Tuliposides H–J and Bioactive Components from the Bulb of Amana edulis

Three new tuliposides H–J (1–3) and 11 known compounds were obtained from the methanolic extracts of the bulbs of Amana edulis for the first time. Their structures were elucidated by NMR, MS, and IR spectroscopic data, optical rotation, and Mosher’s method. The melanogenesis properties of all the isolates were evaluated in B16 melanoma cells. Consequently, tributyl citrate (9) had anti-melanogenesis activity but was cytotoxic toward B16. (+)-Pyroglutamic acid (4), (+)-butyl 5-oxopyrrolidine-2-carboxylate (6), (–)-3-hydroxy-2-methylbutyrolactone (10), and 5-(hydroxymethyl)furfural (12) had increased melanin productions and tyrosinase activities. Those active components could be further studied as the candidates against melanoma and vitiligo for skin diseases or whitening/hypopigmentation for hair.


Introduction
Amana edulis (A. edulis; Miq.) Honda, syn. Tulipa edulis (Miq.) Baker belongs to the Liliaceae family and is a folk medicinal plant used to treat cancer diseases [1,2]. However, there have been few phytochemical and biological studies of this species reported to date. For example, 95% and 50% EtOH extracts could induce apoptosis in human gastric (SGC-7901) [1] and hepatoma (BEL7404, HepG2, and Huh7) [2] carcinoma cells, respectively. The polysaccharides prepared from A. edulis have anti-oxidant properties, including DPPH, OH − , and ABTS + scavenging activities [3][4][5]. In a research of new agents from natural products for skin disorders, we found that the MeOH extracts of the bulbs of A. edulis show potential melanogenesis regulation that has not yet been researched. When exposed to the ultraviolet radiation of solar light or harmful chemicals and pathogenic factors, moderate melanogenesis can protect our skin from reactive oxygen species generation in keratinocytes and melanocytes [6]. Tyrosinase is an important enzyme in melanogenesis to produce more melanin for us to defense against aforementioned hazardous conditions [7]. Therefore, the active components of A. edulis are worthy to be clarified for further medical applications. In this investigation, three new tuliposides H-J (1-3) and 11 known compounds (4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14) (Figure 1) were isolated from A. edulis and structurally identified by NMR, MS, and IR spectroscopic data, optical rotation, or Mosher ester reaction. Compound 9 could decrease melanin contents but be cytotoxic toward B16 melanoma cells. Compounds 4, 6, 10, and 12 had increased melanin productions and tyrosinase activities. Overall, this

Structure Elucidations of Isolates 1-3
The MeOH extract (TEM) of the bulbs of A. edulis was partitioned into EtOAc-soluble (TEE), n-BuOH-soluble (TEB), and H2O-soluble (TEW) fractions separately. The fractionation of the TEE and TEB extracts afforded new isoprenoid glycosides 1-3 and known compounds 4-14 ( Figure 1). Additionally, the major components of the TEW extract were carbohydrates.

Structure Elucidations of Isolates 1-3
The MeOH extract (TEM) of the bulbs of A. edulis was partitioned into EtOAc-soluble (TEE), n-BuOH-soluble (TEB), and H 2 O-soluble (TEW) fractions separately. The fractionation of the TEE and TEB extracts afforded new isoprenoid glycosides 1-3 and known compounds 4-14 ( Figure 1). Additionally, the major components of the TEW extract were carbohydrates.

Chemical Components Elucidation of TEW
The 1 H and 13 C NMR spectra of TEW, a water-soluble crude fraction from TEM extract, showed very similar to that of carbohydrates ( Figures S16 and S17). Polysaccharide prepared from this species have been reported to possess rhamnose, xylose, arabinose, galactose, mannose, glucose, and fructose after monosaccharide analysis [3,8]. The NMR spectroscopic data and the TLC (thin-layer chromatography) retention factor of TEW compared with those of the sugar standards ( Figures S18-S23) suggested D-glucose and D-fructose were the main components in TEW extracts.

Effects of Extracts and Isolates on Melanogenesis
The MeOH extract (TEM) of this species was separated into TEE, TEB, and TEW crude fractions, and the aforementioned four extracts were evaluated for cytotoxicity and melanogenesis effects in B16 melanoma cells at concentrations from 12.5 to 200 µg/mL ( Figure S24). TEE showed obvious cytotoxicity toward B16 at 200 µg/mL and TEB as well as TEW had cytotoxic activities at concentrations from 12.5 to 200 µg/mL ( Figure  S24A-1,B-1,C-1,D-1). In the bioassay, α-melanocyte-stimulating hormone (α-MSH) was used to activate B16 cells to synthesize more melanin, and TEM could moderately stimulate melanogenesis ( Figure S24A-2). Most of all isolates (Figure 1), excluding 13 and 14, were further assayed for the regulation of melanogenesis effects at concentrations from 10 to 40 µM, with arbutin used as a positive control ( Figure 4). As shown in Figure 4, compound 9 obviously and dose-dependently inhibited melanin production that resulted from cytotoxicity toward B16 cells with an IC 50 (half maximal inhibitory concentration) value of 81.9 µM ( Figure S25). Isolates 4, 6, 10, and 12 increased melanin contents dose-dependently up to the maximum percentages of 11.9%, 29.8%, 27.2%, and 17.6%, respectively, at 40 µM with no cellular toxicity. Tyrosinase plays a key role in the first two steps of melanogenesis [6]; therefore, the effects of compounds 4, 6, 10, and 12 on intracellular tyrosinase were further evaluated. α-MSH increased the tyrosinase activity up to 254.3-305.5% when compared with the control group (100%) ( Figure 5 and Table S1). Consequently, 4 (14.1-21.9%), 6 (15.8-21.9%), 10 (8.5-13.1%), and 12 (7.5-11.8%) moderately increased the activity of the enzyme, tyrosinase, in a dose-dependent manner at concentrations of 10 to 40 µM, while compared with the α-MSH group ( Figure 5 and Table S1). A correlation graph between the cellular melanin contents and the effects on the tyrosinase of compounds 4, 6, 10, and 12 is shown in Figure S26. Furthermore, to confirm the melanogenesis-inducing effects of 4, 6, 10, and 12, each compound was added in B16 cells without co-treated α-MSH that was used as a positive control in this assay ( Figure S27). Consequently, individual 4, 6, 10, and 12 did not increase melanin productions alone ( Figure S27) similar to those shown in Figure 4, and it was suggested the abovementioned four compounds could merely enhance melanogenesis effects of α-MSH. Accordingly, additional signal pathways in melanogenesis, such as tyrosinase-related protein-1 (TRP-1), TRP-2, microphthalmia-associated transcription factor, melanocortin 1 receptor, cyclic adenosine monophosphate, protein kinase A, cAMPresponse element-binding protein, c-Jun N-terminal kinases, extracellular signal-regulated kinase, p38, and phosphoinositide 3-kinase/protein kinase B [7,21], should be further tested and confirmed for those active components.
phate, protein kinase A, cAMP-response element-binding protein, c-Jun N-terminal kinases, extracellular signal-regulated kinase, p38, and phosphoinositide 3-kinase/protein kinase B [7,21], should be further tested and confirmed for those active components.

General Experimental Procedures
NMR spectra were measured on a Bruker Avance III 500 MHz instrument (Bruker, Billerica, America) for 1 H and 125 MHz for 13 C-NMR. Chemical shift (δ) values were in ppm, and coupling constants (J) were in Hz with CD3OD, CDCl3, and/or D2O used as solvents. Optical rotations were obtained on a JASCO-P-2000 polarimeter (JASCO, Tokyo, Japan) (cell length 10 mm). IR spectra were recorded on a PerkinElmer Spectrum Two FT-IR spectrometer (PerkinElmer, Waltham, MA, USA). Low-and high-resolution ESIMS (electrospray ionization mass spectrometry) were measured on a Bruker Daltonics Esquire HCT ultra high capacity trap mass spectrometer and an Orbitrap mass spectrometer (LTQ Orbitrap XL, Thermo Fisher Scientific), respectively. TLC was performed on Kieselgel 60

Plant Material
Dry bulbs of A. edulis (formerly T. edulis) were purchased from a traditional Chinese medicine pharmacy in Taichung, Taiwan, in September 2018 and identified by the author Prof. Chang. A voucher specimen (TE201809) was deposited at the Chinese Medicine Research and Development Center, CMUH, Taiwan.

Plant Material
Dry bulbs of A. edulis (formerly T. edulis) were purchased from a traditional Chinese medicine pharmacy in Taichung, Taiwan, in September 2018 and identified by the author, Prof. Chang. A voucher specimen (TE201809) was deposited at the Chinese Medicine Research and Development Center, CMUH, Taiwan.

Extraction and Isolation
The bulbs of A. edulis (5.0 kg) were extracted eight times with MeOH (8.0 L each) at room temperature to obtain a crude extract. The MeOH extract (TEM, 525.0 g) was partitioned three times between ethyl acetate (EtOAc) and H 2 O (1500:1500, v/v) to give an EtOAc-soluble fraction (TEE, 45.0 g) and an aqueous phase, which was further partitioned with n-BuOH/H 2 O (1500:1500, v/v × 3), and then separated into n-BuOH-soluble (TEB, 53.6 g) and H 2 O-soluble (TEW, 410.0 g) fractions.

(R)-and (S)-MTPA Derivatives of 1
The preparation of the (S)-MTPA ester derivative of 1 was carried out by a convenient Mosher ester procedure [22,23]. Compound 1 (3.4 mg, 0.01 mmole) was transferred into a clean NMR tube and then dried completely under vacuum before being full of nitrogen. Furthermore, C 5 D 5 N (0.5 mL) and (R)-(−)-α-methoxy-α-(trifluoromethyl)-phenyl-acetyl chloride (MTPA chloride, 12.2 mg, 0.05 mmole) were added immediately into the sealed NMR tube that was further shaken carefully to mix the sample and MTPA chloride. The 1 H NMR and 1 H-1 H COSY spectra of the mixture after reacting for 24 h at room temperature were recorded. The (R)-MTPA ester of 1 was also prepared similarly by the aforementioned process.

Cell Culture
The murine B16 melanoma cells were cultured in Dulbecco's Modified Eagle Medium (DMEM; GIBCO Invitrogen corporation, New York, NY, USA) supplemented with 10% fetal bovine serum, 100 U/mL of penicillin, and 100 µg/mL of streptomycin at 37 • C in a 5% CO 2 incubator.

Measurement of B16 Cell Viability
The cytotoxicity of B16 cells for the test compounds was measured by MTT with a previously described protocol with slight modifications [6]. The cells were seeded in 96-well plates (1 × 10 3 cells/well) and cultured for 24 h before treated with compounds for other 72 h. After that, the medium was removed, 100 µL of MTT reagent (Thermo Scientific, Waltham, MA, USA) (at the final concentration of 0.5 mg/mL) was added to each well and then incubated at 37 • C for 1 h. The formazan crystal was dissolved in 100 µL DMSO, and the optical density was measured by using a microplate reader (SPECTORstar ® Nano, BMG LABTECH, Ortenberg, Germany) at 570 nm.

Measurement of Melanin Content in B16 Cells
The melanin content in B16 cells was assayed according to a previously described method with slight modifications [6]. The cells were seeded at a density of 5 × 10 3 cells/well in 24-well plates for 24 h. The medium was replaced by a 500 µL fresh culture medium containing 0.5 µM α-MSH with or without compounds for 72 h. Arbutin (1 mM) was used as the positive control. After that, the medium was removed and washed by PBS (pH 6.8) twice. The cells were harvested by NaOH (200 µL, 2 N) and then heated at 85 • C for 30 min. After cooling to room temperature and being centrifuged, the absorbance was measured at 405 nm using a microplate reader.

Intracellular Tyrosinase Activity
Intracellular tyrosinase activity assay was performed by a slightly modified method, which was reported previously [6]. The cells were seeded in 24-well plates at a density of 5 × 10 3 cells/well for 24 h. The cells were treated in DMEM containing 0.5 µM α-MSH with or without compounds for 72 h. After removing the medium, the cells were washed by cold PBS (pH 6.8) twice. The cells were lysed by 100 µL lysis buffer (1% triton X-100 in PBS) and frozen at −80 • C for 15 min. Then, the cell lysates were centrifuged at 13,200× g at 4 • C for 30 min to obtain the supernatant. The total protein content of the supernatant was quantified by BCA protein assay. Next, each quantified lysate (30 µg/90 µL) was mixed with 10 µL of L-DOPA (15 mM) in a 96-well plate and then incubated at 37 • C for 1 h, and the absorbance was measured at 405 nm using a microplate reader.

Conclusions
Overall, 14 pure compounds, including three new isoprenoid glycosides, tuliposides H-J (1-3), were isolated from A. edulis. Citric acid derivative 9, tributyl citrate, had significantly anti-melanogenesis activity but showed cytotoxicity toward B16 melanoma cell that could be further researched for anti-melanoma drugs. While pyroglutamic acid analogues 4 and 6, furanone 10, and furan 12 had dose-dependently increased melanin contents and activated tyrosinase that could be further studied for vitiligo skin disease or anti-whitening and anti-hypopigmentation agents for hair. However, in vitro mechanisms and/or in vivo studies are needed to be investigated in more detail to verify the efficacy of the active components.