Dipeptidylpeptidase-4 Inhibitory Activity of Pergularia Daemia (Forsk) – An In-vitro Estimation

 

Karthik Dhananjayan¹, Arunachalam Sumathy², Sivanandy Palanisamy³

¹Department of Pharmacology, Grace College of Pharmacy, Kodunthirapully, Palakkad, Kerala

²Department of Pharmaceutical Chemistry, Grace College of Pharmacy, Kodunthirapully, Palakkad, Kerala

³Department of Pharmacy Practice, KMCH College of Pharmacy, Coimbatore, Tamilnadu

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

Dipeptidylpeptidase-4 is an enzyme involved in the inhibition of GLP-1 (Incretin), which is necessary for stimulation of insulin release. It is one of the enzyme targets for the treatment of type-2 diabetes mellitus. Ethanolic and aqueous extracts of Pergularia demia were investigated for activity using ellman method. Successive concentrations (50mg/ml, 100mg/ml, 200 mg/ml, 400 mg/ml, and 800 mg/ml) of ethanolic and aqueous extracts were prepared and investigated for their inhibitory activity towards dipeptidylpeptidase-4 as per Kojima et al., method. It was found that both extracts exhibited significant activity towards DPP-4 inhibition. Diprotein-A was used as standard and distilled water as control.

 

 

INTRODUCTION:

Pergularia daemia (Forsk) Chiov, (Asclepiadaceae) is a foetid smelling laticiferous twiner found in the plains throughout the hot parts of India, ascending to an altitude of 1000m in the Himalayas [1] Pergularia species are widely distributed in the world tropics and subtropics from southern and tropical Africa and Asia, have multiple applications in different folk medicine, including the Indian Ayurvedic system, and have been documented for anti-fertility [2], wound healing [3], antidiabetic [4], hepatoprotective [5], cardiovascular effect [6], antibacterial activity [7]. The plant is useful in the diseases of vatha, convulsion, asthma, poisoning; the root is useful in mental disorder, anaemia, leprosy and piles [8]. Plant possesses stomachic, laxative and diuretic properties, useful in cough, biliousness and sore eyes. Leaf paste mixed with castor oil is applied to joints in inflammation, liver complaints, spleen enlargement; leaves have hypoglycemic activity [9]. The juice of the leaves is given in asthma and applied to rheumatic swellings in combination with lime or ginger; it is also used in the preparation of medicinal oil given in rheumatism, amenorrhoea, and dysmenorrhoea [10].

 

The plant has been investigated phytochemically for cardenolides, alkaloids, triterpenes and saponins [11]. Ethanol extract of aerial parts of Pergularia daemia (Forsk.) Chiov. reported for anti-inflammatory, antipyretic, analgesic activity [12]. Earlier reports on biological activities of root extract are scarce. Therefore, the present study was undertaken with the objective to investigate the dipeptidylpeptidase-4 inhibitory activity of the aqueous and alcoholic leaves extracts of Pergularia daemia (Forsk.) Chiov by using Kojima et al., method

 

MATERIALS AND METHODS:

Collection of plant:

The leaves of Pergularia demia (Forsk.) were collected from Coimbatore city in Tamilnadu, India during the month of March 2013. The plant was identified and authenticated by Mr. G.V.S Murthy, Joint director, Scientist, C-I/C, Botanical Survey of India, Tamilnadu Agricultural university campus, Coimbatore bearing the reference no BSI/SRC/5/23/01-13/Tech-653 dated March 15, 2013.

 

Chemicals and Standards:

Dipeptidylpeptidase was purchased from Sigma Aldrich, USA, Tris HCl buffer, Gly-pro-p-nitroanilide (substrate) from Hi-Media Laboratories’ Mumbai, Diprotein-A (Ile-Pro-Ile) was purchased from Biogenuix (Enzo Life Sciences) New Delhi.

 

Table 1. In-vitro dipeptidylpeptidase-4 inhibition using ethanolic extract of Pergularia demia (Forsk)

S.No	Extract	Concentration (mg/ml)	%Inhibition	IC50 (mg/ml)
1	AELPD	50	16.77±0.58	310±0.85
2		100	28.21±0.24
3		200	49.85±0.43
4		400	68.12±0.11
5		800	88.49±0.29

Values are ± SEM (n=3),*P <0.05 when compared with standard

 

Table 2. In-vitro Dipeptidylpeptidase-4 inhibition using aqueous extract of Pergularia demia (Forsk)

S.No	Extract	Concentration (mg/ml)	%Inhibition	IC50 (mg/ml)
1	AELPD	50	14.78±0.44	130±0.26
2		100	32.48±0.14
3		200	64.87±0.69
4		400	75.26±0.21
5		800	92.45±0.58

Values are ± SEM (n=3),*P <0.05 when compared with standard

 

 

Extraction of plant material:

The fresh leaves of Pergularia demia (Forsk.) was collected, dried in shade under room temperature, powdered mechanically and sieved through No. 20 mesh sieve. The finely powdered leaves were kept in air tight container until the time of use. By using 200 ml of ethanol, 15g of dried finely powdered leaves are adopted for a simple maceration procedure at room temperature for seven days, in a conical flask with occasional shaking and stirring.

 

Method:

Dipeptidylpeptidase-4 assays (in-vitro): The assay was performed as per Kojima et al. In brief, the assay was performed in 96 micro well plates. A pre-incubation volume 250µl contained 100mM Tris HCl buffer pH 8.4, 7.5µl of DPP IV enzyme (0.2U/ml) and various concentration of test material/reference inhibitor. This mixture was incubated at 37°C for 30mins, followed by addition of 10µl of 1.4 mM Gly-pro-p-nitroanilide (substrate). The reaction mixture was incubated for 30mins at 37°C and absorbance was measured at 410nm. Diprotein-A (Ile-Pro-Ile) was used as reference inhibitor.

 

RESULTS AND DISCUSSION:

Preliminary phytochemical screening of leaves of Pergularia demia (Forsk) showed the presence of alkaloids, saponin, tannin, terpenoids and flavonoids. It was found that ethanolic and aqueous extract exhibited anti-cholinesterase activity in dose dependant manner. The IC50 values for the ethanolic extract and aqueous extract towards the inhibition of dipeptidylpeptidase-4 was found to be 310mg/ml and 130mg/ml, the details are presented in the tables 1 and 2. Diprotein-A high sensitivity towards DPP-4 and it has maximum inhibition towards the enzyme and also its IC50 value was triple time less than with a value of 45 mg/ml.

 

CONCLUSION:

The ethanolic extract and aqueous extracts of leaves of Pergularia demia showed maximum inhibitory activity towards dipeptidylpeptidase-4. The active constituents of the plant extract can be isolated and further investigation on structural studies may give detailed information on their use on the future for the treatment of diabetes mellitus.

 

REFERENCES:

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2.       Kumar B, Yadav DK, Govindarajan R and Pushpangadan P. Wound healing activity of Pergularia daemia (Forsk.) Chiov. Pharmacog. Ethnopharmacol. 1; 2006: 12-14.

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9.       Sathish CJ, Sharma RA, Jain R, Mascalo N, Capasso F, Vijayvergia R, et al. Ethnopharmacological evaluation of Pergularia daemi (Forsk.) Chiov. Phytotherapy Res. 12; 1998: 378-80.

10.     Bony R Shah, Dhrubo Jyoti Sen and Rajesh Bahekar. Design, Synthesis and Pharmacological Evaluation of Potent and Selective Dipeptidyl-Derived Inhibitors as New Class of Antidiabetic Drugs. Asian Journal of Research in Chemistry. 4 (1); 2011: 50-54.

11.     Kojima K, Ham T and Kato T. Rapid chromatographic purification of Dipeptidylpeptidase IV in human sub-maxillary gland. Journal of Chromatography A. 189; 1980: 233-240.Ghosh MN. Fundamentals of Experimental Pharmacology. Scientific Book Agency, Calcutta. 1984..

 

 

 

Received on 11.04.2013       Modified on 19.04.2013

Accepted on 28.04.2013      © AJRC All right reserved