Bioactive substances inhibiting COX-2 and cancer cells isolated from the fibrous roots of Alangium chinense (Lour.) Harms

Abstract

Alangium chinense has been used as a traditional folk medicine for centuries to treat rheumatism, skin diseases, and diabetes by the people of Southeast Asia. However, the bioactive constituents inhibiting COX-2 and cancer cells (HepG2, Caco-2, HeLa) remain unclear. In this study one new (14) along with twenty-four known compounds (1–13, 15–25) were isolated from the fibrous roots of Alangium chinense by chromatographic methods, and identified by NMR, and Gaussian and CD calculation. Compounds 1, 2, 13, 16, 17, 19, 20, 23, and 24 were isolated from this plant for the first time. Their inhibition effects on COX-2 enzyme and cancer cells were evaluated by MTT assay. Compounds 1–4, 13–14, and 16–18 can be used as good inhibitors against COX-2 enzyme, and compounds 1, 13, 14, and 17 were stronger than the positive control (celecoxib). In addition, molecular docking suggested that compounds 13, 17, and 18 belong to ellagic acids and have good inhibition against COX-2 enzyme. While compounds 1, 5, 13 and 21 showed cytotoxicity against HepG2 cells, compounds 2 and 21 showed cytotoxicity against Caco-2 cells, and compound 20 showed cytotoxicity against HeLa cells.

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1 Introduction

Alangium chinense (Lour.) Harms belongs to the Alangium Lam. in the Alangiaceae, and it is widely distributed throughout the tropical and subtropical regions of the Eastern Hemisphere.¹ A. chinense is rich in alkaloids, sugars, saponins, steroids, triterpenes, anthraquinones, and glycosides,^2–4 and the roots, flowers, and leaves of A. chinense have served as ethnomedicinal treatments for rheumatic arthritis, acroanesthesia, fractures, snake bites, circulatory issues, hemostasis, toxicity, contraception, and wound healing in China.^1,5,6 In addition, the fibrous roots of A. chinense named “Bai Long Xu” or “Ba Jiao Feng” in Traditional Chinese Medicine (TCM), have historically been applied as a treatment for rheumatoid arthritis and traumatic injury by the Miao people of Guizhou province, China.⁷ It is also the principal component of Fengshiding Capsules, which is a traditional Chinese medicine for the treatment of rheumatoid arthritis.^1,8,9

Cyclooxygenase-2 (COX-2) is a very important physiological enzyme playing key roles in various biological functions, especially in the mechanism of pain and inflammation. This makes it a molecule of great interest to the pharmaceutical community as a target.¹⁰ COX 2 enzyme is induced only during inflammatory processes or cancer, so selective COX-2 inhibition can significantly reduce the adverse effects, including GI tract damage and hepatotoxic effects, of traditional NSAIDs.^10,11

Cancer has become a major threat to human health, with increasing mortality around the world.¹² Liver cancer is the fourth leading cause of cancer-related mortality worldwide, despite the liver being the sixth most common site of primary cancer,¹³ and hepatocellular carcinoma (HCC) accounts for 80–90% of primary liver cancers.¹⁴ Colorectal cancer is the third most commonly diagnosed cancer and the fourth most common cause of cancer death worldwide.¹⁵ According to the latest online epidemiological database, there were more than 1.9 million new colon cancer cases in 2020, and 0.9 million deaths were recorded in the same year.¹⁶ The incidence rate and mortality rate have continuously risen in recent years. Even with the rapid development of cancer screening methods, many patients are diagnosed at an advanced stage with multiple symptoms.¹⁶ However, there are only a few effective therapeutic targets for colon cancer patients.¹⁷ Moreover, in the structure of morbidity and mortality, cervical cancer (CC) is one of the most common types of carcinomas among women in all countries of the world, in fourth place after breast cancer, colorectal cancer, and lung cancer.¹³ According to data from the International Agency for Research on Cancer, in 2018, the number of patients with cervical cancer was 570 000, an increase of 7.8% over the past decade, and the number of deaths from this disease amounted to 311 000.^13,14 In 2020, 604 100 new cases of cervical cancer were detected around the world, and the number of deaths was 341 800.^18–21

Therefore, despite the recommended screening programs for cancers, morbidity and mortality rates of cancers have been steadily growing in recent years worldwide. Targeted anticancer drugs, which are useful for the diagnosis of various stages of the tumor process, are very important. Due to its importance in Chinese Traditional Medicine and the necessity to identify its active constituents, we have carried out a phytochemical investigation of the fibrous roots extract of A. chinense. In the study, one new (14) and twenty-four known compounds (1–13, 15–25) were isolated and characterized from the ethyl acetate fraction. Among them, compounds 1, 2, 13, 16, 17, 19, 20, 23, and 24 were isolated from this plant for the first time. Furthermore, inhibition of COX-2 enzyme and cytotoxic activity against three human tumor cell lines (HepG2, Caco-2, and HeLa) have been evaluated with the crude extract and isolated compounds.

2 Results and discussion

2.1 Phytochemical extraction and purification

This study aimed to improve the chemical composition of A. chinense. The spectral data of compounds are basically consistent with the literature. Twenty-five compounds were isolated from ACEE using modern phytochemical techniques and were identified as cis-10-eicosaenoic acid (1), (2S)-5-hydroxy-7-methoxy-dihydroflavone (2), stigmast-4-en-3,6-dione (3), β-daucosterol (4), xanthoxylin (5), lacinilene C (6), (1S)-1-methoxylacinilene C (7), machilin-1 (8), perseal F (9), (−)-kobusin (10), (+)-fargesin (11), tephrosin (12), 3,3′,4′-tetra-O-methylellagic acid (13), (1R,6S)-6-aminocyclohex-2-ene-1-carboxylic acid (14), anabasine (15), (2S)-N-hydroxybenzylanabasine (16), 3,3′,4,4′-tetra-O-methylellagic acid (17), 3,4′-di-O-methylellagic acid (18), 3,4-methylenedioxy-3′-O-methyl-4′-O-glucoside ellagic acid (19), aralia cerebroside (20), uraci (21), chloropyrazine (22), (S)-3,4,5-trihydroxy-1H-pyrrol-2 (5H)-one (23), uvacalol k (24), and salicin (25) (Fig. 1). Among them, compound 14 was a new compound, and compound 1, 2, 13, 16, 17, 19, 20, 23, and 24 were isolated from this plant for the first time.

Fig. 1 Chemical structures of compounds isolated from ACEE.

Compound 1 (cis-10-eicosaenoic acid) was isolated as a white powder in dichloromethane. Its molecular formula was determined as C₂₀H₃₈O₂ with an unsaturation of two. The ¹³C-NMR data (150 MHz, CDCl₃) showed 20 carbon signals, including one carbonyl carbon, one double bond signal, one methyl signal, and sixteen methylene carbons. The NMR data of 1 (Tables 1 and 2) indicated an enoic acid skeleton consistent with ¹H-NMR.²² The ¹H-NMR data (600 MHz, CDCl₃) showed one double bond proton confirmed at [δ_H 5.36 (2H, s, H-10,11)], methylene protons at [δ_H 2.35 (2H, t, J = 7.4, H-2), 2.03 (4H, m, H-9,12), 1.64 (2H, m, H-3), 1.25 (24H, m, H-4–8, 13–19)], and methyl protons at [δ_H 0.90 (3H, t, J = 6.5, H-20)].

Table 1 ¹³C NMR chemical shifts (δ) of compounds 1, 3, 6, and 7 in CDCl₃ (150 MHz)

Position	1	3	6	7
1	180.7	35.3	77.2	81.3
2	34.1	33.8	205.5	200.7
3	24.7	199.5	114.7	116.8
4	29.1	125.4	164.7	162.8
5	29.1	161.1	128.4	128.3
6	29.5	202.3	123.4	123.6
7	29.8	46.8	156.4	157.8
8	29.2	39.1	112.3	112.4
9	29.3	51.0	145.2	143.3
10	129.7	34.2	121.1	121.4
11	129.8	20.9	29.2	28.3
12	27.2	39.8	22.3	22.2
13	29.8	42.5	22.0	21.9
14	29.7	55.9	33.7	31.3
15	29.5	24.0	15.9	15.8
16	29.7	28.0
17	29.7	56.5
18	32.0	11.9
19	22.7	17.5
20	14.0	36.0
21		19.0
22		34.0
23		26.0
24		45.8
25		29.1
26		19.8
27		18.7
28		23.1
29		12.0
OCH3				52.8

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Table 2 ¹H NMR chemical shifts (δ) of compounds 1, 3, 6, and 7 in CDCl₃ (600 MHz)

Position	1	3	6	7
1
2	2.35 t (7.4)
3	1.64 m, 1.64 m		6.02 s	5.84 s
4	1.25 m	6.14 s
5			7.41 s	7.47 s
6
7
8			7.22 s	7.00 s
9	2.03 m, 2.03 m
10	5.36 s
11	5.36 s		3.21 m	3.24 m
12	2.03 m, 2.03 m		1.27 d (7.0)	1.17 d (7.0)
13	1.25 m		1.28 d (7.0)	1.19 d (7.0)
14			1.51 s	1.28 s
15			2.27 s	2.16 s
16				2.83 s
17
18		0.91 d (6.5)
19		1.14 s
20	0.90 t (6.5)
21		0.82 d (7.0)
22
23
24
25
26		0.80 d (6.6)
27		0.70 s
28
29		0.81 d (6.5)

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Compound 2 ((2S)-5-hydroxy-7-methoxy-dihydroflavone) was isolated as a yellow powder in methanol. Its molecular formula was determined as C₁₆H₁₄O₄ with an unsaturation of ten. The ¹³C-NMR data (150 MHz, CD₃OD) showed 15 carbon signals, including twelve aromatic carbons, one carbonyl carbon, one methylene signal, and one methyne carbon. The NMR data of 2 (Table 3 and Table 4) indicated a flavonoid skeleton consistent with ¹H-NMR.²³ The ¹H-NMR data (600 MHz, CD₃OD) showed seven aromatic protons confirmed at [δ_H 7.50 (2H, d, J = 7.7, H-2′, 6′), 7.42 (2H, t, J = 7.7, H-3′, 5′), 7.37 (1H, t, J = 7.7, H-4′), 6.09 (1H, d, J = 2.3, H-6), 6.06 (1H, d, J = 2.3, H-8)], methyne proton [δ_H 5.49(1H, dd, J = 12.8, 3.0, H-2)], and one methylene proton [δ_H 3.13(1H, dd, J = 17.2, 12.8, H-3α), 2.80 (1H, dd, J = 17.2, 3.13, H-3β)].

Table 3 ¹³C NMR chemical shifts (δ) of compounds 2, 5, 15, 16, 22, 23, and 24 in CD₃OD (150 MHz)

Position	2	5	15	16	22	23	24
1		106.9					129.3
2	80.6	164.7	58.2	66.7	149.4	165.9	77.7
3	43.3	94.6	29.6	25.3	144.8	142.1	74.4
4	197.8	168.3	21.7	24.3		152.1	71.8
5	165.3	91.6	22.5	35.5	142.8	100.3	68.7
6	95.9	167.9	45.7	53.0	144.7		128.1
7	169.6						63.6
8	95.0
9	164.5
10	104.1
1′							166.3
2′			149.6	148.5			128.1
3′			136.0	139.5			128.3
4′			133.6	136.1			129.3
5′			124.5	124.2			128.3
6′			148.2	148.0			128.1
1′′				156.7			166.4
2′′				121.8			129.4
3′′				128.0			132.9
4′′				119.0			133.0
5′′				128.6			132.9
6′′				115.1			129.4
7′′				57.4
C=O		204.6
4-OCH3		56.1
6-OCH3		56.2
8-CH3		33.1

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Table 4 ¹H NMR chemical shifts (δ) of compounds 2, 5, 15, 16, 22, 23, and 24 in CD₃OD (600 MHz)

Position	2	5	15	16	22	23	24
1			2.08–1.75 m			7.29 d (7.6)	3.81 t (9.4, 9.5)
2	5.49 dd (12.8, 3.0)		4.46 m	3.35 dd (2.9, 11.2)			4.54 m
3	3.13 dd (17.2, 12.8), 2.80 dd (17.2, 3.13)	6.04 d (2.3)	2.08–1.75 m	1.77 m			5.37 t (9.3, 9.1)
4			3.55 m, 3.27 m	1.53 m, 1.87 m			4.02 t (8.4, 7.9)
5		6.05 d (2.3)	8.71 s	1.83 m, 1.87 m	8.39 s	5.51 d (7.6)
6	6.09 d (2.3)			3.11 d (11.7), 2.17 m	8.34 s		6.60 d (8.3)
7							5.16 d (7.5), 4.75 d (11.4)
8	6.06 d (2.3)
2′	7.50 d (7.7)		8.04 d (8.0)	8.58 s			7.50–8.10 m
3′	7.42 t (7.7)		7.53 dd (7.9, 4.8)
4′	7.37 t (7.7)		8.58 d (3.7)	7.92 d (7.9)
5′	7.42 t (7.7)		2.08–1.75 m	7.43 dd (4.9, 7.8)
6′	7.50 d (7.7)		4.46 m	8.44 d (4.3)
1′′
2′′							7.50–8.10 m
3′′				6.89 d (7.5)
4′′				6.69 t (6.2)
5′′				7.05 t (7.9)
6′′				6.67 t (7.9)
7′′				3.79 d (14.0), 3.13 d (14.0)
8-CH3
4-OCH3		3.88 s
6-OCH3		3.82 s

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Compound 3 (stigmast-4-en-3,6-dione) was isolated as a white crystal in dichloromethane. Its molecular formula was determined as C₂₉H₄₆O₂ with an unsaturation of seven. The ¹³C-NMR data (150 MHz, CDCl₃) showed 29 carbon signals, including two carbonyl carbons, one double bond signal, six methyl carbons, three quaternary carbons, ten methylene carbons, and six methyne carbons. The NMR data of 3 (Tables 1 and 2) indicated a steroid skeleton consistent with ¹H-NMR.²⁴ The ¹H-NMR data (600 MHz, CDCl₃) showed one double bond proton confirmed at [δ_H 6.14 (1H, s, H-4)], and six methyl protons [δ_H 1.14 (3H, s, H-19), 0.91 (3H, d, J = 6.5, H-18), 0.82 (3H, d, J = 7.0, H-21), 0.81 (3H, d, J = 6.5, H-29), 0.80 (3H, d, J = 6.6, H-26), 0.70 (3H, s, H-27)].

Compound 4 (β-daucosterol) was isolated as a white powder in pyridine. Its molecular formula was determined as C₃₁H₅₂O₃ with an unsaturation of six. The ¹³C-NMR data (150 MHz, C₅D₅N) showed 35 carbon signals, including one double bond signal, one glucose carbon, six methyl carbons, and eleven methylene carbons. The NMR data of 4 (Table 5) indicated a steroid skeleton consistent with ¹H-NMR.²⁵ The ¹H-NMR data (600 MHz, C₅D₅N) showed one double bond proton confirmed at [δ_H 5.33 (1H, s, H-5)], one glucose hydrogen proton [δ_H 5.07 (d, J = 7.8, H-1′)], one methyne proton [δ_H 4.02–3.93 (1H, m, H-3)], and six methyl protons [δ_H 0.94 (3H, s, H-19), 0.91 (3H, s, H-18), 0.89 (3H, s, H-21), 0.87 (3H, s, H-29), 0.67 (3H, s, H-26), 1.00 (3H, s, H-27)].

Table 5 ¹³C NMR and ¹H NMR chemical shifts (δ) of compound 4 in C₅P₅N

Position	δC (150 MHz)	δH (J in Hz) (600 MHz)
1	37.9
2	30.7
3	78.5	4.02–3.93 m
4	40.4
5	141.3	5.33 s
6	122.3
7	32.6
8	32.5
9	50.8
10	37.3
11	21.7
12	39.7
13	42.9
14	57.2
15	24.9
16	28.9
17	56.7
18-CH3	12.4	0.91 s
19-CH3	19.8	0.94 s
20	36.8
21-CH3	19.6	0.89 s
22	34.6
23	26.8
24	46.5
25	29.9
26-CH3	19.4	0.67 s
27-CH3	20.4	1.00 s
28	23.8
29-CH3	12.6	0.87 s
1′	103.0	5.07 d (7.8)
2′	75.7
3′	78.9
4′	72.1
5′	79.0
6′	63.2

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Compound 5 (xanthoxylin) was isolated as a white crystal in methanol. Its molecular formula was determined as C₁₀H₁₂O₄ with an unsaturation of five. The ¹³C-NMR data (150 MHz, CD₃OD) showed 10 carbon signals, including one carbonyl carbon, two methoxy carbons, one methyl carbon, and six aromatic carbons. The NMR data of 5 (Tables 3 and 4) indicated an acetophenone skeleton consistent with ¹H-NMR.²⁶ The ¹H-NMR data (600 MHz, CD₃OD) showed two aromatic hydrogen protons confirmed at [δ_H 6.04 (1H, d, J = 2.3, H-3), 6.05 (1H, d, J = 2.3, H-5)], two methoxy protons [δ_H 3.82 (3H, s, H-6), 3.88 (3H, s, H-4)], and one methyl proton [δ_H 2.57 (3H, s, H-8)].

Compound 6 (lacinilene C) was isolated as a yellow powder in dichloromethane. Its molecular formula was determined as C₁₅H₁₈O₃ with an unsaturation of six. The ¹³C-NMR data (150 MHz, CDCl₃) showed 15 carbon signals, including six aromatic carbons, one double bond, one carbonyl carbon, four methyl carbons, one methylene carbon, and one quaternary carbon. The NMR data of 6 (Tables 1 and 2) indicated a sesquiterpene skeleton consistent with ¹H-NMR.²⁷ The ¹H-NMR data (600 MHz, CDCl₃) showed two aromatic hydrogen protons confirmed at [δ_H 7.41 (1H, s, H-5), 7.22 (1H, s, H-8)], one double bond proton [δ_H 6.02 (1H, s, H-3)], one methylene proton [δ_H 3.21 (1H, m, H-11)], and four methyl proton [δ_H 1.27 (1H, d, J = 7.0, H-12), 1.28 (1H, d, J = 7.0, H-13), 1.51 (1H, s, H-14), 2.27(1H, s, H-15)].

Compound 7 ((1S)-1-methoxylacinilene C) was isolated as a yellow powder in dichloromethane. Its molecular formula was determined as C₁₆H₂₀O₃ with an unsaturation of seven. The ¹³C-NMR data (150 MHz, CDCl₃) showed 16 carbon signals, including six aromatic carbons, one double bond, one carbonyl carbon, four methyl carbons, one methylene carbon, one methoxy carbon, and one quaternary carbon. The NMR data of 7 (Tables 1 and 2) indicated a sesquiterpene skeleton consistent with ¹H-NMR.²⁷ The ¹H-NMR data (600 MHz, CDCl₃) showed two aromatic hydrogen protons confirmed at [δ_H 7.47 (1H, s, H-5), 7.00 (1H, s, H-8)], one double bond proton [δ_H 5.84 (1H, s, H-3)], one methylene proton [δ_H 3.24 (1H, m, H-11)], four methyl proton [δ_H 1.17 (1H, d, J = 7.0, H-12), 1.19 (1H, d, J = 7.0, H-13), 1.28 (1H, s, H-14), 2.16 (1H, s, H-15)], and one methoxy proton [δ_H 2.83 (3H, s, 16-OCH₃)].

Compound 8 (machilin-1) was isolated as a colourless oil in dichloromethane. Its molecular formula was determined as C₂₀H₂₄O₅ with an unsaturation of eight. The ¹³C-NMR data (150 MHz, CDCl₃) showed 20 carbon signals, including twelve aromatic carbons, two methyl carbons, two methoxy carbons, and four methylene carbons. The NMR data of 8 (Tables 6 and 7) indicated a lignin skeleton consistent with ¹H-NMR.²⁸ The ¹H-NMR data (600 MHz, CDCl₃) showed six aromatic hydrogen protons confirmed at [δ_H 6.94 (2H, dd, J = 9.1, H-5′′, 6′′), 6.78–6.95 (1H, m, H-2′′), 6.89 (2H, d, J = 8.0, H-2′, 3′), 6.78–6.95 (1H, m, H-6′)], two methoxy protons [δ_H 3.91 (3H, s, 5′-OCH₃), 3.89 (3H, s, 3′′-OCH₃)], four methylene protons [δ_H 5.45 (1H, d, J = 4.2, H-2), 4.64 (1H, d, J = 9.2, H-5), 2.43 (2H, dd, J = 6.3, 3.7, H-3, 4)], and two methyl protons [δ_H 1.00 (3H, d, J = 6.4, 4-CH₃), 0.61 (3H, d, J = 6.9, 3-CH₃)].

Table 6 ¹³C NMR chemical shifts (δ) of compounds 8, 9, 10, 11 and 12 in CDCl₃ (150 MHz)

Position	8	9	10	11	12
1		131.5	54.2	50.2	115.4
1a					111.1
2	84.8	114.3	86.0	82.0	151.1
3	47.6	145.0			144.0
4	43.5	146.2	71.9	71.0	101.1
4a					148.4
5	85.8	108.9	54.4	54.6
6		120.0	85.9	87.7	63.9
6a					76.3
7		95.0
7a					156.7
8		44.9	71.8	69.7	108.7
9		17.8			160.7
10					111.8
11					128.6
11a					109.1
12					191.4
12a					67.5
1′	132.6	131.1	133.0	135.1
2′	108.8	111.8	109.3	106.6
3′	146.7	146.8	149.3	147.9
4′	144.3	153.2	148.8	147.2	109.4
5′	114.0	133.7	111.1	108.2	128.8
6′	118.8	120.0	118.4	119.6	78.0
7′					28.5
8′					28.3
1′′	135.0		135.2	130.9
2′′	108.3		106.6	108.9
3′′	146.3		148.1	148.8
4′′	145.1		147.2	147.9
5′′	113.9		108.3	111.0
6′′	119.3		119.5	117.7
3-CH3	9.4
4-CH3	11.9
CHO		190.6
O–CH2–O			101.2	101.0
OCH3	56.0	56.1	56.0	55.9	56.4
OCH3	55.9	56.0	56.1	55.9	55.9

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Table 7 ¹H NMR chemical shifts (δ) of compounds 8, 9, 10, 11 and 12 in CDCl₃ (600 MHz)

Position	8	9	10	11	12
1			3.09 m	3.27–3.36 m	6.56 s
2	5.45 d (4.2)	6.91 s	4.74 dd (10.5, 5.0)	4.86 d (5.2)
3	2.43 dd (6.3, 3.7)
4	2.43 dd (6.3, 3.7)		3.89 m, 4.25 dd (9.0, 4.0)	4.11 d (9.4), 3.83 t (7.7)	6.51–6.45 m
4-CH3	1.00 d (6.4)
5	4.64 d (9.2)	6.94 s	3.09 m	2.87 d (7.4)
6		6.91 s	4.74 dd (10.5, 5.0)	4.42 d (7.0)	4.63 dd (12.1, 2.4), 4.53–4.46 m
6a					4.57 d (1.5)
7		5.25 d (9.0)
7a
8		3.56 m	3.89 m, 4.25 dd (9.0, 4.0)	3.83 t (7.7), 3.27–3.36 m
9		1.45
9-CH3		1.45 d (6.8)
10					6.51–6.45 m
11					7.73 d (8.7)
2′	6.89 d (8.0)	7.38 s	6.78–6.89 m	6.93 s
3′	6.89 d (8.0)
4′					6.60 d (10.1)
5′	3.91 s		6.78–6.89 m	6.75–6.90 m	5.56 d (10.1)
6′	6.78–6.95 m	7.35 s	6.78–6.89 m	6.75–6.90 m
7′-CH3					1.45 s
8′-CH3					1.39 s
2′′	6.78–6.95 m		6.78–6.89 m	6.75–6.90 m
3′′	3.89 s
4′′
5′′	6.94 dd (9.1)		6.78–6.89 m	6.75–6.90 m
6′′	6.94 dd (9.1)		6.78–6.89 m	6.75–6.90 m
3-CH3	0.61 d (6.9)
CHO		9.85 s
O–CH2–O			5.95 s	5.94 s
OCH3		3.95 s	3.89 s	3.91 s	3.82 s
OCH3		3.89 s	3.88 s	3.87 s	3.73 s

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Compound 9 (perseal F) was isolated as a white powder in dichloromethane. Its molecular formula was determined as C₁₈H₁₈O₅ with an unsaturation of nine. The ¹³C-NMR data (150 MHz, CDCl₃) showed 18 carbon signals, including twelve aromatic carbons, one methyl carbon, two methoxy carbons, one aldehyde group carbon, and two methylene carbons. The NMR data of 9 (Tables 6 and 7) indicated a lignin skeleton consistent with ¹H-NMR.²⁹ The ¹H-NMR data (600 MHz, CDCl₃) showed one aldehyde group proton confirmed at [δ_H 9.85 (1H, s, 2′-CHO)], five aromatic hydrogen protons [δ_H 7.38 (1H, s, H-2′), 7.35 (1H, s, H-6′), 6.94 (1H, s, H-5), 6.91 (2H, s, H-2, 6)], two methylene protons [δ_H 5.25 (1H, d, J = 9.0, H-7), 3.56 (1H, m, H-8)], two methoxy protons [δ_H 3.95 (3H, s, 3′-OCH₃), 3.89 (3H, s, 4-OCH₃)], and one methyl proton [δ_H 1.45 (3H, d, J = 6.8, 9-CH₃)].

Compound 10 ((−)-kobusin) was isolated as a colorless colloid in dichloromethane. Its molecular formula was determined as C₂₁H₂₂O₆ with an unsaturation of ten. The ¹³C-NMR data (150 MHz, CDCl₃) showed 21 carbon signals, including twelve aromatic carbons, three methylene carbons, two methoxy carbons, and four methyne carbons. The NMR data of 10 (Tables 6 and 7) indicated a furofuran lignan skeleton consistent with ¹H-NMR.³⁰ The ¹H-NMR data (600 MHz, CDCl₃) showed six aromatic hydrogen protons confirmed at [δ_H 6.78–6.89 (6H, m, H-2′, 5′, 6′, 2′′, 5′′, 6′′)], two methoxy protons [δ_H 3.89 (3H, s, 3′-OCH₃), 3.88 (3H, s, 4′-OCH₃)], three methylene protons [δ_H 5.95 (2H, s, O–CH₂–O), 3.89 (1H, m, H-4α), 4.25 (1H, dd, J = 9.0, 4.0, H-4β), 3.89 (1H, m, H-8α), 4.25 (1H, dd, J = 9.0, 4.0, H-8β)], and four methyne protons [δ_H 4.74 (2H, dd, J = 10.5, 5.0, H-2, 6), 3.09 (2H, m, H-1, 5)].

Compound 11 ((+)-fargesin) was isolated as colorless colloidal in dichloromethane. Its molecular formula was determined as C₂₁H₂₂O₆ with an unsaturation of ten. The ¹³C-NMR data (150 MHz, CDCl₃) showed 21 carbon signals, including twelve aromatic carbons, three methylene carbons, two methoxy carbons, and four methyne carbons. The NMR data of 11 (Tables 6 and 7) indicated a furofuran lignan skeleton and consistent with ¹H-NMR.³¹ The ¹H-NMR data (600 MHz, CDCl₃) showed six aromatic hydrogen protons confirmed at [δ_H 6.93 (1H, s, H-2′), 6.75–6.90 (5H, m, H-5′, 6′, 2′′, 5′′, 6′′)], two methoxy protons [δ_H 3.91 (3H, s, 3′-OCH₃), 3.87 (3H, s, 4′-OCH₃)], three methylene protons [δ_H 5.94 (2H, s, O–CH₂–O), 4.11 (1H, d, J = 9.4, H-4α), 3.83 (1H, t, J = 7.7, H-4β), 3.83 (1H, t, J = 7.7, H-8α), 3.27–3.36 (1H, m, H-8β)], and four methyne protons [δ_H 4.86 (1H, d, J = 5.2, H-2), 4.42 (1H, d, J = 7.0, H-6), 3.27–3.36 (1H, m, H-1), 2.87 (1H, d, J = 7.4, H-5)].

Compound 12 (tephrosin) was isolated as a white powder in dichloromethane. Its molecular formula was determined as C₂₃H₂₂O₇ with an unsaturation of twelve. The ¹³C-NMR data (150 MHz, CDCl₃) showed 23 carbon signals, including twelve aromatic carbons, two methoxy carbons, one carbonyl carbon, two methyl carbons, one double bond carbon, one methylene carbon, one methyne carbon, and two quaternary carbons. The NMR data of 12 (Tables 6 and 7) indicated an isoflavone skeleton consistent with ¹H-NMR.³² The ¹H-NMR data (600 MHz, CDCl₃) showed four aromatic hydrogen protons confirmed at [δ_H 7.73 (1H, d, J = 8.7, H-11), 6.56 (1H, s, H-1), 6.51–6.45 (2H, m, H-4, 10)], one double bond proton [δ_H 6.60 (1H, d, J = 10.1, H-4′), 5.56 (1H, d, J = 10.1, H-5′)], one methylene proton [δ_H 4.63 (1H, dd, J = 12.1, 2.4, H-6α), 4.53–4.46 (1H, m, H-6β)], one methyne proton [δ_H 4.57 (1H, d, J = 1.5, H-6a)], two methoxy protons [δ_H 3.82 (3H, s, 2-OCH₃), 3.73 (3H, s, 3-OCH₃)], and two methyl protons [δ_H 1.45 (3H, s, 7′-CH₃), 1.39 (3H, s, 8′-CH₃)].

Compound 13 (3,3′,4,4′-tetra-O-methylellagic acid) was isolated as a white needle crystal in methanol. Its molecular formula was determined as C₁₈H₁₄O₈ with an unsaturation of twelve. The ¹³C-NMR data (150 MHz, DMSO-d₆) showed 14 carbon signals, including ten quaternary carbons, two methines, two ester carbonyls, and four methoxy carbons. The NMR data of 13 (Tables 8 and 9) indicated an ellagic acid skeleton consistent with ¹H-NMR.³³ The ¹H-NMR data (600 MHz, DMSO-d₆) showed two characteristic aromatic protons confirmed at [δ_H 7.50 (2H, s, H-5,5′)], and four methoxy confirmed at [δ_H 4.05 (6H, s, OCH₃), 3.96 (6H, s, OCH₃)].

Table 8 ¹³C NMR chemical shifts (δ) of compounds 13, 17, 18, 20, 21, and 25 in DMSO-d₆ (150 MHz)

Position	13	17	18	20	21	25
1	111.8	111.5	111.9	70.4		132.0
2	142.2	141.4	142.0		151.2	155.2
3	140.7	140.7	141.1	73.9		115.3
4	152.9	153.2	152.9	71.0	164.8	127.7
5	112.0	112.2	111.8		142.7	122.2
6	112.5	112.9	113.2		100.7	128.2
7	159.2	158.7	159.1	32.57		58.7
8				130.7
9				130.3
10				34.8, 32.8, 32.6, 31.8
1′	111.9	112.3	107.3	174.24		101.9
2′	141.6	141.8	140.5	70.9		73.9
3′	140.5	141.2	135.9			77.5
4′	150.7	154.2	150.3			70.2
5′	107.1	107.8	107.1			77.0
6′	113.3	113.8	114.0			61.3
7′	158.9	158.9	159.2
1′′				103.9
2′′				71.5
3′′				77.0
4′′				71.0
5′′				77.3
6′′	61.4	61.4	61.4	61.5
4-OMe	57.0
3′-OMe	61.4	61.8
4′-OMe	57.0	57.2	57.0
CH3			14.23
(CH2)n			29.7, 29.7, 29.6, 29.3, 29.2, 26.0, 24.9, 22.6

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Table 9 ¹H NMR chemical shifts (δ) of compounds 13, 17, 18, 20, 21, and 25 in DMSO-d₆ (600 MHz)

Position	13	17	18	20	21	25
1				4.52 dd (6.83, 10.5)
2				5.34 s
3				4.27 s		7.09 d (7.7)
4				4.15 d (7.5)		7.19 dt (8.2, 7.3)
5	7.48 s	7.50 s	7.49 s		5.45 d (7.6)	7.00 t (6.8, 6.0)
6					7.39 d (7.6)	7.36 d (7.4)
7						4.65 dd (14.2), 4.47 dd (14.2)
8				5.34 s
9				5.60 s
10
1′						5.34 d (4.4)
2′				4.57 m		4.47–5.06 m (H-2′–6′)
3′
4′
5′	7.50 s	7.58 s	7.49 s
6′						3.71 m, 3.47 m
7′
1′′				4.93 d (7.86)
2′′				4.06 m
3′′				4.10 s
4′′				4.32 m
5′′				3.86 s
6′′				4.49 dd (2.4, 11.96), 4.36 dd (5.1, 11.62)
3-OMe	4.05 s	4.04 m	4.05 s
4-OMe	3.96 s
3′-OMe	4.05 s	4.06 m
4′-OMe	3.96 s	3.99 s	3.97 s
CH3				0.84 s
(CH2)n				1.48 s
N–H				7.57 d (9.0)	11.01 s, 10.81 s

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Compound 14 ((1R,6S)-6-aminocyclohex-2-ene-1-carboxylic acid) is a white powder with the molecular formula C₇H₁₁NO₂ and an unsaturation of three based on HR-ESI-MS ([M−H]⁻ m/z 140.0717, calcd 140.0790) and NMR analyses (Table 10). It showed a carbonyl group at δ_C 176.6 in ¹³C NMR. The ¹H NMR signals at δ_H 6.11 (1H, m, H-3), 5.74 (1H, m, H-2), 3.91 (1H, d, J = 4.4, H-6), 2.47 (2H, m, H-4), 2.13 (1H, m, H-5) and 1.70 (1H, m, H-5) along with the ¹³C NMR signals resonating at δ_C 48.9 (C-1), 131.3 (C-2), 138.4 (C-3), 34.1 (C-4), 25.7 (C-5) and 59.91 (C-6) were consistent with a cyclohexene skeleton.³⁴ The cyclohexene carboxylic acid was confirmed by the HMBC cross-peaks of H-2 (δ_H 5.74) to C-1 (δ_C 48.9), C-4 (δ_C 34.1), and C-5 (δ_C 25.7), of H-3 (δ_H 6.11) to C-1 (δ_C 48.9), C-4 (δ_C 34.1) and C-5 (δ_C 25.7), of H-4 (δ_H 2.47) to C-2 (δ_C 131.3) and C-3 (δ_C 138.4), of H-5 (δ_H 2.13, 1.70) to C-1 (δ_C 48.9) and C-6 (δ_C 59.9), and of H-6 (δ_H 3.91) to C-1 (δ_C 48.9), C-2 (δ_C 131.3), and C-7 (δ_C 176.6) (Fig. 2).

Table 10 ¹³C NMR and ¹H NMR chemical shifts (δ) of compound 14 in D₂O

Position	δC	δH (J in Hz)	HMBC	H–H cosy	NOE
1	48.9	3.49 br.s		H-6, H-5, H-4	H-2, H-6
2	131.3	5.74 m	C-1, C-4, C-5	H-1, H-4	H-3
3	138.4	6.11 m	C-1, C-4, C-5	H-1, H-4	H-4
4	34.1	2.47 m	C-2, C-3	H-1	H-5
5	25.7	2.13 m, 1.70 m	C-1, C-6
6	59.9	3.91 d (4.4)	C-1, C-2, C-7	H-1	H-1, H-5
7	176.6

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Fig. 2 HMBC (A) and key NOE (B) correlations observed for (1R,6S)-6-aminocyclohex-2-ene-1-carboxylic acid.

In addition, there were H–H cosy of H-1 (δ_H 5.74) to H-6 (δ_H 3.91), H-4 (δ_H 2.47), and H-5 (δ_H 2.13, 1.70), of H-2 (δ_H 5.74) to H-1 (δ_H 3.49) and H-4 (δ_H 2.47), of H-3 (δ_H 6.11) to H-1 (δ_H 3.49) and H-4 (δ_H 2.47), of H-4 (2.47) to H-1 (δ_H 3.49), and of H-6 (δ_H 3.91) to H-1 (δ_H 3.49).

The NOE cross-peaks of H-1 (δ_H 3.49) to H-2 (δ_H 5.74) and H-6 (δ_H 3.91), of H-6 (δ_H 3.91) to H-1 (δ_H 3.49) and H-5 (δ_H 1.70) revealed the relative configuration of the cyclohexene carboxylic acid (Fig. 2). NOE correlations confirmed H-1 and H-6 were β-oriented.

Compound 15 (anabasine) is a white crystal with the molecular formula C₁₀H₁₄N₂ and an unsaturation of five. The ¹³C-NMR data (150 MHz, CD₃OD) showed 10 carbon signals, including five aromatic carbons, four methylene carbons, and one methyne carbon. The NMR data of 15 (Tables 3 and 4) indicated an alkaloid skeleton consistent with ¹H-NMR.³⁵ The ¹H-NMR data (600 MHz, CD₃OD) showed four characteristic aromatic protons confirmed at [δ_H 8.71 (1H, s, H-2′), 8.58 (1H, d, J = 3.6 Hz, H-6′), 8.04 (1H, d, J = 7.8 Hz, H-4′), and 7.53 (1H, dd, J = 7.8, 7.8 Hz, H-5′)], two methylene protons [δ_H 3.17 (2H, m, H-6), 2.03 (2H, dd, J = 12.6, 6.0 Hz, H-3)], one methyne proton [δ_H 3.41 (1H, m, H-2)], and one omino group proton [δ_H 4.37 (1H, m, H-1)].

Compound 16 ((2S)-N-hydroxybenzylanabasine) was obtained as a white oil in methanol, and its molecular formula was determined as C₁₇H₂₀O with an unsaturation of eight. The ¹³C-NMR showed 17 carbon signals, including eleven aromatic carbons, five methylene carbons, and one methyne carbon. The NMR data of 16 (Tables 3 and 4) indicated an alkaloid skeleton consistent with ¹H-NMR.²⁷ The ¹H-NMR data (600 MHz, CD₃OD) showed eight characteristic aromatic protons confirmed at [δ_H 8.53 (1H, s, H-2′), 7.92 (1H, d, J = 8.0, H-4′), 7.43 (1H, dd, J = 8.0, H-5′), 8.44 (1H, d, J = 5.0, H-6′), 6.89 (1H, d, J = 7.5, H-3′′), 6.67 (1H, t, J = 7.5, H-4′′), 7.05 (1H, t, J = 8.0, H-5′′), and 6.69 (1H, t, J = 8.0, H-6′′)], five methylene protons [δ_H 1.69 (1H, m, H-3), 1.53, 1.77 (2H, m, H-4), 1.83, 1.87 (2H, m, H-5), 3.31, 2.09 (2H, m, H-6), and 3.79, 3.13 (2H, d, J = 14.0, H-7′′)], and one methyne proton [δ_H 3.35 (1H, m, H-2)].

Compound 17 (3,3′,4,4′-tetra-O-methylellagic acid) is a white amorphous powder with the molecular formula C₁₇H₁₂O₈ and an unsaturation of twelve. The ¹³C-NMR showed 17 carbon signals, including twelve aromatic carbons, three methoxy groups, and two carbonyl carbons. The NMR data of 17 (Tables 8 and 9) indicated an ellagic acid skeleton consistent with ¹H-NMR.³⁶ The ¹H-NMR data (600 MHz, DMSO-d₆) showed two characteristic aromatic protons confirmed at [δ_H 7.50 (1H, s, H-5), and 7.58 (1H, s, H-5′)], and three methoxy protons [δ_H 4.04 (3H, m, 3-OCH₃), 4.06 (3H, m, 3′-OCH₃) and 3.99 (3H, s, 4′-OCH₃)].

Compound 18 (3,4′-di-O-methylellagic acid) is a white amorphous powder with the molecular formula C₁₆H₁₆O₈ and an unsaturation of nine. The ¹³C-NMR showed 16 carbon signals, including twelve aromatic carbons, two methoxy groups, and two carbonyl carbons. The NMR data of 18 (Tables 8 and 9) indicated an ellagic acid skeleton consistent with ¹H-NMR.³⁷ The ¹H-NMR data (400 MHz, DMSO-d₆) showed two characteristic aromatic protons confirmed at [δ_H 7.49 (2H, s, H-5, H-5′)], two methoxy protons [δ_H 4.05 (3H, s, 3-OCH₃), and 3.97 (3H, s, 3′-OCH₃)].

Compound 19 (3,4-methylenedioxy-3′-O-methyl-4′-O-glucoside ellagic acid) is a white acicular crystal with the molecular formula C₂₂H₁₈O₁₃ and an unsaturation of fourteen. The ¹³C-NMR showed 22 carbon signals, including twelve aromatic carbons, one methoxy group, two carbonyl carbons, one methylene, and one glucose group. The NMR data of 19 (Table 11) indicated an ellagic acid skeleton consistent with ¹H-NMR.³⁸ The ¹H-NMR data (600 MHz, C₅D₅N) showed two characteristic aromatic protons confirmed at [δ_H 7.73 (1H, s, H-5), and 8.44 (1H, s, H-1′)], one methylene protons [δ_H 6.33 (2H, m, H-3a)], one glucose terminal hydrogen proton [δ_H 5.88 (1H, m, H-1′′)], one methoxy group protons [δ_H 4.24 (3H, s, 3′-OCH₃)].

Table 11 ¹³C NMR and ¹H NMR chemical shifts (δ) of compound 19 in C₅P₅N

Position	δC (150 MHz)	δH (J in Hz) (600 MHz)
1	112.5
2	132.0
3	138.8
3a (CH2)	104.6	6.33 m
4	151.1
5	104.6	7.73 s
6	115.8
6a	158.0
1′	113.1
2′	142.0
3′	142.6
4′	153.0
5′	113.4	8.44 s
6′	114.0
6′a	158.6
1′′	102.7	5.88 m
2′′	78.3
3′′	79.0
4′′	74.6
5′′	70.8
6′′	62.1
3′-OMe	61.7	4.24 s

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Compound 20 (aralia cerebroside) is a white amorphous powder with the molecular formula C₄₀H₇₇NO₁₀ and an unsaturation of three. The ¹³C-NMR showed 40 carbon signals, including one acylamine group, one glucose group, twenty-four methylene carbons, four methyne carbons and one double bond signals, and two methyl signals. The NMR data of 20 (Tables 8 and 9) indicated an alkaloid skeleton.³⁹ The ¹H-NMR data (600 MHz, DMSO-d₆) showed one amide hydrogen proton confirmed at [δ_H 7.57 (1H, d, J = 9.0)], one glucose group proton [δ_H 4.93 (1H, d, J = 7.9, H-1′′), 4.06 (1H, m, H-2′′), 4.10 (1H, s, H-3′′), 4.32 (1H, m, H-4′′), 3.86 (1H, s, H-5′′), and 4.49, 4.36 (2H, dd, J = 2.4, 11.96, H-6′′)], four methyne protons [δ_H 5.24 (1H, s, H-2), 4.27 (1H, s, H-3), 4.25 (1H, d, J = 7.5, H-4), and 4.57 (1H, m, H-2′)], one double bond [δ_H 5.34 (1H, s, H-8), and 5.60 (1H, s, H-9)], two methyl protons [δ_H 0.84 (6H, s, CH₃ × 2)], and twenty-four methylene protons [δ_H 1.48 (48H, s, CH₂ × 24)].

Compound 21 (uracil) is a pale yellow needle crystal with the molecular formula C₄H₄N₂O₂ and an unsaturation of four. The ¹³C-NMR showed 4 carbon signals, including two carbonyl carbons and two aromatic carbons. The NMR data of 21 (Tables 8 and 9) indicated a nitrogen heterocyclic skeleton consistent with ¹H-NMR.⁴⁰ The ¹H-NMR data (600 MHz, DMSO-d₆) showed two characteristic aromatic protons confirmed at [δ_H 5.45 (1H, d, J = 7.6, H-5), and 7.39 (1H, d, J = 7.6, H-6)], and two imino group protons [δ_H 11.01 (1H, s, 1-NH), and 11.81 (1H, s, 3-NH)].

Compound 22 (chloropyrazine) is a white needle crystal with the molecular formula C₄H₃ClN₂ and an unsaturation of four. The ¹³C-NMR showed 4 carbon signals, including three aromatic carbons. The NMR data of 22 (Tables 3 and 4) indicated a nitrogen heterocyclic skeleton consistent with ¹H-NMR.⁴¹ The ¹H-NMR data (600 MHz, CD₃OD) showed two characteristic aromatic protons confirmed at [δ_H 8.39 (1H, s, H-5), and 8.34 (1H, s, H-6)].

Compound 23 ((S)-3,4,5-trihydroxy-1H-pyrrol-2 (5H)-one) is a white needle crystal with the molecular formula C₅H₇NO₄ and an unsaturation of three. The ¹³C-NMR showed 4 carbon signals, including one carbonyl carbon, one double bond, and one methyne carbon. The NMR data of 23 (Tables 3 and 4) indicated an alkaloid skeleton consistent with ¹H-NMR.⁴² The ¹H-NMR data (600 MHz, CD₃OD) showed one imino group proton confirmed at [δ_H 7.29 (1H, d, J = 7.6, H-1)] and one methyne hydrogen proton [δ_H 5.51 (1H, d, J = 7.6, H-5)].

Compound 24 (uvacalol k) is a white powder with the molecular formula C₂₁H₂₀O₇ and an unsaturation of twelve. The ¹³C-NMR showed 4 carbon signals, including two aromatic rings, one double bond, one methylene carbon, and four methyne carbons. The NMR data of 24 (Tables 3 and 4) indicated a polyoxygenated cyclohexene skeleton consistent with ¹H-NMR.⁴³ The ¹H-NMR data (600 MHz, CD₃OD) showed ten aromatic hydrogen protons confirmed at [δ_H 7.50–8.10 (10H, m, H-2′–H-6′, H-2′′–H-6′′)], one double bond protons [δ_H 6.60 (1H, d, J = 8.3, H-6)], one methylene proton [δ_H 5.16 d (1H, J = 7.5, H-7a), 4.75 d (1H, J = 11.4, H-7b)], and four methyne protons [δ_H 5.37 (1H, t, J = 9.3, 9.1, H-3), 4.54 (1H, m, H-2), 4.02 (1H, t, J = 8.4, 7.9, H-4), 3.81 (1H, t, J = 9.4, 9.5, H-1)].

Compound 25 (salicin) is a white needle crystal with the molecular formula C₁₃H₁₈O₇ and an unsaturation of five. The ¹³C-NMR showed 13 carbon signals, including six aromatic carbons, one glucose group, and one methylene carbon. The NMR data of 25 (Tables 8 and 9) indicated an aromatic hydrocarbon skeleton consistent with ¹H-NMR.⁴⁴ The ¹H-NMR data (600 MHz, DMSO-d₆) showed four aromatic hydrogen protons confirmed at [δ_H 7.36 (1H, d, J = 7.4, H-6), 7.19 (1H, dt, J = 8.2, 7.3, H-4), 7.09 (1H, d, J = 7.7, H-3), and 7.00 (1H, t, J = 6.8, 6.0, H-5)], one glucose group proton [δ_H 5.34 (1H, d, J = 4.4, H-1′), 4.47–5.06 (4H, m, H-2′–5′), 3.71 (1H, m, H-6′), and 3.47 (1H, m, H-6′)], and one methylene proton [δ_H 4.65 (1H, dd, J = 14.2, H-7), 4.47 (1H, dd, J = 14.2, H-7)].

2.2 Quantum chemical Gaussian and CD calculation

The calculated NMR of compound 14 was conducted with the Gaussian program. The DP4+ data of 1R,6S was 100% and the probabilities of isomer 1 (1S,6R), isomer 3 (1S,6S), and isomer 4 (1R,6R) were 0%. It can be inferred that 1R,6S was the correct relative configuration. In addition, the absolute configuration of compound 14 (1R,6S) was further confirmed with the ECD spectrum. Thus, the absolute configuration of compound 14 was defined as (1R,6S)-6-aminocyclohex-2-ene-1-carboxylic acid (Fig. 3 and Fig. 4).

Fig. 3 Theoretical NMR data (A and B) of compound 14 (isomer 2) and the possibility configurations of four conformations.

Fig. 4 Experimental CD spectra and Gaussian-calculated ECD spectra for compound 14.

2.3 Anti-inflammatory activity of octocarpus COX-2 inhibitors

Prostaglandins (PGs) are metabolites of arachidonic acid via the cyclooxygenase (COX) pathway. COX is a key enzyme that catalyzes the conversion of arachidonic acid to PGs and it includes COX-1 and COX-2.⁴⁵ COX-1 is a structural enzyme that is essential for maintaining human physiological needs,⁴⁶ whereas COX-2 is an inducible enzyme that is highly expressed during inflammation.⁴⁷ COX-2 can enhance PGE2 synthesis during inflammation, which plays a significant role in the subsequent cell inflammatory responses,⁴⁸ eventually producing a series of inflammatory mediators, which participate in the body's physiological and pathological processes through a cascade reaction.

In addition, COX-2 is a very important physiological enzyme playing key roles in various biological functions, especially in the mechanism of pain and inflammation, making it a molecule of great interest to the pharmaceutical community as a target.⁴⁹

Total extract, petroleum ether extract, ethyl acetate extract, N-butanol extract and nineteen compounds were tested for the anti-inflammatory activity of octocarpus COX-2 inhibitors (Fig. 5). Among them, ethyl acetate extract (IC₅₀ = 51.37 ± 7.89 μg mL⁻¹), compounds 1 (IC₅₀ = 10.80 ± 0.30 μM), 2 (IC₅₀ = 34.07 ± 3.57 μM), 3 (IC₅₀ = 67.90 ± 6.21 μM), 4 (IC₅₀ = 69.70 ± 6.55 μM), 13 (IC₅₀ = 16.49 ± 1.76 μM), 14 (IC₅₀ = 19.32 ± 3.58 μM), 16 (IC₅₀ = 24.20 ± 1.20 μM), 17 (IC₅₀ = 13.93 ± 2.44 μM), and 18 (IC₅₀ = 34.73 ± 1.53 μM) showed strong inhibitory activity against COX-2, and the inhibitory activity of compounds 1, 13, 14, and 17 was stronger than that of the positive control drug celecoxib (IC₅₀ = 21.35 ± 5.13 μM or 8.15 ± 3.13 μg mL⁻¹). Compounds 13, 17, and 18 belong to ellagic acids. According to reports, ellagic acids can be used as anti-inflammatory, anticancer and antioxidant agents.^50,51 Moreover, ellagic acids can also inhibit the expression of COX-2 in IL-1β-induced chondrocytes.⁵² This is consistent with our results.

Fig. 5 Results of extracts (A) and compounds (B) inhibiting cyclooxygenase (COX-2) activity (n = 3).

In contrast, compounds 5 (IC₅₀ = 123.14 ± 10.55 μM), 8 (IC₅₀ = 157.33 ± 12.85 μM), 15 (IC₅₀ = 150.00 ± 11.00 μM), 19 (IC₅₀ = 170.00 ± 7.00 μM), 20 (IC₅₀ = 134.00 ± 10.00 μM), 21 (IC₅₀ = 146.00 ± 11.00 μM), 22 (IC₅₀ = 178.00 ± 10.00 μM), 23 (IC₅₀ = 149.00 ± 10.00 μM), 24 (IC₅₀ = 175.00 ± 11.00 μM), and 25 (IC₅₀ = 162.00 ± 10.00 μM) showed relatively weak inhibitory activity against COX-2.

2.4 Cytotoxic activity assay against cancer cells

Cancer refers to a group of some of the most diagnosed and deadliest pathophysiological conditions worldwide. Although cancer is a common disease worldwide, its molecular pathology is characterized by a wide spectrum of biological aggressiveness that makes its control difficult, and thus a life-threatening disease.⁵³

Nineteen compounds were tested for activity against HepG2 cells, Caco-2 cells, and HeLa cells (Fig. 6). Compounds 1, 5, 13 and 21 showed good inhibitory effect on HepG2 cells with IC₅₀ of 25.00 ± 1.08, 8.21 ± 1.12, 78.85 ± 1.06, 78.5 ± 4.41 μM, and there were no significant differences between compounds 1 and 5 and positive control doxorubicin. Compounds 2 and 21 showed good inhibitory effect on Caco-2 cells with IC₅₀ of 85.98 ± 2.96 μM. And compound 20 showed good inhibitory effect on HeLa cells with IC₅₀ of 51.90 ± 2.56 μM. A. chinense showed great anticancer potential, and it could become a good anticancer drug in the near future.

Fig. 6 The effects of compounds against cancer cells (n = 3).

2.5 Molecular docking

Molecular docking is an important tool in computer-aided molecular design, which can be used to predict biological experiments by simulating the interaction of small molecules and biological macromolecules.⁵⁴ The binding energy and binding modes of compounds 13, 17, and 18 with COX-2 were measured (Fig. 7, Table 12). Compound 18 had the lowest binding energy of −8.1 kcal mol⁻¹. Conversely, as the number of hydroxyl groups went up and the number of methoxy groups was reduced, ellagic acids had the lowest binding energy.

Fig. 7 Molecular docking of compounds 13, 17, and 18 with COX-2.

Table 12 Molecular docking mode of compounds 13, 17, 18 and celecoxib with COX-2

Target	Compound	Binding free energy (kcal mol^−1)	Numbers of bonds	Amino acids
COX-2	Compound 1	−7.7	5	His351, Phe580, Ser581, Thr578
	Compound 5	−7.8	6	Gln350, Gly354, Phe580, Ser581, His351
	Compound 6	−8.1	4	Ser581, Phe590, Gln350
	Celecoxib	−10.0	22	Trp387, Ala527, Tyr385, Leu352, Gln192, Ile517, Arg513, Tyr355, Arg120, Val523, Val116, Val349, Ser353

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3 Material and methods

3.1 Chemicals and reagents

Silica gels with 100–200 and 200–300 meshes were purchased from Qingdao Ocean Chemical Co., Ltd (Shandong, China), Toyopearl HW-40F was purchased from Tosoh (Tokyo, Japan), acetonitrile of HPLC grade was purchased from J&K Scientific Inc (Beijing, China). Dichloromethane, ethyl acetate, petroleum ether (Fuyu Fine Chemical Inc, Tianjin, China), a high performance liquid chromatograph (Shimadzu Corporation, Japan), a circulating water multipurpose vacuum pump (Lichen Bangxi Instrument Inc, Shanghai, China), COX-2 LM0168 (LMAI Bio, Shanghai, China), DMEM medium, FBS (Fetal Bovine Serum), and penicillin-streptomycin were purchased from Thermo Fisher Scientific Co., Ltd. MTT reagent, doxorubicin, and cisplatin were acquired from Sigma Aldrich (St. Louis, MO, USA).

3.2 Plant material

The fibrous root of Alangium chinense (Lour.) Harms was purchased from Nayong City, Guizhou Province, China. The morphological characteristics of the plant were compared with those recorded in the Flora of China (Editorial Committee of Flora of China, Chinese Academy of Sciences, 1983) and identified by Qingde Long, an associate professor of Guizhou Medical University, with voucher specimen number 20191010.

3.3 Extraction and isolation

The 30 kg of dried fibrous root of A. chinense were cut into segments, then extracted with 75% ethanol at 25 °C three times, once a week. The obtained extract was filtered through a Brinell funnel. The filtrate was collected and further concentrated with a rotary evaporator. The extract was re-suspended in water with a separation funnel, and then the same amounts of petroleum ether, ethyl acetate, and N-butanol were added for continuous extraction. Then the extraction layer was evaporated using a rotary evaporator to remove the organic reagent to obtain 70.400 g, 159.800 g, and 203.200 g, respectively.

A total of 70.400 g of petroleum ether extract (APEE) was dissolved in dichloromethane and then mixed with silica gel at a ratio of 1 : 3, and then loaded onto a chromatograph silica gel column (75 × 1000 mm) using a gradient of petroleum ether–ethyl acetate solvent system as eluant to obtain fractions Fr. 1 to Fr. 9 based on the TLC analysis. Eluents from the resin column were dried under vacuum. Among them, Fr. 1 (27.900 g) was separated with an aqueous elution of petroleum ether : ethyl acetate (v/v = 1 : 0–1 : 1) with a silica gel column to obtain fractions Fr. 1.1 to Fr. 1.6 based on the TLC analysis. Fr. 1.1 (0.800 g) was separated with an aqueous elution of petroleum ether : ether ethyl acetate (v/v = 50 : 1) with a silica gel column to obtain compound 1 (0.100 g). Fr. 1.2 (1.2 g) was separated with an aqueous elution of dichloromethane : methanol (v/v = 1 : 1) with a silica gel column and further purified by semi-preparative high performance liquid chromatography with methanol : water (v/v = 80 : 20) as the mobile phase to obtain compound 2 (0.016 g, t_R 8.8 min). The precipitated white crystals from Fr. 2 (18.490 g) were recrystallized with methanol and dichloromethane to obtain compound 3 (0.065 g). Fr. 2.3 (1.080 g) was separated with an aqueous elution of petroleum ether : ethyl acetate (v/v = 100 : 1) with a silica gel column and further recrystallized with methanol to obtain compound 4 (0.100 g). Fr. 2.5 (1.080 g) was separated with an aqueous elution of dichloromethane : methanol (v/v = 1 : 1) with a silica gel column and further purified by semi-preparative high performance liquid chromatography with methanol : water (v/v = 80 : 20) as the mobile phase to obtain compound 5 (0.016 g, t_R 8.8 min).

Fr. 3 (22.700 g) was separated with an aqueous elution of dichloromethane : methanol (v/v = 1 : 1) with a silica gel column and further purified by semi-preparative high performance liquid chromatography with methanol : water (v/v = 7525) as the mobile phase to obtain compound 6 (0.008 g, t_R 9.2 min) and compound 7 (0.008 g, t_R 9.7 min). Fr. 3.3 (1.900 g) was separated with an aqueous elution of dichloromethane : methanol (v/v = 1 : 2) with a silica gel column and further purified by semi-preparative high performance liquid chromatography with methanol : water (v/v = 75 : 25) as the mobile phase to obtain compound 8 (0.015 g, t_R 7.5 min). Fr. 4 (17.300 g) was separated with an aqueous elution of dichloromethane : methanol (v/v = 1 : 1) with a silica gel column and further purified by semi-preparative high performance liquid chromatography with methanol : water (v/v = 75 : 25) as the mobile phase to obtain compound 9 (0.010 g, t_R 8.5 min). Fr. 4.3 (3.910 g) was separated with an aqueous elution of dichloromethane : methanol (v/v = 1 : 2) with a silica gel column and further purified by semi-preparative high performance liquid chromatography with methanol : water (v/v = 73 : 28) as the mobile phase to obtain compound 10 (0.010 g, t_R 8.6 min) and compound 11 (0.008 g, t_R 9.1 min). Fr. 5 (15.330 g) was separated with an aqueous elution of dichloromethane : methanol (v/v = 1 : 2) with a silica gel column and further purified by semi-preparative high performance liquid chromatography with methanol : water (v/v = 70 : 30) as the mobile phase to obtain compound 12 (0.012 g, t_R 8.5 min).

In addition, a total of 159.800 g of ethyl acetate extract (ACEE) was dissolved in methanol and then mixed with silica gel at a ratio of 1 : 3, and then loaded onto a chromatograph over a silica gel column (85 × 1200 mm) using a gradient of petroleum ether–ethyl acetate solvent system as eluant to obtain fractions Fr. 1 to Fr. 12 based on the TLC analysis. Eluents from the resin column were dried under vacuum. Among them, Fr. 3 (0.450 g) was separated with an aqueous elution of dichloromethane : methanol (v/v = 1 : 1) with Toyopearl HW-40F and further purified by petroleum ether : ethyl acetate (v/v = 25 : 1) with silica gel to obtain compound 13 (0.004 g) and compound 14 (1.000 g). Fr. 5 (0.430 g) was separated with an aqueous elution of petroleum ether : ethyl acetate (v/v = 1 : 1) with Toyopearl HW-40F and further purified by silica gel with petroleum ether : ethyl acetate (v/v = 15 : 1), and then compound 15 (0.004 g) was obtained. Fr. 7 (0.5 g) was separated with an aqueous elution of dichloromethane : methanol (v/v = 1 : 1) with Toyopearl HW-40F to get five fractions and Fr. 7.3 was further purified by silica gel with dichloromethane : methanol (v/v = 5 : 1) to obtain compound 16 (0.005 g). Fr. 7.4 (0.250 g) was separated with an aqueous elution of petroleum ether : ethyl acetate (v/v = 35 : 1) with silica gel and then recrystallized in methanol to obtain compound 17 (0.004 g). Fr. 10 (0.980 g) was separated with an aqueous elution of petroleum ether : dichloromethane acetate (v/v = 50 : 1) to get five fractions, and then Fr. 10.1 was further purified by semi-preparative high performance liquid chromatography with methanol : water (v/v = 35 : 65) as the mobile phase to obtain compound 18 (0.003 g, t_R 15 min). Fr. 10.3 (0.205 g) was separated with an aqueous elution of petroleum ether : dichloromethane (v/v = 3 : 1) with silica gel and further purified by Toyopearl HW-40F with methanol to obtain compound 19 (0.003 g). Fr. 10.4 (0.245 g) was purified with an aqueous elution of dichloromethane : methanol (v/v = 1 : 1) with Toyopearl HW-40F to obtain compound 20 (0.003 g). Fr. 10.5 (0.220 g) was separated with an aqueous elution of petroleum ether : dichloromethane (v/v = 15 : 1) with silica gel to obtain compound 21 (0.005 g).

A. chinense samples were dissolved in n-butanol and mixed with silica gel at a ratio of 13, and then loaded onto a chromatograph over a silica gel column (85 × 1200 mm, 203.200 g) using a gradient of dichloromethane : methanol acetate solvent system as eluant to obtain fractions Fr. 1 to Fr. 8 based on the TLC analysis, eluents from the resin column were dried under vacuum. Among them, Fr. 2 (0.647 g) was separated with an aqueous elution of dichloromethane : methanol (v/v = 1 : 1) with Toyopearl HW-40F to get seven fractions. Fr. 2.3 was recrystallized with petroleum ether and ethyl acetate to obtain compound 22 (0.004 g). Fr. 2.5 (0.500 g) was separated with an aqueous elution of dichloromethane : methanol (v/v = 1 : 1) with Toyopearl HW-40F to obtain compound 23 (0.003 g). Fr. 3 (0.470 g) was separated using an aqueous elution of dichloromethane : methanol (v/v = 1 : 1) with Toyopearl HW-40F to get four fractions. Fr. 3.3 was further separated with dichloromethane : methanol (v/v = 5 : 1) with silica gel to obtain compound 24 (0.007 g). Fr. 4 (0.400 g) was separated using an aqueous elution of dichloromethane : methanol (v/v = 1 : 1) with Toyopearl HW-40F to get five fractions. Fr. 4.3 was further separated by petroleum ether : ethyl acetate (v/v = 15 : 1) with silica gel to obtain compound 25 (0.005 g).

The compounds isolated from A. chinense were identified with NMR data.

3.4 Compound characterization

Compound (2): amorphous white powder. For ¹H NMR (600 MHz, D₂O) and (150 MHz, D₂O). HR-ESI-MS m/z 141.0790, [M−H]⁻ (calcd for C₇H₁₁NO₂, 140.0718).

3.5 Quantum chemical Gaussian calculation

Quantum chemical Gaussian calculation was conducted according to a previous report with slight modification.⁵⁵ The relative configuration of compound 14 in mol 2 format in Spartan was opened, and the conformation searched for at the molecular force field level of MMFF to obtain the dominant conformation of each compound. Then, under density functional theory (DFT), at B3LYP/6-31G(d) level, these conformations were put into an optimization and vibration analysis calculation, and no virtual frequency was checked after optimization. On this basis, the Boltzmann distributions of each dominant conformation were calculated, and then the non-dominant conformation with Gibbs free energy greater than 1.5 kcal mol⁻¹ was removed, and the optimized dominant conformation was obtained.

In addition, these dominant conformations were put into NMR calculations, and using DFT and the Gaussian default GIAO method, the magnetic shielding value of each carbon atom and hydrogen atom were calculated under gas-phase conditions at rmpw1pw91/6-31+G(d, p) level. According to the proportion of Boltzmann distribution and their respective phases of Gibbs free energy (ΔG), the average values of magnetic shielding values of all conformations of one compound can be obtained. Meanwhile, using tetramethylsilane (TMS) as a reference, the magnetic shielding values of four carbon atoms and 12 hydrogen atoms were calculated. Then all conformations of one compound were subtracted from the average value of these hydrocarbon atoms and the magnetic shielding values of the hydrocarbon atoms to get the theoretical chemical shift value of each hydrocarbon atom we need.

Finally, the calculated chemical displacement value of each hydrocarbon atom calculated by NMR and the actually measured chemical displacement value of each hydrocarbon atom adopted the feasibility analysis of DP4+, which will generate the corresponding probability data.

3.6 Analysis of CD calculation

Circular dichroism (CD) spectra were calculated according to reported methods.⁵⁶ Four kinds of geometric optimization of compound 14 were calculated with the density functional theory (DFT) method and time-dependent DFT (TDDFT) via the Gaussian 09 program. The ECD spectra were optimized at the APFD/6-311+G(2d, p) level in MeOH. The computational data were fitted in OriginPro 2019b (OriginLab Inc., USA).

3.7 Study of the anti-inflammatory activity of octocarpus COX-2 inhibitors

The study of the COX-2 enzyme inhibitory activity of the samples was performed according to the instructions for the kit. Compounds to be tested with 6.25, 25, 50, 100, 200, 400, 800 μM were prepared and celecoxib was used a positive control drug, and the extracts were 5, 25, 50, 100, 200, 400, 800 μg mL⁻¹.

Each sample was provided three times. The procedure was to add samples to wells separately, set up 2 replicate wells for each sample, incubate at 37 °C for 10 min, add 5 μL of COX-2 Probe and 5 μL of COX-2 substrate working solution to each well, centrifuge and mix well. Performed on a culture plate shaker, the incubation was timed for 5 min at 37 °C protected from light and then the fluorometric assay was performed. The excitation wavelength was 560 nm and the emission wavelength was 590 nm. The average fluorescence values were calculated for each sample well and a blank control well, which were recorded as RFU Blank Control, RFU 100% Enzyme Activity Control, RFU Positive Inhibitor Control and RFU Sample, RFU Relative Fluorescence Unit, respectively.

The percent inhibition for each sample was calculated with the following formula: Inhibition (%) = (RFU 100% enzyme activity control − RFU sample)/(RFU 100% enzyme activity control − RFU blank control) × 100% and the IC₅₀ of the inhibitor was calculated.

3.8 Cytotoxic activity assay

The cytotoxic activities of all isolated compounds were tested for their anti-proliferative activity against HepG2, Caco-2, and HeLa cell lines with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay.⁵⁷ Cells were seeded at a density of 5 × 10³ per well into 96-well plates for 24 h. Then the cells were treated with compounds for 48 h. Control cells were exposed to 0.1% DMSO. The test cells were treated with different concentrations of isolated compounds. Adriamycin (10 μM) was used as a positive control. Then, 20 μL of MTT (5 mg mL⁻¹) was added to each well and incubated for 4 h, after which 150 μL of DMSO was added to each well. The reduction in cell viability was determined at 490 nm by using a microplate reader (Bio-Rad iMARK, Berkeley, CA, USA). Three replicates were obtained for each treatment.

3.9 Molecular docking

To investigate the relationship between isolated ellagic acid compounds and COX-2, compounds 13, 17, and 18 were used as ligands in an in silico molecular docking study. The three-dimensional structure of COX-2 (PDB ID 5IKR) was obtained from the online Protein Data Bank (PDB). Optimization of ligand structures was processed with ChemBioDraw (PerkinElmer), and the molecular docking was implemented using AutoDock Tools (version 1.5.6), AutoDock 4.2 package (Autodock 4.2 and Autogrid 4.2), with the help of the Discovery Studio Visualizer (version 16.1.0.15350), which can be used to better visualize the interactions between ligands and receptors. X, Y, and Z were set to 38.889, 1.917, and 61.611 Å in the cubic grid box as the central dimensions of COX-2. Water molecules and persistent inhibitors were removed from the crystal structure of 5IKR. Blind docking over the whole receptor was carried out using the Genetic Algorithm method, and the resultant complex structures were investigated by using the conformations with the most favorable binding energy.

4 Conclusions

It has been reported that in clinical practice, A. chinense is mostly used to treat rheumatoid arthritis, numbness and paralysis, heart failure, strain, low back pain, traumatic injury and other diseases, and its curative effect is accurate. A. chinense is widely used in Miao medicine. Among the prescriptions of Miao medicine for the treatment of rheumatic diseases, A. chinense has been used 44 times, with a frequency of 12.6%, ranking fourth.⁵⁸ Although the star anise maple has good pharmacological activity and rich medication experience in China, there are still many problems that need further research. Modern pharmacology has proved that A. chinense and its extracts have many pharmacological functions. This study enriched the understanding of more compounds in the fibrous roots of A. chinense through the separation of compounds from the fibrous roots of A. chinense, especially using ethyl acetate.

In this study, one new (14) and twenty-four known compounds (1–13, 15–25) were isolated, and seven compounds were isolated from this plant for the first time. After screening the anti-inflammatory and antitumor activities of the isolated compounds in vitro, it was found that compounds 1, 2, 3, 4, 13, 14, 16, 17 and 18 may have potential anti-inflammatory effects and compound 18 showed the lowest binding energy with COX-2 enzyme with molecular docking technology. It is preliminarily speculated that it is one of the main active components with anti-inflammatory effects. The above two components may be the anti-inflammatory active components of the fibrous root of A. chinense. The specific mechanism research group will carry out the next research step to provide a basis for follow-up basic research on active substances. While compounds 1, 5, 13, 20, and 21 showed cytotoxicity against HepG2, Caco-2 and HeLa cells, so they may be at least partly responsible for the antitumor activity of A. chinense. Those compounds can be used as the basis for pharmacological activity research in later stages for anti-inflammatory and anticancer effects.

Author contributions

T. X. and Z. G.: conceptualization, Gaussian and ECD calculations, writing, reviewing and editing; J. H.: purification, isolation of compounds, and COX-2, cancer cells activity assays; X. C.: formal analysis, data curation; L. T.: reviewing and editing; X. S.: conceptualization, reviewing and editing.

Conflicts of interest

The authors declare that there is no conflict of interest.

Acknowledgements

This research was supported by grant from the Basic Scientific Research Project of Guizhou Province (grant number [2021] 535, [2021] 540), the Guizhou Medical University Breeding programs of National Natural Science Foundation of China (grant number 19NSP074), the PhD Start-up Fund of Guizhou University of Traditional Chinese Medicine (grant number 2019-10), the projects of State Key Laboratory of Function and Applications of Medicinal Plants, Guizhou Medical University (grant number FAMP201908K).

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Footnote

† These authors contributed equally to this article.

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