolites that were finally filtered through cellulose filter paper. The extracts were shade dried in large petri plates until the solvents evaporated completely, the residues were scrapped off the plates, weighed to calculate their yield and stored in screw capped sterilized bottles in a deep freezer (-18^oC) until further use.

2. Glucose diffusion inhibition assay:

The protocol used for this assay was adapted from a method described by Edwards et al.^11,12 with modifications. It involved the use of dialysis tubes (6cmX15mm) into which 500µl of a solution of D-glucose (1.65mM) and sodium chloride (0.15M) and 1ml of different plant part extracts (10mg of dried extract powder dissolved in 1mL DMSO) were added and the open end was tied with a thread to seal. The tubes were suspended into 45mL of NaCl solution (0.15M) and incubated at 37^oC for 3 hours after which the concentration of glucose that retained back within the dialysis tube was measured using DNS assay. Dilalysis tube with 500µl of D-glucose (1.65mM) and sodium chloride (0.15M) and 1mL DMSO without plant extract was used as a negative control. Glycomet tablet for diabetes which was locally available was used as positive control. The experiments were performed in triplicates.

(i) DNS assay:

The concentrations of glucose retained within the dialysis tubing after 3 hours without being diffused outside into the external solution due to the activity of different plant extracts added was measured by DNS assay. To 200µL of the dialysis tubing contents after incubation, 250µL of amylase (40mg/mL) in 0.02M sodium phosphate buffer was added in boiling tubes and incubated for 10 minutes followed by the addition of 250µL of starch (1%) in 0.02M sodium phosphate buffer was added. The mixture was incubated further for 10 minutes after which 500µL of DNS reagent (2.18g of DNS in 80ml of 0.5M NaOH, heated at 70^oC, then 30g of potassium sodium tartrate) was added. The boiling tubes were placed in a boiling water bath for 5 minutes. 5mL of water was added to dilute the contents and their absorbance was measured at 540nm. The concentrations of entrapped glucose were calculated from the absorbance using a standard graph for glucose. The percentage of glucose retained within dialysis tube was calculated by the following formula

Glucose dialysis retardation index (GDRI) = (IGcon ÷ FGcon) × 100

where IGcon is the Initial Glucose concentration before incubation and FGcon is the Final Glucose concentration after incubation.

(ii) Statistical Analysis:

Data were expressed as mean±S.E.M. Statistical comparisons between the extracts were performed by one way analysis of variance (ANOVA) and Tukey Kramer’s multiple comparison tests. p<0.001 were considered as significant result.

3. α amylase inhibition assay:

Approximately 400µL of starch (1% heated to dissolve) and 400µL of plant part extracts (10mg/mL) were taken in test tubes. A volume of 200 μL of α-amylase (40μg/mL) was added to each test tube and incubated for 15 min at 37 °C. After the incubation, 100 μL of iodine reagent (200mg iodine crystals with 1200mg KI used after overnight incubation to dissolve) was added to each test tube. Then, 10 mL of distilled water was added. Absorbance of the mixture was taken at 630 nm. Each experiment was done in triplicates. Negative control was starch with sample without amylase, while acarbose was used as positive control. The α-amylase inhibitory activities were calculated as percentage inhibition as per the following equation.

% Inhibition = (Abs_extract ÷ Abs_negative xontrol) × 100

where Abs_extract represents absorbance of extract and Abs_negative control denotes absorbance of negative control.

(i) Calculation of IC50:

The concentrations of the plant extracts that inhibited 50% of the enzyme activity (IC50) were calculated. Extracts with high inhibitory activity were analysed using a series of suitable extract concentrations. IC50 values were determined by plotting percent inhibition (Y axis) versus log10 extract concentration (X axis) and calculated by logarithmic regression analysis from the mean inhibitory values^13,14

(ii) Statistical Analysis:

Data were expressed as mean ± S.E.M. Statistical comparisons between groups were done by one Tukey’s Honest Significant comparison tests to analyze the differences. p<0.05 were considered as significant. Different letters within each bar were used to denote that they are significantly different.

RESULTS AND DISCUSSION:

1. Glucose diffusion inhibition assay:

Glucose diffusion test was conducted to investigate the effect of methanol, ethanol, ethyl acetate, petroleum ether, hexane and aqueous extracts from leaf, stem, seed and root parts of A. viridis with respect to its glucose retardation activity across the dialysis tube (Fig. 1). The fig.1 shows that most of the extracts of A. viridis exhibited inhibition effect with methanol extract of leaf most significantly to retain the highest concentration of glucose of 47.05% (40.125µg/mL) which was almost close to the Glycomet tablet with 59.36% (50.618µg/mL) used as positive control. This was followed by ethanol extract of leaf (20.962µg/mL) and methanol extract of seed (19.85µg/mL).

Fig.1: Effect of different extracts of A.viridis (10 mg/mL) on glucose within dialysis tube after 3 hours of incubation. Initial concentration of glucose was (68µg/mL). The values were expressed as mean ± SEM (n=3). Different letters within each bar were significantly different (P<0.05)

In contrast, the negative control without the plant extract, ethyl acetate and petroleum ether extracts of stem, ethyl acetate extract of seed, ethanol, ethyl acetate and water extract of roots did not show ability to hold glucose within the dialysis tube. This result is in accordance with the study on the entrapment ability of aqueous and ethanolic extract of Teucrium polium on glucose diffusion activity^15,16. In the current study, only the antidiabetic mechanism was tested by diffusion of glucose through a dialysis membrane whereas in the body, there are various transport systems which work in synchronization with other molecules to transport glucose^17,18. Though this is the first report in detail on glucose entrapment potential of 4 parts of A. viridis with 6 solvents in vitro, further studies in vivo is essential to confirm the in vivo action of A. viridis with respect to the glucose diffusion to ease clinical interpretation.

2. Alpha amylase inhibitory assay:

The aim of current study was to establish the inhibitory activity of A. viridis against α-amylase. The percentage inhibition displayed by each extract is shown in Fig. 2 which justifies that A. viridis showed prominent α-amylase inhibitory potential with water extract of stem (100%) for 15 minutes incubation time, which was equally potential to the standard drug acarbose. However since the period of incubation was limited with only 15 minutes, 100% inhibition was accomplished with ease. The IC50 values of the best three extracts with higher alpha inhibitory activity analysed using a series of suitable extract concentrations are shown in Table 1. The water extract of stem which showed 100% amylase inhibition had IC50 value of (5.058±0.41µg/mL) while acarbose showed IC50 value of (4.937±0.26µg/mL).

The percentage of inhibition ranged from 15 to 100. In spite of the fact that the action of the enzyme has not been directly involved in the etiology of diabetes, α-amylase inhibitors have long been thought to enhance glucose resistance in diabetic patients¹⁹. Extensive efforts have been made over the previous decades to discover a clinically effective α-amylase inhibitor with the point of getting better control of diabetes^20,21.

Fig. 2: Effect of different extracts of A.viridis (10 mg/mL) on inhibition of alpha amylase activity after 10 minutes of incubation. The values are expressed as means ± SEM (n = 3)

Table 1: IC50 inhibition values of three A. viridis plant extracts with higher inhibition percentage on alpha amylase for 10 minutes incubation period.

Plant extract	Concentrations (µg/mL)	% Inhibition	IC50 value (µg/mL)
Stem-Water extract	5	49.42	5.058±0.41
	10	100
	20	100
	30	100
	40	100
	50	100
	60	100
Stem-EthylAcetate extract	5	28.83	43.501±0.35
	10	43.47
	20	44.31
	30	46.84
	40	49.19
	50	51.56
	60	54.23
Leaf- MethanolExtract	5	22.85	49.919±0.01
	10	39.13
	20	41.58
	30	45.22
	40	47.79
	50	49.66
	60	50.39

Interestingly, the methanol extract of leaves of A. viridis which sustained the glucose in the dialysis tubing at the highest level of (47.05%), has simultaneously proved to inhibit alpha amylase activity too at a reasonably good rate (39.13%), paving brighter way to isolate promising bioactive compounds from this extract which would inhibit both glucose diffusion and alpha amylase simultaneously. Earlier in 2011, Kumar et al.²² had experimented the effect of whole plant of A. viridis water extract alone on alpha amylase inhibition.

CONCLUSION:

Our results demonstrate that A. viridis is a pharmacologically active plant to exhibit glucose retention and alpha amylase inhibition in vitro. The methanol extract of leaf which showed good inhibition of alpha amylase as well as in the entrapment of glucose could be further studied for isolation and structure elucidation of active compounds and their in vivo assays will be noteworthy for proper innovative clinical drug development.

ACKNOWLEDGEMENTS:

We are grateful to the staff members of Biotechnology Department, Malankara Catholic College for their encouragement throughout this work. We are also thankful to Rev. Fr. Jose Bright, Correspondent/Secretary and Dr. J. Thampi Thanka Kumaran, Principal, Malankara Catholic College, Mariagiri for their constant encouragement and support.

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Received on 18.11.2018 Modified on 31.12.2018

Research J. Pharm. and Tech. 2019; 12(5):2089-2092.

DOI: 10.5958/0974-360X.2019.00346.9