Preliminary Investigation on the Chemical Constituents and Antioxidant Activities of Two Phellinus Mushrooms Collected in Foshan

The Phellinus mushrooms have been known for its immunomodulatory, hypolipidemic, and anticancer activities. In the current work, the chemical constituents of two Phellinus mushrooms, Phellinus pini 141016# and Phellinus sp. 150802#, which were collected in Foshan city, Guangdong province, were investigated preliminarily by pre-test tube method and by comparison with the known natural compounds from Phellinus pini, and their antioxidant activities were evaluated by the biochemical assay of hydroxyl and 2,2-diphenyl1-pricylhydrazyl (DPPH) radicals scavenging activity in vitro. The results show that both the two Phellinus mushrooms contain phenolics, alkaloids, lactones, steroids and terpenoids, and have potent antioxidant activities. The IC50 values of DPPH radicals and hydroxyl radicals for Phellinus pini 141016# were 74.37 μg/mL and 59.69 μg/mL, respectively, and the IC50 values of DPPH radicals and hydroxyl radicals for Phellinus sp. 150802# were 98.95 μg/mL and 165.47 μg/mL, respectively. They are comparable to the antioxidant activity of the standard antioxidant ascorbic acid.


Introduction
Edible and medicinal mushrooms are an important source of natural antioxidants [1].They are ubiquitous in nature and produce various classes of biologically active primary and secondary metabolites.Mushrooms in the genus Phellinus have attracted much attention due to its long history and high medicinal value [2].Among them, Phellinus pini, also known as Huang Zhi, pine white rot fungus, which has anti-cancer, can enhance immunity, lower blood sugar and other activities, is a good medicine for cancer treatment [3] [4] [5].During an ongoing study to discover natural products with antioxidant activity from mushrooms, we collected two mushrooms in Foshan city, Guangdong province, and identified them as Phellinus mushrooms.It was known that the chemical constituents isolated from the fungi of the genus Phellinus are steroids, terpenoids, polysaccharides and fatty acids.In addition, flavonoids and flavanone derivatives, enzymes, pyrone pigments, and alkaloids were also found in the genus of Phellinus [4] [6].But to our best knowledge, the Phellinus mushrooms in Foshan had not yet been studied.Herein we report the preliminary investigation on the chemical constituents and antioxidant activities of the two Phellinus mushrooms.The aim of the study was to provide theoretical basis for the development and efficient utilization of them.

Chemistry
Analytical grade chemicals and reagents were directly used to perform various experiments.

Analytical
UV-2501 PC absorption spectrometer was used to measure the absorbance values.

Mushrooms
The No. 141016# wild fruiting bodies (Figure 1) identified as Phellinus pini were collected in October in 2014 from Leigang Mountain in Foshan city, Guangdong Provice, China.The No. 150802# wild fruiting bodies (Figure 2) were collected in August in 2015 from Leigang Mountain in Foshan City, Guangdong Province, China, and were identified as Phellinus sp., and it maybe is a novel specie of the genus of Phellinus.

Preparation of the Crude Extracts
The fresh fruiting bodies of the two Phellinus mushrooms were dried by air, and grinded into powder.The crude powders were extracted continuously with methanol for 24 hrs.The methanol extracts were combined and the methanol was recovered by vacuum rotary evaporation to obtain the crude extracts.A 1.23 g of crude extracts was obtained from 73 g of crude powders of the fruiting bodies of Phellinus pini 141016#, yielding 1.68%, and a 1.00 g of crude extracts was obtained from 56 g of crude powders of the fruiting bodies of Phellinus sp.150802#, yielding 1.79%.All the crude extracts were solved in methanol and prepared into sample solutions in methanol with concentration of 5 mg/mL.

Preliminary Test on the Chemical Constituents in the Methanol Crude Extracts
The chemical constituents of the methanol crude extracts were determined by preliminary-test tube method [7] [8].The ferric chloride reagent was used to check the phenolic components.Alkaloids were detected by Dragendorff reagent.The hydrochloric acid-zinc powder test was used to check the flavonoids.
The anthraquinones were examined by Bornträger reaction and magnesium acetate test.Cardiac glycosides were examined by Kedde reagent.The lactone and coumarin components were examined by the ferric hydroxamate reaction.
The sterols and triterpenoids were examined by Liebermann-Burchard reaction and Salkowski reaction.

Antioxidant Activity Assay
Scavenging Activity on Hydroxyl Radical The hydroxyl radicals scavenging activity of the crude extracts were evaluated by salicylic acid-FeSO 4 -H 2 O 2 method [8].Spectrophotometric method was used to determine the hydroxyl radicals scavenging ability of different concentrations of sample solution in vitro.A 0 -1.2 mL of sample solution in methanol was added to a clean plug tube, then methanol was added to make up the volume to 5 mL, then 1 mL of 6 mol/L salicylic acid solution in methanol was added.After shaking well, 1 mL of 2 mol/L aqueous ferrous sulfate solution was added, then 1 mL of 1.5% aqueous hydrogen peroxide solution was added.Distilled water instead of 1.5% aqueous hydrogen peroxide solution is as a blank control.After shaking well, methanol was added until reaching the constant volume of 10 mL.Finally, the tubes were placed in 37˚C bath for 60 min.The absorbance values were measured at λ max 510 nm, and calculated as a percentage of radical scavenging activity (%RSA) according to "Equation ( 1)".The experiment was repeated three times.The %RSA values were converted to activity of inhibition concentration at 50 percent (IC 50 ) using a graph plotted between %RSA and the concentration.The standard antioxidant ascorbic acid was used in this experiment.
( ) A 0 is the absorbance value of salicylic acid-FeSO 4 -H 2 O 2 system, and A s is the aborsance value of sample-salicylic acid-FeSO 4 -H 2 O 2 system.

Scavenging Activity on DPPH Radical
The DPPH radical scavenging activity of the crude extracts was measured using the method of Seephonkai [2] and Wu [8].Spectrophotometric method was used to determine the DPPH radicals scanvenging ability of different concentrations of sample solution in vitro.A 1 mL of different concentrations of the sample solution in methanol was mixed with 2 mL of 0.2 mmol/L DPPH solution in methanol．After 30 min at room temperature, the absorbance values of the sample were measured at λ max 517 nm and calculated as a percentage of radical scavenging activity (%RSA) according "Equation ( 2)"．The %RSA values were converted to activity of inhibition concentration at 50 percent (IC 50 ) using a graph plotted between %RSA and the concentration.Standard antioxidant ascorbic acid was used in this experiment.
( ) A DPPH is the absorbance value of 1 mL of methanol mixed with 2 mL of 0.2 mmol/L DPPH solution in methanol, and A s is the absorbance value of l mL of sample solution in methanol mixed with 2 mL of 0.2 mmo1/L DPPH solution in methanol.
In this work, the sample solution of the methanol crude extracts from Phellinus pini 141016 # showed a blue-violet color reaction with ferric chloride, sug- gesting the presence of phenolic components.The sample solution evaporated in water bath to get the residue, the residue was dissolved in 5% sulfuric acid, then filtration, the filtrate reacted with Dragendorff reagents to produce red precipi- The experimental results show that Phellinus pini 141016 # contain phenolics, alkaloids, lactones, steroids and terpenoids, which were consistent with the types of the known natural compounds of Phellinus pini (Figure 3).Since the sample solution of the methanol crude extracts from Phellinus sp.150802# showed the same reaction phenomena as the methanol crude extracts from Phellinus pini 141016# in the chemical constituents preliminary test, it was concluded that Phellinus pini 141016# also contain phenolics, alkaloids, lactones, steroids and terpenoids.

Antioxidant Activity
The Phellinus mushrooms are an important group of medicinal fungi.Cao et al. [15] found all the extracts from fruiting bodies of Phellinus mori, P. baumii and P. vaninii had in vitro antioxidant activities.Seephonkai et al. [2] investigated the antioxidant activity and total phenolic content from the crude extracts and crude fractions of ten species of Phellinus mushrooms collected from northeast Thailand, and the experimental results showed that some of the investigated extracts exhibited potent radical scavenging activity, while the samples with potent antioxidant activity were also determined to have high amount of total phenolics.Wang et al. [16] studied on preparation and detection antioxidant capacities of three polysaccharides, water-extracted polysaccharides (PSI), alkali-extracted polysaccharides (PSII) and sulfate polysaccharides (PSIII), from the fruiting bodies of Phellinus pini, and found PSII and PSIII showed the better result of antioxidant activities with concentration dependence.They [17] also studied the effects of the three polysaccharidies on antioxidant capacity of normal mice, and the results showed that the three polysaccharides significantly enhanced antioxidant capacity in mice, but also increased oxidative stress in vivo.Yang et al.
[18] obtained a polysaccharide designated as PP60-S1 from the fruiting bodies of Phellinus pini, and found PP60-S1 showed a potent antioxidant activity in vitro.
Two heteropolysaccharides, PPM and PPE, were isolated from mycelium and culture medium of Phellinus pini, respectively, and Jiang et al. [19] found PPM possessed better antioxidant activity in vitro than PPE.
In this work, the antioxidant activities of the methanol extracts from the wild fruiting bodies of Phellinus pini 141016 # and Phellinus sp.150802# were evaluated by free radical scavenging test.The results are shown in Figure 4 and Figure 5.The results showed that the two species had significant antioxidant activities, and the free radicals scavenging activity increased with the increase of the concentration of the samples.

Conclusions
The chemical constituents preliminary test results show that both the methanol extracts from the wild fruiting bodies of Phellinus pini 141016# and Phellinus sp.
150802# contain phenolics, alkaloids, lactones, steroids and terpenoids which were consistent with the types of the known natural compounds of Phellinus pini.
Free radicals scavenging activity experimental results show that both the methanol extracts from the wild fruiting bodies of Phellinus pini 141016# and Phellinus sp.150802# have potent antioxidant activities, and the free radicals sca- venging activity has increased with the increase of the concentration of the samples.They are comparable to the antioxidant activity of the standard antioxidant ascorbic acid.isolate and purify natural compounds from these two species, and to evaluate their antioxidant activities in vitro and in vivo, finally to explore the two Phellinus mushrooms as novel antioxidants in functional foods and medicine.
had been isolated from the wild fruiting bodies of the fungus Phellinus pini.In addition, two new ceramides , N-(2'-hydroxynonacosanoyl)-D-erythro-1, 3, 4-trihydroxy-2-aminooctadecane (3) and N-(2'-hydroxytriacontanoyl)-D-erythro-l, 3, that the sample contains alkaloids.The sample solution in the hydrochloric acid-zinc powder test did not occur color reaction, indicating that the sample does not contain flavonoids.In the Bornträger test, the sample solution produced a red reaction with 10% sodium hydroxide, but the red color faded when heating after adding a small amount of 30% hydrogen peroxide solution.Additionally, the sample solution did not occur color reaction with 1% magnesium acetate.All these phenomena suggest that there is no anthraquinone ingredient in the sample.The sample solution did not occur color reaction with Kedde reagent, indicating that no cardiac glycosides in the sample.In the ferric hydroxamate test, the sample solution showed red reaction, indicating the presence of lactones.The sample solution produced a remarkable color reaction in the Liebermann-Burchard reaction and the Salkowski reaction, indicating the presence of sterols and triterpenes.
In conclusion, the results of the present study indicate that the mushrooms Phellinus pini 141016# and Phellinus sp.150802# collected in Foshan have a great potential to be a good source of natural radical scavenger to be used in the food and pharmaceutical industries.Its antioxidant activities could be related to other biological activities for its use in traditional medicine.The next work is to The IC 50 values of DPPH radicals and hydroxyl radicals for Phellinus pini 141016# were 74.37 μg/mL and 59.69 μg/mL, respectively.And the IC 50 values of DPPH radicals and hydroxyl radicals for Phellinus sp.150802# were 98.95 μg/mL and 165.47 μg/mL, respectively.They are comparable to the antioxidant activity of the standard antioxidant ascorbic acid, which IC 50 values of DPPH radicals and hydroxyl radicals were 66.01 μg/mL and 103.34 μg/mL, respectively.