New Bisabolane-Type Sesquiterpenoids from Curcuma longa and Their Anti-Atherosclerotic Activity

To explore the sesquiterpenoids in Curcuma longa L. and their activity related to anti-atherosclerosis. The chemical compounds of the rhizomes of C. longa were separated and purified by multiple chromatography techniques. Their structures were established by a variety of spectroscopic experiments. The absolute configurations were determined by comparing experimental and calculated NMR chemical shifts and electronic circular dichroism (ECD) spectra. Their anti-inflammatory effects and inhibitory activity against macrophage-derived foam cell formation were evaluated by lipopolysaccharide (LPS) and oxidized low-density lipoprotein (ox-LDL)-injured RAW264.7 macrophages, respectively. This study resulted in the isolation of 10 bisabolane-type sesquiterpenoids (1–10) from C. longa, including two pairs of new epimers (curbisabolanones A–D, 1–4). Compound 4 significantly inhibited LPS-induced nitric oxide (NO), interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and prostaglandin E2 (PGE2) production in RAW264.7 cells. Furthermore, compound 4 showed inhibitory activity against macrophage-derived foam cell formation, which was represented by markedly reducing ox-LDL-induced intracellular lipid accumulation as well as total cholesterol (TC), free cholesterol (FC), and cholesterol ester (CE) contents in RAW264.7 cells. These findings suggest that bisabolane-type sesquiterpenoids, one of the main types of components in C. longa, have the potential to alleviate the atherosclerosis process by preventing inflammation and inhibiting macrophage foaming.


Introduction
Atherosclerosis (AS), a common cardiovascular disease, is an important cause of death and morbidity in the developed world [1,2]. Recently, studies have shown that AS is mainly caused by disorders of lipid metabolism and inflammation [3], so lipid metabolism intervention and inflammation intervention are theoretically the two main means of preventing it. Currently, the modulation of lipid metabolism mainly depends on statins, but these drugs have many adverse effects [4]. Therefore, it is necessary to develop more effective treatment strategies and drugs.
As previously reported, blood-activating herbs have special advantages for the treatment of atherosclerotic cardiovascular diseases and have attracted widespread attention [5]. Curcuma longa L., a common medicine for promoting blood circulation and removing blood stasis, has been widely used in traditional Chinese medicine (TCM) for the treatment of hypertension, coronary disease, and peripheral vascular lesions [6,7]. Modern studies have shown that it has anti-inflammatory, vasodilatory, and hypolipidemic activities [8,9], and Modern studies have shown that it has anti-inflammatory, vasodilatory, an hypolipidemic activities [8,9], and its main active components are terpenoids an curcumins [10]. However, previous research has mainly focused on curcumin.
In recent years, our group has conducted chemical and pharmacological studies o the terpenoids of C. longa and obtained several sesquiterpenoids with novel structures an significant vasodilatory activity [11][12][13], which indicated that the sesquiterpenoids of longa deserve further study. Thus, the present study continued the phytochemic investigation of C. longa and explored the anti-inflammatory activity and inhibito activity against foam cell formation in RAW264.7 macrophages. Interestingly, bisabolane-type sesquiterpenoids with similar structures were isolated, including tw pairs of new epimers (1)(2)(3)(4) and a pair of diastereoisomers (5 and 6) ( Figure 1).
Curbisabolanone B (2) had the same molecular formula as compound 1 based on HRESIMS. The 13 C and 1 H NMR data (Table 1) of compound 2 were very similar to tho of compound 1, which implied that compounds 1 and 2 could be epimers. This deductio was further verified by the HSQC, HMBC, and 1 H-1 H COSY data analyses ( Figure 2).
Curbisabolanone B (2) had the same molecular formula as compound 1 based on its HRESIMS. The 13 C and 1 H NMR data (Table 1) of compound 2 were very similar to those of compound 1, which implied that compounds 1 and 2 could be epimers. This deduction was further verified by the HSQC, HMBC, and 1 H-1 H COSY data analyses ( Figure 2).    Curbisabolanone C (3) and Curbisabolanone D (4) were assigned the molecular formula of C 15 H 22 O 2 by HRESIMS. The 1 H, 13 C, and DEPT NMR data (Table 2) of compound 3 suggested that it was the 10,11-dehydration product of 1, which was confirmed by HMBC correlations of H 3 -12 and H 3 -13 with two olefinic carbons (C-10 and C-11) (Figure 2). A comparison of the NMR data of compounds 4 and 3 suggested that they were isomers with the same planar structure. Although this planar structure has been reported in the literature [17], the absolute configurations have not been determined.
Since the relative configurations of compounds 1−4 could not be determined by NOESY experiments due to the bond rotation of the side chain, NMR calculations of the chemical shifts and ECD calculations were used to determine their configurations. As for epimers 1 and 2, NMR calculations at the mPW1PW91/6-311+G(d,p) level were applied to determine their relative configurations based on the PCM model [18]. As shown in Figure 3A,B and Figure 4A Figure 3C) over (6S,7R)/(6R,7S), while the relative configuration of compound 2 was the opposite ( Figure 4C). Consequently, the absolute configuration of compound 1 could be inferred to be (6S,7S)/(6R,7R), and that of compound 2 could be inferred to be (6S,7R)/(6R,7S), which were used for ECD calculations to further determine the absolute configurations using the time-dependent density functional theory (TDDFT) method. As shown in Figure 5A,B, the experimental ECD spectra of compounds 1 and 2 matched well with the calculated ECD spectra of (6S,7S) and (6R,7S), respectively. Hence, compound 1 was determined to be (+)-(6S,7S)-bisabol-2-en-11-ol-1,9-dione, while compound 2 was determined to be (-)-(6R,7S) -bisabol-2-en-11-ol-1,9-dione. Since the relative configurations of compounds 1−4 could not be determined by NOESY experiments due to the bond rotation of the side chain, NMR calculations of the chemical shifts and ECD calculations were used to determine their configurations. As for epimers 1 and 2, NMR calculations at the mPW1PW91/6-311+G(d,p) level were applied to determine their relative configurations based on the PCM model [18]. As shown in Figures  3A,B and 4A,B, the correlation coefficients between the calculated and experimental 13 Figure 3C) over (6S,7R)/(6R,7S), while the relative configuration of compound 2 was the opposite ( Figure 4C). Consequently, the absolute configuration of compound 1 could be inferred to be (6S,7S)/(6R,7R), and that of compound 2 could be inferred to be (6S,7R)/(6R,7S), which were used for ECD calculations to further determine the absolute configurations using the time-dependent density functional theory (TDDFT) method. As shown in Figure 5A,B, the experimental ECD spectra of compounds 1 and 2 matched well with the calculated ECD spectra of (6S,7S) and (6R,7S), respectively. Hence, compound 1 was determined to be (+)-(6S,7S)-bisabol-2-en-11-ol-1,9-dione, while compound 2 was determined to be (-)-(6R,7S) -bisabol-2-en-11-ol-1,9-dione.    To determine the absolute configurations of compounds 3 and 4, the same computational methods as those used for compounds 1 and 2 were carried out. Unfortunately, the DP4+ probability did not distinguish compounds 3 and 4. Therefore, we reviewed the literature to explore methods to solve the absolute configurations of such compounds. From the literature survey, seven pairs of bisabolane-type epimers ( Figure 6, a1/a2-g1/g2) [16,[19][20][21][22][23] structurally similar to compounds 1−4 were found, and their optical rotations are listed in Table 3. It was found that the optical rotation is positive when    To determine the absolute configurations of compounds 3 and 4, the same computational methods as those used for compounds 1 and 2 were carried out. Unfortunately, the DP4+ probability did not distinguish compounds 3 and 4. Therefore, we reviewed the literature to explore methods to solve the absolute configurations of such compounds. From the literature survey, seven pairs of bisabolane-type epimers ( Figure 6, a1/a2-g1/g2) [16,[19][20][21][22][23] structurally similar to compounds 1−4 were found, and their optical rotations are listed in Table 3. It was found that the optical rotation is positive when To determine the absolute configurations of compounds 3 and 4, the same computational methods as those used for compounds 1 and 2 were carried out. Unfortunately, the DP4+ probability did not distinguish compounds 3 and 4. Therefore, we reviewed the literature to explore methods to solve the absolute configurations of such compounds. From the literature survey, seven pairs of bisabolane-type epimers ( Figure 6, a1/a2-g1/g2) [16,[19][20][21][22][23] structurally similar to compounds 1−4 were found, and their optical rotations are listed in Table 3. It was found that the optical rotation is positive when the configuration of C-6 is S (6S,7S or 6S,7R), while the optical rotation is negative when the configuration of C-6 is R (6R,7S or 6R,7R). Thus, based on the optical rotations of compounds 3 ( were proposed for ECD calculations. As illustrated in Figure 5C and D, the calculated ECD spectra of (6S,7S)-3 and (6R,7S)-4 showed good agreement with the experimental curves of compounds 3 and 4, respectively. Thus, the structure of compound 3 was identified as (+)-(6S,7S)-bisabol-2,10-dien-1,9-dione, while that of compound 4 was identified as (-)-(6R,7S)-bisabol-2,10-dien-1,9-dione.

NMR Data and ECD Calculation
The details of NMR data and ECD calculation of compounds 1-4 are shown in Figures S1-S14 and Tables S1-S14; see Supplementary Materials.

Cell Viability
After 24 h of treatment with compounds 1-8 at concentrations of 100, 50, and 25 µM, RAW264.7 cells did not show a significant reduction in cell viability compared with the serum-free DMEM-treated group. Thus, the anti-inflammatory and anti-macrophage foaming activities of these compounds in RAW264.7 cells were not induced by their cytotoxicity.

Anti-Inflammatory Activity
To evaluate the anti-inflammatory activity of compounds 1-8, a model of LPS-induced inflammation in RAW264.7 macrophages were used. Compounds 2-5, 7, and 8 inhibited LPS-induced NO production in RAW264.7 cells in a dose-dependent manner compared to the model group ( Figure 7A). Compound 4 had the highest inhibition rates at 25, 50, and 100 µM (EC 50 = 55.40 ± 14.01 µM) ( Figure 7B). However, the NO inhibitory effects of all these compounds were weaker than those of the positive control (curcumin, EC 50 = 12.50 ± 1.30 µM). Additionally, to further elucidate the anti-inflammatory effect of compound 4, some inflammatory factors were measured. Figure 8 shows that compound 4 at 25, 50, and 100 µM showed a significant reduction in the levels of TNF-α, IL-6, IL-1β, and PGE2 compared with the LPS group.

Anti-Macrophage Foaming Activity
RAW264.7 cells were exposed to 75 μg/mL ox-LDL to induce intracellular lipid accumulation, and they were quantitatively analyzed by Nile Red staining. As shown in Figure 9A

Anti-Macrophage Foaming Activity
RAW264.7 cells were exposed to 75 µg/mL ox-LDL to induce intracellular lipid accumulation, and they were quantitatively analyzed by Nile Red staining. As shown in Figure 9A

Discussion
AS is the leading cause of cardiovascular diseases such as myocardial infarctio coronary artery disease. Growing evidence indicates that AS is an active inflamm process accompanied by lipid infiltration and the repair of endothelial cell da During the lipid infiltration, macrophages phagocytose ox-LDL and turn into foam [27]. Foam cells further migrate to the subintima and secrete inflammatory med such as TNF-α, IL-1β, and IL-6, which aggravate local inflammation in the intim promote the development of AS, eventually leading to the formation and rupture plaques [28,29]. Therefore, inhibiting inflammation, regulating lipid metabolism preventing foam cell formation can effectively suppress the development of AS [30 Bisabolane-type sesquiterpenoids of C. longa have been demonstrated to be a exert anti-inflammatory effects through the NF-κB/MAPK pathway [15,31] vasorelaxant activities through the PI3K/Akt pathway [11]. However, there experimental evidence for the use of such compounds in the prevention and treatm AS. In this study, 10 structurally similar bisabolane-type sesquiterpenoids were ob from the ethyl acetate extract of C. longa. The biological activities of compounds 1-8 further evaluated in terms of cell viability, cell foaminess, and cell inflammat RAW264.7 cells. The results showed that compound 4 observably reduced the lev inflammatory factors (NO, TNF-α, IL-1β, IL-6, and PGE2) and intracellular lipids (T

Discussion
AS is the leading cause of cardiovascular diseases such as myocardial infarction and coronary artery disease. Growing evidence indicates that AS is an active inflammatory process accompanied by lipid infiltration and the repair of endothelial cell damage. During the lipid infiltration, macrophages phagocytose ox-LDL and turn into foam cells [27]. Foam cells further migrate to the subintima and secrete inflammatory mediators, such as TNFα, IL-1β, and IL-6, which aggravate local inflammation in the intima and promote the development of AS, eventually leading to the formation and rupture of AS plaques [28,29]. Therefore, inhibiting inflammation, regulating lipid metabolism, and preventing foam cell formation can effectively suppress the development of AS [30].
Bisabolane-type sesquiterpenoids of C. longa have been demonstrated to be able to exert anti-inflammatory effects through the NF-κB/MAPK pathway [15,31] and vasorelaxant activities through the PI3K/Akt pathway [11]. However, there is no experimental evidence for the use of such compounds in the prevention and treatment of AS. In this study, 10 structurally similar bisabolane-type sesquiterpenoids were obtained from the ethyl acetate extract of C. longa. The biological activities of compounds 1-8 were further evaluated in terms of cell viability, cell foaminess, and cell inflammation in RAW264.7 cells. The results showed that compound 4 observably reduced the levels of inflammatory factors (NO, TNF-α, IL-1β, IL-6, and PGE2) and intracellular lipids (TC, FC, and CE) in RAW264.7 cells. Therefore, bisabolane-type sesquiterpenoids of C. longa may have anti-AS potential, and the mechanism may be related to anti-inflammatory factors, regulation of lipid metabolism, and reduction of macrophage foaminess. However, due to the limited sample quantities of the isolated natural compounds, more in-depth experimental exploration was not conducted.
Interestingly, the structures of compounds 1-8 are very similar, but they differ greatly in terms of anti-inflammatory activity. As depicted in Figure 11, the configuration is an important factor affecting the anti-inflammatory activity. A comparison of compounds 3/4 and 5/6 indicated that the activity was greatly attenuated when the absolute configuration of C-6 was transferred from R to S. In addition to configuration, substitution is another important aspect that affects anti-inflammatory activity. Concretely, a comparison of the activities of compounds 4 and 5 indicated that the introduction of an OH group at C-4 resulted in a significant loss of the anti-inflammatory effect. However, by comparing compounds 4 and 8, it was found that the transfer of the carbonyl group from C-1 to C-4 and the inversion of the C-7 configuration did not affect the intensity of the activity. In addition, the oxidation degree of the side chain may have an impact on such activity. The dehydration of OH-11 in compound 2 to form a double bond [∆ 10(11) ] in compound 4 resulted in a significant increase in the effect. In summary, compound 4 exhibited the most potent anti-inflammatory activity in this study, and the anti-inflammatory activity of the bisabolane-type sesquiterpenoids may be related to the absolute configuration of C-6, the substituent groups, and the degree of oxidation.
carbonyl group from C-1 to C-4 and the inversion of the C-7 configuration did no the intensity of the activity. In addition, the oxidation degree of the side chain ma an impact on such activity. The dehydration of OH-11 in compound 2 to form a bond [Δ 10(11) ] in compound 4 resulted in a significant increase in the effect. In sum compound 4 exhibited the most potent anti-inflammatory activity in this study, anti-inflammatory activity of the bisabolane-type sesquiterpenoids may be related absolute configuration of C-6, the substituent groups, and the degree of oxidation Figure 11. Summary of structure-activity relationships of bisabolane-type sesquiterpeno numbers below the compounds indicate the maximum inhibition rates of the compounds LPS-induced NO production.

General Experimental Procedures
HRESIMS spectra were measured using a Q Exactive instrument (T Scientific™, Waltham, MA, USA). NMR spectra were acquired by a Bruker Avanc 600 NMR spectrometer (Bruker Corporation, Billerica, MA, USA) with TMS as an i standard. The UV and ECD spectra were recorded on an Applied photophysics Ch and Chirascan-plus circular dichroism spectrometer (Applied Photophysic Leatherhead, UK). IR spectra were recorded using a spectrum one FY-IR spectr instrument (Perkin Elmer Inc., Waltham, MA, USA). Optical rotations were me using an Anton Paar MCP 200 automatic polarimeter (Anton Paar GmbH, Graz, A HPLC separations were carried out using an Agilent 1100 instrument ( Technologies Inc., Santa Clara, CA, USA) equipped with a Welch Ultimate XB-C18 (10 × 250 mm 2 , 5 μm). Silica gel (200-300 mesh, Yantai Institute of Chemical Tech Figure 11. Summary of structure-activity relationships of bisabolane-type sesquiterpenoids. The numbers below the compounds indicate the maximum inhibition rates of the compounds against LPS-induced NO production.
Medicine Ingredients of Southwest Specialty Medicinal Materials at Chengdu University of Traditional Chinese Medicine.

Extraction and Isolation
The rhizomes of C. longa (50 kg) were soaked in 95% ethanol overnight and extracted with eight volumes of 95% ethanol under reflux for 3, 2, and 1.5 h, consecutively. Afterward, the EtOH extract was suspended in H 2 O and partitioned successively with petroleum ether and EtOAc to afford a petroleum ether portion of 2 kg and an EtOAc portion of 3 kg. Sequentially, the EtOAc portion underwent silica gel column chromatography and was eluted with petroleum ether and EtOAc (petroleum ether:EtOAc = 1:0, 7:3, and 4:6) and EtOAc and MeOH (EtOAc:MeOH = 1:0, 1:1, and 0:1) to yield six fractions (A-F).

Cell Counting Kit-8 Assay
RAW264.7 cells were seeded in 96-well plates at a density of 2 × 10 5 /mL and treated with various concentrations of compounds. After incubation for 24 h, 10 µL of CCK-8 solution was added to each well, and the cells were incubated for 1 h. The absorbance was measured at 450 nm using a Molecular Devices SpectraMax iD3 Microplate Reader.

Inflammatory Factor Assay
RAW264.7 cells were plated at a volume of 1 mL per well (2 × 10 5 /mL) into 24well plates, incubated for 24 h, and then randomly grouped. The serum-free medium group was used as the control group. The model group was treated with 1 µg/mL of lipopolysaccharide (LPS) alone. The administered groups were treated with 1 µg/mL of LPS combined with different concentrations of compounds (25,50, and 100 µM), and 25 µM curcumin was used as the positive control. The cell supernatants from each group were collected after the cells were incubated for 24 h. NO levels in the cell supernatants were measured by a Griess assay, and the IL-6, IL-1β, TNF-α, and PGE2 levels were measured using ELISA kits.  compound 4). In each group, the cells were incubated for 24 h and fixed for 20 min by 4% paraformaldehyde successively. After the plates were washed with phosphate buffered saline (PBS), the cells were incubated with a 1 µg/mL Nile Red solution for 30 min. The fluorescence was measured at an excitation wavelength of 530 nm and an emission wavelength of 590 nm.

Oil Red O Staining
RAW264.7 cells were plated at 5 × 10 5 cells/well in 24-well plates and randomly divided into groups as described above. After a 24 h treatment, the Oil Red O staining was performed according to the instructions. Then, the Oil Red O was dissolved with isopropanol, and the absorbance was measured at 550 nm. 4.7.3. Intracellular Lipid Content Assay RAW264.7 cells were seeded into 6-well plates at a density of 1 × 10 6 /mL and grouped as described above. After a 24 h treatment, the contents of total cholesterol (TC) and free cholesterol (FC) in each group of cells were measured based on the kit instructions. The cholesterol ester (CE) content was calculated as follows: CE = TC − FC.

Statistical Analysis
Statistical analyses were performed with SPSS 25.0. Data are presented as the mean ± standard deviation (SD). The EC 50 value was analyzed from the cumulative concentration-effect curves by non-linear regression analysis. A one-way ANOVA was used for comparisons between groups. p < 0.05 was considered to be statistically significant.

Conclusions
In summary, 10 bisabolane-type sesquiterpenoids, including two new pairs of epimers (1-4), were obtained from C. longa in this study. Compounds 2, 3, 4, 5, 7, and 8 exhibited anti-inflammatory effects, with compound 4 being the most active. Further studies on compound 4 found that it significantly reduced the levels of NO, IL-1β, IL-6, TNF-α, and PGE2 in RAW264.7 cells. In addition, compound 4 showed an anti-macrophage foaming effect and significantly reduced the intracellular TC, FC, and CE levels in RAW264.7 cells. These results suggest that bisabolane-type sesquiterpenoids, one of the main types of