Evaluation of antioxidant and enzyme inhibition potential of various extracts of mushroom species

Abstract

The present study aimed to evaluate the antioxidant and pancreatic lipase inhibitory potentials of ethyl acetate extracts from five mushroom species: Hypsizygus tessellatus, Lentinula edodes, Schizophyllum commune, Tricholoma matsutake, and Coprinellus disseminatus. The mushrooms were collected and extracted using the maceration method, followed by preliminary phytochemical screening to identify the presence of alkaloids, glycosides, phenols, and tannins. Flavonoids were absent in all extracts. Antioxidant activity was assessed using the DPPH radical scavenging assay, which revealed that none of the extracts exhibited significant free radical scavenging potential. In contrast, all mushroom extracts demonstrated notable inhibition of pancreatic lipase in vitro. Among them, Hypsizygus tessellatus exhibited the highest inhibitory activity with an IC50 value of 4.4 µg, followed by Tricholoma matsutake (IC50 = 6.94 µg), Schizophyllum commune (IC50 = 11.27 µg), Lentinula edodes (IC50 = 17.58 µg), and Coprinellus disseminatus (IC50 = 17.7 µg). These findings suggest that the studied mushrooms, particularly Hypsizygus tessellatus, possess potent pancreatic lipase inhibitory activity, highlighting their potential as natural agents for managing obesity and related metabolic disorders.

Keywords

Hypsizygus tessellatus, Lentinula edodes, Schizophyllum commune, Coprinellus disseminatus, Tricholoma matsutake, Antioxidant activity, Pancreatic lipase inhibition

Introduction

Asia, medicinal mushrooms are traditionally served as both folk remedies and functional foods, with numerous varieties emerging as promising sources of bioactive peptides. Mushrooms are valuable sources of neutraceuticals and have drawn considerable interest as functional foods.Over the last few decades, there has been arising trend in using mushrooms for therapeutic purposes and as health supplements.Mushrooms are known to be a plentiful source of phytonutrients like polysaccharides,dietary fibers and various micronutrients, as well as essential amino acids that are key toforming vital proteins. They are also valued for their delightful taste, aroma, texture, and flavor. Mushrooms provide a nutrient-rich, low-calorie food source with excellentproteins, vitamins and minerals. They also represent a significant and underutilized opportunity for discovering innovative pharmaceutical products.Mushrooms contain a variety of anti-inflammatory substances including polysaccharides,phenolic and indolic compounds, mycosteroids, fatty acids, carotenoids, vitamins, and biometals. Phenolic acids and flavonoids are among the most prevalent phenoliccompounds found in mushrooms and they contribute to a range of bioactivities such as antioxidant, anti-inflammatory, antitumor, antihyperglycemic, antiosteoporotic, anti-tyrosinase and antimicrobial effects. These phenolic compounds are typically present in smaller amounts in mushrooms.

In the present study we discussed about five different mushroom species like

• Hypsizygus tessulates

• Lentinula edodes

• Coprinellus disseminatus

• Schizophyllum Commune

• Tricholoma Matsutake

1. 1. HYPSIZYGUS TESSULATES:

Domain	Eukaryota
Kingdom	Fungi
Division	Basidiomycetes
Class	Agaricomycetes
Order	Agaricales
Family	Lyophyllaceae
Genus	Hypsizygus
Species	H. tessulates

 

It is an edible mushroom, native to East Asia and it is cultivated locally in temperate climates in Europe, North America and Australia and sold fresh in super markets. These are the gilled mushrooms that grown on wood and commonly called as beech mushroom.Cultivation of this mushroom is small and thin in appearance. There are two commercial variations are available such as Buna-shimeji and Bunapi-shimeji and they are originating from Japan. Buna-shimeji is a wild type brown colour mushroom and also known as beech mushroom, brown beech mushroom and brown clamshell mushroom. It consist of phytoconstituents like terpenoids, steroids, polyphenols, polyglucan, flavonoids,alkaloids, polysaccharides and dietary fibres. Bunapi-shimeji is a white colouredmushroom and also known as white beech mushroom, white clamshell mushroom.These mushrooms having various pharmacological activities like anti-tumor activity,immunomodulatory activity, antioxidant activity, anti-inflammatory, antiviral and hypocholestrolemic activity. It is rich in micronutients and polysaccharide fractions, thus making it as a good source of neutraceuticals.

1. 2. LENTINULA EDODES:

Domain	Eukaryota
Kingdom	Fungi
Division	Basidiomycetes
Class	Agaricomycetes
Order	Agaricales
Family	Omphalotaceae
Genus	Lentinula
Species	L. enodes

It is the globally second most widely consumed mushroom and it is well known for its therapeutic potential and commonly used experimental fungus model. Lentinula edodes is the edible mushroom. Shiitake mushroom contains essential macromolecule and micronutrients as well as many bioactive compounds like polysaccharides, antioxidants, dietary fiber and ergosterol. This mushroom contain alkaloids, glycosides, flavonoids, amino acids and analogs, phenolics, polysaccharide, sterols, that play role in bodily functions resulting in improved health of the individuals[12]

1. 3. COPRINELLUS DISSEMINATUS:

Domain	Eukaryota
Kingdom	Fungi
Division	Basidiomycetes
Class	Agaricomycetes
Order	Agaricales
Family	Psathrellaceae
Genus	Coprinellus

Coprinellus disseminatus, also known as the Fairy Ink Cap mushroom, is a species of edible fungus. It belongs to family Psathyrellaceae. Found in the clusters on decaying wood, stumps and tree trunks. Commonly grown in temperate and subtropic regions of Europe, North America and Asia. Coprinellus disseminatus are also recognized for their traditional medical importance in various indigenous societies throughout Asia and Africa. This species was observed growing on fungal comb fragments, which serve as its growth substrate. It consist of phytoconstituents like alkaloids, flavonoids, steroids,saponins, phenol, carbohydrates, proteins, triterpenoids, carotenoids and fatty acids.Corpinellus disseminatus having various medicinal properties like antioxidant, antiinflammtory, antibacterial, antifungal properties and including potential anticancer and immunomodulatory effects.

1.4 SCHIZOPHYLLUM COMMUNE:

Schizophyllum commune is a sap-rot basidiomycetes and cosmopolitan species.

 It is a non edible mushroom. This mushroom vary from creamy yellow to pale white in colour.This fungus is also recognized as plant pathogen that cause wood rot on living trees. It is also known as split-gill mushroom. Schizophyllum commune is found throughout the world. It consist of phytoconstituents like phenolics, flavonoids, vitamin-c, beta-carotene,lycopene, tannins, terpenoids and saponins. This mushroom is found in wild decaying trees after rainy season followed by dry spells, where the mushrooms are naturally Collected.

Domain	Eukaryota
Kingdom	Fungi
Division	Basidiomycetes
Class	Agaricomycetes
Order	Agaricales
Family	Schizophyllaceae

1.4 TRICHOLOMA MATSUTAKE:

Domain	Eukaryota
Kingdom	Fungi
Division	Basidiomycetes
Class	Agaricomycetes
Order	Agaricales
Family	Tricholomataceae
Genus	Tricholoma

The fruiting body of Tricholoma matsutake is the most economically important edible Mushroom.Matsutake mushroom grown in East Asia, South Asia, and Southeast Asia,parts of Europe. Matsutake mushroom are commonly associated with Pinus densiflora.The contents like Ca, Cu, Fe, K, Mg, Na and Zn are present in fruiting bodies of Matsutake mushroom .This mushroom have excellent biological activities but difficultto cultivation. It consist of phytoconstituents like phenols,   flavonoids,alkaloids,steroids,saponins, terpenoids and cardiac glycosides. It is an important edible mushroom that has been artificially cultivated and used as a food and medicinal ingredients in traditional chinese medicines.

MATERIALS AND METHODOLOGY:

COLLECTION AND AUTHENTICATION OF MUSHROOM.

Mushrooms like Hypsizygus tessulates, Lentinula edodes are collected from mushroom shop in Banglore, Karnataka. Coprinellus disseminatus, Tricholoma matsutake and Schizophyllum commune are collected From natural habitat around Malavalli, Mandya district, Karnataka. These different types of mushrooms are authenticated by Dr.Thejas kumar HOD and Assistant professor, Department of Botany, Bharathi college, Bharathinagara, Maddur TQ, Mandya Dist, Karnataka.

 

                       

PREPARATION OF MUSHROOM EXTRACT:

Mushroom extraction by maceration method involves soaking mushroom in a solvent to extract desired compounds. Around 200gm of fresh mushrooms are chopped or grind into small pieces to increase surface area. Place the mushrooms into a glass container, add the solvent ethyl acetate in the ratio of about 1:3 (mushroom to solvent), stir the mixture using magnetic stirrer. Seal the container using aluminium foil and let it for several days (typically 3 days), shaking occasionally this allows the active compounds to dissolve in the solvent. After soaking, strain the mixture using a filter to separate the liquid extract from the solid mushroom material, then liquid extract were allowed to dry, then store and protect it from light and degradation.

ANTIOXIDANT ACTIVITY:

DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical scavanging assay:

Method: 

The radical scavenging activity of different extracts was determined by using DPPH assay. The decrease in the absorption of the DPPH solution after the addition of an antioxidant was measured at 517nm. 

Principle:  

2,2 Diphenyl 2- Picryl Hydrazyl is a stable (in powder form) free radical with red color which turns yellow when scavenged. The DPPH assay uses this character to show free radical scavenging activity. The scavenging reaction between (DPPH) and an antioxidant (H-A) can be written chemically as mentioned below,  

    

                              Figure 13: Principle of DPPH antioxidant assay.

 

Antioxidants react with DPPH and reduce it to DPPH-H and as consequence the absorbance decreases. The degree of discoloration indicates the scavenging potential of the antioxidant compounds or extracts in terms of hydrogen donating ability.    (DPPH) + (H-A) = DPPH-H + (A) 

Reagent preparation:  

0.1mM DPPH solution was prepared by dissolving 4mg of DPPH in 100ml of methanol. 

Materials: Test tubes, volumetric flask, pipette, UV spectrometer. 

Working procedure:

Different volumes (20 - 100μl) of plant extracts were prepared by diluting with DMSO and volume made upto 3ml with DPPH (0.1mM) solution. The reaction mixture was incubated in dark condition at room temperature for 20 min. After 20 min, the absorbance of the mixture was read at 517 nm. 3ml of DPPH was taken as control. The % radical scavenging activity of the plant extracts was calculated using the following formula,

 

       %RSA = Abs control – Abs sample × 100  

                                 Abs control  

Where, RSA is the Radical Scavenging Activity;

             Abs control is the absorbance of DPPH radical+ DMSO; 

             Abs sample is the absorbance of DPPH radical + plant extract. 

              

                                                    Figure 14: DPPH assay.

 4.5 PANCREATIC LIPASE INHIBITION ASSAY:

Principle: The principle of a pancreatic lipase enzyme inhibition assay is to measure the activity of pancreatic lipase and assess how specific inhibitors affect this activity.  Pancreatic lipase is an enzyme that catalyzes the hydrolysis of triglycerides into free fatty acids and glycerol in the digestive system. Inhibitors can interfere with the enzyme's activity either by competing with the substrate (competitive inhibition) or by binding to the enzyme or enzyme-substrate complex (non-competitive inhibition).

Chemicals: p-nitrophenyl butyrate (p-NPB), 0.1 M Tris HCl buffer (p^H 8 ), Pancreatic lipase.

Procedure:

Pancreatic lipase activity was determined by measuring the hydrolysis of p-nitrophenyl butyrate (p-NPB) to p-nitrophenol using a method reported previously. The 0.1 mg/ml of enzyme solution was prepared by reconstituting porcine pancreatic lipase using 0.1 M Tris-HCl buffer (pH 8). Then, 5 μl of test sample was mixed with 90 μl of enzyme buffer and incubated for 15 min at 37°C. After incubation, 5 μl of 10 mM p-nitro phenylbutyrate (p-NPB) was added to enzyme mixture and the reaction was allowed to proceed for further 15 min at 37°C. After incubation, the absorbance of p-nitrophenol released was measured at 405 nm using a UV Vis spectrophotometer. Furthermore, a positive control, Orlistat was used to ensure the reliability of results. 

Relative pancreatic lipase activity (%) was calculated as [(the activity of the compound with the substrate-the activity of the compound without the substrate)/(activity without the compound and with the substrate-negative control without the compound and substrate)] x 100.

 

 

Figure 15: Pancreatic lipase inhibition assay.

RESULT:

 DPP RADICAL SCAVENGING ASSAY:

 The preliminary phytochemical screening showed that Hypsizygus tessulates contains alkoloids but flavonoids are absent. Lentinula edodes contains alkaloids, glycosides but flavonoids are absent. Schizophyllum commune contains tannins but flavonoids, saponins are absent Tricholoma matsutake contains alkaloids glycosides but flavonoids and saponins are absent. Coprinellus disseminatus contains alkoloids and glycosides but flavonoids are absent. The antioxidant property of five types of mushroom is estimated by DPPH radical scavenging assay. By this assay it is confirmed that all five types of  mushroom extracts, don't have free radical scavenging potential.

PANCREATIC LIPASE ASSAY:

The ethyl acetate extracts of above mentioned 5 types of mushroom shows significant inhibition of pancreatic lipase enzyme. Among five different mushrooms, Hypsizygus tessulates shows more percentage of pancreatic lipase inhibitory activity at IC₅₀ value

4.4µg. The increasing order of percentage of pancreatic lipase inhibitory activity is as follows:

Hypsizygus tessulates (IC₅₀=4.4µg) > Tricholoma matsutake (IC₅₀=6.94µg) >

Schizophyllum commune (IC₅₀=11.27µg) > Lentinula edodes (IC50=17.58µg) > Coprinellus disseminatus (IC₅₀=17.7µg).

A) Hypsizygus tessulates:

SI.No	Concentration (µg/ml)	% inhibition of Pancreatic lipase enzyme
1.	2	34.4%
2.	4	45.9%
3.	6	64.5%
4.	8	72.5%
5.	10	79.1%

 

 

 

 

 

 

 

 

 

 

 

Table 6: Pancreatic lipase inhibition activity of Hypsizygus tessulates.

Figure 16: Pancreatic lipase inhibition activity of Hypsizygus tessulates.

B) Lentinula edodes:

SI.No	Concentration (µg/ml)	% inhibition of Pancretic lipase enzyme
1.	2	13.4%
2.	4	36.2%
3.	6	48.7%
4.	8	53.1%
5.	10	66.7%

Table 7: Pancreatic lipase inhibition activity of Lentinula edodes.

Figure 17: Pancreatic lipase inhibition activity of Lentinula edodes.

 C) Coprinellus desseminatus:

SI.No	Concentration (µg/ml)	% inhibition of Pancreatic lipase enzyme
1.	5	26.6%
2.	10	35.8%
3.	15	43.2%
4.	20	54.5%
5.	25	63.9%

 

 

 

 

 

 

 

 

 

 

 

Table 8: Pancreatic lipase inhibition activity of Coprinellus disseminatus.

Figure  18: Pancreatic lipase inhibition activity of Coprinellus disseminatus.

D) Schizophyllum commune:

SI.No	Concentration (µg/ml)	% inhibition of Pancreatic lipase enzyme
1.	2	10.5%
2.	4	19.7%
3.	6	25.7%
4.	8	36.9%
5.	10	44.6%

 

Table 9 : Pancreatic lipase inhibition activity of Schizophyllum commune.

Figure 19: Pancreatic lipase inhibition activity of Schizophyllum commune.

E) Tricholoma matsutake:

Table 10: Pancreatic lipase inhibition activity of Tricholoma matsutake

SI.No	Concentration (µg/ml)	% inhibition of Pancreatic lipase enzyme
1.	2	10.4%
2.	4	37.8%
3.	6	47.6%
4.	8	57.7%
5.	10	65.7%

 

 

 

 

 

 

 

 

 

 

 

 

Figure 11: Pancreatic lipase inhibition of Tricholoma matsutake.

CONCLUSION:

ANTIOXIDANT ACTIVITY BY DPPH RADICAL SCAVENGING ASSAY:

DPPH scavenging assay is most common spectrophotometric method to assess the antioxidant potential of chemical moieties. Advantages of these method include easy performing, having a high sensitivity and rapidity. DPPH is commercially available long lived organic nitrogen radical and freely soluble in organic solvents. It produces stable purple coloured radical, having an absorption maximum at 517nm. The DPPH  assay is believd to involve hydrogen transfer reaction. On the other hand, it is suggested that electron transfer reaction will be the major principle involved in this assay. Therfore, the antioxidant strength of the compound depends upon its hydrogen or electron donating nature. In the phytochemical screening in all the  mushroom extracts, both the phenols and flavonoids are absent. The result obtained by performing DPPH assay indicates above mentioned mushrooms doesn’t possess any antioxidant activity. 

 PANCREATIC LIPASE ENZYME INHIBITION ASSAY:

Enzyme inhibition activity by pancreatic lipase was determined by measuring the hydrolysis of para nitrophenyl butyrate (P-NPB) to p-nitrophenol. The 5 µl of test sample was mixed with 90 µl of enzyme buffer and incubated for 15 min at 37 degree Celsius. After incubation, 5 µl of 10mM  P-Nitro phenylbutyrate was added to enzyme mixture and the reaction was allowed to proceed for further 15 min at 37 degree Celsius. After incubation, the absorbance of P-Nitrophenol released was measured at 405nm using a UV visible spectrophotomete. The ethyl acetate extracts of all 5 types of mushroom tested shows significant inhibition of pancreatic lipase enzyme. Among five different mushrooms, Hypsizygus tessulates shows more percentage of pancreatic lipase inhibitory activity at IC₅₀ value 4.4µg. The potency of pancreatic lipase inhibitory activity  of  mushroom is as follows:

Hypsizygus tessulates (IC₅₀=4.4µg) > Tricholoma matsutake (IC₅₀=6.94µg) > Schizophyllum commune (IC₅₀=11.27µg) > Lentinula edodes (IC50=17.58µg) > Coprinellus disseminatus (IC₅₀=17.7µg).

ACKNOWLEDGEMENT:

I am sincerely thanking you to my dear friends, teachers and family members for their kind support.I am specially thanking you to my friends Ramya S and Sinchana H Gowda for their continous support and motivation to involve in this research.

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