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Alpha Amylase Enzyme Inhibitory and Anti-inflammatory Effect of Lawsonia Inermis

Hasan Imam

Nasir Uddin Mahbub

Md. Forhad Khan

Humayera Kabir Hana

Md. Moklesur Rahman Sarker
Department of Pharmacy, School of Science, Primeasia University, HBR Tower, Kamal Ataturk Avenue, 9 Banani C/A, Dhaka - 1213, Bangladesh

Abstract/ Executive Summary:

Previously it was reported elsewhere that Lawsonia inermis have anti-inflammatory and analgesic effect in experimental animals. The in vitro porcine alpha amylase inhibitory effect was investigated of this plant methanolic extracts and consequently hypoglycemic effect by quantitatively determining the maltose from the maltose standard curve while the anti-inflammatory effect by acetic acid induced writhing test in mice. Acarbose (10 μg mL^-1) and Diclofenac sodium (20 mg kg^-1) were used as reference hypoglycemic and anti-inflammatory drugs, respectively, for this study. The methanolic leaves extract of the plant significantly inhibited (60.97% compared to untreated) enzymatic activity of the amylase at 10 μg mL^-1 dose (p<0.05) also reduced the chemically induced nociceptive pain stimuli significantly at all doses (p<0.01). Carbohydrates, glycosides, flavonoids, saponins and tannins were found to have in phytochemical screening of the extract which are thought to bring these effects. For the conclusive purpose, it is suggesting from the result that the pharmacological properties of this Lawsonia inermis can elicit hypoglycemic effect by inhibiting α-amylase enzyme and can reduce neurogenic pain stimulus. It gives the notion that how this group of patient would be therapeutically benefitted by decreasing both these effects by the same agent which is easy available.

Keywords: Hypoglycemia, Alpha amaylase, Inflammation, Lawsonia inermis

Location: Department of Pharmacy, School of Science, Primeasia University

Start Date:

End Date:

Program Area: Development of Host and Medicinal Plants

Commodity: Plant(s)

Objectives:

The purpose of this study was to investigate the in vitro porcine α-amylase enzyme inhibitory effect and in vivo anti-inflammatory effect of methanolic henna leaves extract.

Methodology:

Identification of plant and preparation of extract: Lawsonia inermis leaves were collected in August from Nikunjo, Dhaka, Bangladesh after botanically identified and authenticated. It was then properly washed with tap water and air dried around 20 days. Dried leaves were powdered using mortar pestle and 60 g powder was socked in 300 mL methanol for 3 days with occasional starring. Filtrate was prepared by filtering through cotton and Whatman filter paper and finally centrifugation. Solvent was evaporated using a vacuum evaporator and reduced to 4.1% w/w which was preserved in -18°C until experiments was done by weighing for desired concentration in water as gram per liter.

Experimental animal: Swiss Albino mice were collected from ICDDRB and housed in plastic case of (28x22x13 cm) dimensions. Soft wood shavings were served as their bedding in side the cages. Standard condition (24±1°C temperature and 55-65% relative humidity, 12 h dark/12 h light) was maintained in the animal house and standard food pellets were supplied ad libitum.

Alpha amylase inhibition assay: In vitro amylase inhibition assay was done by following the chromogenic DNSA method described by Miller (1959), with the following modification. Briefly, 20 μL sample solution was mixed with 1500 μL enzyme solution (8 unit, calculated by maltose standard curve preparation, data not shown) in buffer containing 50 mM Tris-HCl, 20 mM NaCl and 2 mM CaCl₂ at a pH of 7.0. Following mild vortexing, 500 μL of 1% starch solution was added and set the mixture for 5 min at 37°C and reaction was stopped by adding 50 μL of 1 N HCl. Blank was prepared by adding 50 μL of 1 N HCl in the buffer solution. These solutions were mixed with 1 mL DNSA reagent and boiled for 15 min at 100°C in water bath and cooled at room temperature for 10 min and the absorbance was measured at 540 nm. Acarbose (10 μg mL^-1) was used as a positive control. The % of α-amylase inhibition activity was measured by the following formula:

Anti-inflammatory Activity of Lawsonia inermis: Acetic acid induced writhing test is long been used to test the analgesic anti-inflammatory properties for new agents. It consists of first feeding the anti-inflammatory agent or vehicle only as a negative control followed by intra peritoneal administration of 0.6% acetic acid (0.45 mL/mouse), 30 min later. It causes a painful inflammatory effect. The painful sensation of mouse which was manifested as abdominal writhe, were counted after the acetic acid injection.

Maltose standard curve preparation: Standard maltose curve was constructed in order to measure quantitatively the disaccharide formation from the starch in the presence of amylase (data not shown). Standard maltose concentration of 20, 30, 40 and 50 μg mL^-1 was prepared by dilution in water. Unknown disaccharide concentration was calculated by the equation y = mx+c. In this equation, “y” and “x” are the absorbance of sample and the concentration respectively. Whereas, “m” and “c” representing the slope and y-intercept of that equation.

Statistical analysis: Each data point represents the average of three individual experiments and each time duplicate samples were there. Statistical analysis was performed using Microsoft Excel 2007 and data were compared with unpaired two tailed t-test where p-value threshold was used ≤0.05 to indicate a statistical significant.

Source of Information: Pakistan Journal of Biological Sciences, 16: 1796-1800; ISSN 1028-8880

Article Link (Online Journal): DOI: 10.3923/pjbs.2013.1796.1800; URL: http://scialert.net/abstract/?doi=pjbs.2013.1796.1800

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

In the present study, it is found that the methanolic leaves extract of Lawsonia inermis effectively reduced the porcine alpha amylase enzyme and thereby reduced the disaccharide formation also effectively reduced the nociceptive pain induced by acetic acid. These pharmacological properties can attribute the folkloric use of the plant in the management and control of hyperglycemia and arthritic pain. Further investigation in human is essential to reveal its therapeutic potential.

Knowledge Management: Journal