Three New Ionone Glycosides from Rhododendron capitatum Maxim

Six ionone glycosides (1–3 and 5–7), including three new ones, named capitsesqsides A−C (1–3), together with an eudesmane sesquiterpenoid glycoside (4) and three known triterpenoid saponins (8–10) were isolated from Rhododendron capitatum. The structures of these compounds were determined by extensive spectroscopic techniques (MS, UV, 1D-NMR, and 2D-NMR) and comparison with data reported in the literature. The absolute configurations were determined by comparison of the experimental and theoretically calculated ECD curves and LC-MS analyses after acid hydrolysis and derivatization. The anti-inflammatory activities of these compounds were evaluated in the LPS-induced RAW264.7 cells. Molecular docking demonstrated that 2 has a favorable affinity for NLRP3 and iNOS.

Rhododendron capitatum Maxim belongs to the Ericaceae family and is a small deciduous shrub, mainly distributed in the Shaanxi and Qinghai Provinces of China [7].R. capitatum has a high horticultural value due to its bright colours and beautiful flowers and is often grown as an ornamental.As a Tibetan medicine for the treatment of gastric cold, abdominal pain, pharyngalgia, cough, and inflammation [8], previous phytochemical research has shown it contains a variety of structurally diverse meroterpenoids, grayanane diterpenoids, flavonoids, and coumarins [9].These compounds exhibit a variety of biological and pharmacological activities, including anti-inflammatory, antiviral, cytotoxic, and hypoglycaemic activities [7][8][9][10].As part of our continuing studies on chemical components with novel structures and significant pharmacological activities from folk medicinal plants found in the Qinling region [11,12], chemical investigations of the aerial parts of R. capitatum were undertaken, leading to the isolation and identification of three new ionone glycosides (1-3) and seven known compounds (4)(5)(6)(7)(8)(9)(10).This is the first report on the sesquiterpenoid compounds from R. capitatum (Figure 1), wherein compound 1 was a novel ionone with a 6/7 bicyclic skeleton.All the isolated compounds were evaluated for anti-inflammatory activities in the LPS-induced RAW264.7 cells model.
Molecules 2024, 29, x FOR PEER REVIEW 3 of 12 to be 3S, 6S, 9R by comparison of the experimental ECD curve and calculated electronic circular dichroism (ECD) data at the B3LYP/6-311G(d,p) level in MeOH (Figure 4).Therefore, compound 1 was determined as shown in Figure 1, and named as capitsesqside A.   to be 3S, 6S, 9R by comparison of the experimental ECD curve and calculated electronic circular dichroism (ECD) data at the B3LYP/6-311G(d,p) level in MeOH (Figure 4).Therefore, compound 1 was determined as shown in Figure 1, and named as capitsesqside A.  ) correlations of compounds 1 and 2. Table 1. 13 C NMR (100 MHz) and 1 H NMR (400 MHz) data of 1-3 in CD3OD.

Effect of Compounds 1-10 on the LPS-Induced Production of NO
The NOD-like receptor family pyrin domain containing 3 (NLRP3) protein is a member of the inflammatory vesicle protein family, and aberrant activation of this protein has been implicated in the pathogenesis of several inflammatory diseases.Inducible nitric oxide synthase (iNOS) and its product NO play an important role in the inflammatory response and oxidative stress, which is one of the major molecular mechanisms of inflammation.The MTT assay was used to examine the cytotoxicity of compounds 1-10 in RAW 264.7 cells.The results showed no significant cytotoxicity of any compounds at the testing concentrations (Figure 5).Compounds 1-10 were examined for inhibition of NO production in LPS (lipopolysaccharide)-induced RAW 264.7 cells by using the Griess method (Figure 6).In comparison with the positive control, the compounds 2 and 5 exhibited stronger NO inhibitory activity, and other compounds showed a slight inhibitory effect on NO production (Figure 6).The results of the molecular docking analysis indicated that compound 2 exhibited a high affinity for NLRP3 and iNOS, with binding energies of −6.52 and −6.64 kcal/mol, respectively (Figure 7).The carbonyl group at C-3 and hydroxy group at C-9 interact by hydrogen bonding in the NLRP3 pocket with amino acid residues PHE-575 and ALA-228, respectively.Moreover, the hydroxy group at C-9 also forms a hydrogen bonding interaction with PHE-363 in the iNOS pocket.However, compound 5 interacts more weakly than compound 2 with NLRP3 and iNOS.
stronger NO inhibitory activity, and other compounds showed a slight inhibitory effect on NO production (Figure 6).The results of the molecular docking analysis indicated that compound 2 exhibited a high affinity for NLRP3 and iNOS, with binding energies of −6.52 and −6.64 kcal/mol, respectively (Figure 7).The carbonyl group at C-3 and hydroxy group at C-9 interact by hydrogen bonding in the NLRP3 pocket with amino acid residues PHE-575 and ALA-228, respectively.Moreover, the hydroxy group at C-9 also forms a hydrogen bonding interaction with PHE-363 in the iNOS pocket.However, compound 5 interacts more weakly than compound 2 with NLRP3 and iNOS.The molecular interactions of compound 5 with iNOS by molecular docking simulation.

Plant Material
The aerial parts of R. capitatum were collected in Mount Taibai, Shaanxi Province, in June 2019 (GPS coordinates: 107°25′-107°52′ E, 33°55′-33°35′ N) and identified by Dr. Zhen-Hai Wu, College of Life Sciences, Northwest A&F University.A voucher specimen (No.WUK 0480711) could be found in the Herbarium of the College of Life Sciences, Northwest A&F University.

Plant Material
The aerial parts of R. capitatum were collected in Mount Taibai, Shaanxi Province, in June 2019 (GPS coordinates: 107 • 25 ′ -107 • 52 ′ E, 33 • 55 ′ -33 • 35 ′ N) and identified by Dr. Zhen-Hai Wu, College of Life Sciences, Northwest A&F University.A voucher specimen (No.WUK 0480711) could be found in the Herbarium of the College of Life Sciences, Northwest A&F University.

Acid Hydrolysis of Compounds 1-3
Compounds 1-3 (1 mg) were dissolved in 2 N HCl (3 mL) and stirred in water at 90 • C for 2.5 h.The resulting acid aqueous solutions were then concentrated under reduced pressure.To each residue, 1 mL of water was added, and the resulting solutions were extracted with 3 × 1 mL of EtOAc.The residue was dissolved in pyridine (1 mL) containing L-cysteine methyl ester hydrochloride (1 mg, Macklin, Shanghai, China) and stirred at 60 • C for 1.5 h.To each mixture, o-tolyl isothiocyanate (20.0 µL, Macklin, Shanghai, China) was added, and the resulting mixture was stirred at 60 • C for an additional 1.5 h.Finally, the compounds were analysed directly by LC-MS [23].

ECD Calculations
Gaussian 16 software was used to perform ECD calculations.Conformation optimization was carried out in the gas phase using density functional theory (DFT) at the B3LYP/6-31G(d) level.Time-dependent density functional theory was also employed.(TDDFT) ECD calculations were carried out in MeOH (PCM) using the B3LYP/6-311G(d,p) level, and ECD spectra were obtained with SpecDis 1.7 [24].

Cell Culture
The murine macrophage RAW 264.7 cells were obtained from the Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College.They were regularly maintained at 37 • C in DMEM (Cbico, New York, NY, USA) containing 10% FBS (ABW, Frickenhausen, German) and 100 U/mL penicillin (Solarbio, Beijing, China) in a humidified 5% CO 2 atmosphere.

MTT Assay for Cytotoxicity
The MTT assay was used to determine cytotoxicity after pretreatment with compounds 1-10.RAW 264.7 cells were plated at a density of 8 × 10 3 cells/well in a 96-well plate and incubated for 24 h before sample treatment.The cells were then pretreated with various concentrations of compounds (12.5, 25, 50, and 100 µM) for 24 h.Next, 10 µL of 5 mg/mL MTT was added to each well, followed by an additional 4 h incubation.The cultured medium was removed, and the formazan crystals were dissolved in 150 µL of DMSO.The absorbance was measured at 490 nm using a Multidetection microplate reader (BioTek Instruments, Inc., Winooski, VT, USA).

Measurement of NO
The concentration of nitrite was measured to indicate NO production using the Griess reaction.Briefly, RAW 264.7 cells were seeded into 96-well tissue culture plates at a density of 2 × 10 4 cells/mL and stimulated with 1 µg/mL of LPS in the presence or absence of compounds.After incubation at 37 • C for 24 h, 50 µL of cell-free supernatant was mixed with 100 µL of Griess reagent.The mixture was then reacted at 37 • C for 10 min while avoiding light.Absorbance was measured at 550 nm against a calibration curve with sodium nitrite standards.

Molecular Docking
Molecular docking studies were performed to predict the binding interaction of compounds 2 and 5 to NLRP3 (PDB ID: 7PZC) and iNOS (PDB ID: 3E7G) using the software Autodock 4.2 Vina along with AutoDock Tools (ADT 1.5.6)according to the previously described method [25].

Conclusions
In conclusion, three novel ionone glycosides, capitsesqsides A−C (1-3), together with seven known compounds were isolated from R. capitatum.Among them, compound 1 was a rare 6/7 bicyclic skeleton ionone.Compounds 2 and 5 showed potent anti-inflammatory activity in LPS-induced NO production in RAW 264.7 cells.Compared to compounds 2, 3, and 5-7, compound 1 showed reduced activity, suggesting that an α,β-unsaturated ketone group may be an active unit, while compound 4 and three triterpenoids (8-10) showed weak NO inhibitory activity.Molecular docking results suggested that NLRP3 and iNOS may be potential target proteins for the anti-inflammatory activity of compound 2.

Figure 4 .
Figure 4. Experimental and calculated ECD curves of compounds 1 and 2.

Figure 4 .
Figure 4. Experimental and calculated ECD curves of compounds 1 and 2.

Figure 5 .
Figure 5. Effects of compounds 1-10 on cell viability (A: 12.5μM, B: 25μM, C: 50μM, and D: 100 μM).The concentrations of these compounds ranged from 12.5 to 100 μM.Analysis of cell viability was by GraphPad Prism (8.0.2), and data are expressed as the mean ± SD.Three independent experiments were performed.Dexamethasone (DEX) was used as a positive control.

Figure 7 .
Figure 7. (A): The molecular interactions of compound 2 with NLRP3 by molecular docking simulation.(B): The molecular interactions of compound 2 with iNOS by molecular docking simulation.(C): The molecular interactions of compound 5 with NLRP3 by molecular docking simulation.(D): The molecular interactions of compound 5 with iNOS by molecular docking simulation.

Figure 7 .
Figure 7. (A): The molecular interactions of compound 2 with NLRP3 by molecular docking simulation.(B): The molecular interactions of compound 2 with iNOS by molecular docking simulation.(C): The molecular interactions of compound 5 with NLRP3 by molecular docking simulation.(D): The molecular interactions of compound 5 with iNOS by molecular docking simulation.