INTRODUCTION:

The World Health Organization (WHO) has listed 21,000 plants, which are used for medicinal purposes around the World. Among these 2,500 species are in India, out of which 150 species are used commercially on a fairly large scale.^1,2,3

In this growing interest, many of the phytochemical bioactive compounds from medicinal plants have shown many pharmacological activities. There is continuous search for alternative drugs. Natural products, such as plants extract, either as pure compounds or as standardized extracts, provide unlimited opportunities for new drug discoveries because of the unmatched availability of chemical diversity.

The number of diabetes mellitus cases has been increasing worldwide in recent years. Diabetes mellitus is a complex disorder that disturbs the metabolism of carbohydrate, fat and protein.

With a serious complication often resulting in high death rate, the treatment of diabetes spent vast amount of resources including medicines, diets, physical training and so on in all countries. Thus searching for a new class of compounds is essential to overcome diabetic problems. Inflammation is a bodily response to injury, infection or destruction characterized by rubor, calor, tumor and disturbed physiological functions. Inflammation is a normal protective response to tissue injury caused by physical trauma, noxious chemical or microbial agents. It is the body response to inactivate or destroy the invading organisms, to remove the irritants and set the stage for tissue repair. It is triggered by the release of chemical mediators from injured tissue and migrating cells. The management of inflammatory conditions are non-steroidal anti-inflammatory drugs (NSAID), which have several adverse effects especially gastric irritation leading to formation of gastric ulcers.^4,5

Barleria gibsoni Dalz. (Acanthaceae) is widely distributed throughout Africa, India, Sri Lanka and tropical Asia. It is commonly known as Neel Koranti, the juice of the leaf is used in cataract, ulcer and fever. The dried bark is used in cough treatment and the leaves chewed to relieve toothache. The paste of the root is applied to disperse boils and glandular swellings.^6-12 Thus, objective of the present study was to investigate in-vitro antidiabetic and anti-inflammatory activity of ethanol extract of Barleria gibsoni leaves.

MATERIALS AND METHODS:

Collection and Identification of plant material:

The fresh leaves of the B. gibsoni were collected during the month of May-June when flowering, from Satara region, Maharashtra, India. The plant authenticated by Botanical survey of India, Pune, Maharashtra, India. A voucher specimen (BSI/WRC/Tech/2013/FAT 01 dated 27th December, 2013) has been deposited at the herbarium of same place for further reference.

Extract preparation:

The collected fresh matured leaves of B. gibsoni were washed with tap water, air- dried at room temperature for 2-3 weeks at 35-40^oC and then reduced to coarse powder. A 100gm powdered leaves was obtained after defatted with petroleum ether and successively extracted with ethanol using Soxhlet apparatus finally 12.0g of extracts were obtained.¹³

Methods:

Anti-diabetic activity:

α- Amylase inhibition:

Alpha amylase is an enzyme that hydrolyses alpha bonds of large alpha linked polysaccharide such as glycogen and starch to yield glucose and maltose. Alpha amylase inhibitory activity was based on the starch iodine method 6. A starch solution (0.1% w/v) was obtained by stirring 0.1g of potato starch in 100ml of 16mM of sodium acetate buffer. 1ml substrate- potato starch (1% w/v), 1 ml of drug solution (metformin std drug/ethanolic extract) of different concentration such as 20, 40, 60, 80 and 100μg/ml, 1ml of alpha amylase enzyme (1% w/v) and 2ml of acetate buffer (0.1 M, 7.2 pH) was added. The above mixture was incubated for 1 hr. Then 0.1ml Iodine-iodide indicator (635mg Iodine and 1gm potassium iodide in 250ml distilled water) was added in the mixture. Absorbance was taken at 565nm in UV-Visible spectroscopy. % inhibition was calculated as previously published protocol. All the tests were performed in triplicate.¹⁴

Glucose uptake in Yeast cells:

The experimental procedure was carry out by firstly yeast was washed by repeated centrifugation at 3,000g for 5 min in distilled water until the supernatant fluids were clear and a 10% (v/v) suspension was prepared in distilled water. Followed by the addition of various plant extracts in varying concentration 20, 40, 60, 80 and 100 μg/ml and incubated for 10 min at 37°C. Reaction was started by adding 100μl of yeast suspension, vortex and further incubated at 37°C for 60 min. After 60 min, the tubes were centrifuged (2,500 × g, 5 min) and glucose was estimated in the supernatant. metformin was taken as standard drug. The percentage increase in glucose uptake by yeast cells was calculated.¹⁵

Anti-inflammatory activity:

Protein inhibitory action:

The experiment was performed according to previously published protocol. The reaction mixture (1ml) was containing Bovine serum albumin and 1ml test sample of different concentrations. The reaction mixture was incubated at 37°C for 15 min and then the mixture was heated at 57^oC for 30 min. The absorbance of the solution was read at 660nm against blank. The experiment was performed in triplicates. The percentage of inhibition of proteinase inhibitory activity was calculated.^16-20

RESULTS:

Table 1: In vitro amylase inhibition by extract of leaves of Barleria gibsoni

Sr. No	Conc. (µg/ml)	% Inhibition
Sr. No	Conc. (µg/ml)	Standard (Metformin)	Ethanol extract of leaves
1	20	55.16 ± 1.10	53.13 ± 2.13
2	40	64.48 ± 2.11	57.15 ± 2.16
3	60	67.47 ± 3.12	65.23 ± 2.14
4	80	71.20 ± 2.15	67.15 ± 2.12
5	100	73.15 ± 3.11	71.20 ± 2.11

From this Table 1, it is indicate that In vitro amylase inhibition activity ethanol extract of Barleria gibsoni leaves was shown good significant inhibition as compared to standard drug metformin. The extract concentration 100 µg/ml showed 71.20 % inhibition than standard 73.15%.

Table 2: Effect of ethanol extract of Barleria gibsoni leaves in glucose uptake inhibition by yeast cell

		% Inhibition
Sr. no	Conc. (µg/ml)	Standard (Metformin)	Ethanol extract of leaves
1	20	24.15 ± 2.13	20.20 ± 2.35
2	40	55.54 ± 1.40	49.15 ± 2.45
3	60	78.71 ± 3.36	67.61 ± 2.25
4	80	81.19 ± 2.25	71.31 ± 2.15
5	100	87.10 ± 3.15	79.20 ± 2.13

Values were expressed as mean ± SD From this Table 2, it is indicate that In vitro glucose uptake inhibition by yeast cell by ethanol extract of Barleria gibsoni leaves was shown good significant inhibition as compared to standard drug metformin. The extract concentration 100 µg/ml showed 79.20 % inhibition than standard 87.10 %.

Table 3: In Vitro Anti-inflammatory activity of ethanol extract of Barleria gibsoni leaves

Sr. No	Conc. (µg/ml)	% Inhibition
Sr. No	Conc. (µg/ml)	Standard (Metformin)	Ethanol extract of leaves
1	10	43.12 ± 2.14	39.10± 2.14
2	20	58.14 ± 2.13	45.15 ± 1.16
3	30	71.20 ± 2.11	56.21 ± 2.18
4	40	74.59 ± 2.10	61.19 ± 2.12
5	50	78.91 ± 2.09	68.25 ± 2.11

Values were expressed as mean ± SD Table 3 indicates at concentration 10-50µg/ml was effective in inhibiting heat induced albumin denaturation as dose dependent manner. The maximum inhibition 68.25% at the conc. of 50µg/ml was observed from ethanolic extract of Barleria gibsoni.

DISCUSSION:

There are certain problems in using animals in experimental pharmacological research, such as ethical issues and the lack of rationale for their use when other suitable methods are available or could be investigated. Hence, in the present study the protein denaturation bioassay was selected for in vitro assessment of anti-inflammatory property of ethanol Barleria gibsoni extracts production of auto antigens in certain arthritic diseases may be due to denaturation of proteins in vivo. Agents that can prevent protein denaturation therefore, would be worthwhile for anti-inflammatory drug development. Denaturation of proteins is a well-documented cause of inflammation. The inflammatory drugs such as salicylic acid, thermally induced protein denaturation²¹. Similar results were observed from many reports from plant extract.

Diabetes is a metabolic disorder with increasing incidence throughout the world. Insulin is a key player in the control of glucose homeostasis. Lack of insulin affects carbohydrate, fat and protein Metabolism^22,23,24. Management of diabetes without side effects is still challenge to the medical community. It was proposed that inhibition of the activity of such alpha-amylase 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.

The Amylase inhibitory activity and Glucose uptake activity by yeast cells as per table no. 1, and 2 showed that maximum Antidiabetic action at 100μg/ml concentration of extract whereas minimum was observed at 20μg/ml concentration of the extract. Ethanolic extract showed the maximum action 71.20% inhibition.

The mechanism of glucose transport across the yeast cell membrane has been receiving attention as in vitro screening method for hypoglycaemic effect of various compounds/ medicinal plants. Recent studies on the transport of nonmetabolizable sugars and certain metabolizable glycosides suggest that sugar transport across the yeast cell membrane is mediated by stereospecific membrane carriers. It is reported that in yeast cells (Saccharomyces cerevisiae) glucose transport is extremely complex and it is generally agreed that glucose is transported in yeast is by a facilitated diffusion process. Facilitated carriers are specific carriers that transport solutes down the concentration gradient. This means that effective transport is only attained if there is removal of intracellular glucose.

CONCLUSION:

The results also indicate that the ethanolic extracts of leaves of Barleria gibsoni possess anti-inflammatory and antidiabetic properties. This activity may be due to the strong occurrence of phenolic compounds such as flavanoids, tannins, steroids and phenols. The extract serves as albumin denaturation, amylase inhibitory activity and Glucose uptake activity. This study gives an initiative that the compounds of Barleria gibsoni can be used as lead compound for designing a potent antiinflammatory, anti-diabetic a drug which can be used for treatment of various diseases such as cancer, neurological disorder, ageing and inflammation.

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Received on 14.04.2020 Modified on 04.05.2020

Research J. Pharm. and Tech 2021; 14(3):1289-1292.

DOI: 10.5958/0974-360X.2021.00228.6