INTRODUCTION:

Mushrooms are considered to be one of the most health promoting foods because of its nutritional value and rich source of fibre (beta-glucans and chitin) and protein [1, 2]. In addition, it may contained most essential nutrients e.g. vitamins, selenium, potassium, copper etc. Interestingly, humans are more closely associated with fungi group because of its essential molecules that are reported in human diet [1-4]. As per the literature, nutritional and medicinal value or source of mushroom is mainly affected through various factors i.e. species, type of variety including its developmental stage along with environmental conditions [4-7].

In contrast, specific bioactive compounds (polysaccharides, low molecular weight proteins, glycoproteins etc.) are also reported in medicinal mushrooms and showing diverse medicinal properties [1-7]. The most familiar example in case of drugs extracted from mushrooms which may be helpful in the treatment of cancer, diabetics, high cholesterol, depression etc. In short, different varieties of mushroom are available and showed its economic importance as well and it is reported all over the world [7,8]. One of the most costliest species of mushroom i.e. Cordyceps sinensis, endomopathogenic mushroom growing on insects and is mainly reported in the high altitude regions of Indo-Tibet region [9,10]. Similarly, potential some other type of mushrooms are also available and identified its benefit related to human health.

Oyster mushroom (Pleurotus species belongs to class Basidiomycetes and family Agaricaceae), also popularly known as Dhingri in India and is mainly shown as well as reported in temperate along with tropical forests and also found especially on decaying organic matter [11,12]. In literature, major parts of oyster mushrooms i.e. cap spatula shaped like (pileus), central stalk (stipe; short/long) and long ridges along with furrows underneath the pileus (i.e. gills or lamellae) where as mycelium of this species is pure white in colour [11,12]. In contrast, button Mushroom (Agaricus species; class; family Agaricaceae) is another well known variety of mushroom and already reported in almost all over the world [13,14]. In India, production of this particular species earlier was limited up to the winter season, but with advancement and development in technology, these are produced and reported throughout the year. These two particular species are reported in farms and adopting different levels of technology. In this regard, we worked on these two species of mushroom and determined its anti-inflammatory potential of protein against specific protein antigen (typhoid vaccine) and also measuring its antioxidant potential including phenolic content.

MATERIALS AND METHODS:

Sample collection:

Fruiting bodies of two mushrooms were collected from local market of Dehradun and kept in sterile plastic bags washed thoroughly with sterile distilled water and aseptically break lengthwise using sterile blade. The fruiting bodies of three unknown collected mushrooms were identified as Oyster mushroom and button Mushroom on the basis of its morphological characteristics and molecular characterization. For sample preparation, these two different varieties of mushroom i.e. Oyster mushroom and button Mushroom were analyzed the protein content and determined through Lowry test. In addition, these two species were also analyzed for measuring its phenolic content, antioxidant and anti-inflammatory activity.

Extraction of protein:

In this study, mushroom (1 g) species were taken in two different conical flask and then add equal volume of extraction buffer (i.e. 20mM Tris HCl, dissolved in phosphate buffered saline). Incubate mushroom along with extraction buffer for 5-15 minutes and then centrifuged (5000rpm; 10 minutes at 4-5°C). After centrifuging, collect the supernatant and add equal volume of ice cold acetone. Incubate the solution at room temperature and then collect the pellet after centrifuging and washed with ice cold acetone to remove the pigments. Finally, protein concentration of mushroom species were determined through Lowry method and estimate the protein content in case of oyster (1.5mg/ml) and button (1.76mg/ml)

Estimation of phenolic compound:

Protein extract of mushroom species (1000µg/ml) were mixed with folin-ciocalteu reagent (1.5ml) and allowed to incubate at room temperature for one minute followed by the addition of sodium carbonate (60g/l). The tubes were then vortex-mixed for about 15 seconds, and then, allowed to stand for 90 minutes in the dark at room temperature. Absorbance was then measured at 725nm. Gallic acid was used as standard curve with concentration ranges from 1 to 100 µg/ml and the results were expressed in µg/ml. All experiments were performed in triplicates [15].

Antioxidant activity:

The percentage of antioxidant activity was measured in protein extracted from both species of mushroom. This activity was assessed through DPPH ((2,2-diphenyl-1-picrylhydrazyl) free radical assay using protein (0.5 ml) were reacted with stable DPPH radical (0.3ml; 0.5 mM in ethanol) and also add ethanol (3ml). For these studies, ethanol (3.3ml) and sample (0.5mL) serve as blank whereas control was prepared by mixing of both solution (i.e. 3.5ml ethanol and 0.3ml DPPH radical solution). In this study, major observation especially its color (from deep violet to light yellow) and its absorbance was measured at 517nm after 100 min of reaction using UV-visible spectrophotometer [16].

Albumin denaturation assay:

A volume of 5 ml of reaction consist of 0.2ml of egg albumin (from fresh hen’s egg), 2.8ml of phosphate buffer saline (pH 6.4), and 2 ml of varying concentration of protein of each samples so that final concentrations become 100, 500 and 1000µg/ml. The distilled water was used as control. The mixture was then incubated at 37°C in a BOD incubator for 15 minutes and then heated at 70°C for 5 minutes. Finally, its absorbance was measured at 660nm. For these studies, diclofenac sodium was used as reference drug. The percentage inhibition of protein denaturation [15] was calculated using following formula.

% Inhibition= 100 × (Vt/Vc-1)

Vt = absorbance of test sample

Vc = absorbance of control

Anti-inflammatory activity:

In this study, human red blood cells were used for estimating its potential of protein for determining its anti-inflammatory activity in vitro. Human whole blood samples were collected from pathology lab and mixed with equal volume of saline solution (sterilized). Samples were centrifuged (4000rpm; 10 minutes) and pelleted cells were collected. These pelleted cells were washed with saline solution and was used for the estimation of anti-inflammatory property. In this study, using different concentrations of protein containing fixed concentration of typhoid vaccine, standard (typhoid vaccine) and control were separately mixed with phosphate buffered saline and human red blood cell (500 µl) suspension. All these samples were incubated (37 °C, 60 minutes) and then centrifuged at 3000 rpm, 10 minutes. The supernatant was eliminated and hemoglobin content was estimated through spectrophotometer at 570nm [17].

Antimicrobial assay:

To evaluate the potential antimicrobial activity of mushroom species, three bacterial strains were selected namely Bacillus subtilis and Pseudomonas aeruginosa. The Nutrient agar medium (NAM) was used to culture microbes required for antimicrobial susceptibility test. Microbial strains were cultured on to the agar plate and the broth, followed by overnight incubation at 37⁰ C [18]. The antimicrobial activity of the extracts were determined using MHA (Mueller Hinton Agar), developed in 1941 by Mueller and Hinton, has been widely used for antimicrobial susceptibility test because of its non- selective, non - differential properties and high starch content. MHA is a loose agar and usually appear as translucent and light amber in color.

The antimicrobial activity of two different species of protein mushroom were tested using disc diffusion method. Take 100µl of microbial culture of the respective strains is to be spread with the help of a L- shaped spreader containing MHA (20 ml/ plate). The sterile discs (6mm in diameter) containing residues of the protein were independently impregnated on the agar plates which have been previously been inoculated with the selected microbial strain. The plates were then incubated at 37⁰ C for 24 hours and at 30̊ C and for 48 hours for bacterial and fungal strains respectively. Antimicrobial activity was then determined by measuring the diameter of the growth- inhibition zone in millimeters.

Statistical analysis:

The difference between control and mushroom protein species of variable concentration is determined through one way ANOVA test.

RESULTS:

Phenolic compound and antioxidant assay

The results of these studies claimed that protein from oyster mushroom showed higher phenolic content as compared to button mushroom. In addition, oyster mushroom also showed higher antioxidant activity (using DPPH assay) as compared to button mushroom (Fig.1).

Fig.1. Estimation of phenolic content and antioxidant content in mushroom species.

Values were expressed as Mean ± S.E. The difference between control and mushroom species (Oyster and button, 1000 µg/ml) is determined through one way ANOVA (Bonferroni Multiple Comparison Test)

*P<0.05; **P<0.01

Albumin denaturation assay:

The effect of two different species of mushroom (oyster and button) for determining its denaturation activity as shown in Fig.2. The results showed the presence of protein extracted from button mushroom showed higher denaturation activity in case of ovalbumin used as standard for these studies as compared to protein from oyster mushroom.

Fig. 2. Denaturation assay using ovalbumin.

Values were expressed as Mean ± S.E. The difference between control and mushroom species (Oyster and button, 100, 500 and 1000µg/ml) is determined through one way ANOVA (Bonferroni Multiple Comparison Test)

*P<0.05; **P<0.01

Anti-inflammatory activity

The effect of these two species particularly protein content for determining its anti-inflammatory activity as shown in Fig.3. the results of these studies showed that protein at higher concentration showed anti-inflammatory activity against specific vaccine antigen i.e. typhoid vaccine as compared to control.

Fig.3. Anti-inflammatory activity of protein content from mushroom species in human red blood cell suspension.

Values were expressed as Mean ± S.E. The difference between control and mushroom species (Oyster and button, 100, 500 and 1000 µg/ml) is determined through one way ANOVA (Bonferroni Multiple Comparison Test)

*P<0.05; **P<0.01, ***P<0.001

Antimicrobial activity

The results of these studies related to protein were determined by measuring the diameter of zone of inhibition recorded. The results obtained in the evaluation of the antimicrobial activity against Pseudomonas aeruginosa and Bacillus subtilis as shown in Table 1. Overall, protein from mushroom species showed antimicrobial activity against these bacterial strains.

Table 1. Antimicrobial activity of mushroom protein against bacterial strains

Test

Organism/s

Positive control

(Erythromycin)

Negative control

Oyster protein

Button protein

Pseudomonas

Aeruginosa

Bacillus subtilis

*NA- No activity

DISCUSSION:

Consumption of fruits along with vegetables possessed some antioxidant capacity, as well as medicinal herbs and plants which provide some optimal health and nutritional outcomes. These studies are totally based on its quality with respect to nutrition rate may have some beneficial effects in the prevention or declining of various chronic diseases that are highly prevalent in our society [1-3]. In literature, human body has an antioxidant type of defense system, but this system is not sufficient in order to neutralize various multiple attacks that bombard the body day by day. In general, we preferably maintain a balance between various oxidants along with antioxidants with in the body, typically those substances which may act on reactive oxygen species and it is mainly used especially in the form of food supplements [15,16]. However, in recent years, use of some synthetic antioxidants has been restricted because of their toxic and carcinogenic effects. Therefore, alternative type of medicine has been reported and also used in the form of extracts or fractions or pure molecules and fruit with antioxidant properties [16,17]. These plant based properties are now being studied by the scientific community. Various studies show that some medicinal plants including some other metabolites i.e. fungal have more potent antioxidant activity than some fruits and vegetables [15,16]. In view of this, we worked on mushroom species and determining its immunopharmacological properties. In this study, we compare two different species of mushroom and extracted its protein content for determining its anti-inflammatory (using typhoid vaccine) and antioxidant (DPPH assay) potential.

The results of these studies which showed that protein showed anti-inflammatory activity at higher concentration and its activity could be due to declining in antigen (typhoid) specific immune response as compared to standard typhoid vaccine. Further confirmation of these studies, we determined in vitro anti-inflammatory activity (using human red blood cells) and protein denaturation assay (using Ovalbumin) claims that protein showed its activity at higher doses. In addition, whole blood cells were cultured with different concentration of protein exposed with standardized concentration of typhoid vaccine antigen and its studies claims that protein showed anti-inflammatory activity. In addition, denaturation assay was also performed using BSA which may also be considered as one of the major cause of inflammatory disease. Lot of efforts were taken related to development of anti-inflammatory drug and its major task is to prevent or reduce the level of protein denaturation. From these studies, it may found that protein from both species showed less protein denaturation as compared to standard anti-inflammatory drug.

In literature, antimicrobial properties of various medicinal plant products are being studied and reported as well. According to World Health Organization mentioning about the usage of various plant extracts or active constituents are mainly used as medicine in traditional type of therapies [18-27]. In this study, our group worked on protein content extracted from two different species of mushroom and showed strong activity against these tested bacterial strains. In addition, these studies also suggest that these mushroom species showed higher content of phenolic compounds and posses antimicrobial activities against a number of microorganisms. In other words, extraction of phenolic compounds from mushroom species which may also possess both antioxidant and antimicrobial activities.

The adverse health alignments and other side effects of presently used anti-inflammatory medications, naturally comprising anti-inflammatory supplements (generally obtained from plants) are nowadays being preferred [18-27]. Various categories of investigational practices have been reported pertaining to validate its antioxidant and anti-inflammatory effect obtained from natural sources. In an effort in the course of antioxidant and anti-inflammatory formulation from fungal source (mushroom species) for numerous chronic inflammatory ailments. From these studies, we conclude that protein from mushroom species possesses noteworthy anti-inflammatory and antioxidant activity. It is to be further needed to effusively explicate the antioxidants present in mushroom species accountable for this mechanism of action.

CONCLUSION:

This study is totally focused on antioxidant and anti-inflammatory potential of mushroom species and determined their content in terms of phenolic compounds. In other words, species of these mushrooms consist of immunobiologically active substances and showed various medicinal properties. Hence, there is a great need of exploiting these mushrooms to new drug discovery.

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Received on 30.03.2020 Modified on 08.05.2020

Research J. Pharm. and Tech. 2020; 13(12):6017-6021.

DOI: 10.5958/0974-360X.2020.01049.5