Research Articles

2019  |  Vol: 4(1)  |  Issue: 1(January-February) | https://doi.org/10.31024/apj.2019.4.1.5
Antibacterial property of Auricularia polytricha Mont. and Trametes versicolor Linn.

Jiph Daniel L. Sanico1, Manuela Cecille G. Vicencio1,2*

1Department of Biological Science, College of Science, Catarman, Northern Samar

2University Research Office, University of Eastern Philippines, Catarman, Northern Samar, Philippines 6400

*Address for Corresponding author:

Manuela Cecille G. Vicencio

University Research Office, University of Eastern Philippines, Catarman, Northern Samar, Philippines 6400


Abstract

Objective: This study was aimed to determine the antibacterial properties of A. polytricha (Mont.) and T. versicolor (Linn.). Material and methods: Specifically, this study tried to find out if there is a significant difference using the extracts of A. polytricha (Mont.) and T. versicolor (Linn.) compared to positive controls specifically, Cefalexin® and Clindamycin® against Staphylococcus aureus and Escherichia coli. Tests for the confirmation for the presence of alkaloid, cardiac glycoside, cholesterol, flavonoid, saponin, steroid, tannin, and terpenoid were also included in the study to identify the presence of secondary metabolites in the A. polytricha (Mont.) and T. versicolor (Linn.) extract. Determination of antibacterial effect was done using the paper-disc method. By measuring the clear zone of inhibition, you can conclude if the extract of A. polytricha (Mont.) and T. versicolor (Linn.) have significant antibacterial effect against Staphylococcus aureus and Eschirichia coli. Results and conclusion: Results showed that extract of Apolytricha (Mont.) contains cardiac glycoside, steroids and tannins while the extract of Trametes versicolor (Linn.) contains alkaloids and steroids. In the determination for antibacterial effect, Auricularia polytricha (Mont.) and Trametes versicolor (Linn.) extract showed more effective result against Staphylococcus aureus and Escherichia coli compared to Trametes versicolor (Linn.) extract. They can be used as a natural, convenient yet effective alternative in the prevention of diseases caused by Staphylococcus aureus and Eschirichia coli.

KeywordsTrametes versicolor (Linn.), Auricularia polytricha Mont., antibacterial properties


Introduction

Mushrooms have a great potential for the production of useful bioactive metabolites and that they are prolific resource for drug. Mushrooms characteristically contain a tremendous, variety of secondary metabolites that display a broad range of biological activities and the content and bioactivity of these compounds depend on how much the mushroom is prepared and consumed. The vast structural diversity of natural compounds found in mushroom, provide potential opportunities for discovering new drugs that rationally target the abnormal molecular and biochemical signals leading to cancer. Experience from ethnomedicine and extensive basic laboratory findings have shown that mushrooms could play an important role in the prevention and treatment of cancer (Russo et al., 2006).

The use of natural resource in the drug discovery has received much attention nowadays, not only for their potential as source of drugs, but also because they are natural, non-synthetic, and safe and their appreciation by consumers are very favorable. In fact, they have been the most successful source of drug leads for many years. The natural sources usually have a biological or pharmacological activity for use in pharmaceutical drug discovery and drug design. Between 1983 and 1994, 39% of antibacterial and anti-infective drugs were derived from natural products. Also, in that same time period, 39% of all new approved drugs were from either natural products or derived from natural products (http://www.lifepharms.com/).

In Asian countries, edible mushrooms have been valued as functional foods and as medicine resource due to their antioxidative, preventive, and therapeutic properties. A variety of secondary metabolites that includes phenolics, polyketides, terpenes, and other steroids are accumulated in mushroom. The phenolic compounds in Shiitake mushrooms (Lentinusedodes) and straw mushroom (Volvariellavolvacea) are believed to have contributed to their ability to scavenge free radicals, chelate metals, and inhibit lipoxygenase. Mushrooms can be used as natural oxidants in the forms of extracts, concentrates, powders or dietary supplements (Yim and Chye, 2010)

For many years, mankind has benefited from plants as natural source of drugs and herbal remedies. In fact, may studies have been focused on developing biological and pharmaceutical activity in plants, but attempts to explore mushrooms in such a way is much neglected. Hence, studies should be made to explore the potential use of mushrooms and their metabolites for treatment of a variety of human ailments.

Mushrooms, similar to plants, have a great potential for the production of useful bioactive metabolites and that they are prolific resource for drugs. Mushrooms characteristically contain a tremendous, variety of secondary metabolites that display a broad range of biological activities and the content and bioactivity of these compounds depends on how the mushroom is prepared and consumed. The vast structural diversity of natural compounds found in mushrooms, provide potential opportunities for discovering new drugs that rationally target the abnormal molecular and biochemical signals leading to fatal diseases especially cancer. Experience from ethnomedicine and extensive basic laboratory findings have shown that mushrooms could play an important role in prevention and treatment of cancer (Russo et al., 2006).

In the present study, Auricularia polytricha (Mont.) and Trametes versicolor (Linn.) were to be evaluated for its anti-bacterial properties since not many studies had been done on the antibacterial properties of this species. Also, these mushrooms were to be evaluated for the presence of secondary metabolites which may lead to new findings of pharmacologically important substances. However, published studies on these mushrooms are quite limited. So the biological activities of Auricularia polytricha (Mont.) and Trametes versicolor (Linn.) metabolites need to be explored for its use as a source of drugs and functional food to contribute to new therapeutic effects and for the treatment of a variety of human ailments. Hence, this study is to be conducted to provide useful information about the said mushrooms based on the results of this study.

Materials and methods

Collection and authentication of mushroom

A. polytricha (Mont.) and T. versicolor (Linn.) were collected in places where there is an abundant supply of this species of mushrooms. Pictures, using a camera, were taken of each specimen directly from the place where it was located. In picking the specimen, a knife is very useful in cutting its base. Specimen was handled carefully to avoid damage. After gathering the collected A. polytricha (Mont.) and T. versicolor (Linn.), it was placed in a closed container so that the important features will not be lost or dry out.

Preparation and extraction of extracts

About 150 grams of freshly collected A. polytricha (Mont) and T. versicolor (Linn.) were segregated and was washed with distilled water. It was grinded using a blender. The 150 grams of each specimen was mixed with a solvent, specifically ethyl acetate (CH3COOCH2CH3) and was placed for 24 hours in an oven where it has the standard room temperature (32ºC). The ratio of the grinded mushroom against the solvent is 1:2 or for every 1 gram of mushroom, its counterpart was 2 mL of the solvent.

Distillation process was needed to get the pure mushroom extract from the mixture of the solvent and the grinded mushroom. Distillation was used to purify the compound by separating it from a non-volatile or less-volatile material. Due to the difference in their boiling point, the compound which has the lower boiling point was separated first from the mixture. The compound which was retained in the distilling flask is said to be a less volatile material or the pure extract of a substance.

The solvent that was used to get the pure extract of A. polytricha (Mont.) and T. versicolor (Linn.) was ethyl acetate (CH3COOCH2CH3). The liquid part of the mixture was filtered and transferred in a distilling flask which was heated for around 78ºC - 79ºC. The said temperature is the boiling point of ethyl acetate which belongs to the family of esters. When the temperature exceeds 79ºC, the ethyl acetate evaporates and the pure extract is retained in the beaker.

Phytochemical screening of Mushroom (Guevara, 2005)

There was only one (1) level of concentration of A. polytricha (Mont.) and T. versicolor (Linn.) extract which were used in phytochemical screening

100% Concentration = 25 mL pure Mushroom extract

Test for the presence of Alkaloid

In the test, Dragendorff’s reagent and Mayer’s reagent were used in determining the presence of alkaloid. A positive result indicates the presence of orange precipitate in Dragendorff’s reagent and white precipitate with the Mayer’s reagent.

For every 2 mL of the extract, add 1 mL HCl and 6 drops of Mayer’s reagent and Dragendorff’s reagent. Any organic precipitate indicates the presence of alkaloids in the sample.

Test for the presence of Cardiac glycoside

For every 5 mL extract, treat it with 2 mL of glacial acetic acid containing one drop of Ferric chloride solution. This was underplayed with 1 mL of concentrated Sulfuric acid. A brown ring of the interface indicated deoxysugar characteristics of cardenolides. A violet ring might appear below the brown ring whereas acid layer, a greenish ring might form just gradually throughout thin layer.

Test for the presence of Cholesterol

2 mL of the extract and 2 mL of chloroform was added in a dry test tube. Then 10 drops of acetic anhydrides and 2 to 3 drops of concentrated sulfuric acid were added. A red color change to blue green color indicates the presence of cholesterol.

Test for the presence of Flavonoid

For the test of the presence of Flavonoid, Shinoda’s test was followed. Few drops of concentrated HCl and Magnesium fillings were added in 1 mL of ethanol extract. Appearance of pink or magenta red color indicates the presence of flavonoids.

Test for the presence of Saponin

2 mL extract with 20 mL of distilled water was agitated in a graduated cylinder for 15 minutes. The formation of 1 cm of foam indicates the presense of saponin.

Test for the presence of Steroids

2 mL of acetic anhydride was added to 0.5 grams of ethanolic extract of each sample with 2 mL of sulfuric acid. The color change from violet to blue or green indicates the presence of steroids.

Test for the presence of Tannin

The Gelatin test was used to determine the presence of tannin. The formation of the jelly-precipitate indicates the presence of tannin. First, take an equivalent of 2 mL of mushroom extract from the stock extract and evaporate this to incipient dryness over a steam bath. Then extract the residue with 2 mL of hot distilled water. Add 5 drops 10% sodium chloride solution then filter and divide the filtrate into three test tubes. Take one of the three test tubes as the control; take an aqueous solution of tannic acid as reference standard. Take one portion of the filtrate of the mushroom extract with 3 drops of gelatin-salt reagent. Do likewise to the tannic acid solution, the reference standard. Compare with the control and the reference standard.

Test for the presence of Terpenoids

5 mL of extract was added to 2 mL of chloroform and 3 mL of concentrated Sulfuric acid to form a monolayer of reddish brown. Coloration of the interface will show to form positive result for the terpenoids.

Antibacterial Screening

Preparation of Culture Media

Suspend 19 grams of Mueller-Hinton Agar in 500 mL distilled water. Then boil the mixture to completely dissolve the medium. Sterilize it by using oven at 100ºC for 15 minutes. Then allow it to cool down about 50ºC and aseptically pour it to individual petri dishes. Allow it to solidify.

Preparation of Test Organism

The cultured Staphylococcus aureus and Escherichia coli was purchased in Philippine Applied Microbiology (PNCM), National Institute of Molecular Biology and Applied Microbiology (BIOTECH), University of the Philippines Los Baños (UPLB). It was incubated at 37ºC for 24 hours to revive and produce more colony growth.

Preparation of the Positive Control

Clindamycin® was used as positive control for Staphylococcus aureus (Gram positive bacteria) and Cefalexin® was used as positive control for Escherichia coli (Gram negative bacteria).

Pound a commercial tablet of an antibiotic (without removing it from its shelf). After turning it into a powder form, open the sachet and place the powdered tablet in a sterile petri dish. Add 1 mL distilled water to totally dissolve the powered tablet.

Preparation of Sensitivity Disc

Cut the Whattman filter paper no. 1 into round disk shapes using a puncher. The number of filter paper discs is dependent on the number of trials in the study. Place each labeled disks in petri dishes and sterilize at 121ºC for 15 minutes in an oven. After sterilizing those filter disks, soak them in the mushroom extract and commercial antibiotic for 1 hour.

Microbiological Technique

The determination of antibacterial property of the extracts was done using Pour-plate technique. Two sets of suspension of 19 grams Mueller-Hinton agar in 500 mL distilled water, one for Staphylococcus aureus and one for Escherichia coli. It was bring to boiled to completely dissolve the medium while stirring constantly. Allow it to cool about 50ºC.

Using sterile inoculating loops, inoculated cultured from two different bacteria was diluted to each set of Mueller-Hilton agar. It was stirred to distribute bacteria so it can grow more colonies.

Assessment of Antibacterial Property

In determining the antibacterial property of the extract, paper disc technique was used. In each Mueller-Hinton agar treated with bacteria, it was carefully and aseptically impregnated with the soaked filter paper disks. The soaked filter paper discs were placed at the center of each agar plate to maximize the space for bacterial growth and also for the bacterial colony to grow more and be given an allowance for possible antibacterial activity which was demonstrated by producing a clear zone of inhibition around each disks. The plate was incubated at 37ºC for 24 hours. After the incubation, the plates were observed for any zone of inhibition (a clear zone) around the disk which indicates that growth of the organism had been inhibited by the chemical agent that diffuses into the agar from disks. Absence on inhibition indicates the resistance of the organism to the chemical agent present in the disk.

Results and discussion

Determinations of Secondary metabolites present in the extracts of Auricularia polytricha (Mont.) and Trametes versicolor (Linn.)

Table 1. Shows the results for phytochemical screening of A. polytricha and T. versicolor extract

Secondary

 Metabolites

Auricularia polytricha (Mont.)

Trametes versicolor (Linn.)

Alkaloids

Negative

Positive

Cardiac glycosides

Positive

Negative

Cholesterol

Negative

Negative

Flavonoids

Negative

Negative

Saponins

Negative

Negative

Steroids

Positive

Positive

Tannins

Positive

Negative

Terrpenoids

Negative

Negative

As shown in table 1, the secondary metabolites present in the extract of A. polytricha were cardiac glycoside, steroids and tannins. A violet ring was obtained which is an indication that cardiac glycoside was present in the extracts of A. polytricha. In the test for the presence of steroids, a color change from violet to green was observed. It indicates that A. polytrichae extract is positive for the presense of steroids. In the test for the presence of tannins, a formation of jelly-like precipitate was observed. It also indicates that A. polytricha extract is positive for that presence of tannins.

In T. versicolor extract, the secondary metabolites present are alkaloids and steroids. In the test for the presence of alkaloids, the formation of white precipitate using Mayer’s reagent was observed. This means that the T. versicolor extract is positive for the presence of alkaloids. In the test for the presence of steroids, a color change to green was observed. This means that the T. versicolor extract is positive for the presence of steroids.

This implies that edible mushroom specifically Auricularia polytricha (Mont.) have more secondary metabolites present in their extracts compared to the non-edible mushrooms specifically Trametes versicolor (Linn.).

Table 2. Shows the result of Auricularia polytricha (Mont.) extract and T. versicolor (Linn.) extract in inhibiting the growth of S. aureus and E. coli

Mushroom

S. aureus

E. coli

Computed t-value

Tabular t-value

Interpretation

Mean

Variance

Mean

Variance

A. polytricha (edible mushroom)

5

4.5

7

2

-1.94

2.776

Not significant

T. versicolor (non-edible mushroom)

2.5

1.5

4.67

8.67

-1.94

2.776

Not significant

The effectiveness of Auricularia polytricha (Mont.) extract and T. versicolor (Linn.) extract in inhibiting the growth of Staphylococcus aureas and Escherichia coli is presented on table 2. As shown in the table, the Auriculariapolytricha (Mont.) extract and T. versicolor (Linn.) extract showed a good antibacterial against Staphylcoccus aureas and Escherichia coli.

The mean zone of inhibition of A. polytricha on S. aureas (gram-positive bacteria) was 5 mm while the mean zone of inhibition of A. polytricha on E. coli (gram-negative bacteria) is 7 mm. On the other, T. versicolor extract showed a mean zone of inhibition of 2.5 mm on S. aureus (gram-positive bacteria) and a mean zone of inhibition of 4.67 mm on E. coli (gram-negative bacteria).

This implies that A. polytricha and T. versicolor showed a greater mean zone of inhibition on E. coli (gram-negative bacteria) and on S. aureus (gram-positive bacteria).

Comparison between the effectiveness of Auricularia polytricha (Mont.) extract against Trametesversicolor (Linn.) extract based on their antibacterial property in each microorganism

Staphylococcus aureus

Table 3. Difference between A. polytricha and T. versicolor extract in inhibiting the growth of S. aureus and E. coli

Bacteria

A. polytricha

T. versicolor

Computed t-value

Tabular t-value

Interpretation

Mean

Variance

Mean

Variance

S. aureus

5

4.5

2.5

1.5

2.51

2.776

Not significant

E. coli

7

2

4.67

8.67

1.75

2.776

Not significant

As shown in table 3, the mean zone of inhibition of A. polytricha on S. aureus was 5 mm while the mean zone of inhibition of T. versicolor on S. aureus was 2.5 mm. It implies that A. polytricha has greater mean zone of inhibitions on S. aureus compared to T. versicolor.

In table 3, since the t-computed value of 2.51 is less than the t-tabular value of 2.776 at 0.05 level of significance with 4 degrees of freedom. This means that there is no significant difference between A. polytricha extract and T. versicolor extract based on its antibacterial action against S. aureus.

Escherichia coli

As shown in table 4, the mean zone of inhibition of E. coli was 7 mm while the mean zone of inhibition of T. versicolor on E. coli was 2.5 mm. It implies that A. polytricha has greater mean zone of inhibition of E. coli compared to T. versicolor.

In table 4, since the t-computed value of 1.75 is less than the t-tabular value of 2.776 at 0.05 level of significance with 4 degrees of freedom. This means that there is no significant difference between A. polytricha extract and T. versicolor extract based on its antibacterial action against E. coli.

Comparison between the effectiveness of Auricularia polytricha (Mont.) extract versus Trametes versicolor (Linn.) versus Clindamycin® for S. aureus and Cefalexin® for E. coli based on their antibacterial property.

Auricularia polytricha vs. Trametes versicolor (Linn.) vs. Clindamycin® on S. aureus

It is shown in tables 5 and 6 the level of significance among Auriculariapolytricha (Mont.) extract, Trametes versicolor (Linn.) extract and Positive control in inhibiting the growth of each microorganism used in the study.

Table 4. Difference between the A. polytricha extract, T. versicolor extract and Clindamycin® in inhibiting the growth of S. aureus and E. coli

Bacteria

A. polytricha

T. versicolor

Clindamycin®

Computed F-value

Tabular F-value

Interpretation

Mean

Variance

Mean

Variance

Mean

Variance

S. aureus

5

4.5

2.5

1.5

5

2

4.7

5.14

Not significant

E. coli

7

2

4.67

8.67

5.83

1.17

2.07

5.14

Not significant

In table 4 it shows that the F-computed value is 4.7 while the F-tabular value is 5.14 at 0.05 level of significance and with 2, 6 degrees of freedom. Since the computed value is less than the tabular value. This means that there is no significant difference between A. polytricha, T. versicolor, and Clindamycin® in inhibiting the growth of S. aureus.

Auricularia polytricha vs. Trametes versicolor (Linn.) vs. Cefalexin® on E. coli

In table 6 it shows that the F-computed value is 2.07 while the F-tabular value is 5.14 at 0.05 level of significance and with 2 and 6 degrees of freedom. Since the computed value is less than the tabular value. This means that there is no significant difference between A. polytricha, T. versicolor (Linn.) and Cefalexin® in inhibiting the growth of E. coli.

Conclusion

The extract of Auricularia polytricha (Mont.) showed a positive result for the presence of cardiac glycosides, steroids and tannins. On the other hand, the extract of Trametes versicolor (Linn.) showed a positive result for the presence of alkaloids and steroids. Edible mushroom specifically Auricularia polytricha (Mont.) contains more secondary metabolites compared to non-edible mushroom specifically Trametes versicolor (Linn.). There is no significant relationship between the microorganisms used in the study specifically Staphylococcus aureus and Escherichia coli using Auricularia polytricha (Mont.) and also Trametes versicolor (Linn.) extract in terms of antibacterial action. Thus they can be used conveniently yet effective alternative in the prevention of diseases caused by Staphylococcus aureus and Escherichia coli.

Conflicts of interest: None

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