1. INTRODUCTION:

Diabetes mellitus or Diabetes is a complex chronic condition (1) and metabolic disorder of multiple etiology (2) characterized by hyperglycemia over a prolonged period of time and negative nitrogen balance (3). Diabetes is caused if pancreas doesn’t produce sufficient insulin or cells of our body not responding to the produced insulin. Polyuria, Polydipsia, Polyphagia are some of the common clinical presentations of diabetes (4,5). Some acute complications of diabetes include hyperosmolar hyperglycemic state, diabetic ketoacidosis and death (6). If left untreated the long term complications like Heart disease, chronic kidney disease, Stroke, Foot ulcers, Gastro enteritis may occur (7). There are three main types of Diabetes mellitus. They are Type 1 Diabetes mellitus, Type 2 Diabetes mellitus and Gestational Diabetes mellitus (8,9).

In type I diabetes mellitus an individual’s immune system destroys β cells of pancreas that produce insulin. As there is absence of insulin, blood sugar levels increase. It is also called Juvenile diabetes. Children and youngpeople are more prone to this condition (10,11). Some common symptoms of Type 1 Diabetes mellitus are dry mouth, stomach upset, fatigue, blurry vision, frequent skin infections. Some emergency signs of this type include shaking, confusion, rapid breathing, abdominal pain and loss of consciousness (12,13). This type of Diabetes can be treated with Insulin like rapid acting, regular insulin, long acting and slow acting insulin (14,15).

Type 2 Diabetes mellitus is an insulin resistant diabetes mellitus which is also called as adult onset diabetes. In this condition the insulin is produced by β cells of pancreas, but not efficiently utilized by body cells. Defects in GLUT4 and GLUT1may explain the insulin resistance glucose transport (16). Risk factors for this type are obesity, metabolic abnormalities, β cell dysfunction and genetic predisposition. The clinical symptoms include weight loss without any workout, recurrent infections, slow wound healing process, rashes around neck or armpit and being cranky (17,18). Treatment is given with oral hypoglycaemic agents (19) like Sulfonylureas, Meglitinides, Thiazolidinediones, DPP 4 inhibitors, insulin etc, (20).

Holarrhena pubescenscommonly known as Tellicherry bark (English) or Kurchi (Hindi) is a flowering plant native to the Indian subcontinent, central and southern Africa, Indochina and parts of China⁽²¹⁾. It belongs to the Kingdom Plantae, Order Gentianales and Family Apocynaceae⁽²²⁾. Parts of the plant like seeds and barkare used for therapeutic purposes in fever, dysentery, diarrhea and amoebiasis. It has anti-malarial⁽²³⁾, anti-diarrheal⁽²⁴⁾, anti-bacterial⁽²⁵⁾, anthelmintic⁽²⁶⁾, anti-amoebic⁽²⁷⁾, anti-oxidative⁽²⁸⁾, and blood purifying properties. It has no harmful side effects but includes some common effects like nausea and vomiting due to its bitter taste in some people. No adverse drug reactions were reported in pregnant and lactating women⁽²⁹⁾

Since no paper showed any significant studies of anti-diabetic property of Holarrhena pubescens, this experiment was carried out. This research article helps researchers in identifying the anti-diabetic property (type 2) of chloroform and ethanolic extracts of Holarrhena pubescens.

2. MATERIALS AND METHODS:

2.1. EXTRACTION OF PLANT MATERIAL:

The stem bark of Holarrhena pubescens (H.P) wall. ex G. Don was collected from Nirmal district, Telangana, India during November 2016. The plant materials were authenticated by Dr. L. Rasingam, scientist In-charge at botanical survey of India, Deccan regional Centre, Hyderabad, Telangana, India. Voucher specimen No: BSI/DRC/16-17/Tech./681, deposited at the Botanical survey of India.

The plant part was washed and dried under shade. It was powdered using a pulveriser. Then the powder was placed in the soxhlet apparatus (30) and solvents like chloroform and 90% ethanol was used to collect the respective extracts. At 40ºC the extract was concentrated in an evaporater. The extract was stored under required circumstances.

2.2 CHEMICALS:

Cell lines, Media, Reagents, Assay kit and L6 Rat skeletal muscle cells were obtained from Highveld Biological, New Delhi. The (EMEM) Eagle’s Minimum Essential Medium, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-Diphenyl tetrazolium bromide) were obtained from Sigma Aldrich, Banglore. The Fetal Calf Serum (FCS) and Phosphate Buffer Saline (PBS) were from Lonza Biologics. Sigma or Merck Chemicals, Mumbai provided all other chemicals used for this study with analytical grade.

2.3 INVITRO STUDIES:

2.3.1. Cytotoxic screening methodology on cell lines:

L6 rat muscle cells with density of 1×105 cells/ml were used for cytotoxic study. A 96 welled plate was taken and cell suspension of (100µl/well) was seeded into it. It is incubated at 37ºC, 5% CO2, 95% air and 100% relative humidity. After 24 hrs cells were treated with five concentrations of standard drug Metformin (0.1, 0.2, 0.4,0.8, 1𝜇g/ml) and five concentrations of H.P (12.5, 25, 50, 100, and 200𝜇g/ml) in medium free from serum. To make up the final sample concentration, aliquots of 100µl of H.P concentrations were added. After the addition of H.P extract, plates were incubated for 48 hours at 37ºC, 5% CO2, 95% air and 100% relative humidity.

The control well contains medium without the drug substances, and all concentrations of H.P were performed in triplicates. Cytotoxicity of H.P were assessed by MTT (3-(4,5-dimethylthiazolyl-2)- 2,5-diphenyltetrazolium bromide) assay (31), and percentage cell viability was calculated (Table 1).

2.3.2. Glucose Uptake Assay in rat L6 skeletal muscle cells (32):

L6 rat myogenic cells were cultured in Dulebecco’s Modified Eagle Media (DMEM) containing 4.5g/l D-glucose with 10% heat-inactivated fetal calf serum (FCS) at 37ºC, 5% CO2 atmosphere. The cells were seeded into 96-well plate with six wells left as blank wells and let growing to confluence; then cells were fully differentiated in DMEM with 2% fetal calf serum (FCS) for 5 days. Before tests, the medium was replaced by RPMI1640 (2g/l glucose) supplemented with 0.2% phosphate-buffered saline (PBS). The medium was removed after 2 hours, and the same medium containing H.P extracts were compared with vehicle control (DMSO) in the presence and absence of insulin (1 µmol/l). The determination of glucose in the medium was carried out using glucose oxidase method after 48 hours treatment.

The amount of glucose uptake by muscle cells was calculated by using the following formula:

Glucose uptake = Glucose concentration of blank wells −Glucose concentration of cell plated wells (Table 2).

2.3.3. Inhibition of α amylase activity (33, 34)

In a boiling water bath, potato starch (0.125g) was stirred in 20ml Sodium phosphate buffer with 6.7ml Sodium chloride (pH 6.9; 25ml) to make Starch solution (0.5%w/v). 1U/ml of α-amylase was mixed in the same buffer to obtain α-amylase solution. Equal volumes of sodium potassium tartrate tetrahydrate solution and 96mM 3, 5-dinitro salicylic acid (DNS) solution were mixed to prepare colorimetric reagent. Starch solution (1000𝜇l) was mixed with increasing concentration of an enzyme inhibitor such as H.P.C.E (10, 20, 40, 60, 80, 100𝜇g/ml), H.P.E.E (10, 20, 40, 60, 80, 100𝜇g/ml) and Acarbose (1, 2, 4, 6, 8, and 10µg/ml). To these concentrations 1000µl of α-amylase solution was added and incubated at 25ºC for 30 min to react with starch solution. The contents were heated on boiling water bath for 15 min. Distilled water was used to make up the final volume and absorbance was measured at 540nm using spectrophotometer. The % inhibition and 50% inhibitory concentration (IC50) values were calculated (Table 3).

2.3.4. Inhibition of α glucosidase activity (35):

The inhibition of α-glucosidase activity was determined by incubating 100𝜇l of 𝛼-glucosidase enzyme (1 U/ml) solution with 100𝜇l of phosphate buffer (pH 7.0) which contains 100𝜇l of enzyme inhibitor such as H.P.C.E (100, 200, 300, 400, 500𝜇g/ml), H.P.E.E (100, 200, 300, 400, 500g/ml) Acarbose (10, 20, 30, 40, 50𝜇g) in maltose solution at 37ºC for 60 min. The above reaction mixture was kept in boiling water for 2 min to prevent the action on maltose and then cooled. 2ml of glucose reagent was added to this. The absorbance was measured at 540nm to estimate the amount of liberated glucose. The % inhibition and 50% inhibitory concentration (IC50) values were calculated (Table 4).

2.3.5. Statistical significance:

The following formula was used in the calculation of percentage inhibition of α-amylase and α-glucosidase (32):

Absorbance of control – Absorbance of test

Percentage inhibition = ------------------------------- X 100

Absorbance of control

The data of in vitro studies were expressed as mean percentage inhibition ± SD (n=3). The values of IC50 and % inhibition of enzymes was determined using nonlinear regression graph (log10 concentration vs percentage enzyme inhibition). The statistical significance between groups was determined by one-way analysis of variance, in glucose uptake activity , followed by Dunnett’s test for multiple comparisons, and P<0.05 was considered as statistically significant. IC50 value determination and other statistical analysis were done using Graph Pad Prism (Version 5.0) software.

3. RESULTS:

3.1. Table 1: Cytotoxic screening methodology on cell lines (Dose at which showing 50% cell death)

S. No	Drug treatment	Dose (µg/ml)
1	Control	0
2	Metformin	1
3	H.P.C.E	100
4	H.P.E.E	100

The data represented as mean ± SD (𝑛=3)

3.2. Table 2: Glucose uptake in L6 rat muscle cells after 48 h incubation in media with glucose (2 g/L).

S. No	Drug Treatment	Concentration	(Glucose consumption mg/100ml)
			Absence of insulin	Presence of insulin 1µmol/l
1	Control	1%DMSO	3.94±0.06	9.86±0.04
2	Metformin	0.01mM	6.87±0.05	11.45±0.02
3	H.P.C.E	10µg/ml	5.85±0.02	7.89±0.05
4	H.P.E.E	10µg/ml	9.45±0.05	10.54±0.04

The data represented as mean ± SD (𝑛=3). a P < 0.001 metformin and H.P extracts when compared with vehicle control.

3.3. Table 3: Inhibition of 𝛼-amylase enzyme activity by H.P extracts when compared with vehicle control.

Sample	Concentrations (µG/ML)	% Inhibition of enzyme activity	IC 50
Acarbose	1	11.15 ± 0.75
	2	42.99 ± 0.84
	4	53.48 ± 1.40	3.83
	6	62.95 ±1.95
	8	74.60 ± 0.31
	10	85.31 ± 2.02
H.P.C.E	10	48.35±0.45
	20	57.12±0.26	15.35
	40	62.33±0.01
	60	78.22±0.24
	80	89.32±0.45
	100	105.28±0.14
H.P.E.E	10	26.33±0.14
	20	38.21±0.56
	40	47.65±0.21	51.44
	60	55.23±0.56
	80	64.12±0.54
	100	79.11±0.68

The data represented as mean ± SD (𝑛=3).

3.4. Table 4: Inhibition of 𝛼-glucosidase enzyme activity by H.P extracts when compared with vehicle control

Sample	Concentrations (µg/ml)	%inhibition of enzyme activity	IC 50
Acarbose	10	15.15 ± 0.51
	20	45.58 ± 0.41
	30	55.81 ± 0.40	25.32±0.24
	40	68.95 ±0.95
	50	78.50 ± 0.81
H.P.C.E	100	38.15±0.45
	200	47.12±0.26
	300	52.13±0.01	294.21±0.56
	400	68.22±0.14
	500	82.32±0.05
	100	95.28±0.04
H.P.E.E	100	28.23±0.41
	200	33.11±0.61
	300	45.15±0.02	356.21±0.14
	400	58.13±0.16
	500	63.02±0.04
	100	81.01±0.18

The data represented as mean ± SD (𝑛=3).

4. DISCUSSION:

The screening of anti- diabetic activity in Holarrhena pubescens was performed in L6 rat skeletal muscle cell lines and the following results were obtained. Cytotoxic screening was done on cell lines. Cell lines showed 50% cell death when 1µg/ml of Metformin was given. The same result was obtained when 100µg/ml of H.P.C.E. and 100µg/ml H.P.E.E. was given.

After 48 hours of incubation in media with glucose (2g/l), the glucose uptake in L6 rat skeletal muscle was observed as 6.87±0.05mg/100ml in the absence of insulin and 11.45±0.02mg/100ml in the presence of insulin for Metformin at a concentration of 0.01ml. For H.P.C.E. at a concentration of 10µg/ml the glucose uptake into skeletal muscle was 5.85±0.02mg/100ml in the absence of insulin and 7.89±0.05mg/100ml in the presence of insulin. For H.P.E.E. at a concentration of 10 µg/ml the glucose uptake into skeletal muscle was 9.45± 0.05mg/100ml in the absence of insulin and 10.54±0.04 mg/100ml in the presence of insulin.

Inhibition of 𝛼-amylase enzyme activity of H.P.C.E. and H.P.E.E. were compared with vehicle and the results are as follows; When Acarbose concentration of 1µg/ml was given 11.15±0.75% inhibition of 𝛼-amylase enzyme activity was shown. Similarly for 2µg/ml concentration 42.99±0.84% inhibition, 4µg/ml concentration 53.48± 1.40% inhibition, 6µg/ml concentration 62.95±1.95% inhibition, 8µg/ml concentration 74.60±0.31% inhibition and 10µg/ml concentration 85.31±2.02% inhibition was observed.

When H.P.C.E. concentration of 10µg/ml was given 48.35±0.45% inhibition of 𝛼-amylase enzyme activity was shown. Similarly for 20µg/ml concentration 57.12± 0.26% inhibition, 40µg/ml concentration 62.33±0.01% inhibition, 60µg/ml concentration 78.22±0.24% inhibition, 80µg/ml concentration 89.32±0.45% inhibition and 100µg/ml concentration 105.28±0.4% inhibition was observed. When H.P.E.E. concentration of 10µg/ml was given 26.33±0.14% inhibition of 𝛼-amylase enzyme activity was shown. Similarly for 20 µg/ml concentration 38.21±0.26% inhibition, 40µg/ml concentration 47.65±0.56% inhibition, 60µg/ml concentration 55.23±0.56% inhibition, 80µg/ml concentration 64.12±0.54% inhibition and 100µg/ml concentration 79.11±0.68% inhibition was observed.

When Acarbose concentration of 10µg/ml was given 15.15±0.51% inhibition of 𝛼-glucosidase enzyme activity was shown. Similarly for 20µg/ml concentration 45.58±0.41% inhibition, 30µg/ml concentration 55.81± 0.40% inhibition, 40µg/ml concentration 68.95±0.95% inhibition and at 50µg/ml concentration 78.50±0.81% inhibition was observed. When H.P.C.E. concentration of 100µg/ml was given 38.15±0.45% inhibition of 𝛼-glucosidase enzyme activity was shown. Similarly for 200µg/ml concentration 47.12±0.26% inhibition, 300 µg/ml concentration 52.13±0.01% inhibition, 400µg/ml concentration 68.22±0.14% inhibition and 500µg/ml concentration 82.32±0.05% inhibition was observed. When H.P.E.E. concentration of 100µg/ml was given 28.23±0.41% inhibition of 𝛼-glucosidase enzyme activity was found. Similarly for 200µg/ml concentration 33.11±0.61% inhibition, 300µg/ml concentration 45.15 ±0.02 % inhibition, 400µg/ml concentration 58.13±0.16 % inhibition, 500µg/ml concentration 63.02±0.04% inhibition and 100µg/ml concentration 81.01±0.18 % inhibition was observed.

5. CONCLUSION:

Diabetes is a metabolic disorder, its untreated condition may lead to many more complications like Dyslipidemia (36), Diabetic ketoacidosis, Diabetic retinopathy, Diabetic nephropathy, Diabetic neuropathy, Diabetic foot, Alzhimer’s disease and other cardiovascular related diseases (37). The drugs used in treating diabetes may cause some hypoglycemic side effects like Coma, Disturbances in liver and kidney (38) and other adverse drug reactions. So, it is better to take precautionary measures or choose ayurvedic system of medicine for management. This article reveals that Holarrhena pubescens has significant anti-diabetic property its folklore usage in treating diabetic condition can be validated with this scientific results.

6. CONFLICT OF INTEREST

No conflict of interest is declared by the authors.

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Received on 19.03.2020 Modified on 04.05.2020

Research J. Pharm. and Tech. 2020; 13(12):6238-6242.

DOI: 10.5958/0974-360X.2020.01087.2