Part 3 Quantitative Determination of Alkaloids in Lotus Flower (Flower Buds of Nelumbo nucifera) and Their Melanogenesis Inhibitory Activity

3.1. General Experimental Procedures

The following instruments were used to obtain physical data: melting points, Yanagimoto micromelting point apparatus (Yanaco New Science Inc., Kyoto, Japan); specific rotations, SEPA-300 digital polarimeter (Horiba Ltd., Kyoto, Japan, l = 5 cm); UV spectra, UV-1600 spectrometer (Shimadzu Co., Kyoto, Japan); IR spectra, FTIR-8100 spectrometer (Shimadzu Co.); ESIMS and HRESIMS, Exactive Plus mass spectrometer (Thermo Fisher Scientific Inc., Waltham, MA, USA); ¹H-NMR spectra, JNM-ECA800 (800 MHz), JNM-ECA500 (500 MHz), and JNM-ECS400 (400 MHz) spectrometers (JEOL Ltd., Tokyo, Japan); ¹³C-NMR spectra, JNM-ECA800 (200 MHz), JNM-ECA500 (125 MHz), and JNM-ECS400 (100 MHz) spectrometers (JEOL Ltd.) with tetramethylsilane as an internal standard; HPLC detector, SPD-10Avp UV-VIS detector (Shimadzu Co.); HPLC column, Cosmosil 5C₁₈-MS-II (Nacalai Tesque, Inc., Kyoto, Japan), 4.6 mm × 250 mm i.d. and 20 mm × 250 mm i.d. for analytical and preparative studies, respectively.

The following experimental conditions were used for chromatography (CC): ordinary-phase silica gel column chromatography, silica gel 60N (Kanto Chemical Co., Tokyo, Japan; 63–210 mesh, spherical, neutral); reverse-phase silica gel CC, Chromatorex ODS DM1020T (Fuji Silysia Chemical, Aichi, Japan; 100–200 mesh); normal-phase TLC, pre-coated TLC plates with silica gel 60F₂₅₄ (Merck, Darmstadt, Germany; 0.25 mm); reversed-phase TLC, pre-coated TLC plates with silica gel RP-18 F_254S (Merck, 0.25 mm); reversed-phase HPTLC, pre-coated TLC plates with silica gel RP-18 WF_254S (Merck, 0.25 mm), detection was carried out by spraying 1% Ce(SO₄)₂–10% aqueous H₂SO₄ on the plates, followed by heating.

3.2. Plant Materials

The flower buds of Nelumbo nucifera collected in Nakhon Ratchasima, Thailand, in 2011, and were abbreviated as followings: NN-1 (the whole flowers), NN-2 (the petals), NN-3 (the receptacles), and NN-4 (the stamens). The flower buds of N. nucifera collected in Taiwan, in 2011, and were as follows: NN-5 (the whole flowers), NN-6 (the petals), NN-7 (the receptacles), and NN-8 (the stamens). The other samples (NN-9–12) were purchased via Tochimoto Tenkaido Co., Ltd., Osaka, Japan, in 2013 and were abbreviated as follows: NN-9 [the leaves collected from Shandong province, China, in 2010 (Lot. No. 1111C129201)], NN-10 [the fruit collected from Hunan province, China, in 2012 (Lot. No. 1212C026701)], NN-11 [the fruit collected from Hunan province, China, in 2011 (Lot. No. 1206C026701)], and NN-12 [the embryos were collected from Jiangxi province, China, in 2010 (Lot. No. 1207128901)]. These plant materials were identified by one of the authors (M.Y.), and voucher specimens of them are on file in our laboratory. The materials were air-dried in a room under shade for more than a month.

3.3. Extraction and Isolation

Dried flower buds of N. nucifera (NN-1, 1.98 kg) were extracted four times with methanol (10 L) at room temperature for 24 h. Evaporation of the combined extracts under reduced pressure provided a methanol extract (182.75 g, 9.22%). An aliquot (168.51 g) of the methanol extract was partitioned into a mixture of EtOAc and 3% aqueous tartaric acid (1:1, v/v) to furnish an acidic EtOAc-soluble fraction (52.69 g, 2.88%) and an acidic aqueous solution. The aqueous solution was adjusted to pH 9 with saturated aqueous Na₂CO₃ and then extracted with CHCl₃. Removal of the solvent in vacuo yielded a CHCl₃-soluble fraction (17.80 g, 0.97%). The aqueous layer was extracted with n-BuOH, and removal of the solvent in vacuo yielded a n-BuOH-soluble fraction (12.29 g, 0.62). An aliquot (17.70 g) of the CHCl₃-soluble fraction was subjected to normal-phase silica gel CC [450 g, CHCl₃–28% NH₄OH (500:1, v/v) → CHCl₃–28% NH₄OH (500:1, v/v)–MeOH (100:1 → 70:1 → 50:1 → 20:1 → 10:1 → 1:1, v/v) → MeOH] to give 13 fractions (Fr. 1 (441.6 mg), Fr. 2 [= nuciferine (1, 1571.1 mg, 0.1028%)], Fr. 3 (183.2 mg), Fr. 4 (318.3 mg), Fr. 5 (689.7 mg), Fr. 6 [= nornuciferine (2, 782.1 mg, 0.0512%)], Fr. 7 [= asimilobine (4, 508.8 mg, 0.0333%)], Fr. 8 (543.8 mg), Fr. 9 (983.3 mg), Fr. 10 [= norarmepavine (7, 559.2 mg, 0.0366%)], Fr. 11 (464.4 mg), Fr. 12 (3173.1 mg), and Fr. 13 (3212.7 mg)). The fraction 3 (183.2 mg) was purified by HPLC [UV (254 nm), MeOH–0.03% aqueous Et₂NH (60:40, v/v)] to give 1 (83.6 mg, 0.0055%), 2 (15.2 mg, 0.0010%), and pronuciferine (5, 30.0 mg, 0.0020%). The fraction 4 (318.3 mg) was purified by HPLC [UV (254 nm), MeOH–0.03% aqueous Et₂NH (40:60, v/v)] to give 1 (6.3 mg, 0.0004%) and N-methylasimilobine (3, 34.6 mg, 0.0024%). The fraction 5 (689.7 mg) was purified by HPLC [UV (254 nm), MeOH–0.03% aqueous Et₂NH (40:60, v/v)] to give 2 (279.5 mg, 0.0262%), 3 (62.0 mg, 0.0067%), and 5 (181.3 mg, 0.0195%). The fraction 8 (453.8 mg) was purified by HPLC [UV (254 nm), MeOH–0.03% aqueous Et₂NH (40:60, v/v)] to give armepavine (6, 260.3 mg, 0.0170%). An aliquot (599.6 mg) of the fraction 9 was purified by HPLC [UV (254 nm), MeOH–0.03% aqueous Et₂NH (40:60, v/v)] to give 4 (19.2 mg, 0.0012%) and 7 (210.0 mg, 0.0137%). The fraction 11 (464.4 mg) was purified by HPLC [UV (254 nm), MeOH–0.03% aqueous Et₂NH (40:60, v/v)] to give 7 (144.9 mg, 0.0113%), N-methylcoclaurine (8, 56.0 mg, 0.0044%), and norjuziphine (10, 10.1 mg, 0.0003%). An aliquot (512.3 mg) of the fraction 12 was purified by HPLC [UV (254 nm), MeOH–0.03% aqueous Et₂NH (40:60, v/v)] to give coclaurine (9, 14.2 mg, 0.0058%) and 10 (4.5 mg, 0.0018%).

3.4. Preparation of Hydrochlorides of Alkaloids (1–10)

Preparation of the hydrochlorides as standard samples for quantitative analysis was carried out according to the procedure described previously but with a slight modification [10]. Free nuciferine (1, 29.5 mg, 0.1 mmol) was dissolved in a mixture of dichloromethane (1.0 mL) and ethanol (1.0 mL) at 0 °C. Concentrated HCl (150 µL) was added, and after stirring at 0 °C for 1 h, the reaction mixture was suction filtered with a Kiriyama funnel (Kiriyama glass Co., Tokyo, Japan), leaving a residue. The residue was washed with dichloromethane yielded a hydrochloride 1, which was sufficiently pure for analysis. Through a similar procedure, hydrochlorides 2–10 were obtained from the corresponding free alkaloids (2–10).

3.5. Standard Solution Preparation

Accurately weighed 2.00 mg of each hydrochloride salt of alkaloid (1–10) was introduced into a 20 mL volumetric flask, and the volume was made up with methanol; the solution being used as a stock standard solution (100 µg/mL). Aliquots of 50, 100, 500, 1000, and 5000 µL of the stock standard solution were transferred into 10 mL volumetric flasks and the volume was made up with methanol for use as working solutions (0.5, 1.0, 5.0, 10, and 50 µg/mL, respectively) for constructing calibration curves. For calibration, an aliquot of 2.0 µL of each solution was injected into the LC-MS system. Each peak was observed at following retention times: 1 (t_R 43.1 min), 2 (t_R 39.5 min), 3 (t_R 29.7 min), 4 (t_R 21.3 min) and 5 (t_R 13.9 min), 6 (t_R 16.9 min), 7 (t_R 15.9 min), 8 (t_R 9.9 min), 9 (t_R 8.3 min), and 10 (t_R 18.8 min).

3.6. Sample Preparation

An accurately weighed pulverized sample powders (ca. 2 g, conversion with loss on drying) was extracted with 20 mL of three solvent systems (methanol, 50% methanol, or water) under two different conditions (reflux for 120 min or sonication for 30 min, each twice), respectively. After centrifugation of the extracts at 3000 rpm for 5 min, the supernatants were combined and diluted to 100 mL with the extraction solvent. An aliquot (1 mL) of the extract solution was transferred into a 5 mL volumetric flask and made up to the volume with methanol. The solution was filtered through a syringe filter (0.45 µm), and an aliquot of 5.0 µL was subjected to the LC-MS analysis. The remaining extraction solution (90 mL) was evaporated in vacuo to calculate the extraction yields.

3.7. LC-MS Instruments and Conditions

An LC-20A series Prominence HPLC system (Shimadzu Co.) was equipped with a binary pump, a degasser, an autosampler, a thermostated column compartment, a UV detector, and a control module connected with a LCMS-2010EV mass spectrometer (Shimadzu Co.) equipped with an ESI interface. The chromatographic separation was performed on a Cosmosil πNAP column (5 µm particle size, 2.0 mm i.d. × 150 mm, Nakalai Tesque Inc.) operated at 40 °C with mobile phase A (acetonitrile) and B (H₂O containing 0.2% acetic acid). The gradient program was as follows: 0 min (A:B 15:85, v/v) → 20 min (18:82, v/v) → 50 min (50:50, v/v). The flow rate was 0.2 mL/min and the injection volume was 2.0 µL. The detections were performed at 260 nm (UV) and under selected ion monitoring (SIM) by a positive-mode ESI-MS. The operating parameters for MS detection were as follows; nebulizing gas flow: 1.5 L/min, drying gas pressure: 0.15 MPa, CDL temperature: 250 °C, block heater temperature: 250 °C, interface voltage: −3.5 kV, CDL voltage: constant-mode, Q-array DS and RF voltage: Scan-mode.

3.8. Calibration and Validation

The standard curves were prepared over concentration ranges of 0.5–50 µg/mL with five different concentration levels. Standard curves were made on each analysis day. Linearity for each compound was plotted using linear regression of the peak area versus concentration. The coefficient of correlation (R²) was used to judge the linearity. The detection limit and quantitation limit for each analyte were determined by the signal-to-noise (S/N) ratio for each compound by analyzing a series of diluted standard solutions until the S/N ratios were about 3 and 10, respectively, based on a 2 µL injection. Precision and accuracy of the analytical method were tested using a homogeneous extract of NN-1. The intra- and inter-day precisions were determined by estimating the corresponding responses five times on the same day and on five different days, respectively (Table 3). The recovery rates were determined by adding analytes of three different concentrations (10, 15, and 20 µg/mL) to the sample solution (Table 4).

3.9. Reaction of Free Alkaloid (2) with CO₂ in an Air Atmosphere

A free alkaloid 2 (50.0 mg, 0.718 mmol) was dissolved in CDCl₃ (5.0 mL) under an air atmosphere; the resulting solution was kept at room temperature. The reaction was monitored by HPLC analysis: column (Cosmosil 5C₁₈ MS-II (4.6 mm × 250 mm, i.d., Nakalai Tesque Inc.)); detection (UV (254 nm)); mobile phase (MeOH–0.03% aqueous Et₂NH (70:30, v/v)); column temperature (room temperature); and flow rate: (1.0 mL/min). The monitoring results are shown in Figure S1.

After three weeks, the reaction mixture was condensed under reduced pressure to give a crude reddish brown solid (10.0 mg). The analytical sample of 2′′ (2.3 mg) was obtained as colorless needles by recrystallization from a mixture of n-hexane and diethyl ether.

The crude reddish brown solid (39.8 mg), obtained by a similar manner, was purified by preparative HPLC (Cosmosil 5C₁₈ MS-II (20 mm × 250 mm, i.d., Nakalai Tesque Inc.), MeOH–0.03% aqueous Et₂NH (70:30, v/v)) to give 2 (21.1 mg, 42%) and 2a (18.1 mg, 41%).

Compound 2′′: IR (KBr) v_max cm⁻¹: 2927, 2720–2500, 1721, 1450, 1261, 1033; ¹H- and ¹³C-NMR spectroscopic data, see Table 6; positive-ion ESIMS m/z 282 [M − C₁₉H₁₈NO₄]⁺; HREIMS m/z 282.1483 (calcd for C₁₈H₂₀NO₂ , 282.1489).

Compound 2a: An amorphous powder; [α]25D −160.9 (c 0.19, CHCl₃); UV [MeOH, nm (log ε)]: 229 (4.30), 272 (4.12); IR (KBr) v_max cm⁻¹: 2927, 1685, 1446, 1246, 1107; ¹H- and ¹³C-NMR spectroscopic data, see Table 6; positive-ion ESIMS m/z: 362 [M + Na]⁺; HRESIMS m/z: 362.1361 [M + Na]⁺ (calcd for C₂₀H₂₁NO₄Na, 362.1363).

3.10. Reagents for Bioassays

Dulbecco′s modified Eagle′s medium (DMEM, 4.5 g/L glucose) was purchased from Sigma–Aldrich (St. Louis, MO, USA); fetal bovine serum (FBS), penicillin, and streptomycin were purchased from Gibco (Invitrogen, Carlsbad, CA, USA); and other chemicals used in this study were purchased from Wako Pure Chemical Co., Ltd. (Osaka, Japan). The 48- and 96-well microplates (Sumilon) were purchased from Sumitomo Bakelite Co., Ltd. (Tokyo, Japan).

3.11. Cell Culture

Murine B16 melanoma 4A5 cells (RCB0557) were obtained from Riken Cell Bank (Tsukuba, Japan). The cells were grown in DMEM (glucose; 4500 mg/L) supplemented with 10% FBS, penicillin (100 units/mL), and streptomycin (100 µg/mL) at 37 °C in 5% CO₂/air. The cells were harvested by incubation in phosphate-buffered saline (PBS) containing 0.05% ethylenediaminetetraacetic acid (EDTA) and 0.02% trypsin for ca. 5 min at 37 °C and used for the subsequent bioassays.

3.12. Melanogenesis and Cell Viability

Effects on theophylline-stimulated melanogenesis and viability of B16 melanoma 4A5 cells were examined according to the protocol described previously [25,26,27].

3.13. Total Contents (%) of the 10 Alkaloids (1–10) Calculated Based on the Ratio of IC₅₀ Values (µg/mL) Against Melanogenesis Inhibitory Activities

Total contents (%) of the 10 alkaloids (1–10) are presented in reduced values to the content of nuciferine (1), and were calculated out by the following equation.

Total content (%) = [(content (%) of 1)] + [(content (%) of 2) × (IC₅₀ of 1)/(IC₅₀ of 2)] + [(content (%) of 3) × (IC₅₀ of 1)/(IC₅₀ of 3)] + [(content (%) of 4) × (IC₅₀ of 1)/(IC₅₀ of 4)] + [(content (%) of 5) × (IC₅₀ of 1)/(IC₅₀ of 5)] + [(content (%) of 6) × (IC₅₀ of 1)/(IC₅₀ of 6)] + [(content (%) of 7) × (IC₅₀ of 1)/(IC₅₀ of 7)] + [(content (%) of 8) × (IC₅₀ of 1)/(IC₅₀ of 8)] + [(content (%) of 9) × (IC₅₀ of 1)/(IC₅₀ of 9)] + [(content (%) of 10) × (IC₅₀ of 1)/(IC₅₀ of 10)].

3.14. Mushroom Tyrosinase

Tyrosinase activities using l-tyrosine or 3,4-dihydroxyphenyl-l-alanine (l-DOPA) as a substrate were determined according to the protocol described previously [25,26,27].

3.15. Expressions of Tyrosinase, TRP-1, and TRP-2 mRNA

The expressions of tyrosinase, tyrosine-related protein (TRP)-1, and TRP-2 mRNA were assessed according to the previously reported method [25,27].

3.16. Statistical Analysis

Values are expressed as mean ± S.E.M. One-way analysis of variance followed by Dunnett′s test was used for statistical analyses. Probability (p) values less than 0.05 were considered significant.