Secondary Metabolites from the Cultures of Medicinal Mushroom Vanderbylia robiniophila and Their Tyrosinase Inhibitory Activities

Vanderbylia robiniophila (Huaier in Chinese) has been used as a traditional herbal medicine in China for over 1600 years. However, the secondary metabolites of V. robiniophila have not been systematically examined. Corresponding chemical investigation in this study led to the discovery of two new compounds, (22E, 24R)-6β, 7α-dimethoxyergosta-8(14), 22-diene-3β, 5α-diol (1) and vanderbyliolide A (8), along with eight known ones (2–7, 9–10). Their structures were determined by extensive spectroscopic analyses and electronic circular dichroism (ECD) calculations. The tyrosinase inhibitory activity of all isolated compounds was evaluated, and compound 10 showed a potential tyrosinase inhibitory effect with an IC50 value of 60.47 ± 2.63 μM. Kinetic studies of the inhibition reactions suggested that 10 provides the inhibitory ability on tyrosinase in an uncompetitive way.


Introduction
Melanin is the primary pigment responsible for skin color and also protects human skin against harmful effects by absorbing ultraviolet (UV) rays and mitigating oxidative stress [1]. The excessive production of melanin can lead to hyperpigmentation-related disorders and even melanoma in severe cases [2]. Tyrosinase is a reaction rate-limiting enzyme in the process of melanogenesis [3,4]. The skin-lightening products have been investigated as the melanogenesis inhibitors in the medical and cosmetic industry. They are in large demand with a worldwide market of USD 23 billion in 2020 [5]. However, most of the commercially available tyrosinase inhibitors have some drawbacks. For instance, vitamin C is susceptibly degradable and sensitive to temperature and air [6]. Kojic acid and arbutin are reported to cause safety issues such as skin irritation [7], while arbutin is chemically instable and might result in leukemia due to the metabolized products of benzene analogues [8]. Therefore, safe, stable, and effective tyrosinase inhibitors are still in great need for the treatment of dyspigmentation disease and cosmetic applications.
Mushrooms have been traditionally used as food ingredients and folk medicine since antiquity, and recent studies have elucidated their functional benefits, such as anticancer, anti-inflammatory, antiviral, antibacterial and hepatoprotective properties, which have been attributed to various mushroom components [9,10]. Medicinal mushrooms are known for producing specialized molecules with great chemo-diversity that have relevant impacts on human health and diseases [11]. These compounds have proved immensely valuable in the pharmaceutical industry as they provide the structural template and pharmacophores of commercially successful drugs with excellent clinical efficacies and acceptable sideeffects [12]. These include the antibiotic retapamulin from Pleurotus multilus [13] and the first-in-class sphingosine-1-phosphate (S1P) receptor modulator named fingolimod impacts on human health and diseases [11]. These compounds have proved immensely valuable in the pharmaceutical industry as they provide the structural template and pharmacophores of commercially successful drugs with excellent clinical efficacies and acceptable side-effects [12]. These include the antibiotic retapamulin from Pleurotus multilus [13] and the first-in-class sphingosine-1-phosphate (S1P) receptor modulator named fingolimod from Isaria sinclairii [14]. Vanderbylia robiniophila (Murrill) B.K. Cui & Y.C. Dai (Huaier) is a medicinal mushroom mainly parasitized on the trunk of Robinia pseudoacacia L. It has been widely used for more than 1600 years in traditional Chinese medicine (TCM) [15]. Nowadays, "Huaier granule" is recognized by Chinese State Food and Drug Administration as a complementary medicine for the treatment of multiple malignancies, including liver cancer, lung cancer, digestive system cancers, and breast carcinoma [16,17].
The mechanisms behind the significant antitumor effect exerted by Vanderbylia robiniophila are fascinating and intrigue many researchers [18]. It has been revealed that V. robiniophila directly inhibits the growth and proliferation of cancer cells [19], arrests the cell cycle [20], restrains invasion and metastasis [21], interferes with angiogenesis [22], induces cell apoptosis [23] and regulates immune responses [24]. In contrast to the pharmacological activities, there is limited information about the chemical constituents of V. robiniophila. Recent studies have shown that polysaccharides, proteoglycan, and amino acids are the primary ingredients in V. robiniophila extract [25,26]. A metabolomic comparison between the naturally and the artificially cultured Huaier extract using LC-MS analysis showed that the former contains more amino acids, alkaloids, and terpenoids [27]. However, few researchers have been able to conduct any systematic research into the isolation and exact structure elucidation of the secondary metabolites of V. robiniophila [28].
During our investigations on macrofungal resources, a wild-derived strain was isolated from the fruit body of Vanderbylia robiniophila, and the EtOAc fraction of the strain showed significant tyrosinase inhibitory activity. To discover potential tyrosinase inhibitors in V. robiniophila, a detailed chemical investigation of the large-scale fermentation in rice was carried out, which resulted in the purification of two novel secondary metabolites, along with eight known ones, including seven steroids (1-7), two 2(5H)-furanone derivatives (8)(9), and a monoindole alkaloid (10) (Figure 1). The tyrosinase inhibitory activities of the isolated compounds were evaluated. Herein, the details of the isolation, structure elucidation, and bioactivities of compounds 1-10 are described.

General Experimental Procedures
UV spectra were recorded on an ultraviolet-visible spectrometer (UV-1500PC, Macy Instruments, Inc., Shanghai, China) and IR spectra were measured on a Thermo Fisher Nicolet 6700 FT-IR spectrometer (Thermo Scientific, Madison, WI, USA). Optical rotation values were obtained using a JASCO DIP-370 digital polarimeter (JASCO, Tokyo, Japan)

Reagents and Chemicals
The HPLC-grade solvents, such as methanol and acetonitrile, and the analytical reagent solvents, such as petroleum ether, dichloromethane, ethyl acetate, and ethanol, were purchased from Tianjin Yongda Chemical Reagent Co., Ltd. (Tianjin, China). Tyrosinase (1100 U/mg) from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China) was used as the enzyme. L-tyrosine (98%, Biotopped, Beijing, China) served as the substrate and arbutin (98%, Shanghai Macklin Biochemical Co., Ltd., Shanghai, China) was used as a positive control.

Fungal Material
Vanderbylia robiniophila was collected from Liaoning Province, China, in August 2020 and authenticated based on the morphology analysis and ITS gene sequencing (GenBank Accession number OR116090) ( Figure S33) [2]. The fungus was preserved at the Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang.

Fermentation and Extraction
Vanderbylia robiniophila was cultured on malt extract agar (MEA) plates at 28 • C for 7 days. Then, 5 pieces of mycelia were inoculated in a 250 mL conical flask containing 150 mL liquid medium (3% malt extract) and shaking cultured at 28 • C for 7 days at 140 rpm. Afterward, the seed liquid culture (8 mL) was transferred into rice medium (100 g rice and 100 mL water) and fermented at 28 • C statically for 3 months. Subsequently, the cultures were extracted by 90% EtOH 5 times at room temperature. The residue was suspended in water and partitioned with EtOAc.

ECD Calculations
The conformational searches were carried out employing SPARTAN 14 in the MMFF force field. The conformers under the 3.0 kJ/mol energy window were further optimized at the B3LYP/6-31G(d) level by DFT using the Gaussian 09 package [29]. The optimized stable conformers were calculated using TDDFT methods at the B3LYP/6-311+g(d,p) level with an implicit solvent model for CDCl 3 . Then, the calculated ECD curve weighted by the Boltzmann distribution was produced by SpecDis 1.71 [30]. , where A is the absorbance of blank solution after incubation, B is the absorbance of blank solution before incubation, C is the absorbance of sample solution after incubation, and D is the absorbance of sample solution before incubation. The IC 50 values and statistical analyses were performed using GraphPad Prism 7 software, and the results were expressed as means ± SD of triplicate determination.

Kinetic Analysis
The inhibition type was measured by the reaction rate-substrate concentration curve and the Lineweaver-Burk plot. L-tyrosine solutions were diluted to different concentrations (5 mM, 7.5 mM, 9 mM, 12 mM, 15 mM) as the substate. The inhibition constant was determined by the second plot of the y-intercept versus the concentration of the inhibitor. The values of K is were calculated from the following formula:

Anti-Tyrosinase Activities
Tyrosinase inhibitors have been clinically used for diseases associated with melanin hyperpigmentation [47]. In this study, the inhibitory effects of 1-10 on tyrosinase were determined spectrophotometrically compared to the positive control arbutin (Table 2). Among them, compound 10 showed the highest tyrosinase inhibitory activity with IC 50 values of 60.47 ± 2.63 µM, which were comparable to those of arbutin (IC 50 = 58.17 ± 6.09 µM). Compounds 2, 4, 5, and 8 exhibited weak inhibitory activities with IC 50 values ranging from 94.16 to 148.38 µM. The remaining compounds did not show any tyrosinase inhibitory activity at the texted concentration. These observations demonstrated that the indole alkaloid exhibited much stronger inhibitory potency than the other structures. It has been reported that the amino groups such as an indole ring could improve the tyrosinase inhibitory activity, which was consistent with our results [48,49]. Among the isolated steroids, compound 6 has a structural feature of C-ring migration and compound 7 is a highly degraded sterol belonging to the class incisterols. They were inactive even at 200 µM, indicating the importance of the tetracyclic ergostane-type scaffold. Detailed inspection of the structures of compounds 2, 4, and 5 showed the common features of a double bond at ∆ 7(8) within these molecules, which indicated that the position of olefinic bond is crucial for the activity.

Enzyme Kinetic Analysis
To confirm the inhibition mechanism of the most potent compound (10) on the inhibitory activity of tyrosinase, the V-S and Lineweaver-Burk plots were constructed. As shown in Figure 5A,B, both K m and V m values of 10 decreased with the increase in concentration, but the ratio of K m /V m remained unchanged. Thus, 10 belongs to an uncompetitive inhibitor, demonstrating that it inhibits the enzyme by combining with the enzyme-substrate complex. The inhibition constant K is was obtained from the plot of the y-intercept versus the concentration of 10, which was calculated to be 0.04 mM ( Figure 5C). values of 60.47 ± 2.63 µM, which were comparable to those of arbutin (IC50 = 58.17 ± 6.09 µM). Compounds 2, 4, 5, and 8 exhibited weak inhibitory activities with IC50 values ranging from 94.16 to 148.38 µM. The remaining compounds did not show any tyrosinase inhibitory activity at the texted concentration. These observations demonstrated that the indole alkaloid exhibited much stronger inhibitory potency than the other structures. It has been reported that the amino groups such as an indole ring could improve the tyrosinase inhibitory activity, which was consistent with our results [48,49]. Among the isolated steroids, compound 6 has a structural feature of C-ring migration and compound 7 is a highly degraded sterol belonging to the class incisterols. They were inactive even at 200 µM, indicating the importance of the tetracyclic ergostane-type scaffold. Detailed inspection of the structures of compounds 2, 4, and 5 showed the common features of a double bond at Δ 7(8) within these molecules, which indicated that the position of olefinic bond is crucial for the activity.

Enzyme Kinetic Analysis
To confirm the inhibition mechanism of the most potent compound (10) on the inhibitory activity of tyrosinase, the V-S and Lineweaver-Burk plots were constructed. As shown in Figure 5A,B, both Km and Vm values of 10 decreased with the increase in concentration, but the ratio of Km/Vm remained unchanged. Thus, 10 belongs to an uncompetitive inhibitor, demonstrating that it inhibits the enzyme by combining with the enzyme-substrate complex. The inhibition constant Kis was obtained from the plot of the y-intercept versus the concentration of 10, which was calculated to be 0.04 mM ( Figure 5C).

Conclusions
In summary, two new compounds, (22E, 24R)-6β, 7α-dimethoxyergosta-8(14), 22diene-3β, 5α-diol (1) and vanderbyliolide A (8), together with eight known ones (2-7, 9-10), were isolated from the cultures of Vanderbylia robiniophila. All compounds were discovered from the genus for the first time. Compound 10 showed potential tyrosinase inhibitory activity comparable to that of arbutin. The kinetics of the enzymatic reaction indicated that 10 was an uncompetitive inhibitor on tyrosinase. This study provides evidence for the development and utilization of V. robiniophila in skin disorders associated with melanin hyperpigmentation.