Cosmetic potential of Ganoderma lucidum (Reishi) extract in a topical cream formulation

Abstract

Objective

The primary objective of this study was to investigate the biological activities of freeze‐dried Ganoderma lucidum (Reishi) extract obtained from hazelnut pruning waste and to compare its antioxidant, tyrosinase inhibitory and cytotoxic properties with two widely used cosmetic actives—argireline peptide and α‐arbutin. Additionally, the study aimed to develop a topical cream formulation containing Reishi extract and to assess its microbiological safety, preservative efficacy and physicochemical stability under controlled environmental conditions. By integrating bioactivity evaluation with formulation performance, the research sought to determine the potential applicability of Reishi extract as a natural and multifunctional cosmetic ingredient suitable for anti‐aging and skin‐brightening applications.

Methods

The antioxidant capacity of Reishi extract, argireline peptide and α‐arbutin was determined using a copper(II)‐based total antioxidant capacity assay. Tyrosinase inhibitory activity was measured with human tyrosinase sourced from MNT‐1 melanoma cells, while cytotoxicity and safe dose limits were determined in HaCaT human keratinocyte cells via the AlamarBlue® method. A topical cream formulation was prepared using a multi‐phase emulsification technique and characterized physicochemically. Microbiological safety was assessed according to Turkish Cosmetic Regulations, and preservative efficacy was determined through challenge testing. Stability assessments included temperature cycling, long‐term storage at different temperatures and centrifugation tests, monitoring visual, physicochemical and microbiological parameters over a three‐month period.

Results

Reishi extract demonstrated 861 μmol TE/g antioxidant activity, 30% tyrosinase inhibition and a broader safety margin compared with argireline peptide and α‐arbutin, which exhibited cytotoxicity at lower concentrations. The formulated cream retained a skin‐compatible pH and stable viscosity, while showing no signs of phase separation, colour change or integrity loss under all storage conditions. Microbiological analysis confirmed microbial counts below regulatory limits, and challenge testing revealed rapid log reductions in all tested microorganisms, satisfying the acceptance criteria for preservative efficacy. Stability studies supported the physical robustness and microbiological safety of the final product throughout the evaluation period.

Conclusion

Overall, the findings highlight Reishi extract as a bioactive‐rich, safe and stable natural ingredient with strong potential for incorporation into cosmetic formulations. Its antioxidant performance, moderate tyrosinase inhibition and compatibility within the cream matrix support its use in multifunctional anti‐aging and skin‐brightening products.

The biological activities of Reishi mushroom extract (antioxidant, tyrosinase inhibition, cytotoxicity) were evaluated, and the cream formulation developed with the extract was validated through stability and microbiological tests. The results demonstrate that Reishi extract is a promising natural ingredient in cosmetics.

INTRODUCTION

Mushrooms are rich in protein, vitamins and minerals and are excellent sources of glucan, selenium, thiamine, riboflavin, niacin, pantothenic acid and folic acid [1, 2]. There are studies showing that mushrooms have a positive effect on weight control and provide beneficial effects such as supporting longevity and slowing down physiological aging [3, 4]. In recent years, mushrooms have attracted global attention as valuable natural resources, exhibiting diverse bioactivities including immunomodulatory, antioxidant, anti‐inflammatory, antidiabetic, antibacterial, antifungal, antiviral, antitumour, hepatoprotective and lipid‐ and glucose‐lowering properties [5, 6]. As consumers' demand for healthier, organic and ecologically perceived products increases, consumer demand for cosmetics with natural and/or organic ingredients also increases, paving the way for the use of various ingredients derived from mushrooms, such as ceramides, lentinan, schizophyllan, omega 3, 6 and 9 fatty acids, carotenoids and resveratrol in cosmetic products [7]. Many mushrooms possess powerful antioxidant and anti‐inflammatory properties, frequently used to address cosmetic concerns such as fine lines, wrinkles, uneven skin tone and skin dullness. The chemical compounds obtained from the fruiting bodies of macrofungi contain numerous polysaccharides, phenolic compounds, polysaccharide peptides, free amino acids, sterols, proteins, glycosides, triterpenes and alkaloids that can exhibit antioxidant, photoprotective, skin whitening, moisturizing, anti‐inflammatory and anti‐aging effects by stabilizing collagen, elastin and hyaluronic acid levels in the skin [8]. For example, the Reishi mushroom (Ganoderma lucidum) has been used in anti‐aging skincare products in the Far East since the 1980s and rapidly entered the cosmetics market in Western countries in the 2000s, where it is still used in many cosmetic and personal care products. Bath/cleansing gels containing Reishi extract have been reported to reduce toxicity in skin cells, increase hydration, decrease TEWL (transepidermal water loss) and reduce irritation, thereby improving the safety and performance of skincare products [9].

Reishi mushroom is a basidiomycete highly valued as a traditional Chinese herb, and its extracts contain bioactive compounds such as polysaccharides, triterpenoids, alkaloids, enzymes and proteins (including glycoprotein) [10]. Various glycoproteins and glycopeptides have been isolated from Ganoderma species. Glycoproteins are complex proteins formed by the covalent bonding of a peptide chain and a sugar chain. They can be used as enzymes, hormones, transporters, lectins, antibodies and other substances, and different types of glycoproteins have different functions. A glycopeptide is a type of glycoprotein that generally contains a high proportion of polysaccharides. It has various biological functions and plays an important role in cosmetic and dermatological fields [11, 12].

Collectively, Ganoderma proteins exhibit antioxidant, tyrosinase‐inhibitory, anti‐bacterial, anti‐viral, anti‐inflammatory and anti‐allergic activities, highlighting their potential as cosmetic ingredients. Oxidation is the greatest threat to skin aging, and oxidative damage is always accompanied by the cellular aging process. Oxidation causes a decrease in protein activity, including proteases, leading to the accumulation of damaged material, the progressive deterioration of tissues and organs and the formation of free radicals [13]. If the number of free radicals exceeds the reducing power of enzymes and non‐enzymatic antioxidant systems in the body, the body enters a state of stress and leads to aging of the body as the free radical scavenging mechanism deteriorates with age [14]. Excessive accumulation of free radicals in the skin leads to lipid peroxidation, accelerating skin aging [15]. Therefore, reducing the production of free radicals, eliminating aging metabolites and increasing the activity of antioxidant enzymes have become effective methods for delaying skin aging. Polysaccharides, glycoproteins, phenols and proteins in the Reishi mushroom are effective antioxidant components. Reishi extract provides skin protection in a photo‐aging model by reducing UVA‐induced mitochondrial stress and epidermal thickening. This effect is due to the antioxidant activity of triterpenoids [16]. There are also studies demonstrating the anti‐wrinkle activities of Reishi mushroom extract, as well as its free radical scavenging and skin aging delaying properties in cosmetics [17, 18].

Human skin colour is determined by the melanin pigment produced by melanocyte cells located in the basal layer of the epidermis. The accumulation of melanin in visible areas of the skin for various reasons causes brown spots on the skin, a condition known as hyperpigmentation. Tyrosinase, a rate‐limiting enzyme, plays a key role in melanogenesis, the physiological process of melanin production, and whitening agents and tyrosinase inhibitors are widely used in the treatment of hyperpigmentation [19]. Studies have shown that Reishi mushroom exhibits the highest inhibition against tyrosinase activity compared to other basidiomycetes and can be used as a skin whitening agent [20, 21].

Considering these biological activities, Reishi extracts have been increasingly incorporated into anti‐aging, skin‐whitening and anti‐allergic cosmetic products. It is aimed to design an anti‐aging night cream formulation using Reishi mushroom (Ganoderma lucidum) extract, produced from hazelnut pruning waste by Düzce University Industrial Recycling of Agricultural Waste Research and Application Center [22]. This research constitutes a preliminary step toward product development and aims to compare Reishi extract with argireline peptide—widely used for its anti‐aging properties—and α‐arbutin, commonly applied as a skin‐whitening agent in cosmetic products. Argireline peptide and α‐arbutin were not selected as mechanistic equivalents of the Reishi extract, but rather as widely used cosmetic reference ingredients representing distinct functional application categories. α‐Arbutin was included as a reference compound for skin‐lightening activity due to its well‐established tyrosinase inhibitory properties, whereas argireline peptide was considered a representative anti‐aging ingredient commonly employed in commercial cosmetic formulations. Accordingly, the comparison was intended to contextualize the cosmetic relevance of the Reishi extract within established cosmetic benchmarks, rather than to suggest comparable biological mechanisms or overlapping modes of action. Additionally, the study includes the preparation of a cosmetic formulation incorporating Reishi extract, followed by microbiological, anti‐microbial and stability analyses to assess its potential applicability in cosmetic product development.

MATERIALS AND METHODS

Materials

Reishi mushroom, cultivated on hazelnut pruning waste, was obtained from Düzce University Agricultural Waste Industrial Recycling Research and Application Center for extraction. Argireline peptide and α‐arbutin were purchased from cosmetic raw material suppliers.

Preparation of freeze‐dried Reishi mushroom extracts

Reishi mushrooms were obtained from the Agricultural Waste Industrial Recycling Research and Application Center of Düzce University. For extraction, the mushrooms were first ground using a high‐speed blender. The ground material was defatted by washing with petroleum ether and then dried in a hot air oven at 35°C for 16 h to achieve uniform moisture content. The dried mushrooms were mixed with 75% ethanol at a ratio of 1:10 (w/w) and subjected to sonication at 40°C for 30 min. Subsequently, the mixture was incubated in a water bath with orbital shaking at 40°C for 24 h. The ethanol was then removed using a rotary evaporator, and the remaining extract was freeze‐dried using a vacuum lyophilizer (LABFREEZ FD‐10F‐RE) between −50°C and 40°C. The resulting light brown extract was stored at +4°C until further use. For enzymatic and antioxidant activity assays, a stock solution was prepared in dimethyl sulfoxide (DMSO) at 20 mg/mL, incubated at 37°C for 5 h with gentle shaking and centrifuged at 10 000  g . The clear supernatant was collected for subsequent analyses.

Determination of antioxidant activity

The total antioxidant capacity (TAC) of the Reishi mushroom (Ganoderma lucidum) extract was determined using the Antioxidant Assay Kit (MAK334‐1KT; Merck, Germany). In this assay, antioxidants in the extract reduce Cu²⁺ ions to Cu⁺, which subsequently reacts with the kit's dye to form a coloured complex. The colour intensity is proportional to the overall antioxidant activity of the sample. Measurements were performed at 570 nm using a UV–visible spectrophotometer, with Trolox as the standard. The Reishi extract was also compared with arbutin and argireline peptide under the same conditions. All assays were carried out in triplicate, and results were expressed as μM Trolox equivalents (μM TE).

Determination of tyrosinase enzyme inhibitory

The tyrosinase inhibitory activity of the freeze‐dried Reishi mushroom extract was evaluated using human tyrosinase (hsTYR) obtained from MNT‐1 melanoma cell lysates. The assay is based on the enzymatic oxidation of L‐DOPA to L‐DOPAquinone by hsTYR, followed by spectrophotometric monitoring of melanin formation. Samples that inhibit tyrosinase activity result in decreased melanin production, reflected by lower absorbance values measured at 600 nm. Cells were detached using 0.05% trypsin–EDTA, washed with 1X PBS and lysed in PBS containing 1% Triton X‐100. The supernatant was transferred to 96‐well microplates, and Reishi extract solutions were added at increasing concentrations, maintaining a final DMSO concentration of 5% in all wells. After the addition of 4 mM L‐DOPA, the reaction mixtures were incubated at 37°C for 4 h. Absorbance was then measured with a UV–visible spectrophotometer, and the percentage of tyrosinase inhibition was calculated relative to the control. The inhibitory activity of the Reishi extract was also directly compared with that of arbutin and argireline peptide under identical experimental conditions. All experiments were performed in triplicate [23].

Cytotoxicity and dose determination

For cytotoxicity assessment and determination of the safe dose, HaCaT human keratinocyte cells were seeded at 5 × 10³ cells per well in 96‐well plates and allowed to attach for 24 h at 37°C under 5% CO₂. Cells were treated with varying concentrations of Reishi extract, as well as arbutin and argireline peptide for comparison. After 24 h, cell viability was measured using the AlamarBlue® assay (Invitrogen), with fluorescence readings recorded at excitation/emission of 560/590 nm [24]. Cell viability percentages were calculated relative to untreated controls, and the safe concentration range for subsequent experiments was established based on these results. All experiments were performed in triplicate.

Formulation design and characterization of a topical skin cream containing Reishi extract

The formulation presented in Table 1 was used to prepare the topical cream containing Reishi mushroom extract. The cream was prepared using a phase‐based emulsification method. Initially, glycerin and xanthan gum were weighed and mixed, then gradually dispersed into distilled water under continuous stirring to form the aqueous phase. The oil‐phase ingredients were weighed separately. Both phases were heated in a water bath (or on a hot plate) for 10–15 min until all solid components were fully melted, maintaining the temperature at 70–75 °C. The oil phase was then slowly incorporated into the aqueous phase with constant stirring to form a homogeneous emulsion. The mixture was stirred intermittently for 10–15 min while cooling. Once the temperature fell below 40 °C, Phase C ingredients were added, followed by Phase D at temperatures below 30 °C, with gentle mixing after each addition. The final pH of the cream was measured, and the finished product was transferred into suitable containers, labelled and stored at +4 °C. The cream's pH was recorded using a pH meter (Hanna HI98‐192), density was measured with a density kit (Precisa 350–8636). The viscosity of the cream formulations was measured at room temperature (22–25 °C) using a Fungilab Alpha R viscometer at a rotational speed of 50 rpm. Measurements were performed in triplicate, and mean values (in mPa·s) were reported.

TABLE 1.

Formulation of topical Reishi extract cream.

Phase	Function	Component
A	Thickener, Stabilizer	Xanthan Gum
A	Humectant	Glycerin
A	Solvent	Aqua
A	Active	Reishi Extract
B	Emulsifier	Cetearyl Olivate, Sorbitan Olivate
B	Thickener, Stabilizer	Cetyl alcohol
B	Emollient	Coco Caprylate/Caprate
B	Emollient	Caprylic/Capric Triglyceride
B	Occlusive	Shea butter
C	Antioxidant	Tocopheryl acetate
C	Preservative	Glyceryl Caprylate (And) Glyceryl Undecylenate
D	Fragrance	Cedar essential oil

Microbiological analysis of the topical cream

Microbiological quality of the Reishi‐containing topical cream was evaluated in accordance with the guidelines established by the Turkish Medicines and Medical Devices Agency [25]. The presence of specific microorganisms that must not be detected in cosmetic products—namely, Staphylococcus aureus (gram‐positive cocci), Pseudomonas aeruginosa (non‐fermentative gram‐negative rods), Escherichia coli (fermentative gram‐negative rods), Candida albicans (yeast) and Aspergillus brasiliensis (mould)—was assessed. Total aerobic mesophilic microorganisms were determined following TSE EN ISO 21149:2017. The growth of E. coli (ATCC 8739), P. aeruginosa (ATCC 9027), S. aureus (ATCC 6538) and C. albicans (ATCC 10231), as well as mould formation, were evaluated according to the corresponding TSE EN ISO standards [26]. Microbial cultures were first grown in Brain Heart Infusion Broth for 18–24 h. Bacterial suspensions were then standardized to a turbidity equivalent to 0.5 McFarland (approximately 1 × 10⁸ CFU/mL) and subsequently diluted in sterile physiological saline to reach a working concentration of 5 × 10⁵ CFU/mL. Microbiological analyses were conducted using these standardized suspensions, and the methods employed are summarized in Table 2.

TABLE 2.

Microbiological analysis methods.

Analysis	Method	Medium
Staphylococcus aureus ATCC 6538	Enrichment & Inoculation of Culture Media	Baird Parker Agar Medium
Pseudomonas aeruginosa ATCC 9027	Enrichment & Inoculation of Culture Media	Cetrimide Agar Medium
Escherichia coli ATCC 8739	Enrichment & Inoculation of Culture Media	MacConkey Agar Medium + Levine Eosin – Methylene Blue Agar Medium
Candida albicans ATCC 10231	Enrichment & Inoculation of Culture Media	Sabouraud 4% Dextrose Agar + Supplement
Moulds – Yeast	Pour Plate or the Spread Plate Technique	Sabouraud 4% Dextrose Agar + Supplement
Total Aerobic Mesophilic Microorganisms	Pour Plate or the Spread Plate Technique	Tryptic soy Agar with Polysorbate 80 and Lecithin

Anti‐microbial analysis (challenge test) of the topical cream

Challenge testing, also referred to as preservative efficacy testing, was conducted to evaluate whether the Reishi‐containing topical cream formulation provides adequate protection against microbial contamination during consumer use. This procedure was performed following TS EN ISO 11930:2019 and interpreted according to the criteria outlined in the European Pharmacopoeia 5.1.3 (Effectiveness of Anti‐microbial Preservation) [27]. The cream was inoculated with five standard microorganisms: Staphylococcus aureus (ATCC 6538), Pseudomonas aeruginosa (ATCC 9027), Escherichia coli (ATCC 8739), Candida albicans (ATCC 10231) and Aspergillus brasiliensis. Control periods were set according to the product‐specific guidelines. Microbial counts were monitored at defined intervals, and logarithmic reductions of each microorganism were determined on days 7, 14 and 28 following inoculation. All assays were conducted in triplicate, and the methods employed are summarized in Table 3.

TABLE 3.

Anti‐microbial analysis methods.

Analysis	Method	Culture media	Incubation condition
Staphylococcus aureus ATCC 6538	Pour Plate	Tryptic Soy Agar	30°C – 35°C
Pseudomonas aeruginosa ATCC 9027	Pour Plate	Tryptic Soy Agar	30°C – 35°C
Escherichia coli ATCC 8739	Pour Plate	Tryptic Soy Agar	30°C – 35°C
Candida albicans ATCC 10231	Pour Plate	Sabouraud 4% Dextrose Agar	30°C – 35°C
Aspergillus brasiliensis NCPF 2275	Pour Plate	Potato Dextrose Agar	30°C – 35°C

Stability analysis of the topical cream

The stability of the Reishi‐containing topical cream was assessed to determine whether its pH, colour, odour, viscosity, packaging integrity and microbiological quality remained consistent under various stress conditions, simulating the product's shelf life. Stability testing was performed using a stability cabinet device (JSDS‐300C, South Korea) to mimic a 2‐year shelf life. For constant temperature studies, the cream was first stored at −24 °C for 24 h, then thawed and examined. Subsequently, samples were kept at −5 °C for 1 week and evaluated. Room temperature storage at 25 °C in the dark was conducted for 4 months, with assessments performed weekly during the first month and monthly during the second and third months. Additional samples were stored at +45 °C for 3 months, with observations at 3 days, weekly for 4 weeks, and at the second and third months.

To evaluate the effect of temperature fluctuations, the cream was subjected to cyclic storage over 6 weeks, alternating 24 h at −5 °C and 24 h at +25 °C in each cycle. Physical stability under centrifugal stress was tested by centrifuging the samples at −25 °C at 10000 rpm for 10 min and at 3000 rpm for 30 min. At all time points, the cream's pH, viscosity, colour, odour and overall appearance were recorded to monitor stability [28, 29].

Statistical analysis

All experiments were performed in triplicate (n = 3), and the results are expressed as mean ± standard deviation (SD). Due to the exploratory nature of the study and its primary focus on preliminary bioactivity screening and formulation characterization, descriptive statistical analysis was applied. No inferential statistical comparisons were performed.

RESULTS

Antioxidant activity

The total antioxidant capacity (TAC) of the Reishi mushroom extract and the reference compounds was assessed in Figure 1 to compare their overall antioxidant potential. The Reishi extract exhibited a TAC of 861 μmol Trolox equivalents (μmol TE), indicating a considerable antioxidant activity. In comparison, argireline peptide demonstrated a lower TAC value of 375 μmol TE, whereas α‐arbutin showed a considerably higher TAC of 2117 μmol TE. The relatively large standard deviation observed for α‐arbutin compared with the other tested compounds may be attributed to minor variations in solution homogeneity, assay sensitivity or biological variability inherent to cell‐based measurements. These results suggest that while Reishi extract possesses significant antioxidant properties, its activity is intermediate between the tested peptide and α‐arbutin.

FIGURE 1.

Comparison of the total antioxidant capacity (TAC) among Reishi extract, argireline peptide and α‐arbutin.

Tyrosinase enzyme inhibitory

The tyrosinase inhibitory activity of Reishi mushroom extract was evaluated in conjunction with arbutin and argireline peptide as seen in Figure 2. Reishi extract reduced enzyme activity by approximately 30%. Arbutin, a potent tyrosinase inhibitor, inhibited tyrosinase by approximately 80%, while argireline peptide, frequently used in anti‐aging creams and mesotherapeutic applications, did not inhibit tyrosinase but instead increased enzyme activity by 15%. Overall, Reishi extract showed moderate tyrosinase inhibition, although not as much as arbutin.

FIGURE 2.

hsTYR inhibitory activity of Reishi extract (a), argireline peptide (b) and α‐arbutin (c).

Cytotoxicity and dose determination

The cytotoxicity of Reishi mushroom extract was evaluated in HaCaT human keratinocyte cells to determine the dose limit (DLU) for topical formulations, as shown in Figure 3. Reishi extract preserved cell viability up to 25 μg/mL, and concentrations above this threshold resulted in a gradual decrease in viability. In contrast, arbutin exhibited cytotoxic effects at concentrations exceeding 12.5 μg/mL, while the argireline peptide reduced cell viability at doses above 0.8 μg/mL. These results suggest that Reishi extract can be incorporated at relatively higher concentrations without compromising keratinocyte viability and offers a wider safety margin compared to arbutin and, particularly, the argireline peptide, which exhibit cytotoxicity at much lower concentrations.

FIGURE 3.

Cytotoxicity of Reishi extract (a), argireline peptide (b) and α‐arbutin (c) on HaCaT cells. [Colour figure can be viewed at wileyonlinelibrary.com]

Formulation design and characterization of a topical skin cream containing Reishi extract

The resulting Reishi extract‐based topical cream is white, as shown in Figure 4. The fragrance was formulated using cedar essential oil to mask the characteristic mushroom odour, resulting in a light woody scent. The pH, density and viscosity of the cream are presented in Table 4. The pH was measured to be approximately 5.3, which is within the typical physiological range of human skin (pH 4.7–5.5). The logarithmic viscosity of the prepared cream was determined to be 1.42, indicating a suitable consistency for topical application.

FIGURE 4.

Topical Reishi extract cream. [Colour figure can be viewed at wileyonlinelibrary.com]

TABLE 4.

Physicochemical properties of topical cream.

Formulation	pH ± SD	Density ± SD (g/mL)	Viscosity ± SD (cP)	Log viscosity
Cream	5.286 ± 0.014	1.327 ± 0.008	26.257 ± 0.382	1.42

Microbiological analysis of the topical cream

Skin pathogens Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli and Candida albicans are considered to be unacceptable and are not permitted in 1 g/mL of cosmetic product. The results of the microbiological analyses evaluated in this context are shown in Table 5.

TABLE 5.

Microbiological analysis of topical cream.

Parameter	Unit	Microbiological analysis results			Tolerances
		Sample 1	Sample 2	Sample 3
Total Aerobic Mesophilic Microorganisms	CFU/g	< 10	< 10	< 10	< 102
Staphylococcus aureus	/1 g‐mL	Absence	Absence	Absence	Absence/1 g‐mL
Pseudomonas aeruginosa	/1 g‐mL	Absence	Absence	Absence	Absence/1 g‐mL
Escherichia coli	/1 g‐mL	Absence	Absence	Absence	Absence/1 g‐mL
Candida albicans	/1 g‐mL	Absence	Absence	Absence	Absence/1 g‐mL
Total Yeast and Moulds	CFU/g	< 10	< 10	< 10	< 102

Abbreviation: CFU, Colony forming unit.

Anti‐microbial analysis of the topical cream

Anti‐microbial challenge test results are presented in Table 6. All microorganisms, including Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Candida albicans and Aspergillus brasiliensis, showed a significant reduction within the first 7 days. By day 14 and day 28, microbial counts fell below detectable limits (<10 CFU/g), meeting the acceptance criteria defined by the European Pharmacopoeia 5.1.3. These findings indicate that the preservative system consisting of glyceryl caprylate and glyceryl undecylenate provides sufficient anti‐microbial protection during product usage.

TABLE 6.

Anti‐microbial analysis of topical cream.

Microorganism	0. Day		7. Day	14. Day	28. Day
	N	N0	N7	N14	N28
	(cfu/g)	(cfu/g)	(cfu/g)	(cfu/g)	(cfu/g)
Staphylococcus aureus ATCC 6538	1.7 × 108	8.0 × 105	< 10	< 10	< 10
Pseudomonas aeruginosa ATCC 9027	1.6 × 108	2.0 × 104	< 10	< 10	< 10
Escherichia coli ATCC 8739	1.4 × 108	6.5 × 105	< 10	< 10	< 10
Candida albicans ATCC 10231	2.2 × 106	2.5 × 103	< 10	< 10	< 10
Aspergillus brasiliensis NCPF 2275	1.5 × 106	1.3 × 103	< 10	< 10	< 10

Stability analysis of the topical cream

The stability of the topical cream containing Reishi extract was evaluated for 3 months under controlled storage conditions. The formulation maintained its physical integrity throughout the study period, with no observed changes in colour, odour, appearance, viscosity or phase separation (Table 7). The pH remained stable, ranging between 5.29 and 5.41, consistent with skin‐compatible acidity. No microbiological growth was detected during the entire storage period. These results confirm that the formulation is physically and microbiologically stable under the tested conditions.

TABLE 7.

Stability analysis of topical cream.

Parameters	1. Week	1. Month	2. Month	3. Month
	28.06.2025	22.07.2025	22.08.2025	22.09.2025
Colour	White	White	White	White
Odour	Woody	Woody	Woody	Woody
Appearance	No change	No change	No change	No change
pH	5.35	5.29	5.41	5.38
Package	No change	No change	No change	No change
Phase Separation	No change	No change	No change	No change
Microbiological growth	No	No	No	No

DISCUSSION

Reishi mushroom possesses an antioxidant protection system that protects living organisms from the effects of free radicals thanks to the glycoproteins, polysaccharides and phenolic compounds it contains [30]. In one study, different extraction methods were used to extract crude Ganoderma protein from Reishi mushroom, and its antioxidant activity was determined by electromagnetic paramagnetic resonance (EPR). All of them were found to possess antioxidant activity, with the water‐soluble protein crude extract having the highest scavenging activity against hydroxyl radicals and superoxide radicals [31]. In another study, the DPPH radical scavenging activity of Reishi mushroom extract samples obtained with methanol varied between 810 and 998 μM TE [32]. This study supports our results. Another study has shown that GLP2, a polysaccharide obtained from Reishi mushroom, exhibited only 9.5% lower antioxidant activity than butylated hydroxytoluene, which is known to be a strong synthetic antioxidant, and that Reishi mushroom components can be used as a potential antioxidant [33, 34]. In parallel, there are studies confirming the anti‐aging effects of Reishi mushroom extract on aging model mice. In the same study, in vitro analyses of cosmetic products made with the extract on skin fibroblast cells revealed that it stimulated the secretion of hyaluronic acid (HA), hydroxyproline (HYP) and collagen types I and II [35]. Another study examined the wound‐healing efficacy of creams containing different concentrations of Reishi mushroom extract obtained through hot water extraction in rats. The study found that a cream containing 10% Reishi mushroom extract had the fastest wound‐healing effect, while creams prepared at 5%, 15% and 20% concentrations also healed wounds significantly faster than the control group [36]. Reishi mushroom, thanks to its biological functions, is a high‐potential ingredient in various dermocosmetic products. Although the extract is exposed to 70–75°C for approximately 5 min during the emulsification step, its thermal robustness was confirmed in a separate ongoing study in which exposure to 75°C for 15 min (worst‐case condition) did not result in any detectable loss of antioxidant activity (unpublished data). This observation is consistent with previous reports indicating that the major antioxidant constituents of Ganoderma lucidum, particularly polysaccharides and triterpenoids, retain their bioactivity under moderate heating conditions and during extraction processes performed at temperatures up to 80–100°C [30, 34, 37].

Skin spots, which are caused by the accumulation of melanin pigment on the surface of the epidermis due to internal factors such as biological aging, menopause and puberty, or external factors such as UV radiation, nutrition and stress, are one of the biggest skin problems [19]. Skin lighteners used to solve this problem are known to partially inhibit tyrosinase activity, and the most commonly used ingredients in this regard are kojic acid and arbutin [38, 39, 40]. Tyrosinase is a copper‐containing enzyme found in plant and animal tissues that catalyses the production of melanin and other pigments from tyrosine through oxidation [41]. In a study investigating the effectiveness of extracts of Reishi and related basidiomycetes in inhibiting tyrosinase and hence their potential for use as a skin lightener, Reishi mushroom exhibited the highest inhibition of tyrosinase activity compared to other basidiomycetes. While all other basidiomycetes showed inhibition below 25%, the ethanolic extract of Reishi mushroom showed approximately 40% inhibition [21]. Similarly, in a study where the compound ganodermanontriol, isolated from G. lucidum, was examined to determine whether it inhibits tyrosinase activity and melanin production, no tyrosinase inhibition effect was observed up to a certain amount, while approximately 25–50% tyrosinase inhibition was detected as the amount increased [42]. These studies confirm our results. Another study also demonstrated the usability of Reishi mushroom as a skin whitening agent [20].

In addition to all these bioactive effects, the fact that Reishi mushroom is a natural product and has no toxic effects increases its potential in the cosmetic field. Mushrooms are generally known to be non‐toxic, and inotodiol concentrate extracted from the Chaga (Inonotus obliquus) mushroom has not shown toxicity on HaCaT keratinocyte cells [43], and similarly, Reishi extract has not shown any toxicity against human fibroblast Hs68 [21]. Abate et al. reported that Reishi extract protects keratinocytes against H₂O₂‐induced cytotoxicity thanks to its antioxidant activity and therefore accelerates wound healing processes [44]. The apparent discrepancy between the non‐cytotoxic concentration reported in another study, where concentrations of Reishi extract up to 200 μg/mL were tolerated, and the lower threshold observed in our study, can primarily be attributed to differences in extract composition and experimental design. In the study by Wang et al., Ganoderma lucidum was used as part of an extract along with Panax ginseng, a plant known to support cellular metabolism and proliferation; this may have mitigated the cytotoxic responses observed when Ganoderma extracts were evaluated alone. Furthermore, differences in cell viability acceptance criteria, tested concentration ranges, and the lack of detailed extract standardization further limit direct quantitative comparison. In contrast, the present study evaluated a chemically characterized Ganoderma lucidum extract alone and applied a conservative cosmetic safety threshold, which may have led to reporting a lower non‐cytotoxic concentration while providing a more cautious assessment [45]. Based on these results, a topical cream developed from Reishi mushroom extract, which presents a high potential cosmetic active ingredient for anti‐aging cosmetic products, exhibited desirable cosmetic properties such as a smooth and homogeneous texture, good spreadability and rapid absorption upon topical application. Sensory evaluation revealed that the cream formed a light, non‐greasy film on the skin surface and provided a pleasant lasting effect suitable for daily use. The oil‐in‐water emulsion structure contributed to rapid absorption and even distribution, demonstrating good dermal compatibility and delivery potential of the active ingredients. Furthermore, the formulation maintained its physicochemical stability under accelerated and prolonged storage conditions, with no observable phase separation, odour change or pH shift during the study period. Microbiological quality remained within acceptable limits, and preservative efficacy tests confirmed that it provided effective protection against bacterial, yeast and mould contamination. These results collectively demonstrate that Reishi extract can be successfully incorporated into a stable and microbiologically safe cosmetic formulation with favourable sensory and functional properties suitable for commercial application. A direct comparison with a placebo cream was not included in the present study, as the biological activities were evaluated using the Ganoderma lucidum extract itself rather than the final formulation. While the formulation assessments focused on physicochemical stability, microbiological quality and preservative efficacy, placebo‐controlled evaluations of the final product would be valuable in future studies addressing in vivo or functional cosmetic performance.

CONFLICT OF INTEREST STATEMENT

The authors declare that there is no conflict of interest to disclose.

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request.