The present study is to find out the qualitative phytochemical analysis and antioxidant activity of fruiting bodies of Hypsizygus ulmarius (Bull.)

MATERIALS AND METHODS:

Chemical and Solvents:

All chemicals and solvents used were of analytical grade and were obtained from Hi Media chemicals, Mumbai, India.

Collection of Mushroom sample:

Dehydrated Hypsizygus ulmarius fruiting bodies were collected from ‘S’ Mushroom Agritech, Hyderabad, Telangana state, India. Collected mushroom was reported by the supplied to grow at 25℃ and dried properly by solar method. Dried mushroom was powdered in an electric mixer grinder. The powder was than stored for further use.

Preparation of crude extract:

650g of dried powdered mushroom were extracted with 5 L of methanol (40℃–50℃), using Soxhlet apparatus (14 cycles). After extraction, extract was evaporated under reduced pressure and vacuum dried to get viscous residue ^[35].

Fractionation of methanolic extract:

Successive fractionation of methanolic extract illustrated in (Fig.1). The resulting extract and its fractions, methanolic extract, petroleum ether and ethyl acetate fractions, were used for study common properties, estimate phytochemical compounds and antioxidant activity.

Figure 1: Schematic chart of successive fractionation of methanolic extract.

Solubility test:

Solubility of extract and its fractions in various solvents were carried out.

Preliminary phytochemical screening:

Extracts were subjected to phytochemical tests to investigate the presence of the following phytochemicals^[36,37].

Tests for Alkaloids:

Dragendorff’s test:

To 1ml of extract, few drops of Dragendorff’s reagent were added. Appearance of orange brown color indicates the presence of alkaloids.

Mayer’s test:

Few drops of Mayer’s reagent were added to 1ml of extract. Formation of dull white precipitate showed the presence of alkaloids.

Tannic acid test:

1ml of extract was treated with tannic acid solution. Developing of buff colored precipitate indicates the presence of alkaloids.

Picrolonic acid test:

1 ml of extract was treated with picrolonic acid. Presence of yellow precipitate indicates the presence of alkaloids.

Tests for Tannins and Phenolic compounds:

Ferric chloride test:

Few drops of 5% ferric chloride solution were added to 1ml of extract. Appearance of deep blue- black color showed the presence of tannins and phenolic compounds.

Lead acetate solution:

To 1 ml of extract, few drops of lead acetate solution were added. Presence of white precipitate indicates the presence of tannins and phenolic compounds.

Bromine water:

1 ml of extract was treated with few drops of Bromine water. Discoloration of Bromine water indicates the presence of tannins and phenolic compounds.

Acetic acid solution:

Few drops of Acetic acid solution were added to 1 ml of extract. Appearance of red color showed the presence of tannins and phenolic compounds.

Potassium dichromate:

To 1 ml of extract, few drops of Potassium dichromate were added. Appearance of red precipitate indicates the presence of tannins and phenolic compounds.

Test for Flavonoids:

Shinoda test:

To 1ml of extract, 5ml of 95% ethanol, few drops of concentrated HCL and 0.5g Zinc were added. Appearance of a deep red to magenta color indicates presence of flavanols and weak pink to magnetic color or no color indicates presence of flavanones and flavonoids,

Sulphuric acid test:

To 1ml of extract, 80% of Sulphuric acid was added. Appearance of deep yellow solution indicates the presence of flavones and flavonols, red to red- bluish solution indicates chalcones and aurones and orange to red colors indicate the presence of flavanes.

Tests for Glycosides:

Cardiac glycosides test:

Keller- Killiani test:

To 1ml of extract, 0.5ml of Glacial acetic acid, one drop of 5% FeCl3 and concentrated H2SO4 were added, respectively. Appearance of reddish-brown color at junction of the two liquid layers and upper layer appears bluish green indicates the presence of cardiac glycosides

Anthraquinone glycosides test:

Borntrager’s test:

To 1ml of extract, 0.5ml of diluted H2SO4 was added. Boiled and filtered. To cold filtrate, equal volume of chloroform was added and shaked well. The organic solvent was separated. Ammonia was added. Turned of ammoniacal layer to pink or red indicates presence of anthraquinone.

Saponin glycosides test:

Foam test:

To 1ml of extract, 1ml of distilled water was added and shaken well. Appearance of foam indicates presence of saponin.

Coumarin glycosides:

When alcoholic extract made alkaline with NaOH, presence of blue or green fluorescence showed the presence of coumarin.

Steroid test:

Salkowski reaction:

1ml of chloroform and 1ml of concentrated H2SO4 were added to 1ml of extract and shaken well. Appearance of chloroform red layer and greenish yellow fluorescence acid layer indicates presence of steroid.

Catechin test:

Matchstick was dipped in extract, dried, moistened with con. H2SO4 and warmed near a flame. Color of extract turned to pink or red indicates presence of Catechin.

DPPH radical scavenging assay:

Antioxidant activity of extracts were determined using 1,1-Diphenyl-2-picryl hydrazyl (DPPH) with minor modification ^[39]. 0.05ml of extracts dissolved in methanol were diluted to 1.0ml using (ethanol) to attain the concentrations 1-200µg/ml, and were added to DPPH (final concentrations 200µm, in 95% ethanol) and were allowed to stand for 20 min. The absorbance of test extracts was read at 515nm and the percentage of inhibition were calculated by using the formula

Inhibition (%) = [(AControl – ASample)/ (AControl)] × 100

Where AControl is the control absorbance (DPPH solution without extract) and ASample is the sample absorbance (DPPH solution with extract). IC50 values were calculated using linear regression by plotting inhibition % versus concentrations. Ascorbic acid was used as a stander.

Statistical Analysis:

Experimental data are expressed as means ± Stander error. Statistical analyses were performed by one-way ANOVA and t-value. The difference was considered to be statistically significant when the p ≤ 0.05. This analysis was done using SPSS ver. 20.0 software.

RESULTS AND DISCUSSION:

Physical properties of methanolic extract and its fractionation:

The common physical properties of methanolic extract, petroleum ether fraction and ethyl acetate fraction, are showed in Table 1.

Table 1: Properties of methanolic extract and its fractions.

Extract and its	Weight after	Color	Texture
fractionation	extraction
Methanol	133 g	Dark	Semisolid and
extract		brown	viscous
Petroleum ether	8 g	Dark	Semisolid and
fraction		brown	sticky
Ethyl acetate	4 g	Dark	Waxy
fraction		brown

Similar result was also obtained by Shivashankar and Premkumari, 2014 ^[39], in which methanolic extract was brown in color, semisolid and sticky.

Solubility of methanolic extract and its fractionation: Solubility of methanolic extract, petroleum ether and ethyl acetate fractions in different solvents were shown in Table 2.

TABLE 2: Solubility of methanolic extract and its fractionation.

	Different solvents
Extract and its	0.5%	0.5%	Normal	Distilled	PBS
fractionation	DMSO	Acetone	saline	water
Methanol	Soluble	Soluble	Soluble	Soluble	Soluble
extract
Petroleum	Soluble	Soluble	Soluble	Soluble	Soluble
ether fraction
Ethyl acetate	Soluble	Soluble	Soluble	Soluble	Soluble
fraction

Preliminary phytochemical analysis

A qualitative phytochemical analysis tests were performed to investigate the presence of any particular secondary metabolites which have a clinical significant. The results of phytochemical analysis reported in Table 3.

Table 3: Phytochemical analysis of methanolic extract and its fractionation from fruiting bodies of Hypsizygus ulmarius.

Phytochemical compounds	Biochemical tests	Methanolic extract	Petroleum ether fraction	Ethyl acetate fraction
	Dragendroff’s test	+	_	_
Alkaloids	Mayer’s test	_	_	_
Alkaloids	Tannic acid test	_	_	_
	Picrolonic acid test	_	_	_
	FeCl₃ test	_	_	_
Tannins and phenolic	Lead acetate test	+	_	_
Tannins and phenolic	Bromine water test	+	+	_
compounds	Acetic acid test	_	_	_
	Potassium dichromate test	_	_	_
Flavonoids	Shinoda test	+	_	+
	Sulphuric acid test	+	+	+
Cardiac glycosides	Keller-Killiani test	_	_	_
Anthraquinone	Borntrager’s test	+	+	+
Saponins	Foam test	+	+	_
Coumarins	NaOH	+	No	+
Steroids	Salkowski reaction	+	+	+
Catechins	Catechins	_	_	_

Note: (+) Presence, (^_) Absence and (No) Not tested

Figure 2. DPPH radical scavenging activity.

Edible mushroom considered to have a nutritional value and they have many phytochemical compounds and produce different secondary metabolites which have medicinal properties ^[40,41]. One of the main findings of this study was the type of phytochemical compounds in fruiting bodies of Hypsizygus ulmarius. Phytochemical analysis of Hypsizygus ulmarius indicate the presences of many phytochemical compounds in methanolic extract, which are alkaloids, tannins and phenolic compounds, flavonoids, anthraquinone, saponin, coumarin and steroid. Among the two fractions of methanolic extract, petroleum ether fraction showed more number of phytochemical compounds in compare to ethyl acetate fraction. Similar results for methanolic extract of Hypsizygus ulmarius have been reported by Shivashankar and Premkumari, 2014 ^[39], in which alkaloids, saponin, phenolics, tannins and glycosides where found. Aarthy and Eivanal, 2014 ^[42], Also mentioned the presence of phytochemical compounds, phenols and saponins, in aqueous extract of Hypsizygus ulmarius. Extract of this mushroom may will be a good alternative for the treatment of many diseases since it contains a number of phytochemical compounds which possess medical properties such as alkaloids, phenolic compounds and flavonoids.

Antioxidant activity:

DPPH assay of mushroom extracts and ascorbic acid was tested at various concentrations (1-200µg/ml) Fig (2). IC50 values for methanolic extract, petroleum ether fraction, ethyl acetate fraction and ascorbic acid were determined to be 8.4 µg/ml, 7.44 µg/ml, 6.77 µg/ml and 3.77 µg/ml, respectively (Table 4). This value showed that the ethyl acetate fraction possesses the highest scavenging activity than methanolic extract and petroleum ether fraction. Generally, three extracts showed a good activity when compared to stander ascorbic acid.

Table 4: IC₅₀ of methanolic extract, petroleum ether fraction, ethyl acetate fraction and stander ascorbic acid.

Mushroom extract and its fractionation	IC₅₀ value (µg/ml)
Ascorbic acid	3.77 ± 0.007
Methanolic extract	8.4 ± 0.004*
Petroleum ether fraction	7.44 ± 0.002*
Ethyl acetate fraction	6.77 ± 0.005*

All values are expressed as Mean ± SEM (n = 3). * Difference statistically significant when compared with stander ascorbic acid (p ≤ 0.05).

Similar results have been reported by D. R. Babu, 2013 ^[43], in which methanolic extract of H.ulmarius cape and stipe had moderate radical scavenging activity (IC50 was 1.242mg/ml and 1.556mg/ml, respectively). In another study reported by Shivashankar and Premkumari, 2014 ^[39], methanolic extract of H. ulmarius fruiting bodies showed an excellent scavenging activity in which IC50 was 74µg/ml in compare to IC50 of ascorbic acid (68 µg/ml). Greeshma et al., 2016^[44] founded that IC50 of aqueous ethanol extract of mycelia and fruiting bodies of H. ulmarius fruiting bodies were 560µg/ml and 440µg/ml, respectively.

CONCLUSION:

The present study of H. ulmarius fruiting bodies indicate that the methanolic extract and its fractions possess a good scavenging activity in which ethyl acetate fraction showed the highest scavenging activity. This antioxidant activity can be connected to the presence of flavonoids and other phytochemical compounds.

CONFLICT OF INTEREST:

Authors declare no conflict of interest.

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Received on 05.10.2019 Modified on 30.11.2019

Research J. Pharm. and Tech 2020; 13(9):4297-4302.

DOI: 10.5958/0974-360X.2020.00759.3