Antimicrobial Activities of Extracts of Some Species of Mangrove Plants and A New Compound Isolated Towards Some Selected Strains

Introduction

The recent investigations are concentrating on the bio-screening of natural products have revived due to the paucity of safe antimicrobial drugs, anti-reverse transcriptase, anti-HIV and the perilous upsurge of new and re-emerging infectious diseases ^1,2,3. The antibiotics from natural sources are efficacious, biodegradable, less toxic and cost effective and therefore, it could supplement the costly synthesized antibiotic drugs ^4,5,6. Bio-potentiality of mangrove vegetal makes them as a reserved for the development of pharmaceuticals, fish and animal feed additives, agrichemicals and natural pigments ^7,8,9. The mangrove preparations used successfully in the hospitalization of infectious diseases and aliments are envisaged to possess antimicrobial potency ^10,11,12 .

In the present investigation, the different biological parts of four mangrove species namely Aegiceras Corniculatum, Excoecaria agallocha, Rhizophora mucronata and Xylocarpus granatum have been extracted with different solvents like hexane and methanol. These extractes have been screened for antimicrobial activity towards the strains Bacillus puvuilis, Bacillus subtilis, Bacillus coagulans, Staphylococcus aureus, Bacillus licheniformis, Corynebacterium diphtheria, Klebsiella pneumonia, Pseudomonas aeruginosa, Shigella flexneri, Sphingomonas paucimobilis, Escherichia coli and Vibrio cholera  and found to be results are encouraging hence they are presented comprehensively in this article.

Materials and Methods

Collection of Mangrove Medicinal Plants

The different species of mangrove plants viz., Excoecaria agallocha and Xylocarpus granatum, were collected from corangi Mangrove forest near Bhiravapalem in Godavary Estuary ( Latitude 160 15’N and Longitude 820 15’ E) and further ,  Aegiceras Corniculatum and Rhizophora Mucronata ( Latitude 80 99’ N and Longitude 760 87’ E) were collected from Kollam mangrove forest near Krishnapatnam Port, Nellore.

Plant Preparation and Extraction

The fresh plants were washed under running tap water and dried in a warm room for 2 to 6 d. The samples were grinded into fine powder and extracted with n-hexane and methanol successively to get n-hexane and methanol extracts. Then, all the crude extracts were kept at   -20 ℃ until further use. The flavone compound getting By using column chromatography over a column of silica gel (Acme brand, 100-200 mesh, and 450 g) using solvents of increasing polarity from n-hexane through EtoAc. In all 200 Fractions (500 ml) were collected.  The fractions displaying similar spots in TLC were combined and the residues from therein were subjected to re-chromatography over silica gel column to yield one pure compound  Fig.I ¹³ In the form of an off-white solid.

Preparation of a Sample

A sample of 100 mg from each extract and compound was dissolved in 1 mL DMSO. The extract and compound was then sterilized by filtration through sterile syringe filter (0.2 µm pore). Finally the filtered extract and compound was stored as aliquots until it was used.

Microbial Strains

Bacillus puvuilis, Bacillus subtilis, Bacillus coagulans, Staphylococcus aureus, Bacillus licheniformis, Corynebacterium diphtheria, Klebsiella pneumonia, Pseudomonas aeruginosa, Shigella flexneri, Sphingomonas paucimobilis, Escherichia coli and Vibrio cholera.

Agar Ditch Diffusion Method

The agar disc diffusion method was employed for the determination of antimicrobial activities of the extracts according to Qaralleh et al¹⁴ some modification. Briefly, inoculum containing 120⁰(15 lb/in2) was spread on Nutrient agar Medium with the respective bacterial strains of bacteria and medium potato dextrose agar for fungus strains. Testing sterile forceps, the sterile filter papers (6 mm diameter) containing the crude extracts (1 or 1.5 mg), standard antibiotics (30 µg of chloramphenicol or 100 µg of amphotericin B) or negative control (DMSO) were laid down on the coverage of inoculated agar plate. The plates were incubated at 37 ±2℃ for 24 h for the bacteria and at room temperature 28±2℃ for 12 h for yeasts strains. Each sample was tested in duplicate and the zone of inhibition was measured as 50 micro liters diameter.

Table 1: Abbreviation of Mangrove Medicinal Plant Extracts & Compound

Name of the Plant Species	Parts used	Extraction of solvent	Abbreviation
Aegiceras Corniculatum	Fruits	Methanol	AGC
Excoecaria agallocha (compound)	Roots	Hexane	EA
Rhizophora mucronata	Fruits	Methanol	RM
Xylocarpus Granatum	Roots	Methanol	XG

 

Screening for Antimicrobial Activity

The antimicrobial activity was carried out by the employing 24h young cultures with the given compounds by using Agar-well diffusion method. The medium was sterilized by autoclaving at 120⁰c (15 lb/in2). About 20 ml of the medium (Nutrient Agar Medium) with the respective bacterial strains of bacteria and medium (Potato Dextrose Agar) for Fungal strains were transferred aseptically into each sterilized petri Plate. The plates were left at room temperature for solidification. Each plate is made 5 wells with equal distance with of 6mm sterile borer. The test compounds were freshly reconstituted with suitable solvents (DMSO) and tested at various concentrations. The samples and the control along with standard (Ciprofloxacin) were places in 6-mm diameter well. In Antimicrobial assays plates were incubated at 28±20c for fungi about 24h and 37±20C for bacteria 12h. Standard with 5µg/ml used as a positive control for antibacterial activity. Activity diameter of the zone of inhibition was measured using Himedia antibiotic zone scale. Observations and results were represented in Table 2.

Figure 1 Click here to View figure

 

Table 2: Results of Antimicrobial Assay mangrove medicinal plants Gram Positive Bacteria’s

S.No	Plant code	Organism/s	500mg/ml	250mg/ml	100mg/ml	Standard	Control
1	AGC	Bacillus puvuilis	No Activity	No Activity	No Activity	43mm	No Activity
2	AGC	Bacillus subtilis	12mm	10mm	No Activity	40mm	No Activity
3	AGC	Bacillus coagulans	13mm	11mm	10mm	34mm	No Activity
4	AGC	Staphylococcus aureus	7mm	No Activity	No Activity	40mm	No Activity
5	AGC	Bacillus licheniformis	No Activity	No Activity	No Activity	34mm	No Activity
6	AGC	Corynebacterium diphtheriae	No Activity	No Activity	No Activity	36mm	No Activity
7	EA	Bacillus puvuilis	19mm	18mm	17mm	43mm	No Activity
8	EA	Bacillus subtilis	No Activity	No Activity	No Activity	40mm	No Activity
9	EA	Bacillus coagulans	No Activity	No Activity	No Activity	40mm	No Activity
10	EA	Staphylococcus aureus	13mm	12mm	11mm	40mm	No Activity
11	EA	Bacillus licheniformis	14mm	12mm	11mm	32mm	No Activity
12	EA	Corynebacterium diphtheriae	11mm	10mm	No Activity	43mm	No Activity
13	RM	Bacillus puvuilis	12mm	No Activity	No Activity	43mm	No Activity
14	RM	Bacillus subtilis	No Activity	No Activity	No Activity	40mm	No Activity
15	RM	Bacillus coagulans	15mm	13mm	11mm	40mm	No Activity
16	RM	Staphylococcus aureus	No Activity	No Activity	No Activity	38mm	No Activity
17	RM	Bacillus licheniformis	No Activity	No Activity	No Activity	28mm	No Activity
18	RM	Corynebacterium diphtheriae	No Activity	No Activity	No Activity	34mm	No Activity
19	XG	Bacillus puvuilis	No Activity	No Activity	No Activity	43mm	No Activity
20	XG	Bacillus subtilis	12mm	10mm	No Activity	40mm	No Activity
21	XG	Bacillus coagulans	15mm	11mm	No Activity	40mm	No Activity
22	XG	Staphylococcus aureus	14mm	13mm	12mm	40mm	No Activity
23	XG	Bacillus licheniformis	11mm	10mm	No Activity	35mm	No Activity
24	XG	Corynebacterium diphtheriae	No Activity	No Activity	No Activity	34mm	No Activity

 

Gram Negative Bacteria’s

S.No	Plant code	Organism/s	500mg/ml	250mg/ml	100mg/ml	Standard	Control
1	AGC	Klebsiella pneumoniae	No Activity	No Activity	No Activity	32mm	No Activity
2	AGC	Pseudomonas aeruginosa	No Activity	No Activity	No Activity	38mm	No Activity
3	AGC	Shigella flexneri	No Activity	No Activity	No Activity	38mm	No Activity
4	AGC	Sphingomonas paucimobilis	No Activity	No Activity	No Activity	31mm	No Activity
5	AGC	Escherichia coli	No Activity	No Activity	No Activity	40mm	No Activity
6	AGC	Vibrio cholerae	No Activity	No Activity	No Activity	29mm	No Activity
7	EA	Klebsiella pneumoniae	11mm	No Activity	No Activity	28mm	No Activity
8	EA	Pseudomonas aeruginosa	10mm	No Activity	No Activity	38mm	No Activity
9	EA	Shigella flexneri	11mm	No Activity	No Activity	32mm	No Activity
10	EA	Sphingomonas paucimobilis	15mm	No Activity	No Activity	40mm	No Activity
11	EA	Escherichia coli	No Activity	No Activity	No Activity	40mm	No Activity
12	EA	Vibrio cholerae	13mm	12mm	11mm	40mm	No Activity
13	RM	Klebsiella pneumoniae	20mm	13mm	10mm	32mm	No Activity
14	RM	Pseudomonas aeruginosa	15mm	No Activity	No Activity	40mm	No Activity
15	RM	Shigella flexneri	16mm	12mm	No Activity	40mm	No Activity
16	RM	Sphingomonas paucimobilis	19mm	13mm	11mm	30mm	No Activity
17	RM	Escherichia coli	16mm	12mm	No Activity	40mm	No Activity
18	RM	Vibrio cholerae	19mm	13mm	10mm	35mm	No Activity
19	XG	Klebsiella pneumoniae	No Activity	No Activity	No Activity	32mm	No Activity
20	XG	Pseudomonas aeruginosa	No Activity	No Activity	No Activity	40mm	No Activity
21	XG	Shigella flexneri	No Activity	No Activity	No Activity	40mm	No Activity
22	XG	Sphingomonas paucimobilis	No Activity	No Activity	No Activity	31mm	No Activity
23	XG	Escherichia coli	12mm	11mm	No Activity	40mm	No Activity
24	XG	Vibrio cholerae	No Activity	No Activity	No Activity	40mm	No Activity

Diameter of the well = 06mm

Volume of the Compound = 50 Micro liters.

Results and Discussion

The Agar well diffusion method which belongs to Gram positive & Gram negative Bacteria’s of different plant extracts and flavone compound towards different strains have been presented in Table 2. The following observations are significant:

Of all the extracts and compound tested, AGC, EA, RM, XG have shown some remarkable antimicrobial behaviour.

With AGC extract, the antimicrobial activity for strains:  Bacillus puvuilis, Bacillus subtilis, Bacillus coagulans, Staphylococcus aureus, Bacillus licheniformis, Corynebacterium diphtheria, Klebsiella pneumonia, Pseudomonas aeruginosa, Shigella flexneri, Sphingomonas paucimobilis, Escherichia coli and Vibrio cholera.

The Gram Positive Bacteria’s are Bacillus subtilis with the values 12 and 10 respectively, Bacillus coagulans with the values 13, 11 and 10 respectively. And Staphylococcus aureus with the value 7 respectively. These strains have no activity against the Gram Negative Bacteria’s.

With EA flavone compound, the antimicrobial activity for strains:  Bacillus puvuilis, Bacillus subtilis, Bacillus coagulans, Staphylococcus aureus, Bacillus licheniformis, Corynebacterium diphtheria, Klebsiella pneumonia, Pseudomonas aeruginosa, Shigella flexneri, Sphingomonas paucimobilis, Escherichia coli and Vibrio cholera. The Gram Positive Bacteria’s are Bacillus subtilis with the values 19, 18 and 17 respectively. These strains are no activity was found against Bacillus subtilis and Bacillus coagulans. Staphylococcus aureus with the values 13, 12 and 11 respectively. Bacillus licheniformis with the values 14, 12 and 11 respectively.  Corynebacterium diphtheria with the values 11 and 10 respectively.  The Gram Negative Bacteria’s are Klebsiella pneumonia and Shigella flexneri with the value 11 respectively. And Pseudomonas aeruginosa with the value 10 respectively. And Vibrio cholera & Sphingomonas paucimobilis with the values 13 & 15 respectively.

With RM extract, the antimicrobial activity for strains: Bacillus puvuilis, Bacillus subtilis, Bacillus coagulans, Staphylococcus aureus, Bacillus licheniformis, Corynebacterium diphtheria, Klebsiella pneumonia, Pseudomonas aeruginosa, Shigella flexneri, Sphingomonas paucimobilis, Escherichia coli and Vibrio cholera. The Gram Positive Bacteria’s are Bacillus subtilis & Bacillus coagulans with the values 12 & 15, 13, 11 respectely. These strains have no activity was found against Bacillus subtilis, Staphylococcus aureus, Bacillus licheniformis and Corynebacterium diphtheria .

The Gram Negative Bacteri’s  Klebsiella pneumonia with the values 20, 13 and 10 respectively. And Pseudomonas aeruginosa with the value 15 respectively,   Shigella flexneri with the values 16 and 12 respectively,  Sphingomonas paucimobilis with the values 19, 13 and 11 respectively. Escherichia coli with the values 16 and 12 respectively. And Vibrio cholera with the values 19, 13 and 10 respectively.

With XG extract, the antimicrobial activity for strains: Bacillus puvuilis, Bacillus subtilis, Bacillus coagulans, Staphylococcus aureus, Bacillus licheniformis, Corynebacterium diphtheria, Klebsiella pneumonia, Pseudomonas aeruginosa, Shigella flexneri, Sphingomonas paucimobilis, Escherichia coli and Vibrio cholera. The Gram Positive Bacteria’s are Bacillus puvuilis &   Corynebacterium diphtheria these strains are no activity. And Bacillus subtilis with the values 12 and 10 respectively, Bacillus coagulans with the values 15 and 11 respectively, Staphylococcus aureus with the values 14, 13 and 12 etc.  And  Bacillus licheniformis with the values 11 and 10 respectively.  The Gram Negative Bacteria’s are Escherichia coli with the values 12 and 11 respectively. Remaining in all Negative Strains has no activity.

Finally the order of effectiveness is found to be:  EA Hexane > RM MeOH > XG MeOH > AG MeOH.  Finally a new flavone compound is found that more effective than the extracts.

Figure 2: Mangrove plants extracts and a new flavone compound activity on some selected strains   Click here to View figure

 

Figure 3: Mangrove plants extracts and a new flavone compound activity on some selected strains   Click here to View figure

The order of Activity is:  EA Hexane (4) > RM MeOH (1) > XG MeOH (2) > AG MeOH (3).

Conclusion

The extracts and new flavone compound of parts of different species of mangrove plants have been tested for their antimicrobial activity towards the strains Bacillus puvuilis,  Bacillus subtilis,  Bacillus coagulans,  Staphylococcus aureus,  Bacillus licheniformis,  Corynebacterium diphtheria, Klebsiella pneumonia,  Pseudomonas aeruginosa,  Shigella flexneri,  Sphingomonas paucimobilis,  Escherichia coli and  Vibrio cholera  adopting Agar-well diffusion method. It is found that EA Hexane Flavone Compound is effective towards Bacillus puvuilis,  Bacillus subtilis, Bacillus coagulans, Staphylococcus aureus, Bacillus licheniformis, Corynebacterium diphtheria, Klebsiella pneumonia, Pseudomonas aeruginosa, Shigella flexneri, Sphingomonas paucimobilis, Escherichia coli and Vibrio cholera  strains while RM MeOH extract is effective towards the strains  Bacillus puvuilis,  Bacillus subtilis,  Bacillus coagulans, Staphylococcus aureus,  Bacillus licheniformis,  Corynebacterium diphtheria,  Klebsiella pneumonia,  Pseudomonas aeruginosa,  Shigella flexneri,  Sphingomonas paucimobilis, Escherichia coli and  Vibrio cholera . The XG MeOH extract is found to be effective  towards the strains  Bacillus puvuilis,  Bacillus subtilis,  Bacillus coagulans,  Staphylococcus aureus, Bacillus licheniformis,  Corynebacterium diphtheria,  Klebsiella pneumonia,  Pseudomonas aeruginosa,  Shigella flexneri,  Sphingomonas paucimobilis,  Escherichia coli and  Vibrio cholera  strains  while AGC MeOH extract is found to be effective towards the strains Bacillus puvuilis,  Bacillus subtilis,  Bacillus coagulans,  Staphylococcus aureus,  Bacillus licheniformis,  Corynebacterium diphtheria,  Klebsiella pneumonia,  Pseudomonas aeruginosa,  Shigella flexneri,  Sphingomonas paucimobilis,  Escherichia coli and  Vibrio cholera. The order of effectiveness is found to be:  EA Hexane > RM MeOH > XG MeOH > AG MeOH.  Finally a new flavone compound is found that more effective than the extracts.

Acknowledgement

We are thankful to Department of Chemistry K L University, Andhra pradesh, India for making available the necessary facilities. We are grateful to Dr.Varaprasad Bobbarala ADHYA BIOSCIENCE PVT.LTD, Visakhapatnam for his suggestions.

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