Ganoderma lucidum Methanolic Extraction as a Potent Phytoconstituent: Characterization, in-vitro Antimicrobial and Cytotoxic Activity

Background: Ganoderma lucidum has attracted tremendous attention due to its exceptional antimicrobial and anticancer properties that can be delicately tuned by controlling the initial extraction content and concentration. In the present experiment, we detailed the characterization, antimicrobial, and cytotoxic performance of Ganoderma lucidum as a potential multi-functional therapeutic agent. Methods: In this study, we used FTIR, XRD, FESEM, EDX, and HPLC techniques to evaluate the samples, which were then followed by disc diffusion and microdilution broth methods to test its antibacterial effects against four Gram-positive and Gram-negative bacterial strains, Enterococcus faecalis, Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. MTT assay was applied to determine the cytotoxic activity of this natural product against MCF-7 and K562 cancer cell lines. Results: The results revealed that the inhibitory effects of this product had higher antibacterial activity against E. coli and Pseudomonas aeruginosa. The IC50 values of 0.5 and 0.75 mg/mL were obtained for MCF-7 and K-562 cancer cell lines, which conrmed the higher anticancer activity of the GLME against breast cancer cells compared to blood cancer cells. Conclusions: Hence, these data provide pioneer insights into the therapeutic usage of Ganoderma lucidum for treating breast and blood cancers. This work is motivated by research studies looking for pharmacological products to address chronic and acute diseases, where further resources and studies are required to explore such products' adverse effects and toxicity.


Background
Natural products or herbal medicines maybe promising alternatives or supplement for chemotherapy and antibiotic therapy [1,2]. Herbal medicines have extraordinary properties like high antibacterial, antioxidant, and anticancer activities [3,4]. For thousands of years, Ganoderma lucidum (GL) has been used as a major source of pharmacologically active constituents in Chinese and traditional Japanese medicine. It has piqued the attention of researchers and scientists with its numerous medicinal and pharmacological uses since it contains signi cant pharmacologically active compounds [5][6][7]. Proteins, sugars, avonoids, vitamins, minerals, triterpenes, and polysaccharides are among the biologically active compounds found in extractions of GL [8]. The presence of polysaccharides and triterpenoids in GL's structure has numerous pharmacological features. Thence, many experiments have been accomplished to investigate the performance of this natural product against different types of cancer like prostate cancer [9], lung cancer [10], colon cancer [11], and cervical cancer [12]. Also, GL's other pharmacological characteristics such as anti-in ammatory [13], hypoglycaemic [14], hypocholesterolemic [15], antioxidant activity [16], cardio-protective, hepato-protective, and anti-allergic activity, have been evaluated by researchers [5].
This research was planned and carried out in two parts to determine the characterization of GL methanolic extract (GLME) and investigate this natural product's antibacterial and cytotoxic activity. In the rst part, to enhance GLME's properties, sample extraction was done via the standard method. The second portion of this study's antibacterial studies followed the Clinical and Laboratory Standards Institute (CLSI) recommendations. The disc diffusion assay was used to test GLME's antibacterial tolerance against Staphylococcus aureus, E. coli, Enterococcus faecalis, and Pseudomonas aeruginosa. The microdilution broth approach was then used to determine the minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of this substance, with both bactericidal and bacteriostatic effects de ned. In the nal section of this investigation, the cytotoxic activity of GLME against human breast cancer cells and human blood cancer cells was assessed through MTT assay.

Sample extraction and preparation
Pure dried GL was purchased from a traditional market in Shiraz, Iran. The dried GL was grounded to form a ne powder, and then its essence was extracted by pouring it with methanol at room temperature. In this regard, 2.5 g of dried GL was rst blended with 100 mL of methanol and then shook for 24h at 125 rpm. In the next step, the mixture was ltered using lter paper and placed in an oven at 60°C for 12h.
After that, 3 mg of the nal extracted essence was dissolved in MeOH as the stock solution for biological tests.

Characterization
Different analyses were carried out to test the properties of GLME. FTIR spectroscopy with KBr tablets (Bruker model Tensor II) and X-ray diffraction (XRD) (Panalytical model X'Pert Pro, Almelo, Netherlands) was used to explore its crystallinity. The morphology of GLME (Tescan model S Max detector, Brno, Czech Republic) was studied using a eld emission-scanning electron microscope (FE-SEM, Tescan model Mira III, Brno, Czech Republic) and energy dispersive spectroscopy (EDAX). An Azura HPLC device (Knauer, Berlin, Germany) tted with a quaternary gradient pump unit and a UV-vis detector (190-700 nm) were used to study the contents of monosaccharides and disaccharides from GLME. The detector's wavelength was set to 250 nanometers. The components were separated by a Knauer C18 column (4.6mm 250mm i.d., 5m). Solvents A (acetonitrile) and B (benzene) made up the mobile phase (0.045 percent KH2PO4), where 0.8 mLmin-1 and 20 mL were the injection volume and ow rate, respectively.
The mobile process was screened with a 0.45 mm lter and degassed under vacuum until application.
The system was run at ambient temperature.

Disc diffusion assay
Brie y, a 0.5 McFarland scale (1.5 10 8 colony forming units (CFU)/ml)) bacteria culture was used in this experiment, with an optical density of 590 nm. We applied this bacterial culture to nutrient agar with a sterile swab at this stage. Afterward, the blank discs were placed on the solid surface of nutrient agar while all of the discs were soaked in the GLME until saturated. Plates were placed within the incubator at 37ºC for 18 h, and nally, the diameter of inhibition zones was measured [17][18][19]. A solution containing 20% ethanol in water was used as control group because of probable presence of ethanol in nal herbal extract.

Minimum inhibitory concentrations (MICs) assay
Both procedures in this assay were performed according to the Clinical and Laboratory Standards Institute's (CLSI) guidelines for determining GLME's antibacterial susceptibility [20][21][22]. The 96 well-plate was lled with BHI or liquid medium at a concentration of 90 liters for this experiment. The GLME methanolic extraction was then pumped into the culture medium at a concentration of 90 L (with a descending concentration from 1000 g/mL to 7.8 g/mL). In the next step, 10µL of 600 nm OD (0.5 McFarland) microorganisms were transferred into the mentioned wells, which contained the developed samples. The plates were then incubated for 24 hours at 37°C. We used Ampicillin as a standard drug to determine Ganoderma lucidum's antibacterial susceptibility in this method. A solution containing 20% ethanol in water was used as control group because of probable presence of ethanol in nal herbal extract. The optical density was estimated at 600 nm in the nal step (BioTek, Power WaveXS2). This process was carried out three times.

Minimum bactericidal concentrations (MBCs) assay
The value of MBCs was calculated by culturing the media from nutrient agar wells with no bacterial growth. A solution containing 20% ethanol in water was used as control group because of probable presence of ethanol in nal herbal extract. The MBC value is the minor concentration capable of killing 98 percent of microorganisms in culture [23]. This procedure was also done three times. 2.7. MTT assay MCF-7 and K562 cell lines were used to test the cytotoxicity of GLME. The positive control was hydrogen peroxide, and the negative control was the culture medium containing 20% ethanol was used as control group because of probable presence of ethanol in nal herbal extract. A certain amount of each cell line (10000) was placed in each well containing DMEM culture media and incubated to achieve 85 to 90% con uence. The previous media was then used to substitute 100 µL of GLME in a wide variety of concentrations. Then, in each well, 30 mL of MTT (3-(4,5 Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium) stock solution (concentration 4 mg/mL) was transferred and incubated under normal conditions. Purple formazan crystals formed due to the viable cells' mitochondrial function, and we used 100 liters of dimethyl sulfoxide (DMSO) to dissolve these crystals. The plate was shaken in a double orbital manner (for 5 minutes) to dissolve formazan crystals completely. Finally, the optical absorption of the mentioned solution was recorded at 540 nm using an Elisa plate reader (Model 50, Bio-Rad Corp, Hercules, California, USA) [24,25] [29].
The crystallinity of GLME was investigated using XRD, and the resulting pattern is shown in Fig. 1.b. The crystalline plane 002 was assigned to the XRD pattern, which showed an apparent plateau at around 2 = 20°. No peaks existed at higher scattering angles, indicating that the compound was amorphous [30]. FE-SEM images at various scales (i.e., 1 m, 500 nm, and 200 nm) and EDAX analysis of GLME are shown in Fig. 2. GLME had a rod-shaped structure with particle sizes smaller than 60 nm, as seen in Fig. 2. This natural commodity also contained a lot of carbon, nitrogen, oxygen, magnesium, sulfur, potassium, and calcium, according to EDAX research.
The contents of monosaccharides and disaccharides from GLME were calculated using an HPLC method in optimum separation conditions, with acetonitrile-0.045 percent KH2PO4 as the mobile step and a ow rate of 0.8 mLmin-1 at 250 nm as the detector. Polysaccharides from GLME were detected by comparing the retention time of each part with standard curves. Monosaccharides and disaccharides, such as lactose, glucose, sucrose, and maltose, were de ned in Fig. 3C.
The inhibitory action of GLME against selected bacterial strains (i.e., Staphylococcus aureus, Enterococcus faecalis, E. coli, and Pseudomonas aeruginosa) was investigated using the microdilution method (Fig. 3a). GLME was reported to have antibacterial effects against microorganisms at high concentrations (1000, 500 g/ml). It can be noted that the antibacterial effects increased in a concentration-dependent manner as the concentration value increased. The obtained results revealed that the inhibitory effects of this product against gram-negative and gram-positive bacterial strains were not similar. According to the presented data in Table 1, it can be understood that this valuable product had higher antibacterial activity against E. coli and Pseudomonas aeruginosa. The MIC values of the GLME were 125 µg/ml and 250 µg/ml for Gram-negative and Gram-positive microorganisms, respectively. The viability of Staphylococcus aureus and Enterococcus faecalis subjected to GLME was 146 percent and 117 percent, respectively, at the most diluted concentration of the experiment (7.8 g/mL), indicating that the extract had some bene cial effects on bacterial growth. GLME possesses bactericidal and bacteriostatic effects against Gram-negative and Gram-positive strains, mainly due to polysaccharide components in its structure. Figure 3 (d) shows a view of the disc diffusion method after exposure to four different microorganisms.
A common and acceptable approach for assessing cell viability is the MTT assay. This method can also detect and determine biomaterial toxicity [31][32][33]. MTT assay can depict the metabolism and mitochondrial activity of cells. This experiment evaluated the viability or proliferation of human breast and blood cancer cells after 24 h of treatment with methanolic extraction of GLME (see Fig. 3b). The metabolic performance of cells was changed in a dose-dependent manner by the GLME, where the dosage of the sample was varied from 1 to 3000 µg/mL. By increasing the concentration from 1 to 3000 µg/ml, the cell viability percentage was diminished from 108-2.5% for the K-562 blood cancer cell line and 91-6% for the MCF-7 cancerous breast cell line. The IC50 values of 0.5 and 0.75 mg/mL were obtained for MCF-7 and K-562 cancer cell lines, which con rmed the higher anticancer activity of the GLME against breast cancer cells compared to blood cancer cells. The mean zones of inhibition in the disc diffusion (DD) method, with a disc diameter of 6 mm, were measured in millimeters. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were measured mg/mL. The initial doses of both medications were identical (1 mg/mL in 1:1 dilution).

Conclusions
GLME is known to be quite a competent medicinal and nutraceutical agent, owing to its pharmacological, chemical components derived from fruit bodies, mycelium, and spores, which can be health-promoting agents. This work is motivated by research studies looking for pharmacological products to address chronic and acute diseases, where further resources and studies are required to explore such products' adverse effects and toxicity. More clinical studies are needed to validate the e cacy and protection provided by such products. More experiments and clinical research in the future are likely to take place on a wide scale. This research aims to examine the antimicrobial, cytotoxic, and genotoxic activity of the GLME. The extract was evaluated for its antibacterial and antifungal e cacy toward different microorganisms by utilizing a microdilution broth system. GLME was examined using an approach to assess its cytotoxic activity against breast cancer and blood cancer cells. There is no funding for this study.
Authors' contributions SMM and AG developed the idea and structure of the review article. SMM, VRN, SAH and KY wrote the manuscript collecting the materials from databases. MS, AG, W-HC and NO revised and improved the manuscript. AG and NO supervised the manuscript. All the authors have given approval to the nal version of the manuscript. The natural product's FESEM images and EDAX analysis Figure 3 (a) Effects of methanolic GLME extraction on various microorganism's viability percentages in different concentrations (each bar re ects the mean SD (standard deviation) of three independent tests). b) effects of methanolic GLME extraction on human breast and blood cancer cells. GLME blocked cell growth in MCF-7 and K-562 cell lines, as shown by the MTT assay, in which cells were treated with various concentrations of GLME for 24 hours. The results are interpreted by nding the mean and standard deviation of the three separate experiments. (c) HPLC analysis of the GLME. (d) Inhibition zone of GLME against (I) Pseudomonas aeruginosa, (II) Enterococcus faecalis, (III) Staphylococcus aureus, and (IV) Escherichia coli.