INTRODUCTION:

In the Ayurvedic system of medicine, herbal extracts have been used for centuries because many constituents with more than one mechanism of action are considered to be beneficial, while no scientific reports are available for its variety of traditional activities¹. An edible basidiomycete mushroom, Agaricus bisporus (Figure 1) which is obtained from grasslands in Europe and North America was selected here for study.

Fig. 1: Agaricus bisporus

While immature, it has two colour states—white and brown—both of which have various names. When portobello mushroom is immature and white, this mushroom may be known as common mushroom, button mushroom, white mushroom, cultivated mushroom, table mushroom, and champignon mushroom^2-3. But when it is brown in colour, this mushroom may be known variously as Swiss brown mushroom, Roman brown mushroom. Hence, the present study was designed to fill up the lacunae in the literature for some of the in-vitro and in-vivo activities of the extracts of the mushroom A. bisporus with a view to provide scientific evidence. One of the main constituents of the mushroom is Riboflavin, also called as Vitamin B₂, which can act as an anti-inflammatory and analgesic agent^4-5. Flavonoids are also present in greater quantity which may act as an anti-inflammatory and anti-oxidant agent⁶. Alkaloids are also present in greater quantity than other constituents in the extract which may give analgesic and anticancer effect⁷. The present study was an attempt to establish the possible Phytochemical and Biological activities (anti-inflammatory, analgesic, antipyretic, antioxidant and antimicrobial) of alcoholic (methanol) and aqueous extract of A. bisporus.

EXPERIMENTAL PART:

Collection and Preparation of Mushroom Materials:

The fresh Agaricus bisporus (White Button Mushroom) were collected from Kharagpur, West Bengal. From the Department of Pharmacognosy, NSHM Knowledge Campus, Kolkata – Group of Institutions, botanical authentication of the sample was done. The mushroom sample was transported in polythene bags to Pharmacognosy laboratory at NSHM Knowledge Campus, Kolkata – Group of Institutions, where they were sorted out and cut into small pieces. The mushroom materials were air dried at room temperature until dry. The dried sample was ground into fine homogenous powder using an electric mill.

Extraction:

In case of methanol as the solvent, alcoholic extract was prepared using soxhlet apparatus, while aqueous extract was produced by simple cold maceration. Highly concentrated methanolic crude extract and aqueous extract were obtained and preserved. Different phytochemical analysis (qualitative chemical tests and chromatographic studies) was carried out to determine the presence of phytoconstituents.

Laboratory Animals:

Female Swiss albino mice aged between 8 to 12 weeks and weighing between 20-25 grams were used in all the In-vivo studies. The animals were allowed to acclimatize for two days prior to experimentation. The experimental animals were kept in the standard cages in the animal house maintained under standard laboratory condition at an ambient temperature of 25°C with 12 hours daylight and 12 hours darkness cycles. The experimental animals were fed on standard rodent pellets and provided with water ad libitum. The animals were permitted for the study under the Institutional Animal Ethical Committee (IAEC). All the protocols of the study were approved by the IAEC with reference number NCPT/IAEC-11/2018.

Acute Toxicity Study:

This was performed for the extracts to ascertain safe dose by the ‘Oral acute toxic class method’, following the Organization of Economic Cooperation and Development (OECD) 423 guidelines. Swiss Albino mice weighing between 20 and 25g and from 8 to 12 weeks of age were procured for the experimental trial. The selected animals were fed with standard feed and drinking water, and monitored on a regular basis. The animals were selected and grouped, six animals per group. Total 18 animals have been selected for acute toxicity study. They were kept fasting 4 hrs prior to the treatment and the test substance was administered in a single dose by the oral route. A single administration of 2000mg/kg body weight p.o. of the extract (methanolic extract was suspended in Tween 80 solution) was administered to the mice. Responses were noted individually after dosing, at least once during the first 30 minutes, with special attention is given during the first 4 hrs and thereafter for a total of 14days. The mice were observed for 3 days to evaluate considerable changes in body weight and other signs of toxicity. There was no considerable change in body weight before and after treatment and no sign of toxicity was observed. When the experiment was repeated again with the same dose level 2000mg/kg body weight p.o. of the mushroom extract for 7 more days and observed for 14 days, no change was observed from the experiments⁸.

Anti-Inflammatory Activity:

Forty two Swiss albino mice were divided randomly into seven groups of six mice each and treated as follows: Group I (normal control) was not induced with inflammation but received Tween 80. Group II (negative control) was induced with inflammation and received Tween 80. Group III (positive control) was induced with inflammation and received diclofenac (standard drug) at a dose of 15mg/kg body weight. Groups IV, V, VI and VII (experimental groups) were induced with inflammation and received the extracts at the dose levels of 250 mg/kg (methanolic), 500mg/kg (methanolic), 250 mg/kg (aqueous) and 500 mg/kg (aqueous) body weight, respectively. The anti-inflammatory activity of the extracts was assessed using carrageenan-induced right paw edema in mice. Acute inflammation was induced by sub-muscular injection of 0.05 ml 1% carrageenan (sigma-type I) in normal saline 30 minutes after treatment. The change in paw diameter was measured using a digital vernier caliper 30 minutes before injection of carrageenan and at 1, 2, 3 and 4 hours after induction of inflammation. The percentages inhibition in inflammation was calculated using the formula, as follows: Inhibition (%)={(C_t-T_t)/C_t}×100, where, C_t=Paw diameter at 1 hour after carrageenan administration and T_t=Paw diameter after treatment^9-10.

Analgesic Activity:

Method 1: Eddy’s Hot Plate Method:

The animals were divided into 6 groups of 6 animals each and they were given the following doses: Group 1: served as control and received Tween 80 in distilled water as vehicle. Group 2: considered as standard and Diclofenac sodium in Tween 80 with water at a dose of 10 mg/kg body weight was administered orally. Group 3: served as test 1 and received Agaricus bisporus methanolic extract 250 mg/kg body weight orally. Group 4: considered as the test 2 and they were exposed to Agaricus bisporus methanolic extract 500 mg/kg body weight orally. Group 5: served as test 3 and received A. bisporus aqueous extract 250mg/kg body weight orally. Group 6: considered as test 4 and A. bisporus aqueous extract 500 mg/kg body weight administered orally^11-12.

Screening of analgesic activity by hot plate method:

The animals were placed individually in Hot plate, regulated at temperature (45±0.5ºC) before the treatment and its reaction time was determined. After noting the initial reaction time, the treatment should be given to each mice. Then the each animal was placed in the Eddy’s hot plate under regulated temperature to obtain animal response - licking of the forepaws or jump from the Hot plate surface was recorded as the hot-plate latency^11-12.

Method 2: Tail Flick Method:

For evaluation of analgesic activity, mice were divided into six groups. Each group consisted of six animals. The first group served as control (Tween 80). The second group was used as reference standard (Ibuprofen 100 mg/kg p.o.). Two groups received methanolic extract of Agaricus bisporus at two different doses (250, 500 mg/kg p.o.) and other two groups were exposed to aqueous extract of A. bisporus at two different doses (250, 500 mg/kg p.o.). Evaluation of analgesic activity was done using tail flick method in mice^11-12.

Method 3: Acetic Acid Induced Writhing Method:

All mice were divided into six groups for analgesic test. Each group contained six mice. Control group (received Tween 80), Standard Group (received Diclofenac-Na 75mg, 1ml), Test 1 and 2 (received 250mg/kg and 500mg/kg methanolic extract, respectively) and Test 2 and 3 (received 250mg/kg and 500mg/kg aqueous extract, respectively). The analgesic activity of the samples was studied using acetic acid-induced writhing test. One percent acetic acid was administered intra-peritoneally after 30 min of oral administration of test samples. Diclofenac-Na was also administered intraperitoneally after 15 min. Then the mice were observed for specific contraction of body referred to as “writhing” for the next 10 min^13-16.

Antipyretic Activity:

Aqueous suspension of brewer’s yeast was injected to induce pyrexia in Swiss albino mice. The antipyretic activity of mushroom extracts was evaluated after achieving uniform body temperature in the subject. The test animals were divided into six groups (six mice each), i.e. group I received distilled water (control), group II was exposed to paracetamol at a dose of 150 mg/kg b.w p.o (standard), group III and group IV received methanolic extract of Agaricus bisporus at a dose of 250 and 500 mg/kg body weight, respectively and group V and group VI were exposed to aqueous extract of Agaricus bisporus at a dose of 250 and 500 mg/kg body weight, respectively^{13, 17}.

Antioxidant Activity:

Ferric reducing antioxidant power (FRAP) assay:

The total antioxidant potential of a sample was determined using the ferric reducing ability of plasma FRAP assay by Benzie and Strain (1999) as a measure of antioxidant power. The assay was based on the reducing power of a compound (antioxidant).A potential antioxidant will reduce the ferric ion (Fe3+) to the ferrous ion (Fe2+), the latter forms a blue complex (Fe2+/TPTZ), which increases the absorption at 593 nm. Briefly, the FRAP reagent was prepared by mixing acetate buffer (300mM, pH 3.6), a solution of 10 mM TPTZ in 40 mM HCl, and 20 mM FeCl₃ at 10:1:1 (v/v/v). The reagent (3.400μL) and sample solutions (100μL) were added to each well and mixed thoroughly. The absorbance was taken at 593 nm after 30 min. Standard curve was prepared using different concentrations of trolox. All solutions were used on the day of preparation. The results were expressed as μmol trolox equivalent/g. Analyses were performed in triplicate using each extract¹⁸.

ABTS assay:

The ABTS cation radical (ABTS•+) which absorbs at 743 nm (giving a bluish-green colour) is formed by the loss of an electron by the nitrogen atom of ABTS (2,2’-azino-bis (3ethylbenzthiazoline-6-sulphonic acid). In the presence of trolox (or another hydrogen donating antioxidant), the nitrogen atom quenches the hydrogen atom, yielding the solution decolorization. ABTS can be oxidized by potassium persulphate or manganese dioxide, giving rise to the ABTS cation radical (ABTS•+) whose absorbance diminution at 743 nm was monitored in the presence of trolox, chosen as standard antioxidant^19-20.

DPPH Radical Scavenging Activity:

The extract was dissolved in methanol by using sonicator bath. 500μL of the sample solutions of varying concentrations (2-20μg/ml) were mixed with 500μL of freshly prepared methanolic solution of 0.2 mM DPPH. The solution in the test tubes were shaken well and kept in dark for 15 min at room temperature. The reduction in the colour was measured at 517 nm. The control solution consisted of a mixture of appropriate concentrations of methanol and 500μL DPPH. The blank solution contains 500μL of sample and 500μL of methanol. Ascorbic acid was used as a standard. Results were expressed as percentage of inhibition of the DPPH radical. All determinations were done in triplicates. Percentage of inhibition of the DPPH radical was calculated according to the following equation²⁰. Inhibition of DPPH (%)= (Ac - As/Ac) × 100 Where, Ac=Absorbance of control As = Absorbance of samples (or) standard^{14, 21}.

Statistical analysis:

To analyze the different properties, the assays of the three replications were performed in triplicate and the results were expressed by means±SD (Standard Deviations). For the sensory analysis, expressed by means±SE (Standard Errors) were evaluated for 10 samples of each of the treatments. Experimental data were subjected to Analysis of Variance (ANOVA), using the statistical package SAS (Statistic Analysis System) version 9.1.3 and the differences among means were evaluated by Tukey’s test at a level of 5% significance level.

Antimicrobial Activity:

Test Organisms and Inocula Preparation:

The inocula of the test bacteria were prepared using the ‘colony suspension method’. Colonies picked from 24h old cultures grown on nutrient agar were used to make suspensions of the test organisms in saline solution to give an optical density of approximately 0.1 at 600nm. The suspension was then diluted to 1:100 by transferring 0.1mL of the bacterial suspension to 9.9mL of sterile nutrient broth before being used.

Macrobroth Dilution for Determining Minimum Inhibitory Concentration (MIC):

Minimum inhibitory concentration (MIC) deﬁned as the lowest concentration which resulted in maintenance or reduction of inoculums’ viability was determined by ‘serial tube dilution technique’ for the bacterial isolates. Different concentrations 19.5–10000 μg/mL of the crude extract and 0.0195–10 μg/mL of ciproﬂoxacin were diﬀerently prepared by serial dilutions in the Mueller Hinton broth medium. Each tube was then inoculated with 100μL of each of the adjusted bacterial strains. Two blank Mueller Hinton broth tubes, with and without bacterial inoculation, were used as the growth and sterility controls. The bacteria-containing tubes were incubated aerobically at 37^◦C for 24h. After the incubation period, the tubes were observed for the MICs by checking the concentration of the ﬁrst tube in the series (ascending extract and antibiotic concentrations) that showed no visible trace of growth. The ﬁrst tube in the series with no visible growth after the incubation period was taken as the MIC^22-25.

RESULTS AND DISCUSSION:

Phytochemical Potency:

Agaricus bisporus mainly contains a high level of nutrients: dietary fiber (chitin), essential, semi-essential amino acids, unsaturated fatty acids including linoleic and linolenic acids, easily digestible proteins, sterols, phenolic and indole compounds, and vitamins − especially provitamin D2 and B1 , B2 , B6 , B7 , and C.

Anti-Inflammatory Activity:

The methanolic and aqueous extracts of A. bisporus showed potent anti-inflammatory activity on carrageenan induced paw edema in mice. The anti-inflammatory activity of extracts of A. bisporus was comparable to diclofenac (Standard drug). After the third and fourth hour of treatment, the extracts were found to be very active at the dose level of 500 mg/kg body weight (Figure 2).

Fig 2: Diagram shows Anti-inflammatory effects of methanolic and aqueous extracts of Agaricus bisporus on carrageenan-induced inflammation in mice.

The extracts of Agaricus bisporus could, therefore, be an alternative bio-resource for generating anti-inflammatory agents.

Analgesic Activity:

The methanolic and aqueous extracts of A. bisporus showed a challenging analgesic activity in mice. The analgesic activity of extracts of A. bisporus was comparable to diclofenac (Standard drug). The extracts were most active at the dose level of 500 mg/kg body weight after the thirty minutes of treatment (Figure3-5).

Fig 3: Diagram shows Analgesic effects of methanolic and aqueous extracts of Agaricus bisporus in mice by Eddy’s hot plate method.

Fig 4: Diagram shows Analgesic effects of methanolic and aqueous extracts of Agaricus bisporus in mice by tail flick method.

Fig 5: Diagram shows Analgesic effects of methanolic and aqueous extracts of Agaricus bisporus in mice by acetic acid induced writhing method.

The present study scientifically confirms and supports the traditional use of Agaricus bisporus in the management of inflammation.

Antipyretic Activity:

The methanolic and aqueous extracts of A. bisporus showed very potent anti-pyretic activity in mice. The anti-pyretic activities of extracts of A. bisporus were comparable to diclofenac (Standard drug). The extracts were most active at the dose level of 500 mg/kg body weight after the thirty minutes of treatment as anti-pyretic agents (Figure 6).

Fig 6: Diagram shows Anti-pyretic effects of methanolic and aqueous extracts of Agaricus bisporus in mice.

The present study, therefore, scientifically confirms and supports the traditional use of A. bisporus in the management of pyrexia. According to the past report, the methanolic extracts showed analgesic property, but here in this study anti-Inflammatory, analgesic and antipyretic properties have been conducted and reported in comparison with methanolic and aqueous extracts of A bisporus.

Antioxidant Activity:

In the FRAP study, the absorbance of A. bisporus clearly increased due to the formation of the Fe²⁺TPTZ complex with increasing concentration of both the extracts compared to the standard ascorbic acid. Hence, the mushroom should be able to donate electrons to free radicals (Figure7).

Fig 7: Antioxidant activity of methanolic extract and aqueous extract of Agaricus bisporus by FRAP assay using ascorbic acid as standard.

Moreover, the scavenging of ABTS⁺ radical by the extracts of A. bisporus in this study was found to be effective 1 mg/ml. The reduction capability of ABTS radicals was determined by the reduction in its absorbance at 734nm, which was induced by antioxidants. This shows that Agaricus bisporus extract present a good ability to scavenge the ABTS radical (Figure 8).

Fig 8: Antioxidant activity of methanolic and aqueous extracts of Agaricus bisporus by ABTS scavenging activity in percentage using ascorbic acid as standard.

A relationship between the EC50 of DPPH scavenging activity, FRAP assay, phenolic and flavonoid was established among different types of extracts of mushroom. In brief, the antioxidant activity exhibited to a certain extent was probably due to other antioxidant components present in these mushroom extracts besides the phenolic compound. This also indicates that phenolic compounds extracted might cover from moderate polarity to low polarity due to different antioxidant activity (Figure 9).

Fig 9: Antioxidant activity of methanolic and aqueous extracts of Agaricus bisporus by DPPH scavenging activity in percentage using ascorbic acid as standard.

Antimicrobial Activity:

The antibacterial activity of methanolic extracts Agaricus bisporus was evaluated at five different concentrations (100µg/ml, 200 µg/ml, 300 µg/ml, 400 µg/ml, 500 µg/ml). In the present study, methanolic extracts of A. bisporus exhibited considerable antibacterial activity against all tested organisms at high concentrations. The results displayed that the methanolic extracts of 400 µg/ml and 500 µg/ml concentrations had more antibacterial activity. They showed very potent activity against Bacillus subtilis, Escherichia coli and revealed moderate activity against Pseudomonas aeruginosa. But against Staphylococcus aureus, they displayed low activity. The aqueous extracts compared to the methanolic extracts, showed less inhibitory effects against all the tested bacteria. The methanolic extracts of A. bisporus displayed antibacterial activity at 100 µg/ml, 200 µg/ml, 300 µg/ml, 400 µg/ml and 500 µg/ml, while aqueous extracts did not show any antibacterial activity against all the tested bacteria (Table 1).

CONCLUSION:

The extensive literature survey revealed that Agaricus bisporus is important medicinal mushroom with diverse pharmacological spectrum. In the present study, phytochemical and pharmacological investigations of A. bisporus extracts were carried out for different medicinal purpose. The mushroom shows the presence of many chemical constituents which are responsible for varied pharmacological and medicinal property. A. bisporus was observed to be a potential mushroom rich of phenolic and flavonoids could be useful in pharmaceutical and food industry for drugs and additives production. Except these, a rich source of alkaloids and riboflavin may also responsible for the desired pharmacological properties of A. bisporus.

ACKNOWLEDGEMENT:

Authors are thankful to Dr. Subhasis Maity, Director, NSHM Knowledge Campus, Kolkata – Group of Institutions, for providing required facility to complete this work and constant encouragement to perform a better work.

DECLARATION OF INTEREST STATEMENT:

Authors have no conflict of interest in the work and publication of this manuscript in this journal.

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Received on 20.02.2019 Modified on 18.03.2019

Research J. Pharm. and Tech 2019; 12(8): 3811-3817.

DOI: 10.5958/0974-360X.2019.00653.X

Sl. No.	Strains of bacteria (Strain no.)	100 µg/ml	200 µg/ml	300 µg/ml	400 µg/ml	500 µg/ml
1	Bacillus subtilis	+	+	-	-	-
	(ATCC 6051)
2	Staphylococcus aureus	+	+	+	+	-
	(ATCC 12600)
3	Escherichia coli	+	+	-	-	-
	(ATCC 11775)
4	Pseudomonas aeruginosa (ATCC 10145)	+	+	-	-	-