Part 2 Quantitative Determination of Alkaloids in Lotus Flower (Flower Buds of Nelumbo nucifera) and Their Melanogenesis Inhibitory Activity

2.3. Ammonium Carbamate Salt (2′′) 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′′ was observed, when 2 was exposed to the atmosphere in deuterated chloroform (CDCl₃) at room temperature. After three weeks of standing in CDCl₃, compound 2′′ was obtained as a main product (Figure 3). As summarized in Table 6, ¹H- and ¹³C-NMR spectra of 2′′ suggested that there are two kinds of parts derived from the nornuciferine framework in the structure of 2′′. 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 ¹³C-NMR spectrum of 2′. 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 CO₂ uptake from the atmosphere. On the other hand, in the ¹H-NMR spectrum of 2′′ a downfield shift owing to the ammonium ion formation was observed with respect to the signals due to C-5′ methylene (at δ_H 3.24 and 3.87) and C-6a′ 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 (H₂-5), 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′ 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′′ was detected as a Na adduct ion signal at m/z 282.1483 [M − C₁₉H₁₈NO₄]⁺ (calcd for C₁₈H₂₀O₂, 282.1489) in the positive ESI mode; however, a signal due to the carbamate group was not detected in the negative ESI mode. Fortunately, the negative ion part of 2′′ could be confirmed as the corresponding methyl carbamate 2a. Thus, 2′′ 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 ¹³C-NMR spectroscopic properties to those of 2′′ and/or 2, except for a singlet (δ_C 52.6) due to the methyl carbon of the NCO₂CH₃ 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. In the positive ESIMS of 2a, a quasimolecular ion peak was observed at m/z 362.1361 [M + Na]⁺ (calced for C₂₀H₂₁NO₄Na, 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.

2.4. Effects of the Hydrochlorides of These Alkaloids (1–10) and 2a on Theophylline-Stimulated Melanogenesis Inhibitory Activity

To clarify the efficacy of the established quantitative analysis of 10 alkaloids (1–10) as a quality control for lotus flower, correlations between the total alkaloid contents and the melanogenesis inhibitory activities of the corresponding extracts were examined. Previously, we have reported the melanogenesis inhibitory activities of free nuciferine (1, IC₅₀ = 15.8 µM), nornuciferine (2, 62.9 µM), N-methylasimilobine (3, 14.5 µM), asimilobine (4, >100 µM), pronuciferine (5, 47.9 µM), and armepavine (6, 25.6 µM) [2]. Since the hydrochlorides of these alkaloids (1–10), which were of higher purity and stability than those of the corresponding free alkaloids, were used for the standard samples of the present quantitative analysis, the melanogenesis inhibitory activities of these hydrochloride salts were newly examined. It was found that aporphine alkaloids, nuciferine (1, IC₅₀ = 7.1 µM) and nornuciferine (2, 3.9 µM), and benzylisoquinoline alkaloids, armepavine (6, 6.5 µM), norarmepavine (7, 7.5 µM), N-methylcoclaurine (8, 6.5 µM), and coclaurine (9, 3.9 µM), were found to show relatively strong inhibitory activities without notable cytotoxic effects at the effective concentration (Table 7). These alkaloids were more potent than arbutin (IC₅₀ = 174 µM), a commercially used melanogenesis inhibitor used as a positive control [25,26,27]. Among them, 2 and 9 showed especially strong activity, which showed more than 40 times greater than that of arubutin. Previously, the naturally-occurring aporphine alkaloid, 4,5-didehydroguadiscine (IC₅₀ = 4.7 µM) isolated from Hornschuchia oblique, has been reported [28]. To the best of our knowledge, compounds 2 and 9 are the most potent melanogenesis inhibitors within this class of natural products.

2.5. 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.

2.6. Effects on Expression of Tyrosinase, TRP-1, and TRP-2

The TRP enzyme family (tyrosinase, TRP-1, and TRP-2) catalyze the major steps in melanin synthesis [25,26,27]. To clarify the mechanisms of action of these active constituents, we examined the effects of the principal alkaloids (1, 2, 6, 7, and 9) on expression of mRNAs for tyrosinase, TRP-1, and TRP-2 in B16 melanoma 4A5 cells. Except for compound 7, the suppression tendency of mRNA expression for tyrosinase in these alkaloids was observed as presented in Table 9.

2.7. 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 IC₅₀ 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/IC₅₀) 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 (IC₅₀ > 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 (IC₅₀ = 4.5 µg/mL, Table S2). This evidence suggested that other melanogenesis inhibitory active constituents are included in the embryo part.