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The purpose of this study was to explore the amylase inhibitor properties of actinobacteria isolated from salt pans soil, costal area in Kothapattanam, Ongole, Andhra Pradesh, India. Out of 28 colonies, 3 actinobacteria (VITIPB-3, VITIPB-9, VITIPB-23) showed potent amylase inhibition when compare to other strains. The VITIPB-23 and VITIPB-3 potential isolates were inoculated in production media for secondary screening and bioactive compounds are extracted by using different solvents. The ethyl acetate extracts of both strains showed significant amylase inhibition. These strains were further screened for antimicrobial and antioxidant activity. Among three strains, VITIPB-23 and VITIPB-3 showed good inhibitory effect while the other strain showed moderate inhibition. The VITIPB-23 showed antimicrobial effect against all the test organisms. In antioxidant assay the VITIPB-9 showed a 100% free radical scavenging activity along with the other VITIPB-3 which showed more than 95%. Based on these results we concluded that VITIPB-23 possess high amylase inhibition activity and can be further tested for the isolation of new anti-diabetic compounds.
KEYWORDS: Salt pan soil, Marine actinobacteria, amylase inhibitor, antimicrobial and antioxidant activity.
Diabetes is a chronic metabolic disorder in which there is an increased level of monosaccharide in blood plasma. The starch intake in our diet is hydrolyzed by the enzyme amylase to give rise to these sugar moieties which are then transported to the body through blood. Type II diabetes mellitus is a non insulin dependant diabetes mellitus (NIDDM) or adult-onset diabetes (Bahman et al., 2008). It is mainly due to the age factor where the insulin fails to control the glucose level in the blood stream. The level of glucose in blood increases when it is not being absorbed by the body cells and remains within the blood. This leads to a rise in the glycemic index along with an increased oxidative stress. Diabetes in turn leads to many other organ malfunctioning and often shortens the life span of an individual. Improper diet, obesity, lack of sleep is the general reasons which lead to the onset of diabetic conditions. Apart from these, pre exposed genetic traits are a major cause of suffering (Robertson et.al). The other associated disease conditions often observed are Polyuria (frequent urination), Polydipsia (increased thirst), Polyphagia (increased hunger), increased tendency of heart diseases, Peripheral neuropathy, Strokes, Kidney failure, Diabetic retinopathy (eyesight affected), etc. The chances of suffering from such diseases increase with the intake of the commercially available chemotherapeutic drugs, designed in treatment for diabetes because they affect the body metabolism internally.
Actinobacteria are well known for their capability to produce a vast range of antibiotics and enzyme inhibitors as their metabolic by products (Gartner et al., 2011). Since, marine environment shows a variety of stress conditions like salinity, pH and other parameters, they will show different ecologically adapted survival traits. Due to a difference in the atmosphere, these organisms show a variety of metabolic activities which are ought to be different from that in the normal conditions (Saurav et al., 2010). Actinobacteria isolated from marine soil may show production of rare bioactive compounds which are of pharmacologically important to treat various dreadful diseases (Sivasubramanian et al.). As per literature survey, the marine environment is less exploited compared to terrestrial study due to its geographical vastness (Valli et al. 2012). Hence, this study is an attempt to screen for novel bioactive compound (specifically amylase enzyme inhibitor) extracted from marine actinobacteria.
MATERIALS AND METHOD
Sample Collection, Processing and Isolation
Soil samples were collected from salt pans, costal area in Kothapattanam, Ongole, Andhra Pradesh (15°30′ 0″ N, 80°3′ 0″ E), India. The soil samples were collected in plastic bags and immediately transported to the laboratory. The collected samples were processed using different method. To increase the population of actinobacteria, different types of media were selected from the literature. All media were supplemented with a final concentration of 100 µg/L cyclohexamide and 15 µg/L nalidixic acid to inhibit bacterial and fungal growth (kathiresan et al., 2005). A 10 fold serial dilution series was made and plated in triplicate on agar plates. The plates were incubated at room temperature (28°C) and monitored periodically over 3 months for actinobacteria growth (Deepika et al., 2010).
amylase Inhibition (DNSA Assay):
100µl of sample extract was added to 200µl of porcine pancreatic amylase enzyme (1mg/10ml w/v of freshly prepared phosphate buffer), followed by 200µl of freshly prepared 2mM phosphate buffer (pH=6.9). It was then left at room temperature for 20 minutes. To this, 100µl of freshly prepared 1% starch solution (1gm/100ml w/v distilled water) was added, mixed well and left at room temperature for 5 minutes. Finally, 500µl of DNSA (di-nitro salicylic acid) was added and the tubes were kept in boiling water bath for 5 minutes. It was then left to cool down to room temperature. Respective control for the test was prepared by replacing equal volumes of phosphate buffer in place of enzyme. Finally, the percentage inhibition was calculated (Vandana Gulati et al., 2012).
% inhibition = [(Control Absorbance540 - Sample Absorbance540) / (Control Absorbance540)]*100
Fermentation Process and Extraction of Bioactive Compounds
The potent actinobacterial isolates (VITIPB-3, VITIPB-9, VITIPB-23) were inoculated into production broth (SS Media) containing soluble starch-25g, glucose-10g, yeast extract-2g, CaCO3- 3g, trace elements- 1ml, Distilled water -1000ml. Flasks were lodged on the flask shaker at a speed of 120 rpm at room temperature for 7 days. After fermentation, the medium was harvested and centrifuged to remove cell debris and filtrate was collected (Sathish et al., 2012). The bioactive metabolites were recovered from the harvested medium by solvent extraction method. The filtrate was mixed with ethyl acetate, chloroform, n-butanol (1:1 v/v) and shaken vigorously for 1 hour in a solvent extraction funnel. Solvent and filtrate mixture were stabilized for 24-48 hrs. After 48 hrs the solvent phase are separated from aqueous phase. The solvent extracts were concentrated and used for amylase inhibitor assay (Atta et al., 2009).
The clinical isolate of multi drug resistance Moraxiella sp, Micrococcus leuteus, Salmonella typhi and Bacillus subtilis were collected from Narayani Hospital, Ariyur, Vellore District, Tamil Nadu, India, maintained in glycerol stock at -20°C
The test organisms were screened for their sensitivity towards standard antibiotics included, Ampicillin (10mcg/disc) and Streptomycin (10mcg/disc), followed by a Drug sensitivity test performed by disc diffusion method on Mueller Hinton agar (MHA) plates. Bacterial test pathogens were made to grow in nutrient broth overnight and lawn cultured on Mueller Hinton agar plates using sterile cotton swabs. The standard antibiotics discs were placed on the agar surface using a sterile forceps along with well(s) inoculated with the isolates. Plates were incubated at 37°C for 24 hours and were observed for zone of inhibition.
Antibacterial screening of actinobacteria was detected by agar well diffusion method on Mueller Hinton agar (Kumar et al., 2009). Test organisms were inoculated in nutrient broth and incubated for 24 hours at 37°C. The turbidity of the broth was adjusted at 0.5 (optical density) using spectrophotometer. The bacterial cultures were inoculated on MHA plates using sterilized cotton swabs. In each of these plates, wells were cut out using a sterilized gel borer. The actinobacteria crude extracts were used against test pathogen, 100 µL of extracts were loaded into each well. Plates were incubated at 37°C for 24 hours. After incubation, all plates were examined for the presence of zone of inhibition around the wells (Kumar et al., 2010)
2ml of sample along with 1 ml of DPPH (1, 1-diphenyl-2-picrylhydrazyl - light sensitive), was incubated at 20°C for 40 minutes. Absorbance was recorded at 517nm spectrophotometrically (Daljit et al., 2011) with blank as methanol. % scavenging rate was calculated using the formulae:
% scavenging = [(Control Absorbance540 - Sample Absorbance540) / (Control Absorbance540)]*100
RESULT AND DISCUSSION
In support of early reports (karthik et al., 2011), marine sediments are well known for its rich content of actinobacteria. Similarly, the salt pans of Oongole did show a number of actinobacterial colonies on performing serial dilution and plating in triplicates. A total of 28 acclimatized colonies were isolated and sub cultured to obtain them in pure forms. The plating was majorly done in Starch Casein Agar medium, Actinomycetes Isolation Agar Medium and Kuster's Agar Medium (Baskaran et al., 2011) to get enhanced growth rate. These isolates were further assayed for various other parameters. Out of the twenty-eight isolates only 4 showed potency in terms of amylase enzyme inhibition.
The DNSA (3,5-di nitro salicylic acid) assay was carried out to check the inhibition of the enzyme amylase (Raja et al., ). Aromatic DNSA is reduced to a coloured compound 3-amino-5-nitrosalicylic acid in presence of reducing sugars (here glucose) which is capable of absorbing light at 540 nm (λmax) strongly. More the concentration of reducing sugars in the reaction mixture more is the colour developed which directly implies more breakdown of the starch and lesser inhibitory effect on the enzyme. This assay is used for for both Quantitative and Qualitative measure of the glucose moieties present and in other words, to what extent the starch hydrolysis has been inhibited. Denser the colour gradient, lesser is the inhibitory effect of the isolates on the enzyme and vice versa. DNSA assay is the main pathological test to determine the sugar level in the urine of a diabetic patient (Suthindhiran et al., 2009).
In our study, quantitatively, isolate VITIPB-23 shows maximum inhibition followed by VITIPB-3 (53.1% and 51.9% respectively). Isolate VITIPB-9 shows a comparatively less inhibitory effect of 31.6%. Since the colour developed for the isolate VITIPB-23 was less compared to the other two, it can be qualitatively inferred that less of reducing sugar moieties were present in the reaction mixture as a result of the inhibitory effect on the enzyme amylase (Figure 1).
These two isolates (VITIPB-23 and VITIPB-3), showing higher inhibitory effect were further subjected to the solvent extraction process where the cell free extract was mixed thoroughly with different solvents of varying polarity in a ratio 1:1 v/v (gradually from low polar to high polar). On allowing the mixture to stand overnight, two distinct layers of the solvent and the test solution was obtained. The solvent layer obtained was then subjected to evaporation at room temperature (Selvameenal et al., 2009). The extracted compounds are further screened for amylase inhibition. On performing the DNSA assay again it was inferred that only ethyl acetate extract (low polar) showed properties of an enzyme inhibitory compound for both the isolates. VITIPB-3 showed an inhibition of 46.8% while VITIPB-23 showed the same to be 60%.
The antibacterial activity was performed against the drug resistant strains in order to check the cidal effect of the isolates on the clinically obtained, multi drug resistant pathogenic strains. The isolates were primaryly screening and the potent strains were then subjected to secondary screening by the agar well diffussion technique (pickup et al.). Isolate VITIPB-9 shows highest susceptibility toward all the pathogenic strains tested with while VITIPB-3 shows growth inhibitory effect towads Micrococcus alone, that too at nominal levels. Out of the three isolates tested for, isolate VITIPB-23 shows an excellent antimicrobial effect towards all the pathogenic strains used - Moraxiella sp, Micrococcus leuteus, Salmonella typhi and Bacillus subtilis (Table 1).
The antioxidant activity was performed using DPPH (2,2-diphenyl-1-picrylhydrazyl), a light sensitive dark colored crystalline powder composed of stable free radical molecules. It is a brilliant scavenger for other free radicals present in the reaction mixture and hence indicates the radical nature based on the rate of reduction. The rate of decolourisation of the coloured compound to a pale yellowish solution determines the scavenging property of the isolates on the basis of its potential to release hydrogen in the reaction mixture (Sousa et al., 2012). Denser colour represents less of scavenging property while a pale shade implies more of hydrogen in the reaction mixture which gets liberated as a result of its scavenging. Since, DPPH is extremely light sensitive by nature, a quantitative analysis base on this, yields almost accurate results if performed neatly (Figure 2).
Isolate VITIPB-9 shows 100% DPPH reducing property proving it to be an excellent antioxidant by itself getting oxidized. This means that the isolate is capable of releasing almost all the H+ present in solution. Isolate VITIPB-3 is no less with a % scavenging rate of 96%, indicating that almost all of the H+ is liberated with a few remaining in the solution. Similarly, isolate VITIPB-23 shows an antioxidant property of 67.9%. In other words, VITIPB-9 gets completely oxidized, followed by VITIPB-3 and VITIPB-23 which gets partially oxidized (inversely proportional to the %scavenging rate).
Marine actinobacteria being rich producers of secondary metabolite are of major significance in pharmaceutical industry. These metabolites show a varied range of application, each with an unique medicinal purpose. The designing of a naturally obtained drug from various biological sources raises the difficulty level during its manufacture synthetically. To overcome t his problem, the chemically extracted novel compounds from the biological sources are considered to be the basis on which a new antibiotic can be designed. Our current study reveals that the screened actinobacterial isolates of marine origin to posses potential for the same. Since majority of the existing drugs are of terrestrial origin, a rare isolate of marine environment is of great importance..
Figure 1: DNSA Assay for Enzyme Inhibitory Activity.
Table 1: Antimicrobial Zone of Inhibition
Figure 2 : DPPH Assay for antioxidant potential (% scavenging rate)