Screening of Secondary Metabolites and Antioxidant

Wild edible mushrooms produce a variety of bioactive compounds that are known to have antioxidant properties. Natural antioxidants can protect against oxidative induced free radicals without any side effects. Thus, they are consumed by people for food and nutraceutical values. The purpose of this study was to evaluate the phytochemicals and antioxidant activity of three wild edible termite mushrooms ( Termitomyces albuminosus, T. eurhizus and T. robustus ). Different phytochemicals were screened in the 50% ethanol, methanol and water extracts of three termite mushrooms. Total phenolic and flavonoid contents were determined by Folin-Ciocalteau and aluminium chloride method respectively. The antioxidant activity of three termite mushrooms was evaluated by DPPH assay. Qualitative screening of phytochemicals has revealed that alkaloid, steroid, fatty acid, flavonoid, saponin, tannin, carbohydrate and protein are found in the 50% ethanol, methanol and water extracts of three species of termite mushroom. A high amount of total phenolic and flavonoid content was found in the 50% ethanol extract of T. albuminosus , T. eurhizus and T. robustus (TPC: 50.28, 54.56 and 57.63 mg GAE/g extract; TFC: 16.30, 18.43 and 18.80 mg QE/g extract respectively). Due to high phenolic and flavonoid content, 50% ethanol extract of three termite mushrooms has shown high antioxidant activity (i.e., lowest IC 50 : 710.00 - 714.05 µg/ml). These termite mushrooms have antioxidant properties due to the presence of bioactive secondary metabolites that can potentially be used as a source of natural antioxidants in the form of food and nutraceutical.


INTRODUCTION
Termite mushrooms (Termitomyces spp.) are obligatory symbiosis with termites, grow on or near territory built by termites and their excreta.They have pink spores, termite association, subterranean elongated stipes and fruiting bodies. 1 They are tropical gilled mushrooms and good sources of wild food for termites, and mankind for high protein, crude fibre, minerals, vitamins and low fat. 2 They are consumed not only for a dietary supplement but also for therapeutics and nutraceuticals values.[5] Reactive oxygen species (ROS) are highly reactive molecules derived from cellular metabolism as well as from food, drugs, smoke and other pollutants.7][8] Antioxidants inhibit the harmful oxidizing free radicals which damage the living cells. 9Naturally, living organisms are endowed with oxidative defence mechanisms but certainly need external supplements of antioxidants.However, synthetic antioxidants such as butylated hydroxyanisole, butylated hydroxytoluene, tertbutylhydroquinone and propyl gallate have toxic and carcinogenic effects. 10Therefore, the development and utilization of more effective antioxidants of natural origin are desired.][13][14] To find a new natural source of antioxidants, our attention was focused on termite mushrooms.Termite mushrooms are great sources of natural antioxidant compounds they contribute directly to the antioxidative mechanism in living organisms to prevent oxidative damage.The major objectives of this research are to screen different phytochemicals, quantify the total phenolic and flavonoid content, and antioxidant activity of termite mushrooms.So, the present research promotes the sustainable use of mushrooms for food security and health.

Material
The whole part of three species of termite mushroom (Termitomyces albuminosus, T. eurhizus and T. robustus) was collected from the natural habitats in the Chitwan National Park, central Nepal from June to August in 2018.These mushrooms were identified by N.S.Atri (Punjabi University, Patiala, India) and Hari Prasad Aryal (co-author) use their morphological and spore characters.The voucher specimens of these species were deposited at the Natural History Museum of the Tribhuvan University [accession number: T. albuminosus (2-2-1666), T. eurhizus (2-2-1668) and T. robustus (2-2-1672)].The whole mushroom was washed, air-dried and crushed into fine powder.

Extraction
The powder of each species of mushroom was extracted with 50% ethanol, methanol and water.A powdered sample of 20 g of each species was placed ABSTRACT Wild edible mushrooms produce a variety of bioactive compounds that are known to have antioxidant properties.Natural antioxidants can protect against oxidative induced free radicals without any side effects.Thus, they are consumed by people for food and nutraceutical values.The purpose of this study was to evaluate the phytochemicals and antioxidant activity of three wild edible termite mushrooms (Termitomyces albuminosus, T. eurhizus and T. robustus).Different phytochemicals were screened in the 50% ethanol, methanol and water extracts of three termite mushrooms.Total phenolic and flavonoid contents were determined by Folin-Ciocalteau and aluminium chloride method respectively.The antioxidant activity of three termite mushrooms was evaluated by DPPH assay.Qualitative screening of phytochemicals has revealed that alkaloid, steroid, fatty acid, flavonoid, saponin, tannin, carbohydrate and protein are found in the 50% ethanol, methanol and water extracts of three species of termite mushroom.A high amount of total phenolic and flavonoid content was found in the 50% ethanol extract of T. albuminosus, T. eurhizus and T. robustus (TPC: 50.28, 54.56 and 57.63 mg GAE/g extract; TFC: 16.30, 18.43 and 18.80 mg QE/g extract respectively).Due to high phenolic and flavonoid content, 50% ethanol extract of three termite mushrooms has shown high antioxidant activity (i.e., lowest IC 50 : 710.00 -714.05µg/ml).These termite mushrooms have antioxidant properties due to the presence of bioactive secondary metabolites that can potentially be used as a source of natural antioxidants in the form of food and nutraceutical.
Glycosides: Extract of 2 ml was mixed Fehling's solution A and Fehling's solution B and mixture was heated on a water bath for about two minutes.After heating, it gives a brick-red colour that indicates the presence of glycosides.

Polyurenoids:
In a test tube, 2 ml extract and 10 ml acetone were added.If a thick volume was formed, 4-5 drops of hematoxylin were added.The precipitate was separated by filter and if it gives violet precipitate after centrifugation indicates the presence of polyurenoids.
Polyoses: Extract of 2 ml was placed in a porcelain basin and concentrated till to yield a residue.Then, 2-3 drops of conc.H 2 SO 4 was added to the residue and allow to stand for 3-5 minutes.The appearance of red colour in the resulting solution indicates the presence of polyoses after the addition of 3-4 drops of thymolor Molisch's reagent.
Anthocyanin: Extract of 1 ml was heated with an equal volume of 10% HCl on the water bath for about 15 minutes and cool.Then, 2 ml ether was added.If the aqueous part is red and does not turn to violet at neutral pH or blue in an alkaline medium, it shows the presence of anthocyanins.

Carbohydrates:
The extract of 2 ml was mixed with a 10 ml Molisch reagent.Then, 2 ml conc.H 2 SO 4 was added from the side of the test tube.The formation of a violet ring at the junction of two liquids indicates the presence of carbohydrates.
Protein: Extract 0.5 ml when boiled with 2 ml of 0.25 % ninhydrin solution gives violet colour indicating the presence of protein.
Reducing sugar: Extract 0.5 ml was added with 1 ml water and boiled gently with 0.5 ml Fehling's solution A and Fehling's solution B. If it gives reddish-brick precipitate that indicates the presence of reducing sugars.
Starch: Iodine of 0.015 gm and 0.075 gm of potassium iodide were dissolved in 5 ml of distilled water and it was mixed with 2 ml of extract.A blue colour appearance indicates the presence of starch.

Determination of total phenolic content
The total phenolic content was estimated by the Folin-Ciocalteau reagent as the method. 16About 1 ml of extract at concentrations of 100, 80, 50 and 25µg/ml was mixed with 5 ml of 10% Folin-Ciocalteau solution and 4 ml of 7% Na 2 CO 3 .The reaction mixture was incubated for 30 minutes a room temperature and absorbance was measured at 760 nm.Gallic acid was used as a standard for obtaining a calibration curve.The above-mentioned procedure was applied for the gallic acid.

Determination of total flavonoid content
The total flavonoid content was determined by aluminium chloride colourimetric assay. 16An aliquot of 1 ml of extract at the concentration of 100, 80, 50, and 25µg/ml was mixed with 4 ml of double-distilled water in a flask.Immediately, 0.3 ml of 5% sodium nitrite was added to the flask.After five minutes, 0.3 ml of 10% AlCl 3 and at six minutes, 2 ml of 1 M sodium hydroxide was added to the mixture.Immediately, the total volume of the mixture was made up to 10 ml by the addition of 2.4 ml double distilled water and mixed thoroughly.The absorbance of the pink-coloured mixture was measured at 510 nm.Quercetin was used as a standard for obtaining a calibration curve.The abovementioned procedure was applied for quercetin.

Determination of antioxidant activity
Antioxidant activity was determined by using 2, 2-diphenyl-1 picrylhydrazyl free radical (DPPH). 11About 0.5 ml of extract (prepared in methanol) at concentrations of 10, 20 and 30 µg/ml was mixed with 2.5 ml of 0.1 mM DPPH (prepared in methanol).The reaction mixture was shaken vigorously and incubated in dark for 30 minutes a room temperature.The reduction of purple DPPH into yellow in the mixture was measured at 517 nm.A control was prepared by mixing 0.5 ml of distilled water and 2.5 ml of 0.1 mM DPPH.

Phytochemical screening
Qualitative screening of phytoconstituents present in the ethanol, methanol and water extracts have revealed that alkaloid, steroid, fatty acid, flavonoid, saponin, tannin, carbohydrate and protein are found in all extracts of three species of termite mushroom (Table 1).The volatile oil is absent in all the extracts of the three mushrooms.Emodin, glycoside, anthocyanin, reducing sugar and starch are absent in the water extract of three termite mushrooms.Terpenoid and polyurenoid are absent in the methanol extract of T. albuminosus and T. robustus.Polyose is absent in the three extracts of T. albuminosus and T. robustus.

Total phenolic and flavonoid content
The phenolic content in the extract was calculated using the regression equation Y= 0.0165x+0.0184,R 2 = 0.99.The total phenolic content was expressed as mg gallic acid equivalent (GAE) per gram of extract.A high amount of total phenolic content (TPC) was found in the ethanol extract of three termite mushrooms (Table 2).Ethanol extract of T. albuminosus, T. eurhizus and T. robustus has high TPC as 50.28 1a), suggesting that the phenolic content of the extracts contributed to scavenging of 97.24% DPPH radicals.Similarly, the correlation between antioxidant activity and total flavonoid content revealed the coefficient of determination (R 2 ) of 0.928 (Figure 1b), suggesting that the flavonoid content of the extracts contributed to scavenging of 92.8% DPPH radicals.

DISCUSSION
The qualitative phytochemical screening of the extracts of three termite mushrooms has shown the presence of alkaloid, steroid, protein, carbohydrate, tannin, flavonoid, triterpenoid, fatty acid, saponin, glycoside, reducing sugar, starch, emodine, polyurenoid, polyoses and anthocyanin.However, volatile oil has not been found in three species of termite mushroom.8][19][20][21] These phytoconstituents play a significant role in the medicinal properties, so that termite mushrooms are used as food for nutrition, low calories, rich sources of water-soluble vitamins and dietary fibres.Therefore, these wild edible termite mushrooms have been largely used by ethnic people for the treatment of rheumatism, diarrhoea, gonorrhoea, stomach upset and hypertension. 2,17e quantification of more amount of phenolic and flavonoid content in the 50% ethanol extract of three termite mushrooms was revealed due to an increase in its polarity by the addition of 50% water which extracts more compounds. 21The presence of phenolic and flavonoid content exhibits a wide range of spectrum of medicinal properties by reducing the reactive oxygen species which may lead to anticancer, antiinflammatory, antidiabetic, antiallergenic and antiviral properties. 22,23hus, these selected termite mushrooms may be good alternatives for the treatment of diseases associated with excessive free radical generation and damage.and 57.63 mg GAE/g extract respectively as compared to the methanol and water extracts.
The flavonoid content in the extract was calculated using the regression equation Y =0.0042x, R 2 = 0.9987.Total flavonoid content (TFC) was expressed as mg quercetin equivalent (QE) per gram of extract.A high amount of TFC was found in the ethanol extract of three termite mushrooms (see Table 2).Ethanol extract of T. albuminosus, T. eurhizus and T. robustus has high TFC as 16.30, 18.43 and 18.80 mg QE/g extract respectively as compared to the methanol and water extracts.

Antioxidant activity
The antioxidant activity of the extracts of three termite mushrooms was carried out using DPPH free radicals.Inhibition of DPPH radicals by the extracts was expressed in the percentage (Table 3).Fifty percent inhibition of DPPH radicals by the extract (IC 50 ) was calculated from the percentage inhibition of DPPH radicals at various concentrations of the extract.The lowest IC 50 value of the extract indicates the highest antioxidant activity.Ethanol extract of T. albuminosus, T. eurhizus and T. robustus has the lowest IC 50 as 714.05, 712.76 and 710.00 µg/ml respectively as compared to the methanol and water extracts (see Table 3).

Correlation between total phenolic/flavonoid content and antioxidant activity
The correlation between antioxidant activity and total phenolic/ flavonoid content had been determined by plotting IC 50 values for antioxidant activity against the total phenolic/flavonoid content of extracts of three termite mushrooms.The relationship between total phenolic/flavonoid content and antioxidant activity is shown in Figure 1.Correlation between antioxidant activity and total phenolic content revealed the coefficient of determination (R 2 ) of 0.9724 (Figure  DPPH free radicals have been used for the determination of the antioxidant activity of the extracts of termite mushrooms.The extract with an inhibitory concentration (IC 50 ) is less than 10 mg/ml has an antioxidant potential, 24 and it donates a hydrogen atom to scavenge the free radicals.As such, three species of termite mushrooms have IC 50 values ranging from 710.00 to 719.72 µg/ml, therefore they have potent antioxidant activity.The phytochemicals especially phenolic and flavonoids are potential antioxidants. 11It has been demonstrated that a strong correlation between the antioxidant activity of the extracts and their phenolic and flavonoid content.More amount of total phenolic and flavonoid content in the extracts have shown strong antioxidant activity.The termite mushrooms have strong antioxidant activity against tested free radicals.The differences in the antioxidant activity of various solvent extracts may be the result of the extraction of different bioactive substances.0][31] Hence, the importance of antioxidants in food as protective agents to help humans in the reduction of oxidative damage.Hence, in the growing demand for food and pharmaceutical industries, such wild termite mushrooms can be the important candidates. 2,13,32

CONCLUSION
In conclusion, termite mushrooms have several secondary metabolites which have significant antioxidant activity.Hence, termite mushrooms can be widely used in food and their proper utilization for food security.They have also opened the way of their use in the pharmaceutical industry.

Figure 1 :
Figure 1: Correlation between IC 50 values of antioxidant activity and total phenolic content (a) and total flavonoid content (b).

Table 1 : Screening of phytoconstituents in the extract of three species of termite mushroom.
, 54.56 Tharu AK, et al.: Screening of Secondary Metabolites and Antioxidant Activity of Wild Edible Termite Mushroom