In-Vitro Alpha-Amylase Inhibition Action of Isolated Phytoconstituent in Zanthoxylum rhetsa (Roxb) Bark.

Vasudev Pai*, Rudraprabhu V. Savadi and Anant Bhandarkar

Department of Pharmacognosy, KLE’S college of Pharmacy, Vidyanagar, Hubli (Karnataka) India.

*Corresponding Author E-mail: pai_pharmacist@yahoo.co.in

ABSTRACT:

Medicinal plants have curative properties due to the presence of various complex chemical substance of different composition, which are found as secondary plant metabolites in one or more parts of these plants. In present study, in vitro α-amylase inhibition was used to evaluate hypoglycemic effects of various extracts of Zanthoxylum rhetsa (Roxb) bark revealed the presence of alkaloids, tannins, flavonoids, phenolic compounds, steroids. Further, chromatographic isolation of methanolic extract were carried out and direct chloroform extract is used. Isolate from methanolic extracts shown the presence of flavonoidal moiety and is confirmed by analytical techniques. It has shown significant percentage inhibition action +5.67 compared to control whereas chloroform extract has not shown alpha amylase enzyme inhibition.

KEYWORDS: α-amylase inhibition, antidiabetic, postprandial hyperglycemia, Zanthoxylum rhetsa Bark.

1. INTRODUCTION:

Diabetes mellitus is a chronic endocrinal disease there are two main types of diabetes, type I and type II. The most prevalent form of diabetes is non-insulin dependent diabetes mellitus (type II) 90% of cases throughout the world. The control of hyperglycemia is critical in the management of diabetes because in long term, acute and chronic complications can occur if blood glucose concentration is not kept in normal levels.⁽¹⁾ It affected about 171 million people worldwide in 2000 and the number is projected to increase to at least 366 million by 2030.⁽²⁾One therapeutic approach for treating diabetes is to decrease the post‐prandial hyperglycaemia. This is done by retarding the absorption of glucose through the inhibition of the carbohydrate hydrolyzing enzymes, alpha‐amylase and alpha‐glucosidase in the digestive tract. Inhibitors of these enzymes delay carbohydrate digestion and prolong overall carbohydrate digestion time, causing a reduction in the rate of glucose absorption and consequently blunting the postprandial plasma glucose rise.⁽³⁾Pancreatic alpha-amylase is a key enzyme in the digestive system and catalyses the initial step in hydrolysis of starch to a mixture of smaller oligosaccharides.

These are then acted on by alpha-glucosidases and further degraded to glucose which on absorption enters the blood-stream. Degradation of this dietary starch proceeds rapidly and leads to elevated post-prandial hyperglycemia. Many of the synthetic hypoglycaemic agents have their limitations, are non-specific, produce serious side effects and fail to elevate diabetic complications. ⁽⁴⁾

Zanthoxylum rhetsa (Roxb) is a medicinal plant belongs to the Rutaceae family and is traditionally used in diabetes, antispasmodic, diuretic, anti-inflammatory and in various ailments. It is a small or moderate sized tree with pale corky bark which is widely distributed throughout, India, Bangladesh Malaysia and other parts of South Asia.^5-7Literature survey revealed that the plant extract has yet not been screened for its antidiabetic activity. Therefore the present study was carried out to provide pharmacological evidence for the folklore medicinal consideration.

2. MATERIAL AND METHODS:

2.1 Plant materials:

The bark of Zanthoxylum rhetsa. (Roxb) were collected from the local areas of Kumta, Karnataka, and authenticated by Dr. B.D. Huddar, Head, Department of Botany, Shri Kadasiddheshwar Arts College and H.S. Kotambari Science Institute, Vidyanagar, Hubli. A voucher specimen (07PG358, Vasudev Pai) has been deposited in the PG Pharmacognosy laboratory of the college for future reference.

2.2 Preparation extracts:

The shade dried bark was shade dried at room temperature, pulverized, and 100g of coarse powder was successively extracted with petroleum ether (40-60), chloroform and methanol in a Soxhlet extractor. The extracts were concentrated in a rotary flash evaporator and residues were dried in a desiccator over sodium sulfite. After drying, the respective extracts were weighed and percentage yield was determined. The extracts were subjected for phytochemical investigations by qualitative chemical tests.

2.3 Preliminary phytochemical analysis:^8-10

From preliminary phytochemical analysis it was found that Zanthoxylum rhetsa (Roxb) showed positive tests for major primary and secondary plant metabolites like alkaloids, glycosides, tannins, flavonoids, phenolic compounds, steroids.

2.4 Isolation of Phytoconstituents:¹¹

Isolation of phytoconstituents from successive methanolic extract was carried out by using column chromatography using isocratic elution technique, silica gel G was used as adsorbent in preparing TLC Column. Ethyl acetate: Formic acid: Glacial acetic acid: Water (80:8:8:20) were used as mobile phase and UV-254nm as Visualizing agent. One of the isolated phytoconstituents (flavonoid) from methanolic extract and directly the Chloroform extract were used for In-vitro evaluation of α-amylase inhibition activity. Characterization of isolated compound was carried out by FT-IR(Fig.1),¹H-NMR(Fig.2), ¹³C-NMR(Fig.3), Mass spectra (Fig.4) and is named as compound-I.

Fig 04: Mass spectra of COMPI

Table 1: Qualitative chemical analysis of various extracts of Zanthoxylum rhetsa bark.

Extracts	Tannin	Flavonoid	Glycoside	Steroid	Carbohydrate	Phenolic compounds	Alkaloid
Pet-ether	--	--	--	+	--	--	--
Chloroform	--	+	--	+	--	--	+
Methanolic	+	+	+	--	+	+	+

Table01: Mean absorbance, maltose formation and percentage inhibition by methanolic and chloroform extracts.

Sample	Optical density in various time intervals				Maltose (ug/ml) at t = 3 min	Percentage Inhibition
Sample	0 min	1 min	2 min	3 min	Maltose (ug/ml) at t = 3 min	Percentage Inhibition
Control	0.025	0.054	0.085	0.106	28.2	---
Methanol	0.045	0.065	0.090	0.100	26.6	+5.67
Chloroform	0.047	0.064	0.089	0.111	29.4	- 4.25

In vitro α-amylase inhibition:^{12, 13}

Assay method was adopted and modified from Sigma-Aldrich. A starch solution (0.5 % w/v) was obtained by stirring 0.125 g of potato starch in 25 ml of 20 mM sodium phosphate buffer with 6.7 mM sodium chloride, pH 6.9 at 65°C for 15 minutes. 4 unit/ml enzyme solutions was prepared by mixing α-amylase in ice-cold distilled water. Plant extract are dissolved in buffer to final concentration of 1 mg/ml. Colour reagent was prepared by mixing a sodium potassium tartrate solution (12.0 g of sodium potassium tartrate, tetrahydrate in 8.0 ml of 2M NaOH) and 96 mM 3,5-dinitrosalicylic acid solution. Both control and plant extracts were added with starch solution and left to react with α-amyalse solution for 3 minutes. Reaction mixture was mixed with dinitrosalicylic acid solution and kept in water bath at 85°C for 15 minutes. One unit of α-amylase liberates 1.0 mg of maltose from starch. The generation of maltose was quantified by the reduction of 3, 5-dinitrosalicylic acid to 3-amino-5-nitrosalicylic acid (colour change from orange to yellow) and quantified at 540 nm. Plant extract without the amylase was also used as the control and the corrected value of the sample absorbance noted. Standard calibration curve for maltose was plotted against concentration of maltose and absorbance of the solution at 540 nm. The α-amylase inhibition was expressed as percentage of inhibition and calculated by the following equations.

Percentage Reaction=

[Maltose in test / Maltose in control] × 100

Percentage Inhibition= 100- Percentage Reaction

Plant extract used⁹: Methanolic and Chloroform extracts were used for the study. All the extracts were prepared in DMSO to get 20 mg/ml concentration.

Fig. 5: Maltose calibration curve

RESULT:

Alpha-amylase inhibition activity of isolated methanolic extract (Comp-I) and direct chloroform extract of bark of Zanthoxylum rhetsa (Roxb) was studied. There are many enzymes in the human digestive system that help in the digestion of food. It is known that the degradation of starch to glucose in the alimentary canal proceeds rapidly. Alpha-amylase enzymes present in different parts of GIT catalyses the breakdown of polysaccharides (starch) into monosaccharide’s (glucose). A few minutes after the ingestion of starch a marked hyperglycaemia leading to hyperinsulinaemia is observed. As the concentration of α- Amylase increases the rate of reaction is also increases but the time of reaction decreases because of high concentration of α- Amylase will digest the starch rapidly. The present study deals with the inhibition of α- Amylase by both extracts of barks of Zanthoxylum rhetsa. Extracts of barks having α- Amylase inhibition activity which is shown by increase in reaction time i.e. the time taken by α- Amylase to digest the starch. From the observation the methanolic extract was found that as the time of reaction increases the percentage inhibition of Alpha amylase increases when compared control and chloroform extract (Fig. 5 and 6).

Fig 6: Percentage inhibition of alpha amylase enzyme by various extracts at time t = 3 min.

DISCUSSION:

Herbal therapy for diabetes has been followed all over the World successfully for the management of Type 1 and Type II diabetes and their complications. The enzymes responsible for catalysis are denoted as “amylolytic”. α-Amylases are considered as endo-acting enzymes which randomly attack internal α-1,4-glycosidic linkages. Although the activity of these enzymes has not been directly involved in the etiology of the diabetes, however alpha amylase inhibitor has been thought to improve glucose tolerance in diabetic patients. Isolated methanolic extract (Natural flavonoids comp-I) has shown more promising anti-diabetic activity compared to control whereas chloroform extract has not shown alpha amylase inhibition.

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Received on 18.04.2011 Modified on 03.05.2011

Research J. Pharm. and Tech. 4(7): July 2011; Page 1147-1150