GC-MS Analysis of extract for Endophytic fungus Acremonium coenophialum and its Antimicrobial and Antidiabetic

 

Rashid Rahim Hateet

Department of Biology, College of Sciences, University of Misan, Amarah, Maysan, Iraq

 

ABSTRACT:

The aim of the present work was to study antimicrobial and antidiabetic activities of the fungal extract of endophytic fungus Acremonium coenophialum isolated from medicinal plant Myrtus communis was evaluated. The fungal extract was carried out by Ethyl acetate solvent. Column Chromatography, Thin Layer Chromatography (TLC) and Gas chromatography-mass spectrometry (GC-MSS) were used to determine the chemical composition of fungal extract from endophytic fungus A. coenophialum. As a result, a total of nineteen compounds of Fungal extract were demonstrated for antimicrobial activity against seven Gram-Positive Bacteria named Staphylococcus aureus, S. epidermidis, Streptococcus Pneumoniae, Strep. pyogenes, Enterococcus faecalis, Bacillus cereus and B. subtilis which range between (10.0-17.0) mm against seven Gram-Negative Bacteria namely; Escherichia coli, Salmonella typhi, Pseudomonas aeruginosa, Klebsiella pneumoniae, Morganella morganii, Serratia sp. and Proteus vulgaris which range between (10.0-18.0) mm and against both yeasts test was (10.0) mm. Minimum inhibitory concentrations were in the range between (25.0-100), (50-100)μg/ml and (100)μg/ml against Gram-Positive bacteria, Gram-Negative and yeasts test respectively. Minimal bactericidal concentrations ranged from (50-100)μg/ml and (100-200)μg/ ml against Gram-Positive bacteria and Gram-Negative bacteria respectively and minimal fungicidal concentrations (200)μg/ml against both yeasts test. The fungal extract also exhibited antidiabetic potential by inhibition of α-amylase activity. The activity was found to be similar to that of acarbose.

 

 

1.    INTRODUCTION:

Natural products play a major role in the discovery of leads for the development of drugs in the treatment of human diseases. Natural products are an unsurpassed source of bioactive compounds and constitute a relevant economic resource for the pharmaceutical, cosmetic and food industry [1][2]. Endophytes, mainly fungi, and bacteria, colonize the living, internal plant tissues without causing visible symptoms of the disease [3]. There are approximately 300,000 different plant species inhabiting our planet and it can be expected a complex community of many cultivable or uncultivable endophytic microorganisms [4][5].

 

Many strategies have been developed to discover structurally novel natural product leads through available biological approaches; microorganisms play a pivotal role in drug discovery due to their immense structural diversity and a wide variety of biological activities [6]. Medicinal plants provide a unique environment for endophytes and have been known as a repository of endophytes with novel metabolites of pharmaceutical importance [7]. A range of microbial species are known to be endophytic, colonizing inter and intracellular spaces of tissues of higher plants without causing apparent damage on the plants in which they live. Often they have proven to be rich sources of bioactive natural products [8][5]. Endophytic fungi that isolate from medicinal plants are more probable show pharmaceutical potentials. Plant endophytic fungi have been found in each plant species tested, and it is rated that there are over one million fungal endophytes occur in the nature [9]. According to [10] more than 1.5 million endophytic fungi are now thought to live within 270000 species of vascular plants, however, the number of species described is only in the range of 70000–100000, the prospects for additional discoveries of interesting fungal metabolites are bright [11]. The wide span of natural bioactive compounds derived especially from the plant-associated microbes has been largely unexplored [12]. The secondary metabolites produced by endophytes associated with medicinal plants can be exploited for curing diseases [13]. Novel antibiotics, immunosuppressants, antimycotics, anticancer, antidiabetic compounds are few natural products gained from endophytic fungi [14]. The production of bioactive compounds can be increased by biotechnology of endophytic fungi for keeping ecosystem sustainable and biodiversity [15]. In the past few years, Gas chromatography-Mass spectrometry (GC-MS) is used as one of the technological platforms for fingerprint analysis of secondary metabolites in both plant and non-plant species [16]. Diabetes mellitus is one of the major global health and economic problem, characterized by high levels of blood glucose resulting from defects in insulin production, insulin action, or both. Diabetes has affected 6% of the world’s population [17][18]. The present study was aimed to identify the chemical constituents in Ethyl acetate extract of A. coenophialum fungus was analyzed by the GC-MS technique and antimicrobial activity and antidiabetic in vitro.

 

2. MATERIALS AND METHODS:

2.1. Sample collection, isolation and characterization of fungi:

The roots of Myrtus communis collected from Maysan city south of Iraq. After the collection, the roots were washed by water and then by distilled water. The root surfaces were sterilized by consecutive immersion in 70% ethanol for 2sec, sterile distilled water for 2sec, 11% aqueous sodium hypochlorite for 1–5 min and 70% ethanol for 2sec, and then in sterile distilled water. The roots were placed in Petri dishes containing potato dextrose and agar supplemented with 100µg/ml streptomycin and incubated at room temperature. Endophytic fungi growing from the plant tissues was picked and re-cultured on malt extract agar to determine culture purity. The identification of endophytic fungi was performed by analysis of macroscopic and microscopic characteristics of colonies [19][20].

 

2.2. Fungal metabolite extraction:

Five discs (0.5mm diam.) were cut from the isolated fungal culture by using a cork borer, and put it in 500ml of Potato Dextrose broth medium (triplicates) then incubated at 27◦C for 14 days on a rotary shaker. Fungal cultures were filtered through Whatman No 1 filter papers (20–25μm) and the pH was adjusted at 3 by HCl for fungal filtrate. The Fungal filtrate was extracted with Ethyl acetate (1:1 v:v) by using a separating funnel. The organic layer was collected by dehydration of water by using Na2 SO4. The filtrate was filtered again and placed in Petri dishes then left to be dray at room temperature. 100µg of the dried secondary metabolites was dissolved in one ml ethanol as stock secondary metabolites solution to be used for further experiments [19].

 

2.3. Analysis of fermentation product using TLC:

Concentrated EtOAc extract was spotted on TLC Plate and was placed into a chamber filled with eluent mixture petroleum: chloroform (7:3) (v/v) T.L.C analysis was done under UV light at a wavelength of 254 nm. Then, the plate was sprayed with a mixture containing 1% Ce (SO4)₂ and 10% H₂SO₄ and heated for 5 min at 110^oC. Once components of the fraction were successfully separated with good Rf value with appropriate eluent mixture, bioautography was done according to the protocols explained by Choma and Grzelak (2011) with slight modifications. The suspension of tested bacteria and yeasts were prepared (1×10⁸CFU/mL) and inoculated on Muller Hinton agar medium and Sabourauds dextrose agar respectively. The developed TLC plate was then placed on the agar previously seeded with the suspension of tested bacteria for 24 hr. and yeasts for 48 hr. to allow diffusion of bioactive compounds. Then the TLC plate was removed, and the agar layer was incubated at 37°C for 24hr. The inhibition zone was then observed on the agar plate. The spots that exhibited antimicrobial activity were located by compared with the TLC plate that was previously removed.

 

2.4. Column chromatography:

This technique was used a glass column with 1.5 x 35 cm then filled with 15gm of silica gel C60 as a stationary phase, and after a solvent of 0.1gm of fungal extract was used and eluted by petroleum: chloroform ratio (7:3) v\v as a mobile phase with flow rate of 0.5ml/minutes, until the separation was finished. The fractions of fungal extract were collected in vials. These fractions were tested for purity by using thin layer chromatography (TLC) [4].

 

2.5. Susceptibility Disk Method of Fractions:

The antimicrobial activity of fractions from Chromatography Column Analysis (F1-F5) was evaluated using the agar diffusion method. All clinical bacterial isolates were obtained from the Microbiology laboratory of Pharmacy College/Misan University/Iraq.

 

2.5.1. Minimal inhibitory concentration and minimal bactericidal concentration and Minimal fungicidal concentration test:

The minimal inhibitory concentration (MIC), minimal bactericidal concentration (MBC) and minimal fungicidal concentration (MFC) values were determined by the standard serial dilution assay. Fungal extract isolate was selected for this test. The inhibitory test was carried out on Muller-Hinton agar medium for bacteria and Sabourauds dextrose agar for yeasts [19].

 

2.6. Anti-diabetic activity by alpha-amylase inhibition assay method:

Mode of inhibition of crude extract towards α-amylase activity was determined according to the method described by [21] 100μL of the porcine pancreatic amylase (PPA) solution was added to 100μL of tested substance and incubated at room temperature for 20 minutes. The reaction was initiated by the addition of 100μL of 1% soluble starch solution and incubated at 37°C after 10min of incubation were added 200µl dinitrosalicylic acid color reagent. The tubes were kept in a boiling water bath for 10minutes and cooled to room temperature. The reaction mixture was then diluted by adding 5ml of distilled water, and absorbance was measured at 540nm in the ultraviolet (UV)-visible spectrophotometer. The experiments were performed in triplicates, and the alpha-amylase inhibitory activity was calculated as percentage inhibition, using the formula.

 

% Inhibition = ([Abs_control - Abs_samples]/ Abs_control)×100

 

2.7. GC-MS Analysis for Antimicrobial Active Compounds:

The fraction four was subsequently analyzed using a Gas chromatography-mass spectrometer (GC-Ms). The GC-Ms analysis for fraction four of the fungal extract was carried out using gas chromatography-mass spectrometry instrument stands at Al-Mustansiriyah University/College of Science/Department of Chemistry.

 

2.8. Toxicity test:

Cytotoxicity of the fungal extract was examined by using human RBC following a previously described method [22].

 

3. RESULTS AND DISCUSSION:

Fermentation process conducted for 14 days to obtain the maximum amount of secondary metabolites from fungi, fermentation culture was filtered to separate fungal mycelium and media that contained the metabolites. Then media were extracted using Ethyl acetate which has a non-polar characteristic. This solvent was used because MEB media was already contained water, which is polar. GC-MS identification showed that fraction four of fungal extract has 19 detectable compounds (Figure 1) (Table 1).

 

 

Figure 1: The GCMS chromatogram of fraction four of extract A. coenophialum

Table 1: Characteristics of the chemical compounds from GCMS analysis of fraction four extract A. coenophialum. A total of 19 compounds were detected.

 

 

 

 

 

The antimicrobial activity of the fungal extract showed moderate antibacterial effect against Gram-positive bacteria tests with inhibition zone ranging from 10.0mm to 17.5mm and Gram-negative bacteria tests with inhibition zone ranging from 10mm to 18.0mm and antifungal effect against two sets yeast with inhibition zone ranging from 10.5mm to 12.0mm are presented in (Table 2). Endophytes are reported as a novel source of antimicrobial compounds [23]. The fungal extract showed antimicrobial activity with a MIC ranged between 25.0-100ug/ml (Table 2). These results are similar to another study reporting that fungal extract exhibited antibacterial activity against a large number of bacteria reported the MIC [24]. While MBC ranged between 50-100 ug/ml and MFC was 200µg/ml against both test sets yeasts. During long research, only a few numbers of antifungal agents are available for the treatment of various life-threatening fungal infections. The search for new antifungal agents to overcome the growing human problems of drugs resistance in microorganisms is growing. Ongoing global efforts to discover new compounds from EF of medicinal plants are yielding valuable results [25].

 

Endophytic microorganisms are excellent sources of bioactive natural products that can be used to satisfy the demand of pharmaceutical and medical industries), since a single endophyte may be able to produce a variety of bioactive metabolites [26]. The lowest concentration without visible growth was defined as the MIC. (Table1).

 

Gram-Positive:

Bacteria:

Table 2: Growth inhibition zones and MIC and MBC and MFC exhibited fungal extract:

S. aureus	17.0	25	50
S.epidermidis	10.5	25	50
Strep. pneumoniae	14.0	50	100
Strep.pyogenes	10.0	100	200
E. faecalis	10.5	100	100
B. cereus	11.5	50	200
B.subtilis	16.0	25	50
Gram-Negative Bacteria
E. coli	18.0	50	100
Sal. typhi	14.0	50	100
Ps.aeruginosa	10.5	100	200
K. pneumoniae	13.5	100	200
M. morganii	10.0	100	200
Serratia sp	12.0	100	200
P. vulgaris	12.5	100	200
Yeasts			MFC (µg/ml)
C.albicans	10.0	100	200
C.kruzi	10.5	100	200

Inhibition zones (mm)                            MIC(µg/ml)      MBC(µg/ml)

Numbers represent average of three replicates P ≤ 0.05

 

A verification of non-toxicity of the bacterial extract against human blood revealed a negative test. The fungal extract was tested for alpha-amylase inhibitors and they show significant inhibition for alpha enzymes. Percentage inhibitions of alpha-amylase by endophytic fungal extract at various concentrations was calculated and plotted in the (Fig2) the percentage inhibition versus inhibitor concentration was compared for both fungal extract as well as acarbose, a known inhibitor of α-amylase; α-amylase inhibitory studies demonstrated that the fungal extract showed 65.5% of inhibitory activity on α-amylase. There are mechanisms through which medicinal plants can act to control the blood glucose level [27][28]. Such a mechanism is that an alteration of the activity of enzymes involved in glucose metabolism. The α-amylase inhibitors act as an anti-nutrient that obstructs the digestion and absorption of carbohydrates [29]. Inhibition of α-amylase, enzyme that plays a role in digestion of starch and glycogen is considered a strategy for the treatment of disorders in carbohydrate uptake such as diabetes and obesity [17][18] Synthetic inhibitor causes side effect such as abdominal Pain, diarrhea and soft feces in the colon. Similar antidiabetic activity by the endophytic fungal extract was observed by [14].

 

 

Fig. 2: Percent inhibition of alpha-amylase with different concentration of fungal extract

 

4. CONCLUSION:

 Endophytic fungi have proven to be rich sources of novel natural compounds with a wide spectrum of biological activities. Various compounds of pharmaceutical importance are being identified and isolated. The endophytic fungus Acremonium coenophialum isolated from medicinal plant Myrtus communis used for isolation of antimicrobial and antidiabetic compounds by Gas chromatography-mass spectrometry (GC-MSS).

 

5. ACKNOWLEDGMENT:

The author is thankful to the College of pharmacy. Misan University, for the financial support for this project.

 

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Received on 06.06.2019           Modified on 04.07.2019

Accepted on 01.08.2019         © RJPT All right reserved