Immunological evaluation of Artocarpus heterophyllus for determining its antimicrobial and anti-inflammatory activity

 

Sumesh S Shah¹, Amit Gupta²*, Shweta Karne¹, Bharat Shinde^1,2

¹Department of Microbiology, Vidya Pratishthan’s, Arts, Science and Commerce College, Baramati, Maharashtra, India

²Department of Zoology, Vidya Pratishthan’s, Arts, Science and Commerce College, Baramati, Maharashtra, India

 

ABSTRACT:

Objective-The objective of our present study in order to determine its antimicrobial and anti-inflammatory activity of aqueous seed extract of Artocarpus heterophyllus. Methods- In this study, we determined its protein content from aqueous seed extract using Nanodrop method and also analyzing its antibody titre against ovalbumin (OVA) by Elisa method and also estimated its proliferation rate against OVA in lysed human whole blood. In addition, analyzing its antimicrobial activity using variable doses of seed extract against gram positive and gram negative bacteria. Results- The results showed that seed extracts of Artocarpus heterophyllus showed higher amount of protein content at a very lower concentration and also raised its antibody production at higher doses against weak antigen OVA. In contrast, seeds extract showed its anti-inflammatory activity and this activity could be due to reduce its proliferation rate at higher doses and also showed its antimicrobial activity at the same concentration against various bacterial pathogens. Conclusion-Overall, seed aqueous extract of Artocarpus heterophyllus showed its antimicrobial and anti-inflammatory activity.

 

 

 

INTRODUCTION:

In an effort to search for new antimicrobial and anti-inflammatory agents extracted from various medicinal plant products in the form of primary and secondary metabolites [1]. According to the literature, more than eighty percent of people rely on traditional medicines as compared to synthetic based medicines. In the market, lot of antimicrobial drugs are available but it showed several side effects [2].

 

In this regard, researchers focused only on those metabolites extracted from medicinal plant products that are safe, cheap and easy to available. For the last twenty years, number of research papers are already published and mentioned various medicinal plant products especially leaves, roots and stem which showed its antimicrobial and anti-inflammatory activity [3, 4]. In view of this, we focused especially on fruits especially Jack fruit seeds that are also considered as one of the constituents and very few immunological studies were conducted. Artocarpus heterophyllus (Jack fruit; family Moraceae) is an integral part of common Indian diet and is freely available in Indian and adjoining continents. Generally, Artocarpus heterophyllus showed various medicinal properties that are already mentioned in the Ayurveda [5, 6]. In addition, this tree possessed several immunopharmacological activities [7, 8] such as antioxidant, immunomodulatory activity etc. Lot of research work is already done related to this medicinal tree and reported several compounds [5-8] (i.e. morin, cynomacurin, artocarpin/ isoartocarpin/ cyloartocarpin, artocarpesin/ oxydihydroartocarpesin, artocarpetin/ norartocarpetin, cycloartinone, betulinic acid, artocarpanone and heterophylol) that are useful in fever, boils, wounds, skin diseases, convulsions, diuretic, constipation, ophthalmic disorders and snake bite etc. One of the fruits i.e.  Jackfruit, popular food item that are reported throughout the world and also mentioned as national fruit of Bangladesh in the literature. This tree can produce more than 200 fruits in a year. Recently, India is one of the top producer of jackfruits followed by Bangladesh, Thailand, Indonesia and Nepal [9, 10]. In contrast, this tree showed several uses i.e. high nutritive value (potassium, calcium and carbohydrates) of ripe fruits flakes. In Jackfruit, one of the major protein that are reported i.e. Jacalin [11] extracted from the seeds of Artocarpus heterophyllus. In this regard, we focused on aqueous seed extract in order to determine its anti-inflammatory and antimicrobial activity.

 

MATERIALS AND METHODS:

Plant Material and Estimation of Protein Content:

Seeds of Artocarpus heterophyllus were collected from the garden of Vidya Pratishthan’s School of Biotechnology, Arts Science and Commerce College, Baramati, Maharashtra, India. These seeds were dried in a shady area and macerated in liquid nitrogen and then finally dissolved in phosphate buffered saline (PBS, pH 7.4) in order to prepare stock solution (100 mg/ml). Afterwards, protein concentration of aqueous seed extract through Nanodrop method (Fig.1).

 

Fig.1. Estimation of protein content from aqueous seed extract of Artocarpus integrifolia

 

Qualitative analysis of aqueous extract:

Different phytochemical tests were performed for determining its secondary metabolites that are present in aqueous seed extract of Artocarpus heterophyllus. The results clearly indicate its presence of secondary metabolites i.e. saponins (foam test), terpenoids (using chloroform and concentrated sulphuric acid), flavonoids (ferric chloride solution) and tannins (using lead acetate solution) but alkaloids are totally absent.

 

ELISA:

For confirmation of antibody production against specific protein antigen i.e. OVA using variable doses of aqueous seed extract of Artocarpus heterophyllus. For these studies, indirect ELISA was performed in high protein binding plates using OVA (100 µg/well; 96 well plates) as coating antigen. Incubate Elisa plate overnight at 4ºC and then wash with PBS buffer two to three times. After washing, add blocking buffer (100 µl) and then incubated the plate for another 1h at room temperature. Thereafter, add variable concentration of seed aqueous extract of Artocarpus heterophyllus after washing again with PBS buffer. Incubate 96 well plate for another 4-5 h at carbon dioxide incubator. After incubation, add secondary antibody (horse anti-serum; 1:10000 dilution; 100 µl) in 96 well plate. Again, incubate plate for another 1h at carbon dioxide incubator. Finally, TMB substrate solution was added after washing the plate with PBS and then add stop solution. The optical density was measured at 450 nm [12].

 

Antimicrobial and anti-inflammatory activity:

For determining its antimicrobial activity using variable concentration of aqueous seed extract against different bacterial species i.e.  Gram-positive bacteria (Bacillus subtilis) and Gram-negative bacteria (Pseudomonas fluorescens, E. coli, Salmonella typhi). All these bacterial samples were collected from soil sample, Vidya Pratishthan except Salmonella typhi (ATCC strain) and identified on the basis of gram staining method. These studies will be conducted under CLSI guidelines. All these bacterial strains were grown properly in nutritive rich media containing  glucose, yeast extract, sodium chloride, agar and other trace elements with pH 7.4 and maintained at 37ºC for 24 h in incubator. Normally, bacterial colonies were grown and identified through Bergeys manual, determinative bacteriology. One colony of each bacteria is diluted in phosphate buffered saline (PBS, pH 7.4) to reach the final concentration i.e.  10⁶CFU/ml. For these studies, bacterial strains (10⁶ CFU/ml; 50 µl) were taken and then exposed to lysed human whole blood (100 µl) and then treated with variable concentration of aqueous seed extract (0.1- 100 mg/ml, 50 µl). Similarly, lysed human whole blood (virally infected sample) exposed with OVA in presence of variable concentration of aqueous seed extract for determining its anti-inflammatory activity. Incubate (37°C, 24 h) both these plates; media in the wells was removed after centrifuging and then replaced it with fresh medium containing MTT solution (2.5 mg/ml; 10 µl). Again incubate 96 well plate for another 4h at 37°C. Thereafter, media-containing MTT was removed after centrifuging and formazan crystals were appeared at the bottom and dissolved in dimethyl sulphoxide (DMSO). The optical density (OD) was recorded in microplate reader at 570 nm [13, 14].

 

Statistical Analysis:

Data are reported as means ± standard error (SE). The difference between the control and treated samples of seed aqueous extract of Artocarpus heterophyllus is determined by one way ANOVA test (Bonferroni multiple comparison test).

RESULTS:

ELISA:

The results of these Elisa studies as shown in Fig.2 and claimed that aqueous seed extract at higher concentration showed enhancement in antibody production as compared to ovalbumin control. For these studies, OVA containing alum used as standard and showed slightly enhancement in antibody production as compared to control.

 

 

Fig.2.ELISA assay. Indirect Elisa was performed using standard OVA (100 µg/well) as coating antigen. Aqueous extract of seeds of Artocarpus integrifoliawere used for the estimation of anti-OVA antibody titre. Horse anti-serum used as secondary antibody and optical density measured at 450 nm. The difference between the control and standard (alum) is determined by one way ANOVA test. *P<0.05; **P<0.01; ***P<0.001

 

Antimicrobial activity

Antimicrobial studies of aqueous seed extract against different bacterial pathogens in lysed human whole blood as shown in Fig.3. The results showed that aqueous seed extract at higher doses showed decline in proliferation rate as compared to control. Overall, the results claimed that aqueous seed extract showed its antimicrobial activity against different bacterial pathogens.

 

Fig.3.Antimicrobial activity. Bacterial strains (10⁵ CFU/ml; 50 µl) strain were treated with variable concentration (0.01 – 100 mg/ml, 100 µl) of aqueous seeds extract of Artocarpus integrifoliain lysed human whole blood. After incubation, add MTT solution and then observed formazan crystals and finally dissolved in DMSO and then its absorbance was recorded in microplate reader at 570 nm. The difference between the control and variable doses of aqueous seeds extract of Artocarpus integrifoliais determined by one way ANOVA test.*P<0.05; **P<0.01; ***P<0.001

 

Fig.4. Anti-inflammatory activity. Virally infected lysed human whole blood were cultured with variable doses of aqueous seeds extract (as described in materials and methods section). Values are expressed as Mean ± S.E. The difference between control and variable doses of aqueous seeds extract of Artocarpus integrifoliais controlled by one way ANOVA test (Bonferroni multiple comparison test). *P<0.05; **P<0.01 and ***P<0.001

 

Anti-inflammatory activity:

The results of these studies related to its anti-inflammatory activity of aqueous seed extract against weak antigen i.e. OVA as shown in Fig.4. The results of these studies related to aqueous seed extract showed decline in proliferation rate at higher doses as compared to control. In other words, aqueous seed extract showed its anti-inflammatory activity against OVA.

 

DISCUSSION:

As per the literature, more than 50% of drugs derived from medicinal plant products and these are consumed in the form of crude or standardized plant extracts. These plant extracts or molecules that are easy available and showed its efficacy and less side effects. Due to these significant features, medicinal plant products are of the most attractive drug candidate form edication against various pathogens including diseases [15, 16]. In this regard, quantum of research work in the area of drug discovery or development has been undertaken in order to authenticate these medicinal plant products, yet major part of it unexplored. In the present study, we worked on one of the fruits i.e. Jack fruit that are vital requirement for human health. In this study, our group focused on Jack fruits especially seeds of Artocarpus heterophyllus and tried to determine its immuno pharmacological activity against specific protein antigen i.e. OVA. In view of this, we determined its potential of aqueous seed extract against OVA which is determined through Elisa and also determining its antimicrobial activity (using four bacterial strains) and anti-inflammatory (antigen, OVA specific proliferation) activity. For these studies, firstly phytochemical screening of aqueous extracts of seeds was done and its results revealed the presence of saponins, tannins, flavonoids and terpenoids. The phytochemical constituents of aqueous seed extract seemed to have potential as source of useful drugs against different various bacterial pathogens that are vital requirement for human health. As per the literature of Artocarpus heterophyllus, all parts of this tree showed some medicinal properties and also used as medicine against malaria fever. The present studies indicate its antimicrobial and anti-inflammatory properties of aqueous seed extract. These immunological findings were reported and confirmed that these seeds in the form of aqueous extract at higher doses showed decline in the population of gram positive and gram negative bacteria including OVA specific population and considered its antimicrobial and anti-inflammatory properties. These activities could be due to the presence of primary (protein) and secondary metabolites. The anti-inflammatory effects of aqueous seed extract were evaluated by determining their proliferation rate against specific protein antigen i.e. OVA. These seeds exhibited potent anti-inflammatory activity because of sudden decline in proliferation rate at higher doses as compared to control. Thus, OVA used as standard for these studies and may provide a potential therapeutic approach against inflammation-associated disorders [17]. In short, we determined the potential of seeds in the form of aqueous extract that promote human health status and also claimed in order to reduce the burden of various infectious agents. Hopefully, in the future these seeds in the form of aqueous extract from Artocarpus heterophyllus can serve as an important source for developing new anti-inflammatory as well as anti-microbial drugs. Further immunological investigations were focused and determined in vivo assessment of the biological activity of these aqueous extracts and identified its major active components that are responsible for antimicrobial and anti-inflammatory activity in aqueous seed extract.

 

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Received on 11.03.2017       Accepted on 27.05.2017     

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