Litsea cubeba (Lauraceae ) is a 3- to 10 m evergreen tree or shrub widely distributed in Southeastern Asia, Southern China, Japan, and Taiwan [1,2]. L. cubeba can be used as a flavoring or herbal medicine. As a flavoring, it gives a unique flavor resembling that of a mixture of pepper, ginger, and citrus. It is used as a flavor enhancer in foods, cosmetics, and cigarettes [3,4]. The bark of L. cubeba has been used in oriental traditional medicine for the treatment of atopic eczema and coronary heart disease, and its antioxidant activities and antimicrobial activities against Staphylococcus aureus, Salmonella typhi, and Pseudomonas aeruginosa have been reported [5,6,7]. The essential oil from L. cubeba has good fungicidal activities against Sclerotinia sclerotiorum, Thanatephorus cucumeris, Pseudocercospora musae and Colletotrichum gloeosporioides [8,9].Various types of alkaloids have been isolated from this plant [10,11,12]. Litebamine and its N-homologues possess acetylcholinesterase activity [13]. Moreover, litebamine can inhibit platelet aggregation, adenosine 5'-triphosphate (ATP) release and thromboxane B2 formation induced by arachidonic acid and collagen in rabbit platelets [14]. In the present paper, chromatographic separation of an aqueous EtOH extract of the barks of L. cubeba has yielded five novel isoquinoline alkaloids, namely (+)-N-(methoxylcarbonyl)-N-nordicentrin (1), (+)-N-(methoxycarbonyl)-N-nor-predicentrine (2), (+)-N-(methoxyl-carbonyl)-N-norbulbodione (3), and (+)-N-(methoxycarbonyl)-N-nor-isocorydione (4), and (+)-8-methoxyl-isolaurenine N-oxide (5), and one known compound, (+)-N-(methoxycarbonyl)-N-norglaucine (6) (Figure 1). Their structures were established on the basis of their chromatographic properties, chemical and physicochemical methods. Furthermore, all the triterpenoids were evaluated for their in vitro antibacterial, antifungal and cytotoxic properties.

Compound 1 was obtained as a brown amorphous powder. The HRESIMS displayed a pseudomolecular ion at m/z 406.1263 [M+Na]⁺ (calcd for C₂₁H₂₁NO₆Na, 406.1267) consistent with a molecular formula of C₂₁H₂₁NO₆, corresponding to 12 degrees of unsaturation. Its UV absorption at λ_max 303, 284, and 216 nm suggested an aporphine alkaloid skeleton with substituents at C-1, C-2, C-9, and C-10 [15]. Its ¹³C-NMR spectrum showed 21 carbon signals [OCH₃ × 3, OCH₂O × 1, CH₂ (sp³) × 3, CH (sp³) × 1, CH (sp²) × 3 and C (sp²) × 10, Table 1]. The ¹H-NMR spectrum of 1 exhibited three aromatic singlets at δ_H 6.86 (H-3), 6.78 (H-8), and 8.16 (H-11), a methylenedioxy signal at δ_H 6.18, and three methoxys (δ_H 3.69, 3.90, and 3.93). The IR absorption at 1665 cm^–1 and a signal at δ_C 157.2 in the ¹³C-NMR spectrum evidenced the presence of a carbamate moiety [16]. The position of the carbamate group at N-6 was supported by the HMBC correlations (Figure 2) of δ_C 157.2 with δ_H 3.69 (3H, s, OMe), 4.70 (1H, dd, J = 13.8, 3.8 Hz, H-6a), 4.42 (1H, m, H-5a), and 2.98 (1H, m, H-5b). Meanwhile, in the ¹³C-NMR spectra of 1, signals of C-7 and -CO appeared as broad peaks due to the stereo-hindrance effect of methyl ester with C-7 on the NMR time scale at room temperature, and signals sharpened when measured under heating conditions (50 °C). The positions of two other methoxys were assigned based on the NOESY spectrum (Figure 2). The NOE correlations of H-8 and H-11 with the signals of two OCH₃ (δ_H 3.90 and 3.93, respectively) positioned two methoxys at C-9 and C-10, which was further supported by the HMBC of C-9 (δ_C 147.2) with OCH₃ (δ_H 3.90) and C-10 (δ_C 148.0) with OCH₃ (δ_H 3.93) that the two OCH₃ respectively (Figure 2). These data showed similarities to those of (+)-N-(methoxycarbonyl)-N-norglaucine (6). The stereochemistry of C-6a was determined to be S by its positive specific rotation ( = +96.13) [17]. Therefore, compound 1 was identified as (+)-N-(methoxycarbonyl)-N-nordicentrin.

Compound 2 was obtained as a brown amorphous powder. The EIMS afforded a molecular weight of m/z = 385, and its HR-ESI-MS revealed the [M+Na]⁺ peak at m/z = 408.1422 (calcd. for C₂₁H₂₃NO₆Na. 408.1423), corresponding to the molecular formula C₂₁H₂₃NO₆. Comparing the ¹H- and ¹³C-NMR data of 2 with those of compound 1, the data were almost identical. The only significant difference was that the signals of the methylenedioxy moiety at C-1 and C-2 was replaced by a OH and OMe groups in compound 2. The HMBC correlations of C-1 (δ_C 144.8) with OCH₃ (δ_H 3.56) indicated the location of MeO and OH at C-1 and C-2 respectively, which was confirmed by the downfield shift of C-2 from δ_C 146.8 to 150.5. On the basis of the observation of NOESY data similar to those of 1 and the positive specific rotation ( = +258.7 [17], the stereochemistry of 2 was expected to be the same. Accordingly, the structure of 2 was established as (+)-N-(methoxycarbonyl)-N-norpredicentrine.

Compound 3, a violet amorphous powder, exhibited a molecular formula of C₂₀H₁₅NO₇, based on the HRESIMS spectrum which showed a pseudomolecular ion at m/z 404.0743 [M+Na]⁺ (calcd. 404.0746). The ^lH-NMR spectrum showed singlet signals for three aryl protons (δ_H 6.91, 6.88, and 5.92), two OMe (δ_H 3.71 and 3.85), one methylenedioxy (δ_H 6.20), and two triplets for two vicinal coupled methylenes [δ_H 2.63, 2.88 (H-4) and 2.99, 4.43 (H-5)]. The IR spectrum exhibited a conjugated carbonyl absorption (1654 cm⁻¹), and the ¹³C-NMR spectrum displayed two carbonyl signals at δ_C 178.3 and 186.4, similar to the 1,4-dicarbonyl signals in N-norbulbodione [18]. The HMBC correlations of δ_C = 156.2 with δ_H 3.71 (3H, s, OMe), 2.99 (1H, m, H-5a), and 4.33 (1H, m, H-5b) indicated the presence of a N-carbamate group. The methylenedioxyl unit was positioned between C-1 and C-2, and the methoxyl at C-10, respectively, based on the HMBC correlations of the proton signal of methylenedioxy with C-1 and C-2, and of the methoxyl signal (δ_H 3.85) with C-10, respectively. The positive specific rotation ( = +77.8 indicated stereochemistry of C-6a was determined to be S [19] Thus, Compound 3 was elucidated as (+)-N-(methoxycarbonyl)-N-norbulbodione.

Compound 4 was obtained as a violet amorphous powder. Its positive HRESIMS spectrum showed a quasimolecular ion peak at m/z = 420.1055 [M+Na]⁺, consistent with the molecular formula C₂₁H₁₉NO₇, accounting for 13 degrees of unsaturation. The general features of its IR and NMR spectra closely resembled those of 3, except for the chemical shifts of two methoxyls in 4 taking the place of the methylenedioxyl between C-1 and C-2 in 3, which was confirmed by HMBC correlations from the two methoxyl signal (δ_H 3.65 and 3.90) with C-1 (δ_C 143.8) and C-2 (δ_C 151.9), respectively. The stereochemistry of C-6a was established as S as inferred from its positive specific rotation ( = +87.3) [19]. Thus, the structure of 4 was assigned the name (+)-N-(methoxycarbonyl)-N-norisocorydione.

Compound 5 exhibited a quasimolecular ion peak at m/z 378.1315 ([M+Na]⁺) in the high-resolution mass spectrometry, which corresponded to the molecular formula C₂₀H₂₁NO₅. The UV spectrum showed absorptions at λ_max = 304, 284, and 217 nm, characteristic of a 1, 2, 8, 9-substituted aporphine. Its ¹H-NMR spectrum showed a very similar pattern to those of (+)-isolaurenine N-oxide [16], including a methylenedioxyl (δ_H 6.20), two coupled aromatic doublets at δ_H 7.02 (1H, d, J = 9.2, H-10) and 8.66 (1H, d, J = 9.2, H-11), an aromatic singlets at δ_H 6.69 (s), two OCH₃ singlets at δ_H 3.98 and 4.00, and three highly downfield shifts of N-CH₃ at δ_H = 3.35 (3H, s), H-6a at δ_H 4.40(1H, dd, J = 13.8, 3.8 Hz), and H-5 at δ_H 3.57 and 3.71 (each 1H, m) due to the N-oxide. The HMBC correlations of the methylenedioxyl proton signal with C-1 and C-2, and of the two methoxyl signals (δ_H 3.98 and 4.00) with C-8 and C-9, respectively, indicated that methylenedioxyl were located between C-1 and C-2, and the methoxyls at C-8 and C-9 respectively. The NOE correlations of H-10 with H-11, and H-11 with OCH₃ (δ_H 3.98, s) further supported the position of the methoxyl groups at C-8 and C-9. The positive specific rotation ( = +88.2) of 5 indicated an S configuration of C-6a [17], compared with the R configuration of that in (−)-isoboldine β-N-oxide ( = −90.3) [20]. Furthermore, the key NOE correlations of N-CH3 with H-6 indicated the β-N-oxide in 5 (Figure 2). Thus, compound 5 was determined to be (+)-8-methoxyisolaurenine N-oxide.

All compounds were tested for their antimicrobial activities the by disc diffusion method by measuring the inhibition zones and for the most active compounds, minimum inhibitory concentration (MIC) values were also determined. Interesting antimicrobial properties were observed (Table 2), showing that alkaloids 1, 2 and 6 exhibited antimicrobial activity against the bacterium S. aureus and two fungi (A. alternata and C. nicotianae), with MIC values of 0.60–0.80 mM, 0.74–1.04 mM and 1.41–2.14 mM, respectively. Compounds 3,4 have antibacterial activities against S. aureus, while compound 5 showed weak activity against the fungus A. alternata. Moreover, alkaloids 1–4 and 6 possessed higher antibacterial, antifungal activities with lower MIC value than 5. These result suggested that the N-carbamate group may strengthen the antibacterial and antifungal activities of this type of alkaloids.

The in vitro cytotoxic activities of the isolated alkaloids were determined against BGC-823 cells (human gastric carcinoma), HepG2 cells (Human hepatocellular carcinoma), MCF-7 cells (human breast cancer), SGC-7901 cells (human gastric adenocarcinoma), SK-MEL-2 (human skin cancer), and with SK-OV-3 (ovarian) using the revised MTT method. The results are summarized in Table 3. Among the tested compounds, alkaloids 3,4 with two carbonyl groups at C-8 and C-11 exhibited the most potent cytotoxicity against all tested tumor cell lines, with IC₅₀ values of 9.54–12.22 μM and 9.83–11.96 μM, respectively. Compounds 1, 2 and 6 showed moderate cytotoxicity against the six tumor cell lines, while 5 had the weakest activities, with an IC₅₀ value above 70 μM.

Phytochemical investigation of the 70% EtOH extract of L. cubeba led to the isolation of five novel isoquinoline alkaloids: (+)-N-(methoxycarbonyl)-N-nordicentrin (1),(+)-N-(methoxycarbonyl)-N-nor-predicentrine (2), (+)-N-(methoxycarbonyl)-N-norbulbodione (3), (+)-N-(methoxycarbonyl)-N-nor-isocorydione (4) and (+)-8-methoxyisolaurenine N-oxide (5) and one known compound, (+)-N-(methoxycarbonyl)-N-norglaucine (6). All the alkaloids were evaluated for their in vitro antimicrobial activities against two species of bacteria and five species of fungi and cytotoxic properties against BGC-823 cells (human gastric carcinoma), HepG2 cells (Human hepatocellular carcinoma), MCF-7 cells (human breast cancer), SGC-7901 cells (human gastric adenocarcinoma), SK-MEL-2 (human skin cancer), and with SK-OV-3 (ovarian). In the antimicrobial activity screening alkaloids 1, 2 and 6 exhibited activity against the bacterium S. aureus and two fungi (A. alternata and C. nicotianae). Compounds 3, 4 have antibacterial activities against S. aureus, while compound 5 showed weak activity against the fungus A. alternata. In the cytotoxicity bioassays, alkaloids 3,4 withcarbonyl groups at C-8 and C-11 exhibited the most potent cytotoxicity against all tumor cell lines, and compounds 1,2 and 5,6 showed rather moderate cytotoxicity against the six tumor cell lines.