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Comparative Analysis of Lectins Isolated from Seed and Testa of Artocarpus Heterophyllus Lam.

REPON KUMER SAHA*
Department of Pharmacy, East West University, Dhaka, Bangladesh

MAHA JAMIRUDDIN
Department of Pharmacy, East West University, Dhaka, Bangladesh

SRIJAN ACHARYA
Department of Pharmacy, East West University, Dhaka, Bangladesh

Abstract/ Executive Summary:

Artocarpus heterophyllus or jackfruit is the national fruit of Bangladesh. The objective of this study is to characterize and compare the lectin proteins isolated from seeds and testa of Artocarpus heterophyllus. Thin layer chromatography was used to detect the presence of protein in seeds and testa.Hemagglutination assay was done to identify the isolated proteins as lectins. The lectin profiles of seeds and testa were analyzed using SDS-PAGE technique. Antioxidant effects were measured by DPPH scavenging assay. An anti-inflammatory assay was also investigated.Carbohydrate induced and bacterial induced hemagglutination inhibition was done. The seed lectin showed stronger hemagglutination activity and antioxidant activity compared to testa lectin. However, testa lectin showed potent anti-inflammatory activity at low concentration compared to seed lectin. Therefore, in the future these lectins may have the potential to play role as biotechnological tools.

Keywords: Artocarpus heterophyllus Lam., SDS PAGE, Anti-oxidant, Anti-inflammatory, Haemagglutination assay

Location: Reazuddin Bazar, Chittagong, Bangladesh

Start Date: 00-06-2012

End Date:

Program Area: Resource Development and Management

Non-commodity: Comparison

Objectives:

The present work deals with the isolation of the lectins from seeds and testa of Artocarpus heterophyllus and establishment of its antioxidant properties, antiinflammatory activity and hemagglutination activity, including phytochemical evaluation. This would help in future isolation of their active constituent as they have found to contain various important constituents.

Methodology:

Plant Collection and Identification: The seeds of the plant along with the testa were collected from the Reazuddin Bazar, Chittagong, Bangladesh during June, 2012 and identified by the taxonomist of the Bangladesh National Herbarium, Mirpur, Dhaka as Artocarpus heterophyllus Lam. A voucher specimen of the plant has been deposited (Accession No.: 38308) in the herbarium for further reference. Isolation of the lectin: The seeds along with testa were shade-dried and later dried testa was separated from the dried seeds. The testa and seeds were grinded separately. 5g of seed and testa powder was weighed and soaked in 50 ml 0.15 N NaCl solution separately for 3 days at 4°C. The mixture was then filtered and the filtrate was centrifuged at 10,000 rpm for 30 minutes. After centrifugation, the supernatant was collected in a beaker using a pipette. 20 g of Ammonium sulphate was added into each, dissolved, and kept at 4°C for 24 hours. The solution was then centrifuged at 10,000 rpm for 30 minutes at 4^oC. After centrifugation, the supernatant was discarded and the residue was collected. The residue was the isolated protein which was stored at -80°C to prevent protein denaturation. Thin Layer Chromatography(TLC) Analysis: The presence of protein in each extract were analyzed by performing TLC method as previously described. TLC was done under the solvent consisted of n-butanol, glacial acetic acid, and water (8:2:2). For charring, the plates were exposed to 2% ethanolic nin hydrin, dried and then heated to 100ºC for charring purpose. This helped in the fixation of spot and allowed it to be prominently visible. Hemagglutination Inhibition Assay: Agglutination of the red blood cells by the seed and testa protein was carried out using human erythrocytes in a 96- well microtiter plate with slight modifications to previously described. A serial twofold dilution of the protein solution was mixed with an equal volume (50 μl) of a 1% suspension of erythrocytes (A, AB, and O) in 0.9%NaCl at room temperature. The plate was left undisturbed for 1h at room temperature in order to allow for agglutination of the erythrocytes to take place. Smooth button formation in bottom indicated negative activity, while a rough granular deposition at the bottom showed positive activity. The intensity of hemagglutination was determined from the extent of deposition. Carbohydrate Inhibition of Hemagglutination: Hemagglutination inhibition assay with the seed and testa lectin was performed in 96-well microtiter plate with slight modifications to previously described. Serial two-fold dilutions of sugar samples were prepared in 0.9% NaCl. All the dilutions were mixed with an equal volume (50 μl) of the seed and testa lectin solution of known hemagglutination units. The mixture was allowed to stand for 1 hr. at room temperature and then mixed with 100μl of a 1% human erythrocyte (O and A) suspension in 0.9%NaCl. The hemagglutination titers obtained were compared with a non-sugar-containing blank. In this study, the sugars used were: galactose and lactose. The concentration of the sugar in the final reaction mixture which completely inhibited hemagglutination units of the lectin sample were obtained. Bacterial Inhibition of Hemagglutination: This assay was performed in 96-well microtiter plate with slight modifications to previously described. Fifty microlitres of the Salmonella typhi suspension were serially diluted into the successive wells with 0.9%NaCl. Then, 50 L of 1% human erythrocyte suspension was added to all the wells. Sedimentation of erythrocytes in presence of bacteria was visualized in the plate after 30 min. of incubation at 37^oC. In bacterial inhibition of hemagglutination studies in presence of lectin, the serially diluted lectin was previously incubated with bacteria at 37^oC for 30 min. Later, 100 L of 1% erythrocyte suspension was added and the plate was incubated for 30 min at 37^oC.The concentration of lectin at which bacteria inhibited hemagglutinating activity was obtained.

Source of Information: Int. J. Curr.Res.Chem.Pharma.Sci. 2(1): (2015):65–75

Article Link (Online Journal):

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

Therefore, from our present study on the proteins isolated from seeds and testa of Artocarpus heterophyllus can be concluded that the isolated proteins are lectins as they showed significant hemagglutination activity. The lectins have antioxidant properties and anti-inflammatory effects also. They showed hemagglutination activity in presence of bacteria. Hence they have antimicrobial activity too. In the future, the protein isolates may have the potential to play role as biotechnological tools. Further studies may be conducted on the biological activities and mechanisms of action of these lectins so that the production of lectins can be improved and new applications of these lectins can be found.

Knowledge Management: Journal