Optimization and Production of Anti-Inflammatory and Anti-Diabetic Metabolites from Marine Streptomyces sp. VITJS8

 

Elaiyaraja V, Hema Latha M, Subathra Devi. C*

Department of Biotechnology, School of Biosciences and Technology, VIT University,

Vellore -632014, Tamil Nadu, India.

 

ABSTRACT:

Actinomycetes are one of the richest sources for anti-bacterial and anti-inflammatory activity components producing microorganisms. The main focus of the present study was to optimize the culture conditions of Streptomyces sp. VITJS8 for the mass production. The optimization of initial factors pH and temperature was processed and the media was developed for further carbon and nitrogen source alteration. The anti-inflammatory and anti-diabetic activity of ethyl acetate extracts of each parameter was tested. Effect of various nutritional and environmental factors were analysed and optimized. And finally the study reveals that they were growing and producing very active anti-inflammatory components at pH 7.0, temperature 30^◦C with fructose as a carbon source and glycine as nitrogen source. The results obtained from this current study reveals that the ethyl acetate crude extracts can be a potential source for anti-inflammatory drugs.

 

 

 

INTRODUCTION:

Marine bacteria have a diverse range of bioactive metabolites and are used for the treatment of diseases.^1,2,3 Presently, marine bacteria are considered as potential source of therapeutic metabolites. There is an enormous scope to investigate the probabilities of deriving therapeutic metabolites and new products of economic importance from potential marine bacteria for pharmaceutical purposes. Due to unique metabolic capabilities the marine microorganisms are excellent natural resource for screening of novel molecules. These marine microbial metabolites are attracted substantial attention for their novel chemical and stereochemical characteristics that may be exploited in chemical or pharmaceutical synthesis. Several marine microbial metabolites are already being used for industrial and pharmaceutical purposes.Streptomyces sp.,

 

are known as the creators of numerous bioactive metabolites that are beneficial to humans in medicine, such as anti-diabetic, anti-inflammatory, anti-bacterials, antifungals, antivirals, anti-tumor drugs and enzyme inhibitors.²

 

A survey of literature directed no systemic approach has been made to evaluate the anti-inflammatory and anti-diabetic potential of marine actionomycetes by invitro method. The present study involves determination and optimization of anti-inflammatory and anti-diabetic activity of marine Streptomyces sp.VITJS8 by α-amylase inhibition and RBC membrane stabilization and heat induced haemolytic assays.

 

MATERIALS AND METHOD:

Strain selection for optimization:

Streptomycessp VIT JS8.NCIM-5575 (Gen Bank Accession number: KJ725090) isolated from marine sediment⁴ was subjected to optimization process, to enhance the bioactivity .

 

Optimization of media:

Initially the medium was optimized with three different types of media for the media selection. They were ISP 2 medium, SS broth and finally Starch Casein broth (SSB). The culture conditions for this three were same based on the standard as pH 7.2 and temperature 35◦C with as per their composition. Three mediums were prepared and sterilized. Then it was inoculated with the Streptomyces sp. VIT JS8 strain respectively and kept in shaker for growth and production of secondary metabolites for seven days. The flasks were observed and monitored⁵.

 

Process optimization:

Basic parameters, which are pH and temperature, were optimized for further culture condition optimization. SC broth was selected for this process based on the media optimization previously. Initially the pH was optimized in the range of 6.8, 7.0, 7.2, 7.5 and finally 7.7. Among these pH the media was further optimized for temperature altering or optimizing in the manner of 25^◦C, 28^◦C, 30 ^◦C, 38 ^◦C and 42^◦C⁵.

 

Carbon and nitrogen source optimization:

Carbon source are the backbone of the growth of any organism and altering in that results metabolic and growth kinetic changes. So it was begin with starch, glucose, fructose, arabinose and finally lactose for the production of anti-inflammatory and anti-diabetic metabolites. And another important source nitrogen also altered as well as carbon source. So it was selected in the order of casein, arginine, cysteine, glycine and phenyl alanine. And they were inoculated with a loop-full inoculum and incubated in shaker for seven days⁵.

 

Anti-diabetic assay:

Anti diabetic potential of marine actinomycetes was assay in vitroanti­diabetic activity was determined by examining the inhibitory activity of the enzyme α­ amylase which implicates in the breakdown of starch to produce glucose. In this method, 1 mL of extracts were added with 1 mL of the enzyme α­amylase in test tube and incubated for 10 min at 37°C. After 1 mL of 1% starch solution was added into it and once more incubated for 15 min at 37°C. Then 2 mL 3,5­dinitrosalicylic acid mixture was added into it, in order to terminate the reaction. The mixture was then incubated in steaming water bath for 5 min and then cooled. The absorbance of the reaction mixture was then measured at 546 nm in a spectrophotometer. The standard (control) of the reaction without the extract represents the 100% enzyme activity. The % age inhibition of enzyme activity of α­amylase was determined by % inhibition of α-amylase=(enzyme activity of control-Enzyme activity of extract) × 100/ Enzyme activity of control⁶.

 

 

 

Anti- inflammatory assay:

The Blood was collected from healthy human volunteer who has not taken any NSAIDs (Non-Steroidal Anti-Inflammatory Drugs) for 2 weeks prior to the experiment and transported to the centrifuge tubes. The tubes were centrifuged at 3000 rpm for 10min and were washed three times with equal volume of normal saline. The volume of blood was measured and re constituted as 10% v/v suspension with normal saline. The reaction mixture (2ml) contained of 1 mL test sample of different parameters and 1 mL of 10% RBCs suspension, instead of test sample only saline was added to the control test tube. Aspirin was used as a standard drug. All the centrifuge tubes having reaction mixture were incubated in water bath at 56°C for 30min. At the end of the incubation the tubes were chilled under running tap water. The reaction mixture was centrifuged at 2500 rpm for 5 min and the absorbance of the supernatants was taken at 560 nm⁷.

 

Percentage inhibition=(Abs control –Abs sample) X 100/ Abs control

 

RESULTS AND DISCUSSION:

The optimization study of marine Streptomyces sp. VITJS8 was studied by varying the physical and chemical parameters for the maximum activity of anti-inflammation and anti-diabetic.

 

Effect of pH and temperature:

The various pH and different temperature parameters were studied in the production of marine actinomycetes. The results are shown in the table 1 and2,Based on the results, the optimum pH and temperature was found to be pH 7.0 and temperature 30 ◦C.

 

Table 1 Effect of pH

 

Table 2 Effect of temperature

 

Anti-diabetic assay:

The percentage of inhibition of enzyme α­amylase was determined.The results are represented in Figure1. In these results the fructose and cysteine showed the maximum percentage of inhibition of enzyme activity.Fructose and cysteine was found to be the optimum carbon and nitrogen sources for the production of anti-diabetic compounds.(Fig.1)

 

 

Fig.1 Anti-diabetic assay results for different carbon and nitrogen sources (% of inhibition of enzyme activity)

 

Anti-inflammatory assay:

This assay was used for the anti-inflammatory activity because the erythrocyte membrane is similar to the lysosomal membrane and its stabilization infers that the extract may well stabilize lysosomal membranes. The extract was effective in inhibiting the heat induced haemolysis at different concentrations. The results showed that carbon and nitrogen sources were protect significantly the erythrocyte membrane against lysis induced by heat.(Fig.2)The hemolytic percentage for crude extract as calculated from the O.D. readings is 92.45%. Aspirin was used as a positive control. The optimum carbon and nitrogen source was found to be glucose and phenylalanine respectively.

 

 

Fig.2 Effect of different carbon and nitrogen sources in the assay of heat induced hemolysis

 

CONCLUSION:

The present study reveals that the extracts from different parameter and sources can act as potential anti-inflammatory and anti-diabetic activity. Also it gives the data about the optimum pH, temperature, carbon and nitrogen source for the maximum production of anti-inflammatory and anti-diabetic metabolites. In future this study will be useful for the production of anti-inflammatory compounds from the marine actinomycetes and further developed to scale up process using laboratory bioreactor by utilizing the data of this study.

 

ACKNOWLEDGEMENTS:

We are very thankful to VIT University for the encouragement, support and help with all the lab and necessary facilities.

 

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Accepted on 25.08.2017                             © RJPT All right reserved

Research J. Pharm. and Tech 2018; 11(7): 2866-2868.