Anti-inflammatory Activity of Norisoprenoids from the Aerial Parts of Celosia cristata L.

Introduction

Celosia cristata L. (Amaranthaceae) is an herbaceous annual plant widely distributed in the tropical and subtropical regions around the world including Asia, South America, India, and Africa.¹ There are approximately 60 species of genus Celosia, and among them, C. cristata, C. argentea, C. isertii, and C. spicata are considered significantly as dietary leaf vegetables.² C. cristata is an ornamental plant, and is commonly known as cock’s comb flower, since the flower looks like the head on a cock.³ It has traditionally been used to treat hemostasis, eye and mouth inflammation, and gynecological ailments, such as severe menstrual pain and leukorrhea, based on its astringent action.⁴ C. cristata has been reported with various bioactive constituents, such as phenolic compounds, terpenoids, saponins, and carotenoids.^4,5

Norisoprenoids, the carbonyl compounds with 13, 12, 11, 10 or 9 carbon atoms, are the breakdown products of carotenoids. This decomposition process involves enzymatic reactions activated by carotenoid cleavage dioxygenase (CCD) and non-enzymatic reactions such as exposure to light, oxygen, fluctuations in temperature, and acid hydrolysis.⁶ In previous research from plant biotechnology, two classes of CCDs (CCD1 and CCD4) enzymes are involved in species such as petunia, tomato, crocus, grape, citrus, and rose.⁷ Further investigation on food-nutritional studies have shown that norisoprenoids influences the aroma and taste of fruit.⁸ It also has been revealed that it significantly affects the scents produced during the aging process of wine.⁹ Additionally, in a pharmacological study, it was reported various bioactivities such as anti-cancer, anti-tumor, and anti-inflammation.^10,11,12

In the course of phytochemical search of aerial parts of C. cristata, seven known compounds (1–7) (Fig. 1) were isolated from the ethanol extract. The structures were determined by comparing their physical and spectroscopic characteristics with previously published data, particularly 1D and 2D NMR data such as COSY, HSQC, HMBC, and NOESY, in addition to HR-ESI-MS.

Fig. 1.

Chemical structures of the isolated compounds (1–7).

Experimental

General experimental procedures – UV spectra were recorded on a JASCO UV-550 spectrophotometer (JASCO, Tokyo, Japan). 1D and 2D NMR spectra were recorded on Bruker AVANCE 400 spectrometer (Bruker, MA, USA). HR-ESI-MS and LC-HR-MS/MS analyses were conducted with an Orbitrap Exploris 120 mass spectrometer, connected to a Vanquish UHPLC system and diode array detector (Thermo Fisher Scientific, MA, USA). Open column chromatography was performed on Sephadex LH-20 (25-100 μm, Pharmacia, New Jersey, USA). MPLC was conducted utilizing the Biotage Isolera Prime chromatography system. Semi-preparative HPLC was carried out using a Waters HPLC system, equipped with two Waters 515 pumps, a 2996 photodiode array detector, and three semi-preparative columns, such as YMC J’sphere ODS-H80 (4 μm, 250 × 20 mm, I, d., flow rate 10 mL/min), YMC J’sphere ODS-H80 (4 μm, 150 × 20 mm, I, d., flow rate 6 mL/min), and YMC J’sphere ODS-H80 (4 μm, 150 × 10 mm, I, d., flow rate 3 mL/min). TLC was performed on silica gel 60 F₂₅₄ plates (0.25 mm, Merk), and the spots were visualized using a spray regent consisting of 10% vanillin solution in sulfuric acid.

Plant materials – The aerial parts of C. cristata was harvested from wild cultivation at Chungbuk National University, in 2019. A voucher specimen (CBNU-2019-CC) was identified by Professor Moon-Soon Lee and stored in the natural chemistry laboratory of the College of Industrial Plant Science and Technology, Chungbuk National University.

Extraction and isolation – The dried aerial parts of C. cristata (2 kg) were extracted with 80% EtOH (15 × 2 L). The extract was filtered and evaporated under reduced pressure, and the resulting residue (170 g) was suspended in water and sequentially partitioned with n-hexane (4 × 1 L), CH₂Cl₂(4 × 1 L), and EtOAc (4 × 1 L). The CH₂Cl₂-soluble fraction (8.6 g) was fractionated using normal-phase MPLC (silica) with a CH₂Cl₂-MeOH step gradient system (100:0 to 84:16) to give six fractions, CCAC1–CCAC6. CCAC2 (1.58 g) and CCAC3 (2.27 g) were subject to Sephadex LH-20 open column chromatography with n-hexane-CH₂Cl₂ isocratic system (1:4) to obtain fractions CCAC2C, CCAC2D and CCAC3C, CCAC3D, respectively. CCAC3D (1.2 g) was separated using reverse-phase MPLC (C₁₈) with a H₂O-MeOH gradient system (80:20 to 20:80), and six subfractions (CCAC3D-1–CCAC3D-6) were obtained. CCAC3D-3 (149.5 mg) was separated using normal-phase MPLC with a CH₂Cl₂-MeOH step gradient system (99:1 to 91:9), and three subfractions (CCAC3D-3-1–CCAC3D-3-3) were obtained. CCAC3D-3-1 (55.8 mg) was further purified by preparative HPLC [YMC J’sphere ODS-H80 (4 μm, 150 × 20 mm, I, d., flow rate 6 mL/min)] using a H₂O-CH₃CN isocratic system (70:30) to afford compound 1 (1.42 mg; t_R = 18.8 min). CCAC2D (0.81 mg) was separated using semi-prep HPLC [YMC J’sphere ODS-H80 (4 μm, 250 × 20 mm, I, d., flow rate 10 mL/min)] and fraction collector with a H₂O-CH₃CN gradient system (80:20 to 20:80), and twenty-two subfractions (CCAC2D-1–CCAC2D-22) were obtained. CCAC2D-5 (50 mg) was purified by preparative HPLC [YMC J’sphere ODS-H80 (4 μm, 150 × 10 mm, I, d., flow rate 3 mL/min)] using a H₂O-CH₃CN isocratic system (82:18) to afford compounds 2 (4.46 mg; t_R = 15.6 min), 3 (11.59 mg; t_R = 17.5 min), and 4 (7.08 mg; t_R = 22.0 min). CCAC2D-8 (39.6 mg) was purified by preparative HPLC [YMC J’sphere ODS-H80 (4 μm, 150 × 10 mm, I, d., flow rate 3 mL/min)] using a H₂O-CH₃CN isocratic system (75:25), and two subfractions (CCAC2D-8-1–CCAC2D-8-2) were obtained. CCAC2D-8-2 (22.24 mg) was purified by preparative HPLC [YMC J’sphere ODS-H80 (4 μm, 150 × 10 mm, I, d., flow rate 3 mL/min)] using a H₂O-CH₃CN isocratic system (83:17) to afford compounds 5 (3.22 mg; t_R = 65.2 min), 6 (3.57 mg; t_R = 69.9 min), and 7 (1.07 mg; t_R = 75.8 min).

3,9-Dihydroxy-5,7-megastigmadiene (1) – Brown amorphous powder; $$ {\left[\alpha \right]}_{\mathrm{D}}^{20}$$−38.6 (c 0.48, CH₂Cl₂); HR-ESI-MS m/z 211.1694 [M+H]⁺ (calcd. for C₁₃H₂₃O₂, 211.1693); ¹H NMR (400 MHz, CD₃OD): δ 1.02 (3H, s, 12-CH₃), 1.05 (3H, s, 11-CH₃), 1.25 (3H, d, J = 6.3 Hz, 10-CH₃), 1.40 (1H, t, J = 12.0 Hz, H-2ax), 1.65 (1H, dd, J = 12.0, 2.1 Hz, H-2eq), 1.69 (3H, s, 13-CH₃), 1.95 (1H, m, H-4ax), 2.28 (1H, dd, J = 5.9, 16.3 Hz, H-4eq), 3.88 (1H, m, H-3), 4.28 (1H, dd, J = 6.5, 6.3 Hz, H-9), 5.44 (1H, dd, J = 6.5, 16.1 Hz, H-8), 5.99 (1H, d, J = 16.1 Hz, H-7); ¹³C NMR (100 MHz, CD₃OD): δ 21.5 (13-CH₃), 23.9 (10-CH₃), 28.7 (11-CH₃), 30.6 (12-CH₃), 37.7 (C-1), 42.8 (C-4), 48.4 (C-2), 65.5 (C-3), 69.7 (C-9), 126.9 (C-5), 127.2 (C-7), 138.0 (C-6), 140.0 (C-8).

Dehydrovomifoliol (2) – Brown syrup; $$ {\left[\alpha \right]}_{\mathrm{D}}^{20}$$−5.2 (c 0.11, CH₂Cl₂); HR-ESI-MS m/z 223.1328 [M+H]⁺ (calcd. for C₁₃H₁₉O₃, 223.1329); ¹H NMR (400 MHz, CDCl₃): δ 1.02 (3H, s, 12-CH₃), 1.11 (3H, s, 11-CH₃), 1.88 (3H, s, 13-CH₃), 2.30 (3H, s, 10-CH₃), 2.34 (1H, dd, J = 0.9, 17.2 Hz, H-2ax), 2.50 (1H, dd, J = 0.6, 17.2 Hz, H-2eq), 5.96 (1H, s, H-4), 6.46 (1H, d, J = 15.7 Hz, H-8), 6.83 (1H, d, J = 15.7 Hz, H-7); ¹³C NMR (100 MHz, CDCl₃): δ 18.8 (13-CH₃), 23.0 (12-CH₃), 24.5 (10-CH₃), 28.6 (11-CH₃), 41.5 (C-1), 49.7 (C-2), 79.4 (C-6), 127.9 (C-4), 130.4 (C-8), 145.0 (C-7), 160.3 (C-5), 197.0 (9-C=O), 197.5 (3-C=O).

(−)-Loliolide (3) – Yellow amorphous powder; $$ {\left[\alpha \right]}_{\mathrm{D}}^{20}$$−13.3 (c 0.001, CH₂Cl₂); HR-ESI-MS m/z 197.1171 [M+H]⁺ (calcd. for C₁₁H₁₇O₃, 197.1172); ¹H NMR (400 MHz, CDCl₃): δ 1.27 (3H, s, 9-CH₃), 1.47 (3H, s, 8-CH₃), 1.53 (1H, dd, J = 3.7, 14.5 Hz, H-7ax), 1.77 (4H, overlap, H-5ax, 10-CH₃), 1.98 (1H, dt, J = 2.6, 14.5 Hz, H-7eq), 2.46 (1H, dt, J = 2.5, 14.5 Hz, H-5eq), 4.33 (1H, m, H-6), 5.69 (1H, s, H-3); ¹³C NMR (100 MHz, CDCl₃): δ 26.5 (9-CH₃), 27.0 (10-CH₃), 30.7 (8-CH₃), 36.0 (C-4), 45.6 (C-7), 47.3 (C-5), 66.8 (C-6), 86.8 (C-7a), 112.9 (C-3), 172.0 (C-2), 182.6 (C-3a).

(7E)-5,6-Epoxy-3-hydroxy-7-megastigmen-9-one (4) – Brown syrup; $$ {\left[\alpha \right]}_{\mathrm{D}}^{20}$$−13.6 (c 0.39, CH₂Cl₂);HR-ESI-MS m/z 225.1484 [M+H]⁺ (calcd. for C₁₃H₂₁O₃, 225.1485); ¹H NMR (400 MHz, CDCl₃): δ 0.97 (3H, s, 12-CH₃), 1.19 (6H, overlap, 11-CH₃ and 13-CH₃), 1.26 (1H, dd, J = 10.4, 12.9 Hz, H-2ax), 1.65 (2H, overlap, H-2eq and H-4ax), 2.28 (3H, s, 10-CH₃), 2.39 (1H, dd, J = 5.1, 14.1 Hz, H-4eq), 3.90 (1H, m, H-3), 6.29 (1H, d, J = 15.6 Hz, H-8), 7.02 (1H, d, J = 15.6 Hz, H-7); ¹³C NMR (100 MHz, CDCl₃): δ 19.9 (13-CH₃), 25.0 (11-CH₃), 28.3 (10-CH₃), 29.4 (12-CH₃), 35.1 (C-1), 40.6 (C-2), 46.7 (C-4), 64.0 (C-3), 67.3 (C-5), 69.5 (C-6), 132.6 (C-8), 142.4 (C-7), 197.4 (9-C=O).

3-Hydroxy-5,7-megastigmadien-9-one (5) − Yellow syrup; $$ {\left[\alpha \right]}_{\mathrm{D}}^{20}$$−4.3 (c 0.5, CH₂Cl₂); HR-ESI-MS m/z 209.1536 [M+H]⁺ (calcd. for C₁₃H₂₁O₂, 209.1536); ¹H NMR (400 MHz, CDCl₃): δ 1.11 (6H, overlap, 11-CH₃ and 12-CH₃), 1.49 (1H, t, J = 12.0 Hz, H-2ax), 1.79 (4H, overlap, H-2eq and 13-CH₃), 2.08 (1H, dd, J = 9.6, 17.4 Hz H-4ax), 2.30 (3H, s, 10-CH₃), 2.43 (1H, dd, J = 5.1, 17.4 Hz, H-4eq), 4.00 (1H, m, H-3), 6.11 (1H, d, J = 16.5 Hz, H-8), 7.20 (1H, d, J = 16.5 Hz, H-7); ¹³C NMR (100 MHz, CDCl₃): δ 21.6 (13-CH₃), 27.3 (10-CH₃), 28.6 (12-CH₃), 30.0 (11-CH₃), 36.9 (C-1), 42.8 (C-4), 48.4 (C-2), 64.6 (C-3), 132.2 (C-7), 132.4 (C-5), 135.6 (C-6), 142.3 (C-8), 198.5 (9-C=O).

3-Hydroxy-4,7-megastigmadien-9-one (6) − Yellow syrup; $$ {\left[\alpha \right]}_{\mathrm{D}}^{20}$$+1.0 (c 0.21, CH₂Cl₂); HR-ESI-MS m/z 209.1536 [M+H]⁺ (calcd. for C₁₃H₂₁O₂, 209.1536); ¹H NMR (400 MHz, CDCl₃): δ 0.89 (3H, s, 12-CH₃), 1.03 (3H, s, 11-CH₃), 1.41 (1H, dd, J = 6.3, 13.5 Hz, H-2ax), 1.62 (3H, s, 13-CH₃), 1.84 (1H, dd, J = 6.1, 13.5 Hz, H-2eq), 2.26 (3H, s, 10-CH₃), 2.50 (1H, d, J = 10.0 Hz, H-6), 4.27 (1H, m, H-3), 5.63 (1H, br s, H-4), 6.10 (1H, d, J = 15.8 Hz, H-8), 6.54 (1H, dd, J = 10.0, 15.8 Hz, H-7); ¹³C NMR (100 MHz, CDCl₃) δ: 22.7 (13-CH₃), 24.7 (12-CH₃), 27.2 (10-CH₃), 29.7 (11-CH₃), 33.8 (C-1), 43.8 (C-2), 54.3 (C-6), 65.5 (C-3), 125.8 (C-4), 133.6 (C-8), 135.5 (C-5), 147.1 (C-7), 198.0 (9-C=O).

9-Hydroxy-4,7-megastigmadien-3-one (7) – Yellow syrup; $$ {\left[\alpha \right]}_{\mathrm{D}}^{20}$$−6.1 (c 0.42, CH₂Cl₂); HR-ESI-MS m/z 209.1537 [M+H]⁺ (calcd. for C₁₃H₂₁O₂, 209.1536); ¹H NMR (400 M Hz, CDCl₃): δ 0.96 (3H, s, 12-CH₃), 1.03 (3H, s, 11-CH₃), 1.30 (3H, d, J = 6.5 Hz, 10-CH₃), 1.91 (3H, d, s, 13-CH₃), 2.09 (1H, br d, J = 16.5 Hz, H-2ax), 2.33 (1H, br d, J = 16.5 Hz, H-2eq), 2.50 (1H, d, J = 9.2 Hz, H-6), 4.35 (1H, dd, J = 6.0, 6.5 Hz, H-9), 5.53 (1H, dd, J = 9.2, 15.3 Hz, H-7), 5.68 (1H, dd, J = 6.0, 15.3 Hz, H-8), 5.90 (1H, s, H-4); ¹³C NMR (100 MHz, CDCl₃): δ 23.5 (13-CH₃), 23.7 (10-CH₃), 27.2 (11-CH₃), 27.9 (12-CH₃), 36.2 (C-1), 47.5 (C-2), 55.5 (C-6), 68.3 (C-9), 125.9 (C-4), 126.7 (C-7), 138.6 (C-8), 161.8 (C-5), 199.1 (3-C=O).

Assessment of LPS-induced NO production and cell viability – RAW 264.7 cells (ATCC, Manassas, VA, USA) were cultured in DMEM (Sigma-Aldrich, St Louis, MO, USA) supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin. The cells were maintained in a humidified atmosphere with 5% CO₂ at 37°C. As previously described,¹³ the cells were seeded into the 96-well microplates (2 × 10⁵ cells/ well) and incubated for 24 h. Subsequently, the cells were treated with the tested compounds and extracts, initially dissolved in DMSO, and further diluted with the DMEM medium to obtain a range of working concentrations. The cells were then stimulated with LPS (1 μg/mL) to trigger NO production. After 24 h of incubation at 37°C, 100 μL of cell-free supernatant was mixed with 100 μL of Griess reagent (Sigma-Aldrich, St Louis, MO, USA) for 10 min, for nitrite production assessment. The absorbance was measured at 550 nm against a calibration curve established with sodium nitrite standards. The viability of the remaining cells was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay (Sigma Chemical Co., St. Louis, MO).

Results and Discussion

Phytochemical investigation of the aerial parts of C. cristata resulted in the isolation of seven known compounds (1–7), which were identified as 3, 9-dihydroxy-5, 7-megastigmadiene (1), ¹⁴ dehydrovomifoliol (2),¹⁵ (-)-loliolide (3),¹⁶ (7E)-5,6-epoxy-3-hydroxy-7-megastigmen-9-one (4),¹⁴ 3-hydroxy-5,7-megastigmadien-9-one (5),¹⁴ 3-hydroxy-4,7-megastigmadien-9-one (6),¹⁴ and 9-hydroxy-4,7-megastigmadien-3-one (7),¹⁴ respectively, by comparing their physicochemical and spectroscopic data with those of published values.

Inducible NO synthase (iNOS) in macrophages generates NO. The overexpression of iNOS increases NO levels, and excessive NO production may cause various inflammatory disease such as rheumatoid arthritis and inflammatory bowel disease.¹⁷ Therefore, inhibition of NO production is known as one of important strategies in controlling inflammatory disease under various pathological conditions. Although research on the antitumor, antioxidant, and antinociceptive activities of C. cristata has been reported, studies on its anti-inflammatory effects have not been documented.^18-20 In previous study, several C₁₃-norisoprenoids such as 3-oxo-α-damascone (IC₅₀: 2.0 ± 0.9 μM), 4-oxo-β-damascone (IC₅₀ : 1.8 ± 0.9 μM), and β-damascone (IC₅₀: 3.0 ± 0.7 μM) isolated from the apples (Malus sp., Rosaceae), have been reported to inhibit NO production.²¹ Therefore, all isolated norisoprenoids (1–7) were assessed for their anti-inflammatory activity on LPS-induced NO production in RAW 264.7 macrophage cells, with aminoguanidine utilized as a positive control (IC₅₀: 17.6 μM). Among them, only dehydrovomifoliol (2), 3-hydroxy-4,7-megastigmadien-9-one (6), and 9-hydroxy-4,7-megastigmadien-3-one (7), which have a double bond between C-4 and C-5, showed significant activity effects with IC₅₀ values of 17.7, 17.8, and 24.4 μM (Table 1), respectively. Furthermore, the presence of a carbonyl group at C-9 in 2 and 6 instead of a hydroxy group in 7 might enhance the inhibitory effects. This study has confirmed for the first time that C. cristata contains norisoprenoids. Additionally, norisoprenoids isolated from C. cristata were found to exhibit anti-inflammatory activity. These results suggest that norisoprenoids contribute to the anti-inflammatory effects of C. cristata. Therefore, it is suggested that C. cristata might have potential for further research for the anti-inflammatory agents.

Table 1.

Inhibitory effects of compounds 1–7 on LPS-induced NO production in RAW 264.7 Cells a

Acknowledgments

This work was supported by a funding for the academic research program of Chungbuk National University in 2024.

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