Abstract
Nepeta bracteata Benth. is used clinically to treat tracheal inflammation, coughs, asthma, colds, fevers, adverse urination, and other symptoms, along with functions in clearing heat and removing dampness. However, there have been few studies characterizing the material basis of its efficacy. Therefore, the aim of this study was to screen for compounds with anti-inflammatory activities in N. bracteata Benth. Using silica gel, ODS C18, and Sephadex LH-20 column chromatography, as well as semipreparative HPLC, 10 compounds were separated from N. bracteata Benth. extract, including four new diterpenoids (1–4), one amide alkaloid (5), and five known diterpenoids (6–10). The structures of all the isolates were elucidated by HR-ESI-MS, NMR, and CD analyses. Using lipopolysaccharide (LPS)-stimulated RAW 264.7 cells, we investigated the anti-inflammatory activities of compounds 1–10. It is worth noting that all were able to inhibit nitric oxide (NO) production with IC50 values < 50 μM and little effect on RAW 264.7 macrophage viability. Compounds 2 and 4 displayed remarkable inhibition with IC50 values of 19.2 and 18.8 μM, respectively. Meanwhile, screening on HCT-8 cells demonstrated that compounds 2 and 4 also had moderate cytotoxic activities with IC50 values of 36.3 and 41.4 μM, respectively, which is related to their anti-inflammatory effects.
Keywords: terpenoids, Nepeta bracteata Benth., anti-inflammatory
1. Introduction
Inflammation, a common clinical pathological process, is closely related to many diseases such as arthritis, psychosis, cardiovascular and cerebrovascular diseases, and cancer [1,2,3,4]. Current anti-inflammatory drugs, such as glucocorticoids, insulin, and the tyrosinase inhibitor kojic acid, are associated with significant side effects [5,6]. Therefore, finding effective anti-inflammatory drugs with fewer side effects is of great importance. Natural products are an important source for new drug discovery; thus, finding and discovering active components from medicinal plants is a hot topic in pharmaceutical chemistry. Nepeta bracteata Benth. belongs to the genus Nepeta of the Lamiaceae family and is mainly distributed in Pakistan, Nepal, Iran, and other countries. It is a folk medicine used by Xinjiang Uyghurs and a medicinal material imported for use in the Xinjiang Uygur hospital, with the Uyghur name “Zufa” [7]. Clinically, it is used to treat tracheal inflammation, coughs, asthma, colds, fevers, adverse urination, and other symptoms [8,9], along with functions in clearing heat and removing dampness. While modern pharmacology has shown that its extract has significant anti-inflammatory activity, no related research has been conducted to characterize its chemical components [10,11,12]. With the aim of screening for anti-inflammatory active compounds in N. bracteata Benth., a 95% ethanol extract was investigated, and 10 compounds—four new diterpenoids, nepetabrates A–D (1–4), one amide alkaloid, 6-methyl-1,4-oxazocane-5,8-dione (5), and five known diterpenoids, angustanoic acid F (6) [13], 7a-hydroxycallitrisic acid (7) [14], 1-phenanthrenecarboxylic acid (8) [15], angustanoic acid G (9) [16], and jiadifenoic acid K (10) [17]—were obtained (drawn in Figure 1). The structures of the isolates were characterized using comprehensive spectroscopic data analyses. Moreover, the anti-inflammatory activities of the isolated compounds were investigated. In this paper, we describe the structural elucidation of the isolated compounds, as well as their potential anti-inflammatory and cytotoxic effects.
Figure 1.
Structures of compounds 1–10.
2. Results
2.1. Structure Elucidation
Compound 1 was isolated as a white powder. The molecular formula C22H32O3 was established from the HR-ESI-MS spectrum with a positive-ion peak at m/z [M + Na]+ 367.2215 (calculated 367.2249). The IR spectrum of 1 showed absorptions of hydroxyl (3339 cm−1) and ester carbonyl (1759 cm−1) groups. In the 1H-NMR spectrum (Table 1), compound 1 showed peaks for three aromatic protons at δH 7.14 (1H, d, J = 8.4 Hz), 7.17 (1H, d, J = 8.4 Hz), and 7.22 (1H, d, J = 1.8 Hz), which suggests the presence of a benzene moiety. Four methyl protons at δH 1.25 (3H, d, J = 7.2 Hz), 0.97 (3H, d, J = 7.2 Hz), 1.15 (3H, s), and 1.07 (1H, s) indicate the basic diterpenoid skeleton. The downfield methyl signal at δH 2.07 (3H, s) is evidence for the existence of an acetoxyl group. Meanwhile, three oxygenated protons at δH 4.82 (1H, m), 4.00 (1H, d, J = 6.6 Hz), and 4.32 (1H, dd, J = 6.6, 2.4 Hz) suggest the presence of –OCH2– and –OCH– groups in the structure, which is in accordance with the 13C-NMR spectrum showing signals at δC 68.5, 67.3. The 13C-NMR spectrum (Table 1) revealed six aromatic carbon signals at δC 135.9, 147.1, 124.9, 126.9, 146.8, and 127.9. Aside from these aromatic carbons, the 13C-NMR also showed five methyl signals at δC 24.2, 13.9, 24.8, 28.7, and 19.4, five methylene signals at δC 36.2, 37.9, 19.4, 27.4, and 67.3, three methine signals at δC 68.5, 45.3, and 33.7, and two quartus carbon signals at δC 36.8 and 38.6. The proton signals were assigned to the corresponding carbons through direct 1H and 13C correlations in the HSQC spectrum. From the 1H–1H COSY analysis, four substructures (drawn with bold bonds in Figure 2) were established as H2-1/H2-2/H2-3, H-5/H2-6/H2-7, H-11/H-12, and H-15/H3-16/H3-17, suggesting that compound 1 has an abietane diterpene skeleton [16]. In the HMBC spectrum (Figure 2), the correlations from δH 4.82 (1H, m, H-1) to δC 171.6 indicate that the acetoxyl group is attached to C-1. Meanwhile, the HMBC correlations from δH 4.00 (1H, d, J = 6.6 Hz) and 4.32 (1H, dd, J = 6.6, 2.4 Hz) to C-10 (δC 38.6) and C-9 (δC 147.1) indicate that the angular methyl group at C-10 was further oxidized to a hydroxylmethyl unit. Thus, the planar structure of compound 1 was fully elucidated. In the NOESY spectrum, the enhancement between H-1 and H2-20, H-5, and H3-19 suggests the α-orientation of the acetoxyl group at C-1. Considering the identical biosynthetic relationship of abietane diterpenoids, the absolute configuration of 1 can be inferred as 1S, 5S, 10S. The ECD spectra were calculated using density functional theory (DFT) at the APFD/6-311 + g (2d, p) level to further support the deduction (Figure 3). As a result, the structure of compound 1 was determined as shown and given the trivial name nepetabrate A.
Table 1.
NMR spectral data of 1–5 (600 MHz for 1H-NMR and 150 MHz for 13C-NMR).
| No. | 1 a | 2 a | 3 a | 4 a | 5 a | |||||
|---|---|---|---|---|---|---|---|---|---|---|
| δC, Type | δH (J in Hz) | δC, Type | δH (J in Hz) | δC, Type | δH (J in Hz) | δC, Type | δH (J in Hz) | δC, Type | δH (J in Hz) | |
| 1 | 68.5, CH | 4.82, m | 36.2, CH2 | 2.38, m; 1.63, m | 34.4, CH2 | 2.33, m; 1.62, m | 39.1, CH2 | 1.90, m; 1.65, m | 177.4, C | -- |
| 2 | 36.2, CH2 | 1.82, m; 1.21, m | 29.9, CH2 | 2.89, m; 1.27, m | 30.5, CH2 | 2.76, m; 1.23, m | 30.5, CH2 | 1.86, m; 1.29, m | 41.9, CH2 | 3.71, m; 2.87, m |
| 3 | 37.9, CH2 | 2.33, d (12.6);5566771.43, m | 21.1, CH2 | 1.89, m; 1.33, m | 126.5, C | -- | 71.5, C | -- | 60.9, CH2 | 3.79, m; 2.97, m |
| 4 | 36.8, C | -- | 55.5, C | -- | 136.2, C | -- | 153.7, C | -- | 181.5, C | -- |
| 5 | 45.3, CH | 1.87, m | 43.0, CH | 1.87, m | 37.7, CH | 2.32, d (6.0) | 36.9, CH | 2.31, m | 36.6, CH2 | 2.99, m; 2.30, m |
| 6 | 19.4, CH2 | 1.46, m; 1.72, m | 27.2, CH2 | 1.95, m; 1.85, m | 32.8, CH2 | 1.75, m; 1.63, m | 33.7, CH2 | 2.01, m; 1.03, m | 34.9, CH | 2.91, m |
| 7 | 27.4, CH2 | 2.13, m; 2.01, m | 71.9, CH | 3.85, dd (8.4, 4.2) | 69.5, CH | 4.83, t (9.0) | 68.6, CH | 4.86, t (4.8) | 16.9, CH3 | 1.37, d (7.2) |
| 8 | 135.9, C | -- | 134.6, C | -- | 136.4, C | -- | 135.9, C | -- | -- | -- |
| 9 | 147.1, C | -- | 146.5, C | -- | 148.0, C | -- | 146.9, C | -- | -- | -- |
| 10 | 38.6, C | -- | 38.2, C | -- | 36.5, C | -- | 43.0, C | -- | -- | -- |
| 11 | 124.9, CH | 7.14, d (8.4) | 124.7, CH | 7.17, d (9.0) | 124.3, CH | 7.28, d (7.8) | 125.7, CH | 7.25, s | -- | -- |
| 12 | 126.9, CH | 7.17, d (8.4) | 127.2, CH | 6.91, d (9.0) | 126.6, CH | 7.16, dd (7.8, 2.4) | 126.8, CH | 7.15, dd (8.4, 1.8) | -- | -- |
| 13 | 146.8, C | -- | 145.7, C | -- | 144.5, C | -- | 144.7, C | -- | -- | -- |
| 14 | 127.9, CH | 7.22, d (1.8) | 129.0, CH | 7.72, d (3.6) | 128.2, CH | 7.72, d (2.4) | 128.3, CH | 7.22, d (8.4) | -- | -- |
| 15 | 33.7, CH | 2.89, m | 33.7, CH | 2.83, m | 24.4, CH | 1.95, m | 33.7, CH | 1.24, m | -- | -- |
| 16 | 24.2, CH3 | 1.25, d (7.2) | 13.9, CH3 | 1.21, s | 19.4, CH3 | 0.85, s | 24.2, CH3 | 1.26, dd (6.6, 2.4) | -- | -- |
| 17 | 13.9, CH3 | 0.97, t (7.2) | 13.9, CH3 | 1.21, s | 21.5, CH3 | 0.85, s | 24.0, CH3 | 1.26, dd (6.6, 2.4) | -- | -- |
| 18 | 24.8, CH3 | 1.15, s | 25.4, CH3 | 1.22, d (1.8)556677 | 33.7, CH3 | 1.79, s | 21.0, CH3 | 0.88, s | -- | -- |
| 19 | 28.7, CH3 | 1.07, s | 206.6, CH | 9.43, s | 61.9, CH2 | 4.78, d (12.0);5566774.69, d (12.0) | 107.1, CH2 | 5.12, s5566774.74, s | -- | -- |
| 20 | 67.3, CH2 | 4.00, d (6.6)5566774.32, dd (6.6,2.4) | 23.7, CH3 | 1.16, s | 13.9, CH3 | 1.65, s | 28.1, CH3 | 1.45, s | -- | -- |
| 21 | 171.6, C | -- | -- | -- | -- | -- | -- | -- | -- | -- |
| 22 | 21.2, CH3 | 2.07, s | -- | -- | -- | -- | -- | -- | -- | -- |
a Spectraal data were recorded in CDCl3.
Figure 2.
Key 1H–1H COSY (in bolds) and HMBC (arrows) correlations of compounds 1–5.
Figure 3.
Experimental and calculated ECD spectra of 1.
Compound 2 was obtained as a white powder with its molecular formula assigned as C20H28O2 according to the positive HR-ESI-MS peak at m/z [M + Na]+ 323.2013 (calculated 323.2089), exhibiting seven degrees of unsaturation. Through the 1H- and 13C-NMR spectra, we inferred that the basic mother nucleus of compound 2 was an abietane diterpene, which was further confirmed by the 1H–1H COSY and HMBC spectra (Figure 2). In fact, the NMR data of compound 2 were similar to those reported for 1-phenanthrenecarboxylic acid, except for an additional aldehyde signal at δH 9.43 (s), δC 206.6, and oxygenated methine carbon at δC 71.9 [15]. The presence of the aldehyde group was due to the oxidation of a methyl group at C-19, as supported by the correlations between H-19 (δH 9.43) and C-4 (δC 55.5) in the HMBC spectrum. In addition, one hydroxyl group placed at C-7 led to the downfield chemical shift of C-7 (δC 71.9), as confirmed by HMBC correlations. The relative configuration of compound 2 was established by analysis of its NOESY data. The key NOE correlations between H-5 and H-7 and between H3-18 and H-19 supported the β-orientations of both the aldehyde and the hydroxyl groups. Combined with the experimental and calculated CD curves (Supplementary Materials. Figure S31), the absolute configuration of compound 2 was identified as established and given the trivial name nepetabrate B.
Compound 3, purified as a white powder, has a molecular formula of C20H28O2, deduced from the HR-ESI-MS quasimolecular ion at m/z 323.2021 [M + Na]+ (calculated 323.2089). The 1H- and 13C-NMR spectroscopic data (Table 1) of 3 were similar to those of compound 2, except for the additional hydroxymethyl group at δC 61.9 and double bond at δC 126.5 and 136.2, indicating that compound 3 is an analogue of compound 2. In the HMBC spectrum, the correlations from H-5 (δH 2.32) to C-3 (δC 126.5) and C-4 (δC 136.2), from H3-20 (δH 1.65) to C-4 (δC 136.2), and from H2-19 to C-3 (δC 126.5) (Figure 2) implied a double bond at C-3/C-4, as well as substitutions of its methyl and hydroxymethyl groups. Compound 3 is the product of methyl migration and dehydrogenation of compound 2, which is not common in abietane diterpenes. The similar NOESY spectra of compounds 2 and 3 suggest their identical relative configurations. Together with the experimental and calculated CD curves (Supplementary Materials, Figure S32), compound 3 was elucidated as nepetabrate C.
Compound 4 was obtained as a white powder with a molecular formula of C20H28O2 based on the positive-ion HR-ESI-MS peak at m/z 323.2023 [M + Na]+ (calculated 323.2089). The 1H- and 13C-NMR spectroscopic data (Table 1) of 4 were quite similar to those of 3, except for the appearance of olefin protons at δH 5.12 and 4.74 and the disappearance of two oxygenated protons in 4. Further analysis of the NMR data of compound 4 revealed the presence of an outer ring double bond at C-4/19, which was confirmed by the HMBC correlations from H-19 (δH 4.78, 4.69) to C-3 (δC 71.5) and C-5 (δC 36.9). Furthermore, the quartus carbon of C-3 (δC 71.5), together with the molecular formula above, implies the presence of hydroxyl substitution in the structure. The HMBC correlations from H3-18 (δH 0.88) to δC 71.5 reveal the location of CH3-18 at C-3. The key NOESY enhancements between H-5 and H-7 and between CH3-18 and CH3-20 revealed the opposing configurations of 3-OH and 7-OH. The absolute configuration was determined by DFT calculations of the ECD spectra (Supplementary Materials, Figure S33), which confirmed the 3R, 5R, 7S, and 10S configurations. Thus, compound 4 was assigned as nepetabrate D.
Compound 5 was obtained as a yellow amorphous powder, with its molecular formula assigned as C7H11NO3 on the basis of its positive HR-ESI-MS (m/z 180.0723 [M + Na]+ (calculated 180.0739), implying three degrees of unsaturation. The IR spectrum of 5 showed carbonyl (1724 cm−1) and methyl (2938, 2924 cm−1) groups. In the 1H-NMR spectrum (Table 1), compound 5 showed four downfield chemical signals at δH 3.79, 2.97, 2.99, and 2.39, one methyl proton at δH 1.37 (3H, d, J = 7.2 Hz), and one methine proton at δH 2.91 (1H, m). The 13C-NMR spectrum (Table 1) revealed the presence of two carbonyl groups at δC 177.4 and 181.5, three methylene carbons at δC 41.9, 60.9, and 36.6, one methine carbon at δC 34.9, and one methyl carbon at δC 16.9. Analysis of the 1H–1H COSY correlations revealed the presence of two partial structures of –(CH2)2– and –CH2–(CH)CH3–, as shown in Figure 2. In the HMBC spectrum, the correlations from δH 3.79, 2.97 (2H, m, H-3) and δH 2.99 (1H, H-5a), 2.39 (1H, dd, J = 4.2, 4.2 Hz, H-5b) to δC 181.5 (C-4), δH 3.71, 2.87 (2H, m, H-2), and δH 1.37 (3H, d, J = 7.2 Hz, H3-7) to δC 177.4 (C-1) demonstrate that the two fragments were connected through ester carbonyl carbons. Further analysis of the carbon signal of C-2 (δC 41.9) and the molecular formula above confirmed an amide unit between C-2 and C-6. The absolute configuration of C-6 was assigned as S on the basis of a comparison of its experimental and calculated CD curves. From all the above data, compound 5 was established as shown and named 6-methyl-1,4-oxazocane-5,8-dione.
2.2. Bioactive Activity
The anti-inflammatory activities of compounds 1–10 were evaluated in RAW 264.7 macrophages with aspirin as the positive control [18,19]. As shown in Table 2, in the RAW 264.7 macrophage viability test, all compounds showed mild or inavtive effects for RAW 264.7 macrophages. On the contrary, compounds 1–10 displayed different degrees of anti-inflammatory activity against RAW 264.7 macrophages, with IC50 values ranging from 18.0 to 46.3 μM. Among the abietane diterpenes, compounds 2 and 4 displayed the most anti-inflammatory activity with IC50 values of 19.2 and 18.8 μM, respectively. Compound 5, as an amide alkaloid, also showed a remarkable inhibition effect with an IC50 value of 18.0 μM, as compared with the IC50 values of 15.9 for the positive control, aspirin. Cytotoxic testing on HCT-8 cells showed that compounds 2 and 4 had moderate activity with IC50 values of 36.3 and 41.4 μM, respectively, while the other compounds were inactive or mildly active when compared with the positive control, adriamycin.
Table 2.
Anti-inflammatory and cytotoxic activities of the isolated compounds.
| Compounds | Cell Survival Rate (%) | IC50 (μM) | |
|---|---|---|---|
| RAW 264.7 Macrophages | RAW 264.7 Macrophages | HCT-8 | |
| 1 | 97.93 ± 0.26 | 38.2 ± 1.15 a | >50 |
| 2 | 88.26 ± 0.32 | 19.2 ± 1.25 | 36.3 ± 1.10 |
| 3 | 99.84 ± 0.21 | 22.3 ± 1.26 | >50 |
| 4 | 92.21 ± 0.19 | 18.8 ± 0.75 | 41.4 ± 0.91 |
| 5 | 99.21 ± 0.15 | 18.0 ± 1.13 | >50 |
| 6 | 99.89 ± 0.13 | 36.2 ± 1.21 | >50 |
| 7 | 98.75 ± 0.45 | 37.1 ± 0.81 | >50 |
| 8 | 99.12 ± 0.28 | 37.5 ± 0.92 | >50 |
| 9 | 98.26 ± 0.18 | 42.3 ± 0.56 | >50 |
| 10 | 98.75 ± 0.20 | 46.3 ± 1.02 | >50 |
| Aspirin b | 15.9 ± 0.38 | ||
| Adriamycin | 1.78 ± 0.14 | ||
| DMEM (PBS) c | 100.00 | ||
a Values are means ± SD of triplicate experiments. b Positive control substance. c Negative control substance.
3. Discussion
Although Nepeta bracteata Benth. is widely used clinically and has promising curative effects, there are few studies about this medicinal plant. Previous research found that N. bracteata Benth. has certain free-radical-scavenging ability and an anti-inflammatory effect in vitro, and its alcohol extract displayed certain DPPH free-radical-scavenging ability, providing a theoretical basis for further research [20,21]. Therefore, we first investigated the active substances of N. bracteate Benth. and obtained nine abietane diterpenoids including four new ones, nepetabrates A–D (1–4), and one new amide alkaloid (5). Compared with previous studies, we explored the anti-inflammatory activities of compounds 1–10 on RAW 264.7 macrophages cells. The cell viability of the RAW 264.7 macrophages experiment displayed that all the isolated compounds had mild toxicity to cells at 50 μM. The anti-inflammatory activity test showed that all the abietane diterpenoids displayed different degrees of inhibition effect. Among them, compounds 2 and 4 displayed the greatest anti-inflammatory activities with IC50 values 19.2 and 18.8 μM, as well as moderate cytotoxic activities with IC50 values of 36.3 and 41.4 μM, further proving the correlation between inflammation and cancer. Morever, the results also showed that compound 5 had significant anti-inflammatory activity but had no significant advantage over diterpenes. Accordingly, we believe that diterpenes represent the material basis for the plant to exert its clinical anti-inflammatory effect, which deserves further study.
4. Materials and Methods
4.1. General Experimental Procedures
Optical rotation data were measured using a Perkin-Elmer 341 digital polarimeter (PerkinElmer, Norwalk, OH, USA). UV (1.0 mg of sample was dissolved in 3 mL of chromatographic grade methanol for each sample) and IR (1.0 mg of sample was pressed in KBr for each sample) spectral data were recorded on Shimadzu UV2550 and FTIR-8400S spectrometers (Shimadzu, Kyoto, Japan). CD spectra were obtained using a JASCO J-815 spectropolarimeter. NMR spectra were obtained using a Bruker AV III 600 NMR spectrometer with chemical shift values presented as δ values using TMS as the internal standard (samples dissolved in an appropriate amount ofdeuterated chloroform). HR-ESI-MS was performed using an LTQ-Orbitrap XL spectrometer (Thermo Fisher Scientific, Boston, MA, USA); samples were dissolved in chromatographic methanol and treated through a membrane, single pump. Colurmn chromatography (CC) was performed using silica gel (100–200 and 200–300 mesh, Qingdao Marine Chemical Plant, Qingdao, China). Semi-preparative HPLC was performed using an HPLC PUMP K-501, LC3000 high-performance liquid chromatograph (Beijing Tong Heng Innovation Technology Co., Ltd, Beijing, China), and Kromasil 100-5C18, 250 × 10 mm, E108850. Precoated silica gel GF254 plates (Zhi Fu Huang Wu Pilot Plant of Silica Gel Development, Yantai, China) were used for TLC. All solvents used (petroleum ether, ethyl acetate, dichloromethane, methanol (analytical grade and chromatographic grade), and deuterated chloroform) were of analytical grade (Beijing Chemical Plant, Beijing, China).
4.2. Plant Material
Nepeta bracteata Benth. was purchased from Xinjiang Uygur hospital (Urumqi, China) and identified as Nepeta bracteata Benth. by Professor Leiling Shi. A voucher specimen (M20191025) was deposited at the Medical Laboratory of Xinjiang Institute of Chinese and Ethnic Medicine (Urumqi, China).
4.3. Isolation and Purification of Compounds 1–10
The aerial part of Nepeta bracteata Benth. (6.0 kg) was soaked in ethanol at room temperature (3 × 40 L, 3 h each time) and extracted three times under reflux. Removal of the ethanol under reduced pressure yielded the ethanol extract (437.0 g). The ethanol extract was dissolved in water and successively extracted with petroleum ether (3 × 1000 mL), dichloromethane (3 × 1000 mL), and ethyl acetate (3 × 1000 mL). The petroleum fraction (134.8 g) was subjected to CC (12.0 cm × 40.0 cm, 300.0 g) over a silica gel (100–200 mesh), eluting with a stepwise gradient of petroleum ether/EtOAC (from 1:0 to 0:1; i.e., 1:0, 100:1, 50:1, 25:1, 8:1, 5:1, 1:1, and 0:1, v/v) to yield fractions A–H. Fr.F was subjected to CC (5.0 cm × 15.0 cm, 70.0 g) over a silica gel (100–200 mesh), eluting with a stepwise gradient of petroleum ether/EtOAC (from 20:1 to 1:1; i.e., 20:1, 10:1, 3:1, and 1:1, v/v) to yield four fractions (Fr.F 1–4). Fr.F 3 was subjected to CC (3.0 cm × 20.0 cm, 65.0 g) over a silica gel (200–300 mesh), eluting with a stepwise gradient of petroleum ether/EtOAC (from 10:1 to 3:1; i.e., 10:1, 5:1, and 3:1, v/v) to yield three fractions (Fr.F 3-1–3). Fr.F 3-2 was purified using semi-preparative HPLC with MeOH/H2O (90:10, v/v) as the mobile phase to yield compound 1 (9.0 mg, tR = 42.4 min). Fr.F 3-3 was purified using semi-preparative HPLC of MeOH/H2O (85:15, v/v) as the mobile phase to yield compounds 2 (5.2 mg, tR = 27.0 min) and 3 (9.2 mg, tR = 28.6 min). Fr.H was subjected to CC (4.0 cm × 15.0 cm, 30.0 g) over a silica gel (100–200 mesh), eluting with a stepwise gradient of petroleum ether/EtOAC (from 15:1 to 1:1; i.e., 15:1, 5:1, 3:1 and 1:1, v/v) to yield four fractions ( Fr.H 1–4 ). Fr.F 3 was subjected to CC (3.0 cm × 20.0 cm, 70.0 g) over a silica gel (200–300 mesh), eluting with a stepwise gradient of petroleum ether/EtOAC (from 4:1 to 1:1; i.e., 4:1, 2:1, and 1:1, v/v) to yield three fractions (Fr.H 3-1–3). Fr.F 3-3 was purified by semi-preparative HPLC of MeOH/H2O (85:15, v/v) as the mobile phase to yield compound 4 (3.2 mg, tR = 25.6 min). The dichloromethane fraction (67.4 g) was subjected to CC (8.0 cm × 40.0 cm, 240.0 g) over a silica gel (100–200 mesh), eluting with a stepwise gradient of CH2Cl2/MeOH (from 1:0 to 0:1; i.e., 1:0, 100:1, 50:1, 30:1, 20:1, 5:1, 1:1, and 0:1, v/v) to yield fractions I–O. Fr.I was subjected to CC (8.0 cm × 15.0 cm, 105.0 g) over a silica gel (100–200 mesh), eluting with a stepwise gradient of petroleum ether/EtOAC (from 15:1 to 1:2; i.e., 15:1, 8:1, 4:1, 2:1, 1:1, and 1:2, v/v) to yield six fractions (Fr.I 1–6). Fr.I 3 was isolated through ODS MPLC elution with MeOH/H2O (50:50, 70:30, 90:10, and 100:0, v/v), and purified using semi-preparative HPLC to give compounds 6 (2.0 mg, tR = 23.2 min), 7 (2.3 mg, tR = 26.4 min), and 8 (1.4 mg, tR = 12.7 min). Fr.J was subjected to CC (6.0 cm × 20.0 cm, 70.0 g) over a silica gel (100–200 mesh), eluting with a stepwise gradient of petroleum ether/EtOAC (from 15:1 to 1:2; i.e., 15:1, 8:1, 4:1, 2:1, 1:1, and 1:2 v/v) to yield six fractions (Fr.J 1–6). Fr.J 2 was purified by semi-preparative HPLC of MeOH/H2O (85:15, v/v) as the mobile phase to yield compounds 9 (3.6 mg, tR = 15.9 min) and 10 (1.4 mg, tR = 19.3 min). Fr.J 6 was purified by semi-preparative HPLC of MeOH/H2O (38:62, v/v) as the mobile phase to yield compound 5 (3.6 mg, tR = 9.5 min).
4.4. Characterization of Compounds 1–5
Nepetabrate A (1), white powder (MeOH); UV (MeOH) λmax (logε) 291 (3.52) nm; IR (film) νmax 3339, 2962, 2871, 1759, 1467, 1213, 1144 cm–1; 1H- and 13C-NMR data (CDCl3), see Table 1; HR-ESI-MS m/z 367.2215 [M + Na]+ (calculated 367.2249, C22H32O3).
Nepetabrate B (2), white powder (MeOH); UV (MeOH) λmax (logε) 294 (3.76) nm; IR (film) νmax 3338, 2994, 2936, 2872, 1460, 1221, 1099 cm–1; 1H- and 13C-NMR data (CDCl3), see Table 1; HR-ESI-MS m/z 323.2013 [M + Na]+ (calculated 323.2089, C20H28O2).
Nepetabrate C (3), white powder (MeOH); UV (MeOH) λmax (logε) 292 (3.93) nm; IR (film) νmax 3342, 2958, 2875, 1456, 1230, 1138 cm–1; 1H- and 13C-NMR data (CDCl3), see Table 1; HR-ESI-MS m/z 323.2021 [M + Na]+ (calculated 323.2089, C20H28O2).
Nepetabrate D (4), white powder (MeOH); UV (MeOH) λmax (logε) 294 (3.53) nm; IR (film) νmax 3351, 2940, 2866, 1445, 1227, 1115 cm–1; 1H- and 13C-NMR data (CDCl3), see Table 1; HR-ESI-MS m/z 323.2023 [M + Na]+ (calculated 323.2089, C20H28O2).
6-Methyl-1,4-oxazocane-5,8-dione (5), yellow amorphous powder (MeOH); UV (MeOH) λmax (logε) 291 (3.76) nm; IR (film) νmax 3350, 2938, 2874, 1724 cm–1; 1H- and 13C-NMR data (CDCl3), see Table 1; HR-ESI-MS m/z 180.0723 [M + Na]+ (calculated 180.0739, C7H11NO3).
4.5. RAW 264.7 Macrophage Viability Test
The MTT colorimetric method was used to detect the effect of compounds 1–10 on the viability of RAW 264.7 macrophages. The RAW 264. 7 macrophages in the logarithmic growth phase were digested with trypsin to prepare a single-cell suspension, which was seeded in a 96-well plate at a density of 1 × 104 cells per well and cultured in a 5% CO2 incubator for 24 h at 37 °C, before discarding the supernatant. The blank control group was cultured with 10% FBS-containing DMEM, and the drug group was treated with aqueous solutions of compounds 1–10, with six replicate wells for each concentration. Incubation was continued in 5% CO2 at 37 °C. After 24 h of incubation, 10 μL of 5 mg/mL MTT was added to each well. The culture solution was removed after culturing for 4 h. Then, 100 μL of DMSO was added to each well, before shaking for 10 min to achieve complete dissolution. The optical density (OD) was measured at 492 nm using a microplate reader to calculate cell viability.
4.6. Anti-Inflammation Assay
The anti-inflammatory activity of the isolated compounds was evaluated in lipopolysaccharide-stimulated RAW 264.7 macrophages using the MTT colorimetric method. The RAW 264.7 macrophages were seeded in 96-well plates at a density of 1 × 104 cells per well for 24 h, followed by treatment with different extracts of identical purity for another 24 h. The compounds were dissolved in dimethyl sulfoxide (DMSO) and diluted appropriately just before cell treatments. Cells were incubated with the extract at indicated concentrations, with DMSO not exceeding 0.1% in all experiments. The cells were cultured in DMEM with 10% FBS and antibiotics (100 U/mL penicillin and 100 µg/mL streptomycin) at 37 °C with 5% CO2. NO release was measured as an indicator of the nitrite concentration.
4.7. Cytotoxicity Test
The cytotoxic activities of compounds 1–10 against HCT-8 cells were tested using the MTT colorimetric method. HCT-8 cells were cultivated on DMEM medium at 37 °C and 5% CO2. After diluting the DMEM medium, cells were seeded into 96-well sterile microplates (6 × 104 cells/well) and cultured with a series of various concentrations of tested compounds or adriamycin (positive control) for 24 h at 37 °C. After incubation, all compounds were tested at five concentrations (10−100 μM) for 1 h. Following this, the supernatant was removed, and all components were dissolved in 100% DMSO, at such an amount that there was a final DMSO concentration of 0.1% added to each well. The absorbance was measured using a microplate reader at a wavelength of 570 nm. Data are displayed as the means ± SD (n = 3). The cell growth assay was repeated three times, and the IC50 values were calculated using Microsoft Excel software.
5. Conclusions
Nine abietane diterpenoids, including four new ones and one new amide alkaloid, were obtained from the ethnic medicine Nepeta bracteata Benth. for the first time, which clarified the active substances of N. bracteata Benth. and laid the foundation for its further clinical application. Furthermore, the anti-inflammatory and cytotoxic activities of all isolates were tested. Compounds 2 and 4 displayed potential biological activities with IC50 values of 19.2 and 18.8 μM in the anti-inflammation assay and IC50 values of 36.3 and 41.4 μM in the cytotoxicity test, respectively. Both compounds are active molecules with potential research value.
Acknowledgments
The authors especially thank Xiaoling Ma for her guidance and help in the process of revising the article.
Supplementary Materials
Supplementary data associated with this article can be found in the online version.
Author Contributions
L.S., G.M. and J.Z. conceptualized and designed the experiments; M.Z. performed the experiments; M.C. and Y.H. helped with data analysis; C.F. and H.W. helped with structure elucidation. All authors have read and agreed to the published version of the manuscript.
Funding
The work was financially supported by the sub-project of the National Key R&D Program (2019YFC1712303), the Special Regional Collaborative Innovation Project of Xinjiang Uygur Autonomous Region (2020E01011), and the Major Science and Technology projects of Xinjiang Uygur Autonomous Region (202107638).
Institutional Review Board Statement
Not applicable.
Informed Consent Statement
Not applicable.
Data Availability Statement
The data of the NMR and cellular anti-inflammatory and toxic activity presented in this study are available in supporting information.
Conflicts of Interest
The authors declare no conflict of interest.
Sample Availability
Samples of the compounds 1–10 are available from the authors.
Footnotes
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Data Availability Statement
The data of the NMR and cellular anti-inflammatory and toxic activity presented in this study are available in supporting information.