Determination of In vitro antidiabetic potential of Aqueous and Ethanol extracts and isolated fraction of Zanthoxylum armatum DC stem bark

K. Keerthana¹, G. Jothi²

¹Research Scholar, Department of Biochemistry, Srimad Andavan Arts and Science College (Autonomous), (Affiliated to Bharadidasan University), Tiruchirappalli, Tamil Nadu, India – 620005.

²Assistant Professor in Biochemistry, Srimad Andavan Arts and Science College (Autonomous), (Affiliated to Bharadidasan University), Tiruchirappalli, Tamil Nadu, India – 620005.

*Corresponding Author E-mail: jothi173@yahoo.com

ABSTRACT:

Background: Diabetes mellitus is a chronic metabolic disorder affecting 25% of the world population. It occurs due to deficiency or insensitivity of insulin leading to increased blood glucose level and its consequent complications. Inhibition of carbohydrate hydrolyzing enzymes such as α – amylase, α – glucosidase and α- sucrase can be one of the important strategies to lower blood glucose levels. But these inhibitors that are used in clinical practice for management of diabetes are known to be associated with various gastrointestional side effects. Therefore, it is the need of time to identify and explore the drugs from natural sources without side effects. Aim: In the present study, a commonly available species of Rutaceae - Zanthoxylum armatum DC was selected and explored for its anti-diabetic potentials employing in-vitro methods. Methods: Inhibitory potential of aqueous extract, ethanol extract and isolated fraction of bark of Z. armatum on α – amylase, α – glucosidase and α- sucrase enzyme was determined. Result: Aqueous bark extract exhibited potential inhibitory effect against glucose hydrolysing enzymes in a concentration dependent manner compared to ethanol extract and isolated fraction. The isolated fraction showed significantly higher glucose binding capacity and strong hindrance to diffusion of glucose across a dialysis membrane. Conclusion: The results indicated that aqueous bark extract and isolated fraction of Z.armatum possess good glucose absorption inhibitory activity.

KEYWORDS: α – amylase, α – glucosidase, α- sucrase, glucose adsorption, glucose bounding capacity, glucose diffusion retardation index, Zanthoxylum armatum DC.

INTRODUCTION:

Diabetes mellitus is a chronic disease characterized by impaired carbohydrate, fat and protein metabolism. Diabetes if left untreated produces macrovascular and microvascular complications, which account for considerable morbidity and mortality throughout the world¹. It is the most prevalent disease in the world affecting 25% of population and affects 150million people and may rise to 300million by 2025 with one- third of affected individuals being from India and China².

Existing lines of treatment for diabetes with the administration of synthetic drugs or insulin therapy has its own shortcomings like ineffectiveness on oral administration, short shelf life, fatal hypoglycemia on excess dosage and development of resistance on prolonged usage. Because of these limitations, many diabetic patients are inclining towards the alternative therapies that include diet, food supplements and herbal medicines.

Traditional antidiabetic plants are considered to be useful sources for developing new oral hypoglycemic drug. There are many hypoglycemic plants known through the folklore but their introduction into the modern therapy system awaits in vitro and in vivo testing³. Literature review showed that plant species belonging to Rutaceae such as Zanthoxylum zanthoxyloides⁴, Aegle marmelos⁵, Murraya koienigii^6,7, Clausena anisa⁸ and Limonia acidissima Linn⁹ and alkaloids isolated from the Rutaceae plants are well documented for their antidiabetic activity. Hence the present investigation is aimed to identify and develop a novel antidiabetic herbal drug from common plant sources belonging to the family Rutaceae. Zanthoxylum armatum DC. (Z.armatum) commonly called as ‘Timur’ in Tamil, and ‘Tejohva’ in Ayurveda were selected. In traditional system of medicine (Ayurveda), stem bark of Z.armatum is used for treating Amavata, Kasa and Aruci¹⁰. Aqueous and ethanol extract of test drug had high extractive value compared to other extracts (results not presented) and as isolated fraction is rich in alkaloid – known for its antidiabetic potential, the present investigation aims in evaluating the in vitro antidiabetic potential of aqueous, ethanol and isolated fraction of stem bark of Z.armatum.

MATERIALS AND METHODS:

Identification and Authentification:

Stem bark of Z. armatum was collected from Palampur District of Himachal Pradesh, identified with the help of Flora of Presidency of Madras¹¹ and authenticated with the help of CSIR - Institute of Himalayan Bioresource Technology, Palampur, Himachal Pradesh.

Sample Preparation:

Preparation of Aqueous Extract:

The stem bark of the plant was collected during their blooming period, cleaned, shade dried and coarsely powered. 200g of coarse powder of stem bark Z.armatum was boiled in 1200ml of water. The contents were reduced to one third and filtrate was evaporated to dryness. Paste form of the extract obtained was stored in an airtight container at 4°C. The aqueous extract was then subjected to further analysis.

Preparation of Ethanol Extract:

The stem bark of the plant materials under study were shade dried and powdered coarsely. About 500gms of plant material was soaked in ethanol for 48hrs. After 48 hrs of soaking the solvent was distilled off under reduced pressure at 50°C and dried in vacuum. The ethanol extract was then subjected to further analysis.

Isolation of bioactive fraction¹²

Coarsely powdered plant material (Bark) was soaked in Petroleum ether for 48 hours. Filtered and the residue was soaked in acetone for 48 hours. Again it was filtered and the extract was evaporated to dryness which was further packed in column for isolation of bioactive fraction. The concentrated acetone extract after removal of the solvent was loaded on a preparative column using silica gel wetted with petroleum ether as the stationary phase. A gradient elution starting with 100% petroleum ether to 100% ethyl acetate was chosen as the eluting medium. At mobile phase Pet.ether: Ethyl acetate - 75:25, an orange fraction obtained were checked for the presence of alkaloid using TLC using mobile phase - chloroform: methanol in ratio of 10:1 on spraying with Dragondroff’s Reagent. A single orange colour spot was identified with Rf value of 0.35 confirms the presence of alkaloids.

In Vitro antidiabetic studies:

In vitro antidiabetic assay of extracts and isolated fraction (Skimmianine) of Z. armatum were determined by evaluating following parameters a-amylase inhibitory activity¹³, a-glucosidase inhibitory activity¹⁴, a -sucrase inhibitory activity¹⁵, glucose adsorption capacity¹³ and glucose diffusion¹³ using standard textual procedure.

Statistical Analysis:

All determinations were done in triplicate and results were expressed in mean ± standard error mean (SEM).

Table – 1 Inhibition of alpha amylase enzyme by aqueous, ethanol extract and isolated fraction of Z. armatum DC. (Stem bark).

S. No	Aqueous extract		Ethanol extract		Isolated fraction
S. No	Concentration (mg/mL)	% Inhibition	Concentration (µg /mL)	% Inhibition	Concentration (µg /mL)	% Inhibition
1.	2	11.76 ± 1.96	100	18.09 ± 1.45	20	15.15± 1.75
2.	4	33.33 ± 2.99	200	36.19 ± 1.98	40	27.27± 3.03
3.	6	52.94 ± 1.96	300	59.04 ± 1.45	60	42.42± 1.75
4.	8	68.62 ± 1.13	400	69.52 ± 1.09	80	60.61± 6.31
5.	10	78.43 ± 1.13	500	74.28 ± 0.95	100	78.79± 4.63
	IC50 Value - 5.8mg		IC50 Value- 280 µg		IC 50 Value - 69 µg