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Ganoderma pfeifferi Bres. 1889 and
Ganoderma resinaceum Boud. 1889 as
Potential Therapeutic Agents: A
Comparative Study on Antiproliferative
and Lipid-Lowering Properties
Milena Ra
š
eta * , Marko Kebert , Jovana Mi
š
kovi
ć
, Sa
š
a Kosti
ć
, Sonja Kaisarevic , Nebojsa Stilinovic ,
Sa
š
a Vukmirovi
ć
, Maja Karaman
Posted Date: 19 June 2024
doi: 10.20944/preprints202406.1364.v1
Keywords: Ganoderma; natural products; health benefits; fungal extracts; anticancer activity; liver
protection; kidney protection; mineral composition
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Article
Ganoderma pfeifferi Bres. 1889 and Ganoderma
resinaceum Boud. 1889 as Potential Therapeutic
Agents: A Comparative Study on Antiproliferative
and Lipid-lowering Properties
Milena Rašeta 1, 2,*, Marko Kebert 3, Jovana Mišković 2, Saša Kostić 3, Sonja Kaišarević 4, Nebojša
Stilinović 5, Saša Vukmirović 5 and Maja Karaman 2
1 Department of Chemistry, Biochemistry and Environmental Protection, Faculty of Sciences, University of
Novi Sad, Trg Dositeja Obradovića 3, 21000 Novi Sad, Serbia; milena.raseta@dh.uns.ac.rs (М.R)
2 ProFungi Laboratory, Department of Biology and Ecology, Faculty of Sciences, University of Novi Sad, Trg
Dositeja Obradovića 2, 21000 Novi Sad, Serbia; jovana.maric@dbe.uns.ac.rs (J.M.);
maja.karaman@dbe.uns.ac.rs (M.K.)
3 Institute of Lowland Forestry and Environment, University of Novi Sad, Antona Čehova 13d, 21000 Novi
Sad, Serbia; kebertmarko@gmail.com (M.K.); sasa.kostic@uns.ac.rs (S.K.)
4 Department of Biology and Ecology, Faculty of Sciences, University of Novi Sad, Trg Dositeja Obradovića
2, 21000 Novi Sad, Serbia; sonja.kaisarevic@dbe.uns.ac.rs (S.K.)
5 Department of Pharmacology, Toxicology and Clinical Pharmacology, Faculty of Medicine, University of
Novi Sad, Hajduk Veljkova 3, 21000 Novi Sad, Serbia; nebojsa.stilinovic@gmail.com (N.S.);
sasavukmirovic99@gmail.com (S.V.)
* Correspondence: milena.raseta@dh.uns.ac.rs (М.R); Tel.: +381-21-485-2762
Abstract: Medicinal mushrooms, especially Ganoderma species hold immense promise for
production of a wide range of bioactive compounds with various effects. The biochemical potential
of indigenous fungal strains, specific to a region, could play a critical role in the continuous search
for novel strains with superior activities on a global scale. This research focused on the ethanolic
(EtOH) and hot-water (H2O) extracts of fruiting bodies of two wild-growing Ganoderma species: G.
pfeifferi and G. resinaceum with an aim to assess its nutritional (total carbohydrate content-TCC), and
mineral composition in relation to bioactive properties: antioxidant, antiproliferative and lipid-
lowering. Atomic absorption spectrophotometry (AAS) revealed that G. pfeifferi is a promising
source of minerals that are essential for numerous physiological functions in the human body like
bone health and muscle and nerve function, with calcium (Ca 4.55 ± 0.41 mg/g d.w.) and magnesium
(Mg 1.33 ± 0.09 mg/g d.w.), being the most abundant macro element present. Zinc (Zn), manganese
(Mn), and chromium (Cr) were particularly notable, with concentrations ranging from 21.49 to 41.70
mg/kg d.w. The EtOH extract of G. pfeifferi demonstrated significantly elevated levels of TCC,
essential macromolecules for energy and structural functions in the body, with higher quantities of
all three standard carbohydrates detected in this type of extract. Similar to revealed composition the
same species, G. pfeifferi stood out as the most prominent antioxidant agent, with H2O extract being
stronger than EtOH in ABTS assay (86.85 ± 0.67 mg TE/g d.w.), while EtOH extract displayed the
highest anti OH• radical scavenging ability (IC50 = 0.18 ± 0.05 μg/mL) as well as the most notable
reducing potential among all. The highest antiproliferative effect against the breast cancer cell line
(MCF-7), demonstrated the H2O extracts from G. resinaceum with the most pronounced activity after
24 hours (IC50 = 4.88 ± 0.50 μg/mL) which surpasses that of the standard compound, ellagic acid (IC50
= 33.94 ± 3.69 μg/mL). Administration of both Ganoderma extracts mitigated diabetic lipid
disturbances and exhibited potential renal and hepatic protection in vivo on white Wistar rats by
preservation of kidney function parameters in G. resinaceum H2O pre-treatment (urea: 6.27 ± 0.64
mmol/L, creatinine: 50.00 ± 6.45 mmol/L) and the reduction of ALT levels (17.83 ± 3.25 U/L)
compared to diabetic control groups treated with saline (urea: 46.98 ± 6.01 mmol/L, creatinine: 289.25
± 73.87 mmol/L, and ALT: 60.17 ± 9.64 U/L). These results suggest that pre-treatment with G.
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© 2024 by the author(s). Distributed under a Creative Commons CC BY license.
2
resinaceum H2O extracts may have potential antidiabetic properties. In summary, detected
microelements are vital for maintaining overall health, supporting metabolic processes, and
protecting against various chronic diseases. Further research and dietary assessments could help
determine the full potential and applications of two underexplored Ganoderma species native to
Serbia in nutrition and health supplements.
Keywords: Ganoderma; natural products; health benefits; fungal extracts; anticancer activity; liver
protection; kidney protection; mineral composition
1. Introduction
The integration of traditional knowledge with modern scientific research continues to uncover
valuable natural compounds with potential health benefits. The rising incidence of chronic diseases
due to aging and lifestyle changes underscores the need for effective treatments [1]. Fungi, with their
rich nutritional profile and therapeutic properties, offer promising avenues for addressing global
health concerns, particularly oxidative stress. Leveraging ancient folk remedies, many of which form
the basis of modern medications, can enhance our approach to managing and preventing these
prevalent health issues [2]. For instance, recent research has focused on natural products for the
prevention and treatment of metabolic syndrome, with particular attention to medicinal mushrooms
[3,4]. Among these, the Ganoderma species, known for their extensive use in traditional medicine,
have shown promising potential [5,6]. Ganoderma species, such as G. resinaceum, have been
documented to possess various bioactive compounds, including nortriterpenoids (lucidone A–F, H,
I–K, ganosineniol B–C, ganoderense F, and ganosineniol), which exhibit a wide range of beneficial
biological activities [7]. These activities include anti-inflammatory, antioxidant, and antidiabetic
effects, which are particularly relevant to combating the multifaceted aspects of metabolic syndrome
[5,7].
Species belonging to the genus Ganoderma (P. Karst.) have been utilized as a natural resource in
Traditional Chinese Medicine (TCM) for over two hundred years [2,8]. Its utilization has primarily
relied on observations and accounts of its efficacy in cancer prevention and treatment, combating
infections, modulating the immune system, and regulating blood pressure, among other medicinal
benefits [5,6]. The medicinal properties of Ganoderma spp. primarily stem from the abundance of
various bioactive compounds they produce, notably triterpenoids like ganoderic acids, which have
been reported to exhibit anti-hypercholesterolemic, anticancer, hepatoprotective, antioxidant, anti-
inflammatory, antimicrobial, and hypoglycemic properties [9,10]. G. resinaceum extracts have shown
inhibitory effects against enzymes such as acetylcholinesterase, tyrosinase, α-amylase, and α-
glucosidase [11,12]. Also, nortriterpenoids extracted from Ganoderma species display a diverse range
of biological activities, such as antitumor, anti-inflammatory, neurotrophic, hepatoprotective, and
anti-HIV-1 protease effects [7]. These properties are noteworthy due to their structural diversity and
potential as models in pharmaceutical research. Additionally, polysaccharides such as β-glucans and
phenolic compounds play significant roles, exhibiting anti-inflammatory, antimicrobial, antioxidant
and antiproliferative [5,6,11–17], while ergosterol peroxide has been shown to induce tumor cell
death [18]. Wang et al. [8] proposed that the bioactive compounds from G. pfeifferi and other
Ganoderma species may exert a synergistic effect, indicating that their medicinal properties typically
result from the combination of multiple ingredients.
Ganoderma species, renowned for their medicinal properties, have been extensively studied for
their various health benefits [5,6,12,15,16,19–21]. In 1889, Patouillard expanded the genus to include
48 species, while data from 2022 indicate that the genus now comprises 181 species [22]. Among them,
G. lucidum is globally recognized, owing to its use in TCM as the “Mushroom of Immortality” [23].
The genus is widely distributed across the globe, particularly in tropical and subtropical areas
spanning Africa, America, Asia, and Europe [24]. In Europe, the genus consists of seven species [20],
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with G. pfeifferi and G. resinaceum being among the less studied species compared to G. lucidum, G.
applanatum, and G. adspersum, among others.
Regarding taxonomy of these species, Rašeta et al. [12] claimed that G. pfeifferi and G. resinaceum,
along with G. subamboinense and three G. lucidum strains from the United States and Taiwan, form a
single monophyletic cluster based on phylogenetic analysis. This group is distinguished by its ability
to produce chlamydospores in culture. Two selected species, G. pfeifferi and G. resinaceum are from
genus Ganoderma, which is the major genus of the Ganodermataceae family (Polyporales,
Basidiomycota), known for producing bioactive compounds such as triterpenoids, polysaccharides,
sterols and phenolics [6,20].
Studies have also highlighted the efficacy of Ganoderma extracts in improving insulin sensitivity,
regulating lipid metabolism, and reducing inflammation, as key factors in the management of
metabolic syndrome [25–28]. The bioactive compounds in Ganoderma species can modulate multiple
metabolic pathways in the body [29], making Ganoderma a promising candidate for the prevention
and treatment of various chronic diseases [30].
As of today, the focus on antioxidant, antimicrobial, and enzyme inhibition including in vitro
antidiabetic activity [11–14,16,17,19,31,32] has focused on new medicinal species G. pfeifferi,
originated from Serbia but also presents an autochthonous European species, and G. resinaceum,
distributed in Asia, and North Africa as well.
In our previous study, we presented findings on the use of (EtOH) and hot-water (H2O) extracts
in suspension form derived from G. pfeifferi and G. resinaceum, which showed potential for application
in diabetes [16]. Hence, the objective of this study was to conduct a thorough comparative assessment
of the in vitro antiproliferative and in vivo lipid-lowering, and hepato- and nephroprotective
characteristics of G. pfeifferi and G. resinaceum, an area notably deficient in current literature.
2. Materials and Methods
2.1. Fungal Material and Extracts Preparation
Fruiting bodies of two Ganoderma fungal species, namely G. pfeifferi and G. resinaceum, were
gathered in September 2010 from Nature Park Begečka Jama and the University of Novi Sad Campus
in Serbia, respectively. The determination and identification of the collected specimens took place at
the Department of Biology and Ecology, University of Novi Sad and voucher specimens have been
archived in the ProFungi Laboratory, Department of Biology and Ecology, University of Novi Sad
(Serbia), under the numbers 12-00723 and 12-00722.
All experiments utilized ethanol (EtOH) and hot-water (H2O) extracts, prepared according to
previously established methods [15,16]. Both extracts were dissolved in distilled water (dH2O),
achieving a final concentration of 100 mg/mL dry weight (d.w.) and were stored at temperatures of
−20 °C before undergoing analysis.
2.2. Mycochemical Characterization
2.2.1. Quantification of Macro- and Microelements by Using Atomic Absorption Spectrophotometry
(AAS)
Macro- (Ca and Mg) and microelements (Cu, Ni, Cd, Pb, Cr, Mn, Fe and Zn) were analyzed in
powdered fungal samples following the procedure outlined by Kebert et al. [33] using a flame
technique within Atomic Absorption Spectrophotometry (AAS) [model FS AAS240/GTA120, Agilent]
(Santa Clara, California, USA). Approximately 0.3 g of dried material (oven-dried at 70 °C for 24
hours) was ground, homogenized, and digested in a mixture of 10 mL of nitric acid and 2 mL of 30%
(w/v) hydrogen peroxide using a microwave-assisted digestion system (D series; Milestone,
Bergamo, Italy) at 180 °C (900 W) for 45 minutes. The resulting homogenates were filtered and diluted
to 25 mL with deionized water. The prepared samples were analyzed by using with the acetylene/air
burner flame technique for Cr, Cu, Mn, Fe, and Zn and the nitrous oxide (N2O)–acetylene flame for
Ca content determination. Concentrations of each element were determined using single-element
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hollow-cathode lamps at specific wavelengths and expressed as mg/kg dry weight (d.w.) of fungal
material.
2.2.2. Total Carbohydrate Content (TCC)
The total carbohydrate content (TCC) of the fungal extracts was determined using the phenol–
sulfuric acid method outlined in Rašeta et al. [16]. Initially, 50 μL of each fungal extract or glucose
was mixed with 150 μL of concentrated H2SO4 (or dH2O for correction) and shaken for 30 minutes at
room temperature in an incubator shaker (IKA KS 4000i control, Staufen, Baden-Württemberg,
Germany). Subsequently, 30 μL of 5% phenol in water was added, and the mixture was heated for 10
minutes at 70 °C in the same incubator shaker. After allowing the microplate (Thermo Fisher
Scientific, Waltham, Massachusetts, USA) to cool to room temperature for 5 minutes in a water bath,
it was dried, and absorbances (Thermo Fisher Scientific, Waltham, Massachusetts, USA) were taken
at 490 nm for glucose (hexose), 750 nm for sucrose as a disaccharide, and 480 nm for xylose (pentoses).
A calibration curves using glucose, sucrose and xylose (13.59–2173.91 μg/mL), were prepared to
determine the total carbohydrate content of the fungal extracts, expressed as mg glucose equivalents
(mg GluE), mg sucrose equivalents (mg SucE), and mg xylose equivalents (mg XylE), per gram of dry
weight (d.w.).
2.3. In Vitro Examination of Biological Activities
2.3.1. Antioxidant Activity
The antioxidant activity was assessed through the ABTS radical scavenging activity assay [34],
OH radical scavenging activity assay [35], while the reducing power of fungal extracts was
determined using the ascorbate equivalent antioxidant capacity (A.E.A.C.) assay [36]. The scavenging
activity in the case of ABTS assay and reducing power of the extracts were determined based on the
standard curve equation of Trolox and ascorbic acid, respectively, while results of scavenging OH
assay was expressed as IC50 values (concentration that inhibits 50% of hydroxyl radicals). The results
were quantified as milligrams of Trolox equivalents per gram of dry weight (mg TE/g d.w.) and
milligrams of ascorbic acid equivalents per gram of dry weight (mg AAE/g d.w.).
2.3.2. Antiproliferative Activity
The antiproliferative activity of the analyzed fungal extracts was assessed using the estrogen-
dependent breast cancer cell line (MCF-7) following the method outlined by Mosmann [37]. Ellagic
acid was utilized as positive control agent. Cancer cell viability was monitored over a 24-hour (acute)
and 72-hour (chronic) incubation period for extract concentrations ranging from 50 to 250 μg/mL.
Cell cytotoxicity was determined as the IC50, which represents the concentration that inhibits 50% of
cell growth extrapolated from concentration–response curves.
2.4. In Vivo Procedures and Assays
2.4.1. Laboratory Animals
In vivo part of the research was conducted on white Wistar rats of both genders, obtained from
the Military Medical Academy of Belgrade, Republic of Serbia. The rats, weighing between 210 and
340 grams and aged up to four months, were accommodated in UniProtect airflow cabinets (Ehret
GmbH, Emmendingen, Germany) with standard plexiglass cages. The housing conditions
maintained a constant room temperature of 22 ± 1 °C, 55% ± 1.5% humidity, and a regular circadian
rhythm (12-hour day/night cycle). Throughout the entire experiment, the rats were provided with
unrestricted access to tap water and standard pelleted laboratory rodent feed from the Veterinary
Institute Subotica, Serbia. All experimental procedures adhered to the guidelines of the European
Directive (2010/63/EU) for animal experiments and were subject to review and approval by the Ethics
Committee for the Protection and Welfare of Experimental Animals at the University of Novi Sad,
Serbia.
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2.4.2. Experimental Procedures
The animals were randomly allocated into ten groups, each comprising six individuals. Among
these, five groups underwent no alloxan pre-treatment (normoglycemic), while the remaining five
groups were subjected to alloxan-induced hyperglycemia (diabetic). In order to induce
hyperglycemia alloxan was dissolved in saline and applied intraperitoneally at a dose of 100 mg/kg.
Hyperglycemia was confirmed 48 hours following the application of alloxan and animals with a
glycemia higher than 15 mmol/L were selected for subsequent experiment. Both, normoglycemic and
diabetic, control groups of animals were treated with saline at a dose of 1 mg/mL. Similarly,
experimental groups of normoglycemic and diabetic animals were subjected to identical treatment,
receiving an oral aqueous suspension (1 mg/mL) containing the EtOH and H2O extracts of the two
analyzed fungal species. The extract suspensions were administered perorally by nasogastric probe
over a 5-day period. Two hours after the last dose of fungal extracts or saline, the rats were
anesthetized using a 25% solution of urethane at a dose of 5 mL/kg via intraperitoneal injection. Once
the righting reflex was lost, the animals were exsanguinated through intracardial puncture to obtain
blood and tissue samples for further analysis.
2.4.3. In Vivo Biochemical Parameters Analysis
The concentration of lipids (lipid status) was determined in the serum of animals. The
concentrations of total cholesterol, total triglycerides and high-density lipoprotein (HDL) cholesterol
and low-density lipoprotein (LDL) were measured using clinical biochemistry methods.
The enzymatic activity of aspartate aminotransferase (AST) and alanine aminotransferase (ALT),
as well as the concentrations of urea and creatinine, were assessed in order to monitor hepatic and
renal function using serum of the examined animals.
All analyses were performed using standard spectrophotometric methods on an automatic
chemical analysis system, Olympus AU 400 (Hamburg, Germany).
2.4. Statistical Analysis
The study utilized an array of statistical methods, including descriptive statistics, one-way
Analysis of Variance (ANOVA), t-tests, Principal Component Analysis (PCA), dendrogram
hierarchical clustering, and Pearson correlation analysis. The differentiation among analyzed fungi
species in the one-way ANOVA was assessed using the Fisher (F) test, with statistical significance
denoted by “p-values.” The results of the t-tests were depicted with box-plot diagrams. All statistical
analyses were performed using the R programming environment. The “rstatix” R package was
employed for descriptive statistics, two-way ANOVA, and t-tests, while dendrogram clustering was
carried out with the “dendextend” R package. Various data visualizations were created using the
“ggplot2” R package [38–40].
3. Results and Discussion
3.1. Mycochemical Characterization
3.1.1. AAS Quantification of Macro- and Microelements
AAS analysis was utilized to evaluate the multi elemental composition of both G. pfeifferi and G.
resinaceum, encompassing a total of 11 metals. These included macroelements such as Ca, K and Mg,
and microelements like Cu, Ni, Cd, Pb, Cr, Mn, Fe and Zn. The summarized results can be found in
Figure 1a,b.
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Figure 1. a) Composition of macroelements (mg/g d.w.) and b) microelements (mg/kg) in G. pfeifferi
and G. resinaceum samples.
Figure 1a,b display the outcomes of the mineral composition analysis, highlighting the
prevalence of three major macroelements, namely Mg, Ca and K. In G. pfeifferi, the predominant
macroelements were Mg and Ca, constituting a substantial portion of the overall mineral composition
[32], which aligns with previous studies highlighting the essential role of Mg and Ca in various
physiological processes, including the immune regulating actions of Mg and its crucial role in
regulating inflammation and immune response to infectious agents and malignancies [41,42].
Contrastingly, G. resinaceum exhibited a distinctive macroelement profile, with a notable abundance
of K. The significance of potassium in cellular activities, particularly in maintaining osmotic balance
and regulating enzyme functions, suggests potential therapeutic implications associated with G.
resinaceum consumption [43,44].
Beyond macroelements, the AAS analysis also unveiled the presence of essential microelements
in both Ganoderma species (Figure 1b). It is noteworthy that elements such as Zn and Cu were detected
in appreciable amounts in both tested species, with higher concentration of Zn in G. pfeifferi (41.70 ±
1.11 mg/kg d.w.), in contrast to G. resinaceum, where Cu was a more dominant microelement (22.22 ±
0.48 mg/kg d.w.). Moreover, the concentration of Cu in G. resinaceum from Poland was two times
lower (11.00 ± 3.00 mg/kg d.m.) [45] compared to concentration determined in this study, while Zn
was not quantified. These microelements play crucial roles in enzymatic activities, oxidative stress
defense, and overall metabolic processes [46,47].
The comparative assessment of mineral composition between G. pfeifferi and G. resinaceum
unveils species-specific variations and prevalence of tested elements in G. pfeifferi. This aligns with
the previous report where Marek et al. [45] suggested that among tested group of Ganoderma species,
fruit bodies of wild growing G. resinaceum and cultivated G. pfeifferi were characterized by a higher
level of all elements jointly than the other analyzed Ganoderma species. Rašeta et al. [48] highlighted
that the mineral content of edible fungi after consumption is influenced by cooking or processing
methods, often resulting in mineral leakage into water or brine. While these minerals offer nutritional
benefits, excessive intake may pose risks, especially considering fungus’ ability to accumulate toxic
elements and radionuclides. Therefore, it’s crucial to assess metal content in wild fungi and consume
them in moderation.
Considering this information and as depicted in Figure 1b, it can be inferred that G. pfeifferi and
G. resinaceum exhibited negligible accumulation of toxic elements such as the examined Cd and Pb
(ranging from 2.84 to 4.93 mg/kg and 2.84 to 3.45 mg/kg d.w., respectively). Such distinctions may
influence the therapeutic potential of these fungi, as the interplay between different elements could
contribute to their observed biological activities.
In summary, the metal composition analysis of G. pfeifferi and G. resinaceum indicated significant
concentrations of biogenic metals alongside minor levels of heavy metals, potentially attributable to
bioaccumulation processes, which is in accordance with Yalcin et al. [32]. Consequently, there arises
a necessity for the regulated cultivation of these fungi with promising medicinal attributes. Moreover,
understanding the mineral composition of Ganoderma species is pivotal for unraveling the potential
therapeutic benefits associated with these fungal species.
3.1.2. Total Carbohydrate Content (TCC)
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Regarding the TCC analysis, it is important to highlight that EtOH extracts of G. pfeifferi
demonstrated a notably higher concentration of glucose and sucrose (303.87 ± 54.80 mg GluE/g d.w.
and 44.51 ± 9.49 mg SucE/g d.w., respectively) compared to the same extract of G. resinaceum,
indicating a substantial disparity in their carbohydrate composition (Figure 2). Conversely, G.
resinaceum EtOH exhibited increased xylose content (233.18 ± 11.37 mg XylE/g d.w.), whereas all hot-
water extracts displayed statistically significant lower TCC in both species (Figure 2).
Figure 2. Total carbohydrate content: (a) glucose, (b) sucrose, and (c) xylose. Different small letters
indicate significant differences among different analyzed extracts of G. pfeifferi and G. resinaceum;
Tukey’s significant difference (HSD) post hoc test (p ≤ 0.05). Data represent the mean ± standard
deviation (SD).
These findings align with our earlier investigation, wherein EtOH extracts demonstrated
superiority in TCC compared to water extracts, accompanied by a higher TCC content measured in
glucose equivalents in G. pfeifferi [12]. On the other hand, higher TCC was determined in the study of
G. resinaceum from Serbia, where 52.1 ± 3.2 g GluE/100 g was quantified in hot water extract [31]. This
discrepancy in carbohydrate levels between the two species and type of solvent used, suggest that
extraction method, together with geographical origin i.e., ecological factors, may have implications
for their nutritional value and potential applications, as suggested for other fungal species [49].
Quantification of monosaccharide content in analyzed species represent valuable report, since
generally fungal polysaccharides are composed of glucose, galactose, and mannose, but other
carbohydrates can also be found (e.g., xylose, arabinose, fucose, ribose) [50], as demonstrated in this
study as well. Since ratio of monosaccharide composition in fungi is very important and it has been
shown that polysaccharides from G. lucidum, have positive effects as hypoglycemic agents [51], future
research should be based on detailed identification of polysaccharides including monosaccharide
composition from the analyzed two Ganoderma species as well.
3.2. Biological Activities of Examined Extracts
3.2.1. Antioxidant Activity
The antioxidant activity of Ganoderma species was assessed through ABTS, OH, and A.E.A.C
assays, revealing varying degrees of efficacy across the tested species (Figure 3).
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Figure 3. Antioxidant activity in hot-water (H2O) and ethanolic (EtOH) extracts of G. pfeifferi and G.
resinaceum: (a) RSC against ABTS radical (2,2′-azinobis-3-ethylbenzothiazoline-6-sulfonic acid); (b)
radical scavenger capacity against OH radical; (c) ascorbate equivalent antioxidant capacity
(A.E.A.C.) assay. Distinct lowercase letters denote significant differences observed among different
analyzed samples including standard compound, propyl gallate (PG), as determined by Tukey’s
honestly significant difference (HSD) post hoc test (p ≤ 0.05). The data are presented as the mean ±
standard deviation (SD).
Results from the ABTS assay indicated potent antioxidant capacity in G. pfeifferi H2O extracts
(86.85 ± 0.67 mg TE/g d.w.), while G. resinaceum exhibited a comparatively lower neutralization of
ABTS radical. Furthermore, there was no statistically significant distinction observed in the
neutralization of this radical between the EtOH and H2O extracts of G. resinaceum. This similarity is
also evident in the extracts of G. pfeifferi, albeit in the neutralization of the OH radical, where extracts
demonstrated notable antioxidant potential in comparison with the analyzed standard compound
(PG) (Figure 3). Interestingly, the A.E.A.C assay also highlighted G. pfeifferi as possessing the highest
reduction power activity, compared to reduction ability of G. resinaceum extracts.
In samples of G. resinaceum obtained from Turkey as well Zengin et al. [11] determined lower
antioxidant potential in comparison with results of this study, underscoring the diverse antioxidant
properties among Ganoderma species. Results from this study are in accordance with the study of
Yalcin et al. [32], where extracts of G. pfeifferi from Turkey showed higher reduction potential
compared to neutralization of ABTS radical. Moreover, compared to our results, H2O extracts of G.
pfeifferi exhibited two times higher ability of ABTS radical neutralization (170.32 ± 3.17 mg TE/g) [32].
Recently, Sułkowska-Ziaja et al. [19] conducted antioxidant analyses on both studied species. In
comparison with our findings, methanolic extracts from mycelial cultures (Lublin, Poland) exhibited
lower activity in the ABTS assay (9.77 ± 0.13 mg TE/g for G. pfeifferi and 11.60 ± 0.36 mg TE/g for G.
resinaceum, respectively), while the reduction potential of the samples analyzed by Sułkowska-Ziaja
et al. [19] was also lower (ranging from 8.10 to 31.78 mg TE/g). This suggests that the choice of extract
solvent and preparation method could influence experimental outcomes.
PCA analysis was conducted to revile a connection between the antioxidant activity and the
detected TCC, alongside our previous research (Table S1) [16], where the phenolic profile was
determined in the tested extracts. PCA analysis revealed 60.40% variance of PC1 and 25.97% of PC2,
while distinct clustering may be observed among two analyzed Ganoderma species (Figure 4).
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Figure 4. Principal Component Analysis (PCA) of the observed antioxidant and antiproliferative
activity along with phenolic compounds (Table S1) [16] and total carbohydrate content (TCC) in the
four examined fungal extracts of G. pfeifferi and G. resinaceum. The following are the abbreviations of
the examined parameters: EtOH - ethanolic extract; H2O - hot-water extract; ABTS - 2,2′-azinobis(3-
ethylbenzothiozoline)-6-sulfonic acid; OH - hydroxyl radical; A.E.A.C. - ascorbate equivalent
antioxidant capacity; IC50 24h/72h- antiproliferative inhibitory concentration in the case of incubation
of 24h and 72h; GluE - glucose equivalents; SucE - sucrose equivalents; XylE - xylose equivalents;
Tukey’s honestly significant difference (HSD) post hoc test (p ≤ 0.05). Data represent the mean ±
standard deviation (SD).
Evidently, G. resinaceum separated in the positive quadrant of both PCs, opposite to G. pfeifferi
extracts and all quantified compounds, suggesting negative correlation between phenolics (Table S1),
TCC and antioxidant properties of G. resinaceum extracts [16] (Figure 4). On the contrary, in G. pfeifferi
extracts clustered together with TCC and all identified phenolics, suggesting they role as strong
antioxidative agents in this species. Moreover, this is in accordance with correlation analysis where
strong positive correlation between antioxidant properties except in the case of neutralization of OH
radical and phenolic compounds is evident (Figure 5). Also, this assumption was confirmed in
previous research as well [11–13,15,16,49]. However, separation of G. resinaceum extracts in PCA
graph, opposite to phenolics (Table S1), could be related to lower levels of these compounds and thus
lower antioxidant properties observed.
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Figure 5. Pearson’s coefficient of the correlation matrix of the examined parameters in ethanolic
(EtOH) and hot-water (H2O) extracts of G. pfeifferi and G. resinaceum extracts collected on the territory
of the Republic of Serbia. Blue squares represent a highly significant correlation of inspected
parameters, while red squares present low interactions, assessed according to the corresponding
Pearson’s coefficient. The following are the abbreviations of the examined parameters: ABTS—radical
scavenger capacity against 2,2′-azinobis(3-ethylbenzothiozoline)-6-sulfonic acid, ABTS•+; OH—
radical scavenger capacity against hydoxyl radical, OH•; A.E.A.C. - ascorbate equivalent antioxidant
capacity; IC50 24h/72h- antiproliferative inhibitory concentration in the case of incubation of 24h and
72h; GluE - glucose equivalents content of TCC; SucE - sucrose equivalents content of TCC; XylE -
xylose equivalents content of TCC; All.Sal - alloxan + saline; CHOL - cholesterol; T.TGC - total
triglycerides; CREA - creatinine; ALT - alanine aminotransferase and AST - aspartate
aminotransferase.
3.2.2. Antiproliferative Activity
The MTT assay was utilized to assess the in vitro antiproliferative activity of the two analyzed
crude extracts and standard compound (ellagic acid). According to data from Table 1, it is evident
that all extracts demonstrated antiproliferative effects during subacute incubation (after 24 hours),
with the most potent activity observed in the H2O extract of G. resinaceum species (IC50 = 4.88 ± 0.50
μg/mL), comparable to the activity of standard compound (IC50 = 33.94 ± 3.69 μg/mL for ellagic acid).
In general, EtOH extracts showed less inhibition of MCF-7 cells, except for the G. pfeifferi EtOH
extract, which exhibited significant subacute inhibition (IC50 = 154.05 ± 12.92 μg/mL).
Table 1. The antiproliferative effects of Ganoderma extracts and standard compounds on the MCF-7
cell line the MTT assay, IC50 values (μg/mL).
Incubation
period
Analyzed samples
G. pfeifferi
EtOH
G. pfeifferi
H
2
O
G. resinaceum
EtOH
G. resinaceum
H
2
O
Ellagic acid
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24h
154.05 ± 12.92
c
653.35 ± 10.19
e
363.87±1.51
d
4.88±0.50
a
33.94±3.69
b
72h
78.33±1.89
b
49.25±1.72
a
181.07±0.21
d
113.33±0.62
c
43.06±1.22
a
IC50 – extract concentration required to inhibit cell growth by 50%. Values are expressed as mean ± SD of
triplicates. a,b,c,d,e – different letters in the same row in comparison with ellagic acid as a standard
compound indicate significant difference between extracts (ANOVA, Tukey post hoc, p < 0.05).
PCA analysis was performed to establish a link between the antiproliferative activities observed
in this study and detected TCC, together with our prior research findings (Table S1) [16] where
phenolic profile was determined in the tested extracts. The PCA analysis demonstrated distinct
clustering patterns among the Ganoderma species based on their antiproliferative activity. The initial
pair of principal components, PC1 and PC2, explained 86.37% of the overall variance, demonstrating
a significant portrayal of the dataset. Specifically, G. resinaceum which exhibited the highest
antiproliferative activity, separated in the positive quadrant of both PCs, opposite to G. pfeifferi and
all quantified compounds [16] (Figure 4). This is in accordance with obtained results, since lower
levels of phenolic compounds (Table S1) and TCC were observed in G. resinaceum EtOH and H2O
extracts. On the other hand, correlation matrix revealed positive correlation among xylose and
antiproliferative activity (Figure 5), suggesting that maybe carbohydrate compounds are important
for cytotoxic effect. In contrast, extracts from G. pfeifferi exhibited diminished antiproliferative
activity, yet demonstrated elevated levels of TCC (Figure 4) and phenolic content presented in Table
S1, clustering together within the I and III quadrants. Moreover, correlation matrix showed that only
protocatechuic acid, one of the most abundant phenolic in G. pfeiferi extracts (Table S1), has a
significant positive correlation with antiproliferative activity (Figure 5).
Rašeta et al. [15] summarized that extensive research conducted in recent decades has identified
a broad spectrum of bioactive compounds extracted from Ganoderma species, including phenolic
acids, isoflavones, polysaccharides, triterpenes, sterols, nucleosides, proteins, and polysaccharide-
protein complexes, all with potential antiproliferative effects. Considering the analysis of the MCF-7
cell line with unpurified extracts, it is hypothesized that interactions among various biomolecules
present in the tested extracts may synergistically demonstrate antiproliferative activity, potentially
mitigating the toxicity of individual components. This suggests that the interaction between different
biomolecules could enhance the therapeutic efficacy of Ganoderma crude extracts [15]. However,
drawing on previously reported data, it is theorized that the polysaccharides present in the analyzed
hot-water extracts are responsible for the antiproliferative activity, while terpenoids potentially
present in the EtOH extracts contribute primarily to a proliferative effect on MCF-7 cells [15].
Consequently, it can be inferred that this effect is linked to the direct antiproliferative treatment
against tumor cells [52]. The same group claimed that high-molecular weight fungal compounds such
polysaccharides and polysaccharide–protein complexes are significant for the exhibition of antitumor
activity, due to their increased solubility in water [52]. The most active polysaccharides of Ganoderma
belong to β-(1-3)-D-glucans, well known in promotion antitumor activity in animals and humans by
acting as immune modulators – biological response modifiers, because they promote natural and
acquired immunity of host organism itself [15].
In contrast, our results indicate that identified phenolic compounds (Table S1) and TCC are
probably not responsible for detected antiproliferative activity, especially in G. resinaceum. This
suggests that terpenoid compounds could play the main role in this activity [10], since various
extracts from Ganoderma lucidum enriched with triterpenoids inhibit the growth of hepatoma cells by
suppressing protein kinase C and activating mitogen-activated protein kinases [53]. Also, various
types of ganoderic acids, including ganoderic acid T and its C-3 epimer compound, isolated from G.
orbiforme, showed cytotoxic effect on MCF-7 cells [54], indicating that in this study triterpenoids could
be responsible for high antiproliferative activity of G. resinaceum, as well. The results of the study of
Rikame et al. [55] support this assumption, since terpenoids were quantified as major components in
G. resinaceum extracts and exhibited cytotoxic activity against human colon HCT 116 cancer cells,
while ganoresinoid A from this species alleviated LPS-induced apoptosis as described by Kou et al.
[56], but further research should be conducted.
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3.2.3. Lipid-Lowering Properties
Diabetes mellitus was induced in the animals, leading to dyslipidemia characteristic for this
disease, as best observed in the group receiving a physiological solution for five days after diabetes
induction. In this group and based on Table 2, a drastic increase in serum total triglyceride
concentration (4.19 mmol/L) is evident, which is significantly higher compared to the total
triglyceride values of the control group (1.14 mmol/L) and all other experimental groups treated with
fungal extracts. Another laboratory parameter supporting diabetic dyslipidemia is the elevated level
of LDL cholesterol fraction, significantly higher in the group of animals with diabetes treated with
physiological solution compared to all other investigated groups.
Table 2. In vivo biochemistry parameters; The concentration of total cholesterol (mmol/L),
triglycerides (TGC; mmol/L), HDL (mmol/L), and LDL (mmol/L) cholesterol, urea (mmol/L), and
creatinine (mmol/L), as well as the enzymatic activity of aspartate aminotransferase (AST; U/L) and
alanine aminotransferase (ALT; U/L) (mean value ± SD) in the serum of normoglycemic and diabetic
(alloxan) rats treated with saline, EtOH and H2O extracts of the species G. pfeifferi and G. resinaceum.
Parameter
Control Alloxan +
saline
Alloxan +
saline +
G.p.
EtOH
G.p.
EtOH
Alloxan +
saline +
G.p. H2O
G.p.
H2O
Alloxan +
saline +
G.r.
EtOH
G.r.
EtOH
Alloxan +
saline +
G.r. H2O
G.r.
H2O
Lipid
status
Total
cholesterol
1.54±0.24a
1.73±0.24a 1.78±0.25
a
1.56±0.34
a
1.90±0.26
a
1.71±0.19
a
1.55±0.23
a
1.63±0.32
a
1.49±0.18
a
1.55±0.21
a
Total TGC
1.14±0.60a
4.19±0.94b 1.09±0.20
a
0.89±0.19
a
1.71±0.23
a
1.090.40a
1.01±0.30
a
1.47±1.09
a
0.75±0.21
a
1.15±0.51
a
HDL
0.90±0.17a
0.75±0.17a 1.01±0.16
a
0.90±0.21
a
0.97±0.25
a
0.98±0.15
a
0.79±0.17
a
0.78±0.10
a
0.77±0.15
a
0.740.12a
LDL
0.21±0.10a
0.85±0.40b 0.27±0.14
a
0.25±0.12
a
0.06±0.06
a
0.30±0.13
a
0.32±0.12
a
0.35±0.10
a
0.37±0.16
a
0.27±0.15
a
Renal
function
Urea
7.40±0.56a
46.98±6.01b
76.64±7.63
c
8.50±1.37
a
74.32±10.
97
c
8.73±1.21
a
6.23±0.73
a
8.02±1.49
a
6.27±0.64
a
8.00±0.85
a
Creatinine
52.25±.62a
289.25±73.8
7
b
379.00±79.
09
c
51.00±3.0
3
a
395.40±97
.39
c
48.50±2.4
3
a
48.50±4.7
2
a
50.40±9.0
2
a
50.00±6.4
5
a
52.83±8.4
7
a
Liver
function
AST
268.00±44
.62a
310.50±33.2
9a
320.80±41.
41a
305.40±66
.41a
308.17±52
.32a
288.83±4
4.59a
224.67±70
.21a
319.83±10
.76a
207.50±38
.87a
284.67±1
8.33a
ALT
98.00±13.
00
c
60.17±9.64b
50.17±14.1
6
b
83.60±18.
35
c
51.83±12.
59
b
82.17±15.
54
c
23.20±3.9
0
a
34.60±10.
78
a
17.833.25
a
24.208.64
a
Data represent the mean ± standard deviation (SD). a, b, c Results that do not share the same superscript in the
same column are statistically significant. p<0.01 (Tukey test, ANOVA).
The animal model of alloxan-induced diabetes effectively mirrors lipid metabolism disturbances
in humans with hyperglycemia. This primarily leads to an increase in blood triglycerides as an
alternative energy source, resulting in the elevation of very-low-density lipoproteins (VLDL) and
subsequently the highly atherogenic LDL fraction. Therefore, considering the clinical therapeutic
goal, the focus is primarily on lowering LDL cholesterol and secondarily on total triglycerides [57].
The valuable nutritional profile and benefits of using fungi in hyperglycemic conditions have
been demonstrated in our previous studies [16,58,59]. In this study, it is evident that treatment with
extracts from both fungal species of the genus Ganoderma prevented the disturbance in lipid status
common in diabetes mellitus. There is even a tendency towards an increase in HDL cholesterol in
certain groups of diabetic animals treated with fungal extracts, although unfortunately, statistical
significance was not reached.
Furthermore, clinical studies have indicated a connection between dyslipidemia and the risk of
cancer development, emphasizing that anything favorably impacting the lipid profile can be
beneficial in prevention. In addition to showing antiproliferative potential, the examined Ganoderma
species demonstrate a positive effect on in vivo lipid status, representing their additional
pharmacological value [60,61].
In addition to lipid metabolism disturbances in diabetes, changes in renal and hepatic tissues
also occur, as observed in this study. Serum levels of urea and creatinine, biochemical indicators of
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kidney function, were significantly elevated in the group of diabetic animals treated with
physiological solution compared to the control group of healthy animals (urea – 46.98:7.40 mmol/L;
creatinine – 289.25:52.25 mmol/L). In all normoglycemic animals treated with fungal extracts, the
serum concentration of urea and creatinine remained at the level of the control group values, which
was not the case for diabetic animals. Neither the H2O or EtOH extract of G. pfeifferi succeeded in
preventing the increase in kidney function parameters; however, both extracts of G. resinaceum
achieved this. Although it has been demonstrated that meroterpenoids from fungi of the genus
Ganoderma exhibit renoprotective effects [62,63], we consider that the treatment duration with
extracts in this study is too short for a direct impact on renal tissue. Nevertheless, it is possible that
indirectly, through the regulation of glycemia, as shown in our previous research [16], there has been
a positive influence on serum levels of urea and creatinine in the case of the G. resinaceum.
Regarding liver function, the ANOVA test indicated no statistically significant differences in
AST values when comparing all groups of animals. In contrast to AST, the values of ALT, which is a
more specific indicator of liver function, differ significantly. It is observed that serum ALT values are
lower in all groups of animals treated with the extract of the G. resinaceum compared to all G. pfeifferi
groups, as well as the control groups of normoglycemic and diabetic animals. Additionally, the ALT
concentration in groups of diabetic animals treated with a physiological solution or G. pfeifferi extracts
is significantly lower compared to the control normoglycemic group. However, it cannot be
confidently claimed that a hepatoprotective effect has been demonstrated, only by considering the
result that compared to normoglycemic control in the most groups of animals treated with fungal
extracts a decrease in ALT values was evident. In our previous study, using the same model and
experimental design, we showed that there were no noticeable histo-morphological changes in liver
tissue indicating damage. The same study described that treatment with G. pfeifferi and G. resinaceum
fungal extracts has a positive effect on liver biochemical parameters, with lipid peroxidation in the
first place [16].
PCA analysis and correlation matrix for antidiabetic activity were also conducted to determine
the correlation levels among this activity and determined bioactive compounds. PCA total variance
was (76.07%), where PC1 was 58.66% and PC2 was 17.41% (Figure 6).
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Figure 6. Principal Component Analysis (PCA) of the lipid-lowering properties along with phenolic
compounds (Table S1) [16] and total carbohydrate content (TCC) in the four examined fungal extracts
of G. pfeifferi and G. resinaceum. The following are the abbreviations of the examined parameters: EtOH
- ethanolic extract; H2O - hot-water extract; Allox.Sal - alloxan + saline; CHOL - cholesterol; T.TGC -
total triglycerides; CREA - creatinine; ALT - alanine aminotransferase and AST - aspartate
aminotransferase; Tukey’s honestly significant difference (HSD) post hoc test (p ≤ 0.05). Data
represent the mean ± standard deviation (SD).
Regarding PCA analysis, a similar pattern emerged as seen with antioxidant and
antiproliferative activity (Figure 6), where extracts from G. resinaceum distinctly separated from those
of G. pfeifferi, along with all previously identified phenolics (Table S1) and TCC. This implies that the
potent antidiabetic activity observed in G. resinaceum extracts cannot be solely attributed to phenolics
and carbohydrates, suggesting that other compounds may likely play a pivotal role. To corroborate
this, findings from other authors illustrate hepatoprotective effect of terpenoid compounds isolated
from Ganoderma fungi, such as ganomycin, fornicatin A, D, and F [8,63]. However, the role of phenolic
compounds in antidiabetic activity should not be ignored, since correlation matrix revealed that
detected phenolic compounds (Table S1) showed significant positive correlation with measured
parameters regarding lipid-lowering properties of examined extracts. This is also in accordance with
our previous study [16] and Prabhakar [64], suggesting the necessity of further research and
investigation of synergistic effects.
4. Conclusions
By comprehending the unique chemical compositions and therapeutic potentials of G. pfeifferi
and G. resinaceum, these species could offer opportunities for the creation of innovative
biopharmaceutical treatments, particularly for conditions which encompass both irregular cell
growth and irregular lipid metabolism. The AAS quantification of micro and macroelements in G.
pfeifferi and G. resinaceum provides valuable insights into the nutritional and therapeutic aspects of
these medicinal fungal species. Further research exploring the biological activities associated with
specific mineral compositions is warranted, as it could enhance our understanding of the potential
health benefits conferred by these fungi.
Our findings underscore the therapeutic potential of Ganoderma extracts in ameliorating
dyslipidemia associated with diabetes mellitus. Moreover, these extracts did not lead to organ
damage; rather, they resulted in a decrease or no change in the biochemical parameters indicative of
liver and kidney function compared to the control group. Further investigation into the underlying
mechanisms and long-term effects of Ganoderma extracts on lipid metabolism and organ function is
warranted for comprehensive understanding and clinical translation.
Supplementary Materials: The following supporting information can be downloaded at the website of this
paper posted on Preprints.org. Table S1: LC-MS/MS detection of phenolic compounds in examined extracts.
Author Contributions: Conceptualization, M.R.; methodology, M.R., M.K. (Marko Kebert), S.K. (Sonja
Kaišarević), N.S. and S.V.; validation, M.R., M.K. (Marko Kebert), S.K. (Sonja Kaišarević), N.S. and S.V.; formal
analysis, M.R., M.K. (Marko Kebert), N.S. and S.V.; investigation, M.R., J.M. and M.K. (Marko Kebert); resources,
M.R., M.K. (Marko Kebert), S.K. (Sonja Kaišarević), N.S. and S.V.; data curation, M.R., M.K. (Marko Kebert), S.K.
(Saša Kostić), N.S. and S.V.; writing-original draft preparation, M.R. and J.M.; writing-review and editing, M.R.,
J.M., M.K. (Marko Kebert), S.K. (Saša Kostić), S.K. (Sonja Kaišarević), N.S., S.V. and M.K. (Maja Karaman);
visualization, M.R. and S.K. (Saša Kostić); supervision, M.R. and M.K. (Maja Karaman); project administration,
M.R., N.S., and S.V. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by the Ministry of Science, Technological Development, and Innovation of
the Republic of Serbia, grant numbers 451-03-66/2024-03/200125, 451-03-65/2024-03/200125 and 451-03-47/2023-
01/200114.
Institutional Review Board Statement: The experimental procedures were carried out following the guidelines
of the European Directive (2010/63/EU) on animal experiments and received approval from the Ethics Commiee
for Protection and Welfare of Experimental Animals at the University of Novi Sad, Serbia.
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15
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments: The authors express their gratitude to retired professor Dr. Mira Popović for her invaluable
assistance, knowledge transfer, and mentorship during the doctoral dissertation of Dr. Milena Rašeta.
Conflicts of Interest: The authors declare no conflicts of interest.
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