Inflammatory and Cytotoxic Activities of Abietane Terpenoids from Nepeta bracteata Benth.

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.


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 2 of 10 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. 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.
a Spectraal data were recorded in CDCl 3 .
Molecules 2021, 26, 5603 5 of 10 Compound 2 was obtained as a white powder with its molecular formula assigned as C 20 H 28 O 2 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 1 H-and 13 C-NMR spectra, we inferred that the basic mother nucleus of compound 2 was an abietane diterpene, which was further confirmed by the 1 H-1 H 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 δ H 9.43 (s), δ C 206.6, and oxygenated methine carbon at δ C 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 (δ H 9.43) and C-4 (δ C 55.5) in the HMBC spectrum. In addition, one hydroxyl group placed at C-7 led to the downfield chemical shift of C-7 (δ C 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 H 3 -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 C 20 H 28 O 2 , deduced from the HR-ESI-MS quasimolecular ion at m/z 323.2021 [M + Na] + (calculated 323.2089). The 1 H-and 13 C-NMR spectroscopic data (Table 1) of 3 were similar to those of compound 2, except for the additional hydroxymethyl group at δ C 61.9 and double bond at δ C 126.5 and 136.2, indicating that compound 3 is an analogue of compound 2. In the HMBC spectrum, the correlations from H-5 (δ H 2.32) to C-3 (δ C 126.5) and C-4 (δ C 136.2), from H 3 -20 (δ H 1.65) to C-4 (δ C 136.2), and from H 2 -19 to C-3 (δ C 126.5) (Figure 2) implied a double bond at C-3/C-4, as well as substitutions of its methyl and hydroxymethyl groups.  (Table 1) of 4 were quite similar to those of 3, except for the appearance of olefin protons at δ H 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 (δ H 4.78, 4.69) to C-3 (δ C 71.5) and C-5 (δ C 36.9). Furthermore, the quartus carbon of C-3 (δ C 71.5), together with the molecular formula above, implies the presence of hydroxyl substitution in the structure. The HMBC correlations from H 3 -18 (δ H 0.88) to δ C 71.5 reveal the location of CH 3 -18 at C-3. The key NOESY enhancements between H-5 and H-7 and between CH 3 -18 and CH 3 -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 C 7 H 11 NO 3 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 1 H-NMR spectrum (Table 1), compound 5 showed four downfield chemical signals at δ H 3.79, 2.97, 2.99, and 2.39, one methyl proton at δ H 1.37 (3H, d, J = 7.2 Hz), and one methine proton at δ H 2.91 (1H, m). The 13 C-NMR spectrum (Table 1) revealed the presence of two carbonyl groups at δ C 177.4 and 181.5, three methylene carbons at δ C 41.9, 60.9, and 36.6, one methine carbon at δ C 34.9, and one methyl carbon at δ C 16.9. Analysis of the 1 H-1 H COSY correlations revealed the presence of two partial structures of -(CH 2 ) 2 -and -CH 2 -(CH)CH 3 -, as shown  -7) to δ C 177.4 (C-1) demonstrate that the two fragments were connected through ester carbonyl carbons. Further analysis of the carbon signal of C-2 (δ C 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.

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

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 antiinflammatory activity test showed that all the abietane diterpenoids displayed different degrees of inhibition effect. Among them, compounds 2 and 4 displayed the greatest antiinflammatory activities with IC 50 values 19.2 and 18.8 µM, as well as moderate cytotoxic activities with IC 50 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.

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). Semipreparative 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 GF 254 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).

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).

Isolation and Purification of Compounds 1-10
The aerial part of Nepeta bracteata Benth.

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 × 10 4 cells per well and cultured in a 5% CO 2 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% CO 2 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.

Anti-Inflammation Assay
The anti-inflammatory activity of the isolated compounds was evaluated in lipopolysa ccharide-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 × 10 4 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% CO 2 . NO release was measured as an indicator of the nitrite concentration.

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% CO 2 . After diluting the DMEM medium, cells were seeded into 96-well sterile microplates (6 × 10 4 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 IC 50 values were calculated using Microsoft Excel software.

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 IC 50 values of 19. 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.