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

A quantitative analytical method for five aporphine alkaloids, nuciferine (1), nornuciferine (2), N-methylasimilobine (3), asimilobine (4), and pronuciferine (5), and five benzylisoquinoline alkaloids, armepavine (6), norarmepavine (7), N-methylcoclaurine (8), coclaurine (9), and norjuziphine (10), identified as the constituents responsible for the melanogenesis inhibitory activity of the extracts of lotus flowers (the flower buds of Nelumbo nucifera), has been developed using liquid chromatography-mass spectrometry. The optimum conditions for separation and detection of these 10 alkaloids were achieved on a πNAP column, a reversed-phase column with naphthylethyl group-bonded silica packing material, with CH3CN–0.2% aqueous acetic acid as the mobile phase and using mass spectrometry equipped with a positive-mode electrospray ionization source. According to the protocol established, distributions of these 10 alkaloids in the petal, receptacle, and stamen parts, which were separated from the whole flower, were examined. As expected, excellent correlations were observed between the total alkaloid content and melanogenesis inhibitory activity. Among the active alkaloids, nornuciferine (2) was found to give a carbamate salt (2′′) via formation of an unstable carbamic acid (2′) by absorption of carbon dioxide from the air.


Introduction
A Nymphaeaceae plant Nelumbo nucifera Gaertn. (common name "lotus" in English) is extensively cultivated in Eastern Asian countries [1][2][3]. All parts of this plant, including the leaves, stamens, flowers, seeds, and rhizomes, have been used as traditional medicines or vegetables for thousands of years [2][3][4]. The lotus flower, the flower buds of N. nucifera, has been used for the treatment of vomiting blood, bleeding caused by internal and external injuries, and various skin diseases, and also as a sedative and an anti-inflammatory agent in traditional Asian medicines [2]. In the course of our studies on the bioactive constituents from the flower buds of N. nucifera, we have isolated several alkaloids, e.g., nuciferine (1), nornuciferine (2), N-methylasimilobine (3), asimilobine (4), pronuciferine (5), and armepavine (6), with melanogenesis inhibitory activities in theophylline-stimulated murine B16 melanoma 4A5 cells [2]. As a result of the increasing interest in lotus flower as a possible cosmetic for skin whitening, there is a strong demand for efficient quality control measurements to ensure the authenticity and content of the active constituents in such products, and to verify the labeled claims. In this paper, we propose a simple, rapid, and precise analytical method for liquid chromatography-mass spectrometry (LC-MS) simultaneous quantitative determination of five aporphine alkaloids (1-5) and five benzylisoquinoline alkaloids, (6), norarmepavine (7), N-methylcoclaurine (8), coclaurine (9), and norjuziphine (10), using a one-step sample preparation procedure.

Isolation of Principal Alkaloids (1-10) from Lotus Flower
To obtain the principal alkaloids (1-10), an isolation procedure from this plant material was newly developed in this study by modifying the previously reported method [2]. Thus, dried flower buds of N. nucifera were extracted with methanol under reflux to obtain a methanol extract (9.22% from the dried material). 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 (2.88%) and an acidic aqueous solution. The pH of the aqueous solution was adjusted to 9 with saturated aqueous Na 2 CO 3 and then extracted with CHCl 3 to obtain a CHCl 3 -soluble fraction (0.97%). The aqueous layer was further extracted with n-BuOH to obtain an n-BuOH-soluble fraction (0.62%). As shown in Table 1, the methanol extract was found to inhibit theophylline-stimulated melanogenesis (IC 50 = 5.6 µg/mL) without cytotoxicity (cell viability in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay: 103.3˘7.1%) at 100 µg/mL. Through bioassay-guided separation, the CHCl 3 -soluble fraction (IC 50 = 0.37 µg/mL) was found to be more active than the EtOAc n-BuOH-soluble fractions (IC 50 = 11.1 and 13.7 µg/mL, respectively). Each value represents the mean˘S.E.M. (n = 4); asterisks denote significant differences from the control group, ** p < 0.01; a Bioassay-guided separation study was carried out using the flower buds of N. nucifera originating in Thailand (NN-1).

Simultaneous Quantitative Analysis of 10 Alkaloids (1-10) in Lotus Flowers
To provide sufficient purity for quantitative analysis, the hydrochlorides of these alkaloids (1-10) were prepared by reported method [10]. As shown in Figure 2

Simultaneous Quantitative Analysis of 10 Alkaloids (1-10) in Lotus Flowers
To provide sufficient purity for quantitative analysis, the hydrochlorides of these alkaloids (1-10) were prepared by reported method [10]. As shown in Figure 2    Extraction efficiently was tested using NN-1 (loss of drying 10.33%); a relative value (%) against the content obtained by methanol under reflux is given in parentheses; b less than the quantitation limit. Table 3. Linearities, detection and quantitation limits, and precisions for alkaloids (1-10) in lotus flower.

Intra-Day Inter-Day
Nuciferine ( x is the concentration of the analyte solution (µg/mL), and y is the peak area of the analyte; b values are the amount of the analyte injected on-column and c precision of the analytical method were tested using the methanol extract of NN-1 (n = 5).  Prior to analysis, extraction conditions were examined to optimize the extracts 1 quality in association with the contents of the alkaloids (1-10). The extraction efficacies were compared for three solvent systems (methanol, 50% aqueous methanol, and water) under two different conditions (reflux for 120 min or sonication for 30 min, each twice). As shown in Table 2, "reflux in methanol" afforded the highest contents of the active alkaloids (1-10). Therefore, all the analytical samples were prepared by employing the method "reflux in methanol for 120 min".
Some analytical parameters, such as linearity and limit of quantitation of the developed method, were evaluated as shown in Table 3. The calibration curve was linear in the range studied (0.5-50 µg/mL) showing a correlation coefficient (R 2 ) of greater than 0.9996 for each constituent. Linear regression equations of their calibration curves for each constituent are described in Table 3, where y is the peak area and x is the concentration of the analyte. The detection and quantitation limits were estimated to be 0.17-0.90 and 0.51-2.65 ng, respectively, indicating sufficient sensitivity of this method. The relative standard deviation (RSD) values were 0.25%-1.36% for intra-day and 0.39%-1.40% for inter-day assays. Accuracy was determined in recovery experiments using the methanol extract of NN-1. As shown in Table 4, recovery rates of 92.3%-105.8% were obtained, with RSD values of lower than 1.6%.
According to the protocol thus established, contents of the alkaloids (1-10) collected in two different regions (NN-1 in Thailand; NN-5 in Taiwan) were measured. The assay was found to be reproducible, precise, and readily applicable to the quality evaluation of lotus flower 1 s extracts. As shown in Table 5, N-methylcoclaurine (8, NN-1: 5.73 mg/g in dry material; NN-5: 2.88 mg/g) was the richest constituent among the alkaloids (1-10). The total alkaloid content in the Thai (NN-1: 14.96 mg/g) and Taiwanese (NN-5: 3.53 mg/g) samples were quite different. However, a more extensive study would be required to confirm that this result was due to differences between regions. To characterize the distribution of the alkaloids (1-10) in the flower, the whole flower parts (NN-1 and NN-5) were separated into petals (NN-2 and NN-6), receptacles (NN-3 and NN-7), and stamens (NN-4 and NN-8); then, quantitative analysis of each separated sample was performed. It was found that the alkaloids (1-10) were mainly contained in the petal part. Furthermore, other parts of the lotus plant (e.g., leaf (NN-9), fruit (NN-10 and 11), and embryo parts (NN-12), which are used for traditional medicines) were also examined. It was found that the total alkaloid content of the leaf (NN-9: 1.20 mg/g), fruit (NN-10 and NN-11: each less than the quantitation limit), and embryo parts (NN-12: 0.64 mg/g) of N. nucifera were lower than those of the flower buds (NN-1 and NN-5) (Table S1). (2 11 ) Formation from the Free Alkaloid (2) The gradual transformation of one of the alkaloids isolated in this study, nornuciferine (2), into a highly polar material 2 11 was observed, when 2 was exposed to the atmosphere in deuterated chloroform (CDCl 3 ) at room temperature. After three weeks of standing in CDCl 3 , compound 2 11 was obtained as a main product ( Figure 3). As summarized in Table 6, 1 H-and 13 C-NMR spectra of 2 11 suggested that there are two kinds of parts derived from the nornuciferine framework in the structure of 2 11 . Thus, with respect to one of the nornuciferine parts, five carbons α and β to the nitrogen atom appeared as pair signals [δ C : 29.9/30.2 (C-4), 41.8/44.4 (C-5), 54.9/55.8 (C-6a), 33.9/35.9 (C-7), 124.8/124.9 (C-11c)] in the 13 C-NMR spectrum of 2 11 . Additionally, a pair of signals, which corresponded to an amide type carbonyl carbon, was also observed at δ C 157.5 and 160.1. The chemical shift of the signals suggested that the nitrogen atom of 2 was functionalized as a carbamate anion by the CO 2 uptake from the atmosphere. On the other hand, in the 1 H-NMR spectrum of 2 11 a downfield shift owing to the ammonium ion formation was observed with respect to the signals due to C-5 1 methylene (at δ H 3.24 and 3.87) and C-6a 1 methine (at δ H 4.29) protons of the other nornuciferine parts as compared with those of 2 [δ H : 3.01 and 3.40 (H 2 -5), 3.85 (H-6a)]. Two broad singlets, which appeared at the highly-deshielded regions (δ H 9.96 and 10.84), were due to acidic protons, which also support the ammonium ion structure depicted in Figure 3. The anticipated structure of 2 1 was strongly supported by the IR spectrum, which showed N + -H and C=O stretching absorptions at 2720-2500 and 1721 cm´1, respectively. Moreover, the positive ion part of 2 11 11 could be confirmed as the corresponding methyl carbamate 2a. Thus, 2 11 easily gave a 1:1 mixture of methyl carbamate 2a and original amine 2 by treatment with methanol at room temperature ( Figure S1). As shown in Table 6, compound 2a showed similar 13 C-NMR spectroscopic properties to those of 2 11 and/or 2, except for a singlet (δ C 52.6) due to the methyl carbon of the NCO 2 CH 3 moiety, which was confirmed by the correlation between the singlet at δ H 3.76 due to the methyl protons and a singlet at δ C 156.0 due to carbonyl carbon in the HMBC of 2a.

Ammonium Carbamate Salt
In the positive ESIMS of 2a, a quasimolecular ion peak was observed at m/z 362.1361 [M + Na] + (calced for C 20 H 21 NO 4 Na, 362.1363).  It is well known that ammonia, primary amines, or secondary amines (A) absorb CO2 to transform into the corresponding carbamic acids (B), which easily react with the original amines to produce stable carbamic acid ammonium salts and (C) as shown in Figure 4 [15][16][17][18][19][20][21][22][23][24]. Therefore, it is reasonable to anticipate that the product 2′′ forms via an acid-base reaction between original amine 2 and an unstable carbamic acid (2′), which was obtained by the CO2 absorption reaction with the nitrogen atom of 2.

Effects on Mushroom Tyrosinase
To characterize the mode of action of melanogenesis inhibitory activities of the alkaloids, inhibitory effects on (i) enzymatic tyrosinase activity and (ii) expressions of tyrosinase-related proteins (TRPs) e.g., tyrosinase, TRP-1, and TRP-2 were examined.
A copper-containing enzyme tyrosinase is a key enzyme in melanin biosynthesis involved in determining the color of skin and hair. It catalyzes oxidation of both L-tyrosine and L-DOPA, following another oxidation of L-DOPA to dopaquinone and, finally, oxidative polymerization via several dopaquinone derivatives to yield melanin. Tyrosinase inhibitors are being clinically used for the treatment of several dermatological disorders associated with melanin hyperpigmentation. The tyrosinase inhibitor kojic acid is commonly used as an additive in cosmetics for skin whitening and/or depigmentation [25][26][27]. As shown in Table 8, none of the alkaloids showed inhibitory activities when using both L-tyrosine and L-DOPA as substrates. This suggests that tyrosinase inhibition is barely involved in the mechanisms of action of these melanogenesis inhibitors. Each value represents the mean˘S.E.M. (n = 4); asterisks denote significant differences from the control group, * p < 0.05, ** p < 0.01; commercial kojic acid was purchased from Nakalai Tesque Inc., (Kyoto, Japan); a each alkaloid was evaluated by its hydrochloride salt.

Correlation between the Melanogenesis Inhibitory Activity and Total Contents of Alkaloids (1-10) in Lotus Flower Extracts
The inhibitory effects of the methanol extracts of the lotus flowers (NN-1-8) on theophylline-stimulated melanogenesis were examined. As a result, the IC 50 values were detected in all the lotus flower samples in the ranges of 5.8-78.9 µg/mL (Table 10). In Figure 5, correlations between the total content of 10 alkaloids (value reduced to 1) and the melanogenesis inhibitory activities (1/IC 50 ) of the corresponding extracts were plotted. As expected, excellent correlations were observed between the total content and the inhibitory activities (R = 0.9632). As the minimum involvement, these correlations were shown between the content of two principal alkaloids (1 and 2) and the activities (R = 0.9657). In addition, the methanol extracts from the leaf and fruit parts of N. nucifera (NN-9-11) did not show melanogenesis inhibitory activities (IC 50 > 100 µg/mL, Table S2). On the other hand, despite the fact that the methanol extract from the embryo of N. nucifera (NN-12) contained scarcely any alkaloids (1-10) (vide supra), potent melanogenesis inhibitory activity was observed (IC 50 = 4.5 µg/mL, Table S2). This evidence suggested that other melanogenesis inhibitory active constituents are included in the embryo part.

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

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 Na 2 CO 3 and then extracted with CHCl 3 . Removal of the solvent in vacuo yielded a CHCl 3 -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. 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.ˆ150 mm, Nakalai Tesque Inc.) operated at 40˝C with mobile phase A (acetonitrile) and B (H 2 O containing 0.2% acetic acid). The gradient program was as follows: 0 min (A:B 15:85, v/v) Ñ 20 min (18:82, v/v) Ñ 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.

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 (R 2 ) 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).
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 11 (2.3 mg) was obtained as colorless needles by recrystallization from a mixture of n-hexane and diethyl ether.