Screening Of Bioactive Substances Isolated Biology Essay

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Abstract

Despite the advances mankind has made in science and technology, drug-resistant strains are posing a major deadly threat to human life. There is an ever-growing need for the outlook of new antibiotics to defeat the drug-resistant strains or control them before they become resistant. This study was undertaken to screen for novel bioactive substances present in moss growing around plum trees from the hill-station in Palani Hills. The collected moss sample was air-dried and then pulverized for further processing. Now the active substances will be separated from the sample by solvent extraction method using the Soxhlet's apparatus. The solvents to be employed are acetone, methanol, hot water and cold water. The filtrate thus obtained will be allowed to evaporate in order to remove the traces of the solvent, leaving behind the crude extract. The crude extract will be used to determine the Minimum Inhibitory Concentration (MIC) and Anti-microbial Activity against clinical pathogens - Escherichia coli, Staphylococcus aureus, Klebsiella, Enterococcus, Shigella, Salmonella, Proteus and Pseudomonas by the agar method.

Keywords: Drug-resistant strains. Antibiotics. Bio active substances. Soxhlet's apparatus. Minimum Inhibitory Concentration.

Introduction

Mosses are important members of the division Bryophyta. They are soft, small plants, typically 11 cm in length. They generally are found in clumps or mats in damp and ill-lit areas. They lack flowers or seeds. Mosses, at times, produce capsules containing spores. Since mosses lack vascular systems, they require damp environments for survival, and need water for reproduction.  Moss species can be classified as growing on: rocks, exposed mineral soil, acid soil, stream sides, shaded humus soil, downed logs, burnt stumps and growing on trees. Specific mosses grow on specific trees. Mosses may survive desiccation, for months, but can be restored to life on rehydration.

There are approximately 12,000 species of moss classified in the Phylum Bryophyta. Mosses have been a source of medicines and anti-microbial agents since ancient times. It is one of the most renowned sources of ancient Chinese and Indian medicines. In the World War I, Sphagnum mosses were used as first-aid dressings on soldiers' wounds, as these mosses are highly absorbent and have mild antibacterial properties. [1]

Sphagnum is easily recognized by its habit of growth and has a typical pale-green colour. Preparations of calcined sphagnum have been used as effective and economical germicides. Sphagnol, a distillate of Peat Tar, is authoritatively recognized as an extremely useful application in eczema, psoriasis, pruritus, haemorrhoids, chilblains, scabies, acne and other forms of skin diseases, while it is very beneficial for allaying irritation arising from insect bites. For the latter purpose it is a preventative no less than a cure.

Now with the advent of more and more numbers of new drugs and anti-microbial agents for the treatment of diseases, the numbers of resistant forms of the pathogens are also on a rise. Indiscriminate uses of drugs and self-administration have led to the development of various multi-drug resistant forms which are difficult to deal with. Hence, workers are now looking at the possibilities of developing newer ways of tackling disease causing organisms by using various plant extracts. Mosses are a promising source of medicinal compounds that have been used in the olden times for similar purposes. Now, scientists are hence re-considering the prospects of using extracts from mosses for curing diseases and solving many of the difficult complications.

A study was conducted by Irudayaraj et al., (2011)[2] to screen the anti-cancer spike-mosses for the presence of various bioactivities and to identify the important bioactive chemicals present in Selaginella inaequalifolia (Hook and Grev). Results of preliminary phytochemical screening on five different extracts (petroleum ether, benzene, chloroform, ethanol and distilled water) of the spike-mosses. S. inaequalifolia show the presence steroids, triterpenes, phenolic group, tannin, sugars and catechin. Alkaloids, amino acids, anthraquinone and reducing sugar did not show any positive result. Among the five different extracts, ethanol and chloroform extracts show the presence of maximum number of compounds. The results on antimicrobial studies show that all the three microbes, Escherichia coli and Candida albicans tested are resistant to the ethanol extract and susceptible to petroleum ether extract. The petroleum ether extract shows maximum inhibition with 45 mm of inhibition zone in C. albicans. The inhibition zone in S. aureus and E. coli are 26 mm and 22 mm respectively.

Another study undertaken by Elibol et al., (2010)[3] indicated that ethanolic and methanolic extracts of Synthrichia ruralis had inhibition effect against Salmonella, E. coli and Saccharomyces cerevisiae, while acetone and chloroform extracts were inactive against all test microorganisms. The extract of Bryum capillare was active against only five strains (Salmonella, Staphylococcus aureus, Bacillus cereus, Psuedomonas aeruginosa and S. cerevisiae). The extract of Pholiota squarrosa showed moderate activity against some strains but, chloroform extract had prohibitive effect against only B. cereus strain. The acetone extract of Grimmia anodon was active against only C. albicans strain. Orthotrichum rupestre extracts were inactive against Salmonella, P. aeruginosa and C. albicans.

Table 1: Anti-microbial activity of Bryophytes in different solvents [3]

Materials and methodology

During this study, the anti-microbial activity as well as the Minimum Inhibitory Concentration (MIC) of moss collected from Palani Hills was studied.

Sample Collection: The moss sample was collected from a hill-station in Palani Hills which was found growing at the base of pine trees. The moss was placed in sterile polythene bags and transported to the laboratory. It was then dried in the sun and finally ground into a fine powder using a mortar and pestle. The powder was used to obtain the acetone and methanol extracts. The crude extract from these solvents was assumed to contain bioactive compounds that would have an inhibitory effect on clinical pathogens. To test this, the Minimum Inhibitory Concentration was determined by the well diffusion method.

Materials Required: The following materials and reagents were used- Ground Moss Sample, Acetone, Methanol, Acetone Extract of Sample, Methanol Extract of Sample, Mueller Hinton Agar (MHA), Nutrient Agar (NA), Nutrient Broth (NB), 95% Ethanol (for sterilization), DMSO (as a solvent for the dried extract and Antibiotic Discs (Streptomycin). Clinical pathogens were also were procured from the Microbial Biotechnology Laboratory, Hexagon, VIT University. These were: Escherichia coli, Staphylococcus aureus, Klebsiella pneumonia, Enterococcus spp., Shigella spp., Salmonella spp., Proteus spp., Pseudomonas aeruginosa.

Figure2: Pathogens used for determining MIC

Soxhlet Extraction: The powdered moss was used to obtain the methanol and acetone extract using Soxhlet's apparatus. The temperature was maintained at below 56oC (boiling point of the solvents). The extraction was continued for around 48 hours. Then extract was then collected and dried under reduced pressure to remove all traces of the solvent.

Figure1: Acetone Extract Figure 4: Methanol extract

Cold Water Extract: 6g of ground dried moss was added to an Erlenmeyer flask and mixed with 200ml of cold distilled water. The flask was then kept on a rotary shaker for 24 hours at __ rpm speed. After 24 hours, the solution was filtered into a beaker using a filter paper and plastic filter. The filtrate obtained was used as the water extract during the study of the synergistic effects along with various antibiotics.

Antimicrobial Assay: For both the assays, the dried extract was scraped from the container and weighed. 300mg of the extract was dissolved in 6ml of DMSO (di-methyl sulphoxide). This gave a concentration of 50mg/ml. plates containing Mueller Hinton Agar were prepared and were swabbed with the clinical pathogens listed above. Wells were made in the agar with a cork borer and the extract was added in different volumes. The volumes added were 25u, 50ul, 75ul and 100ul. An antibiotic disc of Streptomycin was also added to each plate as a positive control. A separate plate was made as the negative control; only DMSO was added to this plate. The plates were then incubated for 24 hours at 37oC. After incubation, the plates were checked for zones of inhibition and they were measured in millimetres (mm).

Figure3: Plates inoculated with the extract

Synergistic Effect: After the zones of inhibition were measured, the synergistic effects of 4 antibiotics along with each of the extracts; i.e. water extract, acetone extract and methanol extract were studied. To determine whether the antibiotics were in synergy with each of the extracts, the following assay was conducted. Plates containing Mueller-Hinton Agar were prepared. From the results obtained previously, it was observed that E. coli, Klebsiella and S. aureus showed inhibition with acetone as well as methanol extracts. Therefore, only these 3 micro-organisms were considered. Next, 4 antibiotics were selected viz. Streptomycin, Vancomycin, Penicillin and Tetracycline. The antibiotic discs were taken and soaked in the water, acetone and methanol extract. Then they were placed at appropriate distances on the swabbed plates. Controls for all the antibiotics were also made. The plates were then incubated at 37oC for 24 hours. The zones of inhibition were measured and the fold-increase percentages were calculated for each extract.

Results and Discussion

The aim of this study was to assess the antimicrobial activity of the moss sample collected from the Palani hills. To do this, well diffusion test was performed. The active ingredients extracted from the moss using a Soxhlet apparatus with different solvents viz. acetone and methanol. 50mg.mL-1 concentration of the extract was loaded in the wells made on a culture medium previously inoculated with eight different pathogenic organisms. Inhibition zones were observed due to the presence of the active ingredients against the pathogens in the extracts. The diameters of the inhibition zones recorded in millimetres are given in Table 2 and 3.

Figure4 and Figure5: Zones of inhibition in Staphylococcus spp. and E. coli

Table 2: Zones of inhibition obtained with Acetone Extract

Micro-organisms

Diameter of Zone (mm)

25µl

50µl

75µl

100µl

E.coli

13

18

18

22

Salmonella spp

-

-

-

-

S.aureus

-

-

-

-

Enterococcus

-

-

-

-

K.pneumoniae

16

14

13

18

P.monocytogens

-

-

-

-

P.aeruginosa

-

-

-

-

Shigella spp

12

12

12

20

Key for table 2: '-': No activity observed

Negative control (DMSO): No Zone

According to the readings obtained, the acetone extract showed antimicrobial activity on Escherichia coli, Klebsiella pneumoniae and Shigella spp.

Table 3: Zones of inhibition obtained with Methanol Extract

Micro-organisms

Diameter of Zone (mm)

25µl

50µl

75µl

100µl

E.coli

10

12

14

15

Salmonella spp

12

11

13

12

S.aureus

7

11

12

13

Enterococcus

-

-

-

-

K.pneumoniae

11

12

12

15

P.monocytogens

-

-

-

-

P.aeruginosa

-

-

-

-

Shigella spp

-

-

-

-

Key for table 3: '-': No activity observed

Negative control (DMSO): No Zone

From the table, it was observed that methanol extract showed antimicrobial activity on Escherichia coli, Salmonella spp., Staphylococcus aureus and Klebsiella pneumoniae.

As E. coli, S.aureus and Klebsiella showed zones of inhibition with both acetone and methanol extracts, they were used for calculating the fold-increase percentage. To do this, a combination of antibiotic discs and extracts were used. The results have been summarized in Table 4.

Synergistic Values:

Micro-organism

Antibiotic Used

Diameter of Zones of Inhibition (mm)

Fold-Increase Percentage (%)

= [ (b-a) / a] *100

Antibiotic Only (a)

Antibiotic + Extract (b)

Antibiotic Control

Water Extract

Methanol Extract

Acetone Extract

Extracts

W

M

Staphylococcus

aureus

Penicillin

30

36

36

33

20

20

Streptomycin

24

30

30

29

25

25

Tetracycline

28

33

36

32

17.8

28.5

Vancomycin

21

29

28

28

38.1

33.3

Escherichia coli

Penicillin

-

-

-

-

-

-

Streptomycin

14

12

14

17

-

0

Tetracycline

-

9

10

11

-

-

Vancomycin

-

-

-

11

-

-

Klebsiella pnuemoniae

Penicillin

-

-

-

-

-

-

Streptomycin

10

12

13

14

20

30

Tetracycline

22

25

25

30

13.6

13.6

Vancomycin

-

-

-

-

-

-

Table 4: Fold-Increase Percent for Water Extract, Acetone Extract and Methanol Extract along with Antibiotics

Key: W- Water Extract; M- Methanol Extract; A- Acetone Extract

The above values were used to calculate the fold-increase percentage by using the formula:

Fold increase %= [ (b-a) / a] *100 ;

Where- a: Zone of inhibition of the antibiotic only

b: Zone of inhibition of antibiotic + extract

For S. aureus, Penicillin showed a fold-increase of 20% with water extract, 20% with methanol extract and 10% with acetone extract. Streptomycin showed 25%, 25% and 20.8% with each of the extracts respectively. Tetracycline showed 17.8%, 28.5% and 14.2% while Vancomycin showed 38.1%, 33.3% and 33.3% fold-increase with water extract, methanol extract and acetone extract.

For E. coli, Penicillin showed no fold increase with any extract. Streptomycin showed no fold increase with water extract and methanol extract and 21.4% with acetone extract. Tetracycline and Vancomycin did not show any fold-increase.

For Klebsiella pnuemoniae, Penicillin did not show any fold-extract. Streptomycin showed 20%, 30% and 40% with each of the extracts respectively. Tetracycline showed 13.6%, 13.6% and 36.3% with each of the extracts while Vancomycin did not.

Conclusion

The results were interpreted and it was determined that the methanol extract was more effective against clinical pathogens than the acetone extract. This was because it succeeded in inhibiting Salmonella spp. and Staphylococcus aureus in addition to Escherichia coli and Klebsiella pnuemoniae. The reason for this is that, methanol is more polar than acetone and thus was able to extract the bioactive compounds from the moss more effectively. This implies that further studies using different solvents of increasing polarity will be able to determine the solvent which is most effective in extracting the bioactive compounds from the moss. It was also observed that antibiotics in combination with extracts from moss show enhanced anti-microbial activity against clinical pathogens. This study can hence be considered during the development of anti-microbial drugs using moss extracts in combination with antibiotics against antibiotic-resistant micro-organisms.

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