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Valorization of Polypore Mushroom Phellinus fastuosus by Analyzing Antioxidative, Antiproliferative and Apoptosis Induction Potential

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Abstract

Purpose

Phellinus fastuosus (Lév.) S. (Hymenochaetaceae, Hymenochaetales, Agaricomycetes, Basidiomycota) is a member of wood-rotting polyporoid fungi that contains numerous metabolites reported with many medicinal properties and has been used in traditional medicine for the treatment of various diseases. Inspired by the medicinal properties of this polypore the present study on the antioxidant and antiproliferative potential of methanolic extract of Phellinus fastuosus using various in vitro assays was proposed.

Methods

The extraction of the basidiocarp of Ph. fastuosus was done sequentially in hot water (Pfaq), methanol (Pfme) and ethyl acetate (Pfea) to obtain the respective extracts. The antioxidant potential of different extracts was examined with 2,2-Diphenyl-1-picrylhydrazyl (DPPH) assay, Ferric ion reducing antioxidant power and Phosphomolybdate assay. The cytotoxicity activity was determined by using MTT assay in human epidermoid carcinoma cells (A431), human cervical cancer (HeLa cells), human osteosarcoma (MG-63) and normal epidermoid cells (L929). For the assessment of changes in cell morphology, and apoptotic induction in A431 cell line was further investigated using phase-contrast microscopy, Hoechst 33342 staining and AO/EtBr dual staining. Flow cytometry was used for the estimation of production of reactive oxygen species (ROS) andmitochondrial membrane potential (MMP).

Results

Among all, Pfme extract showed effective free radical scavenging potential in DPPH assay, as compared to the other extracts. Therefore the Pmfe extract was further evaluated for the antiproliferative activity in A431, HeLa and MG-63 cell lines. This extract was very effective in A431 with GI50 (growth inhibitory dose 50%) value of 81.39 compared to its effect in HeLa and MG-63 cells with GI50 values of 173.47 and 191.53 μg/ml respectively. The Pfme extract was further investigated to explore its role in apoptosis induction in A431 cell line. Phase-contrast and fluorescence microscopic studies exhibited all the characteristics indicative of apoptosis, viz., shape change, cell shrinkage, cell rounding-off and nuclear condensation. To understand the cause of effectiveness of Pfme extract, HPLC analysis was carried out which showed the presence of different polyphenols.

Conclusions

A critical examination of results highlighted that the Pmfe extract induced apoptosis in A431 cells via ROS-mediated apoptotic pathway which may be ascribed to the presence of polyphenols in it.

Graphical Abstract

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Data Availability

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.

Abbreviations

A431:

Human epidermoid carcinoma cells

AIF:

Apoptotic inducing factor

AO:

Acridine orange

ANOVA:

Analysis of variance

CO2 :

Carbon dioxide

CTAB:

Cetyltrimethylammonium bromide

CytC:

Cytochrome C

DCFH-DA:

2′,7′-Dichlorofluorescein diacetate

DCF:

Dichlorofluorescein

DMEM:

Dulbecco’s modified Eagle medium

DMSO:

Dimethyl sulfoxide

DPPH:

2,2-Diphenyl-1-picrylhydrazyl

EA:

Early apoptosis

EtBr:

Ethidium bromide

FBS:

Fetal bovine serum

GAE:

Gallic acid equivalents

HeLa:

Human cervical cancer cells

HPLC:

High pressure liquid chromatography

L929:

Normal epidermoid cells

LA:

Late apoptosis

L:

Live

MEM:

Minimum essential medium

MG-63:

Human osteosarcoma cells

MMP:

Mitochondria membrane potential

MTT:

3-(4,5-Dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide

N:

Necrotic

NCCS:

National Centre for Cell Science

PBS:

Phosphate buffer saline

PDA:

Photodiode array

ROS:

Reactive oxygen species

Rh123:

Rhodamine123

rpm:

Revolutions per minute

SE:

Standard error

TAC:

Total antioxidant activity

TCA:

Trichloroacetic acid

TFC:

Total flavonoid content

TPC:

Total phenol content

UV:

Ultraviolet

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Acknowledgements

The authors are grateful to the Head, Department of Botany, Punjabi University, Patiala and Department of Botanical and Environmental Sciences, Guru Nanak Dev University, Amritsar for providing necessary laboratory facilities. The author is also thankful to UGC DSA-1 and DST FIST level-1 for providing financial assistance.

Funding

This study was supported by UGC-DSA-1 and DST-FIST-level-1.

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Authors and Affiliations

  1. Department of Botany, Punjabi University, Patiala, Punjab, 147002, India

    Avneet Kaur, Ellu Ram, Gurpaul Singh Dhingra & Avneet Pal Singh

  2. Department of Botanical & Environmental Sciences, Guru Nanak Dev University, Amritsar, Punjab, 143005, India

    Shivani Attri, Ajay Kumar, Pallvi Mohana, Sharabjit Singh, Prabhjot Kaur & Saroj Arora

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  1. Avneet Kaur
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  2. Shivani Attri
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Contributions

AK (Avneet Kaur): conceptualization, methodology, validation, writing original draft, investigation and software. SA (Shivani Attri) and PK: conceptualization, investigation. PM and SS: investigation and formal analysis. ER: Collection of the raw material. AK (Ajay Kumar): investigation, formal analysis, reviewing. APS, SA (Saroj Arora) and GSD: supervision, validation, methodology, conceptualization, validation and original drafting. All authors have read and agreed to the published version of the manuscript.

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Correspondence to Avneet Pal Singh.

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Kaur, A., Attri, S., Kumar, A. et al. Valorization of Polypore Mushroom Phellinus fastuosus by Analyzing Antioxidative, Antiproliferative and Apoptosis Induction Potential. Waste Biomass Valor 14, 2659–2672 (2023). https://doi.org/10.1007/s12649-023-02046-2

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