INTRODUCTION

Fungi are extremely diverse in expression of morphology, ecology, metabolism, and

phylogeny. 1 Fungi produce a diversity of bioactive compounds, making them ideal for
natural goods.2 Mushrooms are the fleshy, spore-bearing fruiting bodies of fungi belonging to
the phyla Ascomycota and Basidiomycota. They are responsible for producing and dispersing
spores and typically grow above soil or on nutrient-rich substrates. While some mushroom
species are edible, others are toxic and pose significant health risks. 3 Mushrooms are rich
sources of structurally diverse secondary metabolites many of which exhibit bioactive
properties with potential therapeutic applications in disease treatment. 4 However, certain
toxic mushrooms, referred to as poisonous or toxic fungi, produce natural compounds known
as toxins. Understanding and classifying these toxic mushrooms is crucial for ensuring the
safe consumption of wild mushrooms and advancing scientific research. 5 There are around
1600 species of mushrooms, however only100 species have been recognized to be consumed
for edible purposes. About 33 species of edible mushrooms are under cultivation throughout
the world.6
The medicinal and nutritional properties of fungus Ganoderma (Lingzhi, Reishi or
Mannentake) has been used in traditional Chinese herbal medicine since nearly 2,000 years
ago and their use has been extended to worldwide. 7 Ganoderma curtisii is a wood-decaying
basidiomycete characterized by distinctive macroscopic and microscopic morphological
features.7 The basidiocarp is annual to perennial, sessile, and generally fan-shaped to
semicircular with a reddish-brown to orange-brown pileus typically having a laccate,
varnished appearance and with visible zones of concentric growth, sometimes with a
yellowish edge. The context (flesh) is yellow-brown, corky to woody, and of variable
thickness depending on age. The underside has a white to creamy pore surface, which
darkens with age or bruising, and is covered with tiny round pores, usually 4–6 per mm.
Microscopically, the fungus possesses a trimitic hyphal system comprising thin-walled
generative hyphae with clamp connections, thick-walled unbranched skeletal hyphae, and
binding hyphae. The basidiospores are ellipsoid to broadly ovoid in form, golden-brown,
truncate at one end, double-walled, and 9–13 × 6–9 μm, the inner wall ornamented by
echinulations (spiny projections). Ecologically, G. curtisii is a white-rot fungus, lignin- and
cellulose-degrading, and prevalent on hardwood stumps and logs in tropical and subtropical
regions of Asia and the Americas. These morphological characteristics, especially the shiny
reddish pileus, trimitic hyphal structure, and echinulated basidiospores, differentiate it from
other closely related species such as Ganoderma lucidu. 8 Some pharmacological properties of
Ganoderma have been associated with its ability to reduce the risk of heart disease, cancer
and stimulate the immune system.9 Beneficial properties for the health of Ganoderma species
are attributed to the bioactive components such as polysaccharides, triterpenes, sterols, lectins
and some protein.10 Traditional Asian Medicine for more than two centuries. Its use has been
based mainly on observations and testimonies of its effects. 11,12 In the prevention and
treatment of cancer, fighting bacterial infections, regulating the immune system, and blood
pressure, among other medicinal effects. Today, Ganoderma is consumed as a nutraceutical,
in soup/tea or other drinks.12

MATERIALS AND METHODS

Freshly collected basidiocarps of Ganoderma curtisii were obtained from hardwood logs and
isolated by morphological characteristics.13,14 The fruiting bodies were washed with water,
dried at 40–45 °C using a hot-air oven, and cut into small pieces by using sterile scalpel. The
sample was kept in closed containers at room temperature until extraction.
SUCCESSIVE SOXHLET EXTRACTION
An approximate 50 g of small pieced G. curtisii was filled into a thimble and was subjected to
Soxhlet extraction in succession with increasing polarity solvents: ethyl acetate, methanol,
and water.
1. Ethyl acetate extraction: Sample was extracted in Soxhlet apparatus with 250 mL of ethyl
acetate for 6–8 hours until the solvent became clear. The extract was then filtered and
concentrated under vacuum using a rotary evaporator at 40 °C, and stored in amber vials at 4
°C.
2. Extraction with methanol: The residues were next extracted with 250 mL methanol under
identical conditions. The methanol extract was concentrated, filtered, and stored.
3. Extraction with water: Finally, the remaining residue was extracted with 250 mL distilled
water for 6–8 hours. The aqueous extract was lyophilized and stored at 4 °C.
STORAGE OF EXTRACTS
All crude extracts were weighed for calculation of yield percentage and kept in airtight amber
vials at 4 °C until further phytochemical and bioactivity assays.15

Phytochemical screening:
Proteins and Amino Acids
Proteins and amino acids were tested in the Ganoderma curtisii extracts by routine colour
reactions. Free amino acids were tested by the Ninhydrin test in which heating with
ninhydrin reagent gave a purple-blue colouration. Peptide bonds were tested by the Biuret
test that resulted in violet or purple colour after alkaline copper sulfate treatment.
Furthermore, Xanthoproteic reaction was conducted to detect aromatic amino acids like
tyrosine and tryptophan, resulting in a coloration ranging from yellow to orange when
treated with concentrated nitric acid.
Carbohydrates
Qualitative carbohydrate analysis was done by classical tests. Benedict's and Fehling's tests
were used for reducing sugars as evidenced by green, yellow, or brick-red cuprous oxide
precipitates. Molisch test was conducted as a general carbohydrate assay that gave rise to a
violet ring at the junction on reaction with concentrated sulfuric acid. For distinguishing
ketoses, Seliwanoff's test was utilized, where resorcinol in concentrated HCl produced a
bright cherry red coloration.
Anthraquinone Glycosides
Anthraquinone glycosides were analysed using Bornträger's reaction, in which chloroform
extract treated with ammonia gave rose-pink to cherry-red colour, showing the occurrence
of free anthraquinones. The Bornträger's test was also modified to establish bound
anthraquinones following hydrolysis with hydrochloric acid and ferric chloride, followed by
chloroform and ammonia extraction.
Plant Acids
Plant acids were detected by the reaction with sodium bicarbonate solution, where carbon
dioxide liberation producing stable froth was taken as an indication of free carboxylic acids.
Phenols
Phenolic substances were identified by colour and precipitation tests. The Ferric chloride
test gave blue, green, or black coloration on complex formation with hydroxyl groups,
whereas the Lead acetate test gave a white precipitate, establishing the presence of
phenolic constituents.
Quinones
Quinones were detected by treating with concentrated sulfuric acid, which produced a
characteristic red colour.
Tannins
Tannins and phenolic glycosides were tested using the Ferric chloride test, in which
hydrolysable tannins gave blue coloration and condensed tannins gave green. Tannins were
verified by the Gelatin test through the production of a gelatinous precipitate in the
presence of sodium chloride.
Coumarins
Coumarins were identified by alkaline reagent test where sodium hydroxide produced yellow
coloration that assured their existence.
Flavonoids
Flavonoids were investigated by several confirmatory tests. Magnesium turnings and
hydrochloric acid yielded pink to red coloration in Shinoda test. Alkaline reagent test gave
yellow colour reversibility with acid, and lead acetate precipitation assured flavonoid
existence.
Terpenes and Terpenoids
Terpenes and terpenoids were screened by Salkowski’s test, in which chloroform extracts
treated with concentrated sulfuric acid produced a reddish-brown or golden-yellow
coloration at the interface. In addition, specific tests such as the copper acetate reaction
yielded green coloration in diterpenes.
Steroids and Phytosterols
Steroidal components were established using the Liebermann–Burchard test, during which
extracts after treatment with acetic anhydride and concentrated sulfuric acid underwent
colour changes from pink to green, establishing the presence of unsaturated sterols and
triterpenoids.
Saponins
The froth test was used to test saponins, during which vigorous shaking with water resulted
in a stable froth that lasted for more than 30 minutes, establishing their presence.
Cardiac Glycosides
Cardiac glycosides were tested by the Keller–Killian test, in which a reddish-brown ring at the
junction and bluish-green coloured top layer showed the presence of deoxy sugars of
cardenolides. Further confirmation reactions like Baljet and Legal tests were employed to
identify the lactone ring of cardiac glycosides.
Alkaloids
Alkaloids were tested by reacting acidified extracts with traditional reagents like Mayer's,
Wagner's, Dragendorff's, Hager's, and Valser's, which yielded white, reddish-brown, orange,
yellow, or creamy precipitates, respectively, indicating the presence of alkaloidal
compounds.16

Antimicrobial activity:
The antibacterial potential of the ethyl acetate, methanolic, and aqueous extracts of
Ganoderma curtisii were assessed employing the agar well diffusion assay. The test
organisms were both Gram-positive and Gram-negative bacteria like Staphylococcus aureus,
Bacillus subtilis, Escherichia coli, and Pseudomonas aeruginosa from a microbial culture
collection. The bacterial strains were initially activated in nutrient broth and incubated at 37
°C for 18–24 h. Standardized inoculum corresponding to 0.5 McFarland turbidity standard,
~1×10^8 CFU/mL, were evenly spread on sterile Mueller–Hinton agar plates using a sterile
swab. Aseptically 6 mm diameter wells were punched in the agar using a sterile cork borer,
and 50–100 µL of each extract (concentration from 25 to 200 mg/mL, dissolved in respective
solvents) was added to individual wells. Plates were left for 30 min at room temperature to
enable diffusion of the extracts, and subsequently incubated at 37 °C for 24 h. Antibacterial
activity was determined by measuring the diameter of clear inhibition zones surrounding the
wells with a Vernier caliper. Positive controls were provided by standard antibiotics (e.g.,
streptomycin or ampicillin, 10 µg/mL), while solvent blanks were used as negative controls.
All the assays were conducted in triplicate and the results were presented as mean inhibition
zone (mm) ± standard deviation.17
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