Methods and applications for extracting various monomeric compounds with anti-inflammatory activity from *Tea japonica*.

Abstract

This invention relates to the field of traditional Chinese medicine extraction and separation technology, specifically disclosing a method and its applications for extracting various monomeric compounds with anti-inflammatory activity from *Tetrapanax papyriferus*. The method includes steps such as maceration, extraction, macroporous resin separation, silica gel column chromatography, and preparative HPLC purification. Using this method, 23 monomeric compounds can be extracted and separated from *Tetrapanax papyriferus*, among which compounds 1-8 are new compounds, and compounds 9-13, 16-18, and 20-23 are compounds isolated from this plant for the first time. This invention also screens the 23 extracted monomeric compounds for anti-inflammatory activity, disclosing the applications of compounds 1-13, 16-18, and 20-23 in the preparation of anti-inflammatory drugs. This invention provides a material basis for further enriching the research data on the chemical composition of *Tetrapanax papyriferus* and better utilizing its medicinal value.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine extraction and separation technology, specifically to a method and application for extracting various monomeric compounds with anti-inflammatory activity from *Tea japonica*. Background Technology

[0002] Berchemia lineata (L.) DC., belonging to the genus Berchemia in the family Rhamnaceae, is one of the medicinal species of *Berchemia lineata*, also known as Wulonggen, Makouzi, and Laoshuer. It is distributed in Wuming, Pingguo, Cenxi, Cangwu, Hexian, Tiandeng, and Xiangzhou counties (cities) in Guangxi. *Berchemia lineata* is a commonly used ethnic medicine distributed in the Zhuang and southwestern minority areas of Guangxi, possessing analgesic, anti-inflammatory, hepatoprotective, and anti-tumor biological activities. It has a bland, slightly sweet taste and neutral properties; it has the effects of clearing heat and toxins, dispelling wind and dampness, and possessing anti-inflammatory, anti-swelling, hemostatic, analgesic, and anti-cancer properties. It is used for respiratory infections, cardiovascular diseases, digestive diseases, rheumatic immune diseases, tumors, lung abscesses, and traumatic injuries, and externally for burns, scalds, and snake bites.

[0003] The chemical composition of Berchemia lineata is complex, with its main components including flavonoids, anthraquinones, dimers, phenylpropanoids, terpenes, glycosides, and organic acids. Regarding the study of the chemical composition of Berchemia lineata medicinal materials, relevant literature was retrieved, such as the article titled "Study on the Chemical Composition of Berchemia lineata Roots," authored by Shen Yuxia, Teng Hongli, Chen Xiaolong, Yang Guangzhong, and Mei Zhinan. This study investigated the chemical composition of the roots (iron-clad gold) of Berchemia lineata, using silica gel, HPLC, and Diaion HP-20 chromatography to separate and purify the chemical components. The structures of the compounds were identified based on their spectral data. The results showed that the structures of eight compounds were isolated and identified: (-)-eugenol (1), (+)-podophyllin (2), (+)-cantharis (3), (+)-lyoniresinol-3α-O-β-D-glucopyranoside (4), chrysophanol (5), and floribundiquinone. C(6), polyanthoceratoquinone A(7) and ferneneol(8), wherein compounds 1-4 were isolated from this genus for the first time, and compounds 5-8 were isolated from this species for the first time.

[0004] Studies show that the pharmacological effects of *Houttuynia cordata* mainly include anti-tumor, anti-inflammatory, hepatoprotective, enzyme-lowering, blood-activating, and anti-respiratory infection effects. Clinically, it is commonly used to treat respiratory diseases, cardiovascular diseases, digestive diseases, rheumatic immune diseases, tumors, lung abscesses, burns, and other diseases. However, the extraction and research of chemical components in *Houttuynia cordata* are currently insufficient. To further enrich the research data on the chemical components of *Houttuynia cordata*, to find new lead compounds with good activity in anti-tumor, anti-inflammatory, hepatoprotective, enzyme-lowering, and anti-respiratory infection activities, and to better utilize the medicinal value of *Houttuynia cordata*, the development and isolation of monomeric compounds from *Houttuynia cordata* and the study of the medicinal effects of the extracted monomeric compounds are urgently needed. Summary of the Invention

[0005] The purpose of this invention is to provide a method for extracting various monomeric compounds with anti-inflammatory activity from *Tetrapanax papyriferus* and its application in the preparation of anti-inflammatory drugs. Using this method, 23 monomeric compounds can be extracted and isolated from *Tetrapanax papyriferus*, of which compounds 1-8 are new compounds, and compounds 9-13, 16-18, and 20-23 are compounds isolated from this plant for the first time. This provides a material basis for further enriching the research data on the chemical composition of *Tetrapanax papyriferus*, finding new lead compounds with good anti-tumor, anti-inflammatory, hepatoprotective, enzyme-lowering, and anti-respiratory infection activities, and better utilizing the medicinal value of *Tetrapanax papyriferus*.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A method for extracting multiple monomeric compounds with anti-inflammatory activity from *Tea japonica* var. *mongolica* includes the following steps:

[0008] (1) The aerial parts of the fine-leafed tea were dried and crushed, and extracted three times with 70% ethanol-water for 24 hours each time to obtain ethanol extract; the extracts were then extracted three times with equal volumes of petroleum ether, ethyl acetate and n-butanol, and dried completely to obtain petroleum ether extract, ethyl acetate extract and n-butanol extract.

[0009] (2) After the n-butanol extract was dissolved in water, it was separated by adsorption of D-101 macroporous resin. The gradient eluent was 30%, 50%, 70%, and 95% ethanol-water. After drying, the extracts were obtained as 30%, 50%, and 70% ethanol extracts.

[0010] (3) The 50% ethanol extract was subjected to normal phase silica gel column chromatography with dichloromethane / methanol at volume ratios of 20:1, 9:1, 8:2, 7:3, and 1:1. After TCL detection, similar fractions were combined to obtain fractions A to L in sequence.

[0011] (4) Component C was repeatedly prepared by HPLC. The ratio of CH3CN:H2O = 32:68 was used to obtain compound 9. Compound 12 was obtained by recrystallizing component H. Component F was subjected to reversed-phase silica gel column chromatography with 30%, 50%, 70%, and 90% methanol-water as gradient eluents. After TCL detection and merging, components F.1 to F.11 were obtained.

[0012] (5) Component F.2 was repeatedly prepared by HPLC under the condition of CH3CN:H2O = 19:81 to obtain compounds 6 and 7; after recrystallization of component F.3, compound 10 was obtained. The mother liquor after filtration was separated by gel column chromatography with methanol as isocratic eluent, and then repeatedly prepared by HPLC with CH3CN:H2O = 19:81 to obtain three compounds: compounds 4, 5 and 8; after recrystallization of component F.4 with methanol as solvent, compound 11 was precipitated.

[0013] (6) The J component was prepared by reversed-phase silica gel column chromatography with 30%, 50%, and 70% methanol-water as gradient eluents, and the combined TCL was detected and then repeatedly prepared by HPLC with CH3CN:H2O = 18:82 to obtain compound 13.

[0014] (7) The ethyl acetate extract was separated by normal-phase silica gel column chromatography using petroleum ether / ethyl acetate volume ratios of 15:1, 10:1, 9:1, 8:2, 7:3, 6:4, and 1:1 as the mobile phase. The combined fractions were detected by TCL and obtained as Fr.1 to Fr.16. Fr.6 fraction was recrystallized to obtain white crystals, which was compound 23. Fr.15 fraction was recrystallized to obtain yellow powder, which was compound 14.

[0015] (8) Fr.9 fraction was separated by normal-phase silica gel column chromatography with petroleum ether / ethyl acetate volume ratios of 15:1, 10:1, 9:1, 8:2, 7:3, 6:4, and 1:1 as mobile phases. After TCL detection and combination, it was repeatedly prepared by HPLC. Compound 19 was obtained under CH3CN:H2O = 50:50, and compound 22 was obtained under CH3CN:H2O = 22:78. Fr.14 fraction was separated by reverse-phase silica gel column chromatography with 30%, 50%, 70%, and 90% methanol-water as mobile phases. After TCL detection and combination, Fr.14.1 to Fr.14.14 fractions were obtained.

[0016] (9) Fr.14.7 was repeatedly prepared by HPLC with CH3CN:H2O = 25:75 to obtain three compounds, namely compounds 17, 20 and 21; Fr.14.10 was repeatedly prepared by HPLC with CH3CN:H2O = 35:65 to obtain compound 16; Fr.14.11 was repeatedly prepared by HPLC with CH3CN:H2O = 30:70 to obtain compound 1; Fr.14.14 was repeatedly prepared by HPLC with CH3CN:H2O = 40:60 to obtain compounds 2, 3, 15 and 18;

[0017] (10) Of the 23 monomeric compounds obtained by the above extraction and separation, compounds 1-8 were identified as new compounds by structural identification, among which compound 1 is a novel skeleton compound with the molecular formula C. 16 H 16 O5; Compounds 2 and 3 are naphthoquinones, with molecular formulas of C10, C20, C30, and C40, respectively. 17 H 14 O4, C 16 H 12 O4; Compounds 4 and 5 are naphthoquinones, with molecular formulas of C10, C20, C30, C40, C50, C60, C70, C80, C9 ... 21 H 22 O8, C 22 H 24 O9; Compounds 6 and 7 are flavonoids, both with the molecular formula C09. 23 H 26 O 12 Compound 8 is a bibenzyl group with the molecular formula C. 21 H 26O8; Compound 9 is Vittarin-B; Compound 10 is Demethylflavasperone-10-O-β-D-glucopyranoside; Compound 11 is Rubrofusarin-6-O-β-D–glucopyranoside; Compound 12 is Rubrofusarin-6-O-α-L-rhamnosyl-(1→6)-O-β-D-glucopyranoside; Compound 13 is Kaempferol-3-O-α-L-rhamnopyranosyl-(1→6)-β-D-glucopyranoside; Compound 14 is quercetin; Compound 15 is kaempferol; Compound 16 is naringenin; Compound 17 is dihydrokaempferol; Compound 18 is 2-hydroxyemodin 1-methyl ether; Compound 19 is emodin; Compound 20 is trans-cinnamic acid; Compound 21 is 7-hydroxy-2,5-dimethylchromon; Compound 22 is methyl 3,4-dihydroxybenzoate; compound 23 is Isoarborinol.

[0018] The above method for extracting various monomeric compounds with anti-inflammatory activity from *Tetrapanax papyriferus* yielded 23 monomeric compounds. Among them, compound 1 is a novel skeleton compound; compounds 2 and 3 are naphthopyranones; compounds 4, 5, 10, 11, and 12 are naphthopyranones; compounds 6 and 7 are flavonoids; compounds 8 and 9 are bibenzyl groups; compounds 13-17 are flavonoids and dihydroflavonoids; compounds 18 and 19 are anthraquinones; compounds 20-22 are simple phenols; and compound 23 is a terpene. Compounds 1-8 are new compounds, and compounds 9-23 are known compounds. Compounds 9-13, 16-18, and 20-23 are compounds isolated from this plant for the first time. The names and molecular formulas of the 23 isolated monomeric compounds are shown in Table 1; the classification and structural formulas of the compounds are shown in the appendix. Figure 1 .

[0019] Table 1. 23 compounds isolated from *Tea styrax*.

[0020]

[0021] The present invention also provides the application of compounds 1-13, 16-18, and 20-23, obtained by the method described above for extracting various monomeric compounds with anti-inflammatory activity from *Tea japonica*, in the preparation of anti-inflammatory drugs.

[0022] This invention screened 23 monomeric compounds obtained from extraction and separation for their anti-inflammatory activity. The drug toxicity of the monomeric compounds was detected using the CCK-8 assay. A classic inflammatory response model was established using LPS-induced RAW264.7 mouse macrophages. The NO content in the cell supernatant was detected using the Griess method, the NO inhibition rate was calculated, and the anti-inflammatory activity gradient of individual compounds was detected. Compounds 14, 15, and 19 were known compounds and were not analyzed. The study found that the novel skeleton compound 1 achieved a NO inhibition rate of 70.81% after 12 hours of administration at 50 μM. New compounds 2-8 and the first-isolated compounds 16-18 and 20-23 also showed varying degrees of anti-inflammatory effects. The novel skeleton compounds, as well as bibenzyl, dihydroflavonoids, and anthraquinone compounds, exhibited good anti-inflammatory activity, suggesting that these compounds may be the pharmacodynamic basis for the anti-inflammatory effects of *Gynostemma pentaphyllum*.

[0023] The beneficial effects of this invention are as follows:

[0024] 1. This invention provides a method for extracting various monomeric compounds with anti-inflammatory activity from *Tea japonica*. The method employs ultraviolet spectroscopy, optical rotation spectroscopy, nuclear magnetic resonance, high-resolution mass spectrometry, quantum chemical calculations, and other chemical methods for structural identification. Using this method, 23 compounds can be extracted and isolated from *Tea japonica*, exhibiting diverse structural types. Compound 1 possesses a novel 3,4-dihydro-11H-benzo[b]pyrano[4,3-e]oxepin-11-one skeleton, while the remaining seven new compounds belong to three structural types: naphthopyranones (2-5), flavonoid glycosides (6-7), and bibenzyl compounds (8). Compounds 2 and 3 are relatively rare α-naphthopyranones in natural products, previously only found in marine organisms; this is the first time such components have been discovered in plants. Compounds 4 and 5 are angular naphthopyranones, and compound 8 is a bibenzyl compound; all are structural types isolated from *Tea japonica* for the first time. In addition, compound 9 is a bibenzyl group, compounds 13-17 are flavonoids and dihydroflavonoids, compounds 18 and 19 are anthraquinones, compounds 20-22 are simple phenols, and compound 23 is a terpene. This invention enriches the research data on the chemical composition of *Tetrapanax papyrifer*, providing a material basis for further development of new lead compounds with good anti-tumor, anti-inflammatory, hepatoprotective, enzyme-lowering, and anti-respiratory infection activities, and for better utilization of the medicinal value of *Tetrapanax papyrifer*.

[0025] 2. This invention screened 23 monomeric compounds extracted from *Tetrapanax papyrifer* for their anti-inflammatory activity. Compounds 14, 15, and 19 were known compounds and were not analyzed. Experimental results showed that the new skeletal compounds, novel compounds, and compounds isolated from this plant for the first time all inhibited LPS-induced NO release from LPS-induced mouse macrophages RAW264.7 to varying degrees, exhibiting different levels of anti-inflammatory effects without significant drug toxicity. This invention has identified effective anti-inflammatory components in *Tetrapanax papyrifer*, providing a sufficient theoretical basis for modern research and clinical application of *Tetrapanax papyrifer* in anti-inflammatory applications. Attached Figure Description

[0026] Figure 1 The classification and structural formulas of the 23 monomeric compounds obtained by the method of this invention. Detailed Implementation

[0027] Example 1

[0028] I. Methods for extracting various monomeric compounds with anti-inflammatory activity from *Tea japonica* and structural identification of the compounds 1. Instruments and materials

[0029] D-101 macroporous resin (Anhui Samsung Resin Technology Co., Ltd.); silica gel (Qingdao Ocean Chemical Plant, 200-300 mesh); thin-layer silica gel plate (Qingdao Ocean Chemical Plant); ODS silica gel (50μm, YMC Corporation, Japan); dextran gel LH-20 (GE, USA); rotary evaporator (RE-5298, Shanghai Yarong Biochemical Instrument Factory); UV-Vis spectrophotometer (UH5300, Hitachi High-Tech Corporation); semi-preparative high-performance liquid chromatograph (Ulitimate 3000, Dionex, USA, DAD detector); nuclear magnetic resonance spectrometer (ADVANCE III™ 600, 500MHz, Bruker, Germany); circular dichroism chromatograph (Chirascan Plus, Applied Photophysics, UK); polarimeter (AutopolIV-T, Rudolf, USA); liquid chromatography-mass spectrometry (Q... Exactive (Thermo Fisher Scientific); semi-preparative chromatographic column (250mm×10mm, 5μm, YMC, Japan); chromatographic grade reagents (Hubei Futon Biochemical Technology Co., Ltd.); other analytical grade reagents (Sinopharm Group).

[0030] The stems and leaves of *Berchemia lineata* were purchased from Nanning, Guangxi, and identified as *Berchemia lineata* (L.) DC. by Professor Teng Hongli (Guangxi International Zhuang Medicine Hospital). The raw material specimens of its stems and leaves are currently stored in the specimen room of the School of Pharmacy (South-Central University for Nationalities).

[0031] 2. Separation and extraction

[0032] The aerial parts of *Tea japonica* (10 kg) were dried and pulverized, and extracted three times with 70% ethanol-water for 24 hours each time, yielding 700 g of ethanol extract. Then, it was extracted three times each with equal volumes of PE (petroleum ether), EtOAc (ethyl acetate), and n-butanol (n-butanol). After complete drying, 57 g of petroleum ether extract, 100 g of ethyl acetate extract, and 300 g of n-butanol extract were obtained. The n-butanol extract was dissolved in water and separated by adsorption using D-101 macroporous resin (gradient eluent: 30%, 50%, 70%, 95% ethanol-water). The extracts were combined and dried to obtain 30%, 50%, and 70% ethanol extracts.

[0033] The 50% ethanol extract was subjected to normal-phase silica gel column chromatography with dichloromethane / methanol (20:1, 9:1, 8:2, 7:3, 1:1) as the mobile phase. After TCL detection, similar fractions were combined to obtain fractions A to L in sequence.

[0034] Fraction C (CH3CN:H2O = 32:68) was repeatedly prepared by HPLC to obtain compound 9 (tR = 50.1 min, 3 mg). Compound 12 (4 g) was then obtained by recrystallization of fraction H. Fraction F was subjected to reversed-phase silica gel column chromatography using 30%, 50%, 70%, and 90% methanol-water as gradient eluents. The fractions were combined by TCL analysis to obtain fractions F.1–F.11. Fraction F.2 was repeatedly prepared by HPLC under CH3CN:H2O = 19:81 conditions to obtain compound 6 (tR = 31.0 min, 10 mg) and compound 7 (tR = 34.9 min, 5 mg). Recrystallization of F.3 yielded compound 10 (4.5 mg). The mother liquor after filtration was separated by gel column chromatography using methanol as the isocratic eluent, followed by repeated HPLC preparation (CH3CN:H2O = 19:81) to obtain three compounds: compound 4 (tR = 34.0 min, 1 mg), 5 (tR = 46.7 min, 1.1 mg), and 8 (tR = 25.6 min, 1 mg). Recrystallization of fraction F.4 using methanol as the solvent precipitated compound 11 (4 mg).

[0035] Fraction J was eluented with 30%, 50%, and 70% methanol-water gradients, and then subjected to reversed-phase silica gel column chromatography. The fractions were combined by TCL detection and then repeatedly prepared by HPLC (CH3CN:H2O = 18:82) to obtain compound 13 (tR = 42.1 min, 3.89 mg).

[0036] The ethyl acetate extract was separated by normal-phase silica gel column chromatography using petroleum ether / ethyl acetate ratios of 15:1, 10:1, 9:1, 8:2, 7:3, 6:4, and 1:1 as the mobile phase. TCL detection and combining yielded fractions Fr.1–Fr.16. Recrystallization of Fr.6 yielded white crystals, compound 23 (4.4 mg), and recrystallization of Fr.15 yielded a yellow powder, compound 14 (4.5 mg). Fr.9 was also separated by normal-phase silica gel column chromatography (using petroleum ether / ethyl acetate volume ratios of 15:1, 10:1, 9:1, 8:2, 7:3, 6:4, and 1:1 as the mobile phase). TCL detection and combining yielded fractions, followed by repeated HPLC preparation. Compound 19 (tR = 43.9 min, 1 mg) was obtained under CH3CN:H2O conditions of 50:50, and compound 22 (tR = 27.8 min, 1 mg) was obtained under CH3CN:H2O conditions of 22:78. Fraction Fr.14 was subjected to reversed-phase silica gel column chromatography using 30%, 50%, 70%, and 90% methanol-water as mobile phases, and the fractions were then combined by TCL detection to obtain fractions Fr.14.1–Fr.14.14. Fr.14.7 was repeatedly prepared by HPLC (CH3CN:H2O = 25:75) to obtain three compounds: 17 (tR = 36.9 min, 4.9 mg), 20 (tR = 42.6 min, 7.1 mg), and 21 (tR = 47.0 min, 5.5 mg). Fr.14.10 was repeatedly prepared by HPLC (CH3CN:H2O = 35:65) to obtain compound 16 (tR = 22.9 min, 1.2 mg), and Fr.14.11 was repeatedly prepared by HPLC (CH3CN:H2O = 30:70) to obtain compound 1 (tR = 67.2 min, 2 mg). Fr.14.14 was repeatedly prepared by HPLC (CH3CN:H2O=40:60) to give compounds 2 (tR=40.5min, 4.5mg), 3 (tR=33.5min, 2mg), 15 (tR=14.5min, 12mg) and 18 (tR=22.9min, 1.2mg).

[0037] 3. Structural identification

[0038] Compound 1bercheminol A is a brown amorphous powder, [α]20D -27.3 (c 0.05, MeOH); UV (MeOH)λmax (log ε) 210 (2.87), 295 (2.63) nm; ECD (c 0.5, MeOH)λ (θ) 227 (+14.58), 259 (+7.72), 307 (-11.78) nm; 1H NMR (500 MHz, CD3OD) and 13C NMR (125 MHz, CD3OD); HRESIMS m / z 289.10695 [M+H]+ (calcd for C16H17O5, 289.10705). The product is a brown amorphous powder. Based on the quasi-molecular ion peak m / z of compound 1 (289.10705 [M+H]+, calculated value: 289.10705) obtained by HR-ESI-MS, the molecular formula is presumed to be C16H16O5, with an unsaturation degree of 9. ¹H-NMR (500MHz, CD3OD) shows that compound 1 has a pair of meta-coupled aromatic protons [δH 6.15 (¹H, d, J = 2.5Hz, H-6), 6.40 (¹H, d, J = 2.5Hz, H-8)], an isolated alkene proton [δH 6.07 (¹H, s, H-4)], a pair of methylene groups [δH 2.64 (¹H, dd, J = 13.5, 3.5Hz), 2.35 (¹H, dd, J = 13.5, 8.5Hz), and a hydroxymethyl group [δH...]. [4.45 (1H, m)], a singlet methyl [δH 2.31 (3H, s)], a bimodal methyl signal [δH 1.34 (3H, d, J = 6.5 Hz)], and a methoxy signal [δH 3.81 (3H, s)]. Based on the information provided by 13C-NMR (125MHz, CD3OD), DEPT, HSQC, and HMBC spectra, compound 1 has a total of 16 carbon atoms, including a 1,2,3,5-tetrasubstituted phenyl group [δC 132.4(s), 106.5(d), 156.2(s), 100.8(d), 153.6(s), 132.8(s)], a trisubstituted double bond [δC 122(d), 1132.0(s)], an enolized double bond [δC 172.9(s), 105.1(s)], an ester carbonyl group [δC 171.9(s)], an oxymethimide group [δC 77.3(d)], two methyl groups [δC 21.9(q), 20.7(q)], a methoxy group [δC 56.7(q)], and a methylene group [δC 21.9(q), 20.7(q)]. 39.1(t)]. Based on the information given in the 1H-1H COSY and HSQC spectra, we can deduce that compound 1 has the CH3(14)CH(O)(13)CH2(12)- structural fragment.According to HMBC, H2-12 (δH 2.64, 2.35) is associated with δC 105.1 (s, C-2), 132.0 (s, C-3), and 122.2 (s, C-4), H-4 (δH 6.07) is associated with δC 105.1 (s, C-2), and CH3-11 [H 2.31 (s)] is associated with δC 105.1 (s, C-2) and δC 173.1 (s, C-1), indicating that the trisubstituted double bond is attached to C-12, the trisubstituted double bond and the enolized double bond are connected through C-2 and C-3, and the remaining ester carbonyl group should be attached to C-2. Furthermore, HMBC shows a correlation between H-4 (δH 6.07) and δC 106.5 (d, C-6) and 132.8 (s, C-10), proving that the aforementioned structural fragment should be attached to C-5. Based on the ROESY spectrum, the correlation between the methoxy group and H-8, combined with the correlation between the hydrogen atom of the methoxy group and δC 153.6 (s, C-9) shown in the HMBC spectrum, suggests that the methoxy group should be attached to C-9. Based on the molecular formula, compound 1 has 9 degrees of unsaturation. Besides the two double bonds, one carbonyl group, and one benzene ring already shown, two more degrees of unsaturation are missing, suggesting that the compound also has two rings. Because the correlation information provided by HMBC is insufficient, it is impossible to determine the connection positions of the two rings solely based on the NMR data; it is also impossible to determine which ring is formed by an ether bond and which ring is formed by an ester bond. Therefore, compound 1 has two possible structures: one is an ether bond between C-1 and C-10, with C-2 linked to C-13 via a carbonyl group at C-15 to form an ester bond, resulting in structure 1a; the other is an ester bond formed by C-2 linked to C-10 via C-15, while an ether bond forms between C-1 and C-13, resulting in structure 1b. To further determine the connection positions of the two rings, we performed NMR calculations on 1a and 1b using the Gauge-Including Atomic Orbitals (GIAO) method with methanol as the IEFCM solvation model, and used DP4+ analysis to determine the two possible cyclization modes. According to the calculation results, 1b has a larger correlation coefficient R², and its CMAD, CLAD, and RMSD values ​​are all smaller, indicating that the theoretical chemical shift value of 1b is in better agreement with the experimental value, with smaller errors and higher precision. DP4+ analysis shows that the probability of 1b is as high as 100%, therefore, the cyclization mode of compound 1 should be 1b. DP4+ analysis determined that compound 1 has a cyclic configuration of 1b, containing one chiral carbon atom. Its absolute configuration was determined by ECD calculations. Its structure was ultimately determined to be 13S-1b′, and it was named bercheminol A.Compound 1 belongs to the 3,4-dihydro-11H-benzo[b]pyrano[4,3-e]oxepin-11-one skeleton, suggesting its synthesis via the acetic acid-malonic acid (AA-MA) pathway, a possible biosynthetic route. One molecule of acetyl-CoA and five molecules of malonyl-CoA undergo Claisen condensation and decarboxylation to yield polyketide i. i then reacts with acetoacetyl-CoA through a series of reactions including Claisen condensation, hydrolysis, and decarboxylation to obtain key intermediate ii. Key intermediate ii undergoes aldol condensation, dehydration, enolization, and reduction to yield phenolic compound iii. Finally, iii is etherified, oxidized, and methylated to obtain compound 1.

[0039] Compound 2bercheminol B is a brown amorphous powder, [α]20D+15.6 (c 0.05, MeOH); UV (MeOH)λmax(logε)220(2.87),280(2.61),365(2.10)nm; ECD (c 0.5, MeOH)λ(θ)212(+26.79),228(-14.00),247(+1.39),286(-5.44)nm; specific 1H NMR (500MHz, CD3OD) and 13C NMR (125MHz, CD3OD); HRESIMS m / z 283.09594 [M+H]+ (calcd for C17H15O4, 283.09649). High-resolution mass spectrometry (HR-ESI-MS) revealed the quasi-molecular ion peak of compound 2 at m / z: 283.09594 [M+H]+ (calculated value: 283.09649), thus inferring its molecular formula as C17H14O4, which has 11 degrees of unsaturation. Based on 1H-NMR (500MHz, CD3OD), compound 2 exhibits a set of 1,2,3-trisubstituted benzene rings [δH 7.44 (1H,d,J=8.0Hz,H-9), 7.58 (1H,t,J=8.5Hz,H-10), 7.08 (1H,d,J=8.0Hz,H-11)]. An isolated aromatic proton signal [δH 7.50 (1H,s,H-8)], an isolated olefin proton signal [δH 5.79 (1H,s,H-3)], a set of doublet signals [δH 3.03 (1H,dd,J=12.0,17.5Hz,H-7), 3.27 (1H,dd,J=3.0,16.5Hz,H-7)] are the splitting signals of the methylene group, and there is also a set of multiplet signals [δH 4.59 (1H,m,H-6)], suggesting that compound 2 contains an oxygen-bound methine group. Analysis of the 13C-NMR (125MHz, CD3OD) and DEPT spectra revealed that compound 2 has 17 carbon atoms, including 5 sp2-hybridized methines, 1 methyl group, 1 methoxy group, 4 sp2-hybridized quaternary carbons, 3 oxygen-bonded sp2-hybridized tertiary carbons, 1 oxygen-bonded methine, and 1 ester carbonyl group. These NMR data suggest that compound 2 likely belongs to the naphthylpyranone class. Comparison of its spectral data with pannorin B obtained by Kaur et al. from marine organisms revealed a high degree of similarity, with only two differences: First, the 1,2,3-trisubstituted benzene ring signal in compound 2 replaces the pair of meta-coupled aromatic proton signals in pannorin B, indicating that the methoxy and hydroxyl groups at C-10 and C-12 are replaced by hydrogen and methoxy groups, respectively. This is further supported by the HMBC spectrum.Secondly, the chemical shift of C-6 in compound 2 is 24.7 ppm higher in the field compared to the C-6 position of pannorin B, indicating that the hemiacetal carbon at the C-6 position in pannorin B has been replaced by an oxygen-containing methine. This is corroborated by the CH3CH(O)CH2- information provided by 1H-1H COSY and HMBC information. Finally, ECD calculations were performed, and the good agreement between the 6R-2a and 6S-2b curves and the experimental ECD curves was compared. The fitting results show that the absolute configuration at the C-6 position is (6S), and compound 2 is named bercheminol B.

[0040] Compound 3bercheminol C is a brown amorphous powder, [α]20D+9.6 (c 0.05, MeOH); UV (MeOH)λmax(logε)220(2.64),285(2.35)nm; ECD (c 0.5, MeOH)λ(θ)212(+2.65),231(-1.96)nm; specific 1H NMR (500MHz, CD3OD) and 13C NMR (125MHz, CD3OD); HRESIMS m / z 269.08072 [M+H]+ (calcd for C16H12O4, 269.08084). HR-ESI-MS shows that the quasi-molecular ion peak m / z of compound 3 is 269.08072 [M+H]+ (calculated value: 269.08084). Therefore, we speculate that its molecular formula is C16H12O4, with 11 degrees of unsaturation, and 14 fewer mass units than compound 2. Its NMR data are very similar to those of compound 2, also exhibiting a set of 1,2,3-trisubstituted benzene ring signals [δH 7.34 (1H,d,J=7.8Hz,H-9), 7.48 (1H,t,J=7.8Hz,H-10), 7.00 (1H,d,J=7.8Hz,H-11)], an isolated aromatic proton signal [δH 7.50 (1H,s,H-8)], an isolated alkene proton signal [δH 5.77 (1H,s,H-3)], a set of doublet signals [δH 3.00 (1H,dd,J=10.8,16.3Hz,H-7), 3.23 (1H,dd,J=2.4,16.2Hz,H-7)], and a set of multiplet signals [δH The result of 4.61 (1H, m, H-6) indicates that the parent nucleus of compound 3 is essentially the same as that of compound 2, except that the former lacks a methoxy group signal. Therefore, it is speculated that compound 3 is the demethylated product of compound 2, and the information provided by the HMBC spectrum further confirms this conclusion. Its absolute configuration was determined to be 6S-3b by ECD calculation, and it was named bercheminol C.

[0041] Compound 4bercheminol D is a white amorphous powder, [α]20D -8.3 (c 0.02, MeOH); UV(MeOH)λmax(logε) 225 (3.11), 255 (2.98), 360 (2.37) nm; specific 1H NMR (500MHz, CD3OD) and 13C NMR (125MHz, CD3OD); HRESIMS m / z 403.13870 [M+H]+ (calcd for C21H23O8, 403.13874). As a white amorphous powder, HR-ESI-MS revealed a quasi-molecular ion peak of 403.13870 [M+H]+ (calculated value: 403.13874), thus suggesting its molecular formula is C21H22O8 with 11 saturations. According to NMR data, compound 4 exhibits a set of glucose signals [δC 102.3(C-1'), 75.4(C-2'), 78.5(C-3'), 71.4(C-4'), 78.7(C-5'), 62.6(C-6')]. Based on its terminal proton signal [δH 5.26(1H,d,J=7.5Hz,H-1')] and a coupling constant of 7.5Hz, it is presumed to be a β-configuration glucose. Acid hydrolysis experiments, supplemented by HPLC analysis of the retention times of the L-cysteine ​​methyl ester hydrochloride derivatization product and standard sugar of compound 4, confirmed the presence of a D-form glucose. In addition to the glucose signal, the aglycone of 4 also includes an isolated aromatic proton signal [δH 7.50(1H,s,H-6)], a set of 1,2,3-trisubstituted benzene ring signals [δH 7.62(1H,t,J=8Hz,H-8),7.34(1H,d,J=8Hz,H-9),7.50(1H,d,J=7.5Hz,H-7)], an isolated alkene proton signal [δH 6.31(1H,s,H-3)], and two tertiary methyl signals [δH 2.55(3H,s,2-CH3),2.86(3H,s,5-CH3)]. Based on the combined analysis of the information from 13C-NMR (125MHz, CD3OD) and DEPT, compound 4 has a total of 21 carbon atoms. In addition to the carbon atom signals of glucose, the aglycone also contains 5 sp2 hybridized methines, two methyl groups and 8 deprotonated carbons, including 4 sp2 hybridized quaternary carbons, one conjugated carbonyl carbon [δC 182.6(C-4)], and three oxygen-bonded quaternary carbons [δC 167.6(C-2), 158.1(C-1a), 156.4(C-10)].The above NMR information indicates that compound 4 may belong to the angular naphtho-γ-pyranone group (References: Shen JW, Jiang JS, Zhang XF, et al. Two new benzochromone glycosides from the stem of Berchemia racemosa[J]. Journal of Asian Natural Products Research, 2007, 9(6-8): 499-503. and Chovolou Y, Ebada SS,). W, et al. Identification of angular naphthopyrones from the Philippine echinoderm Comanthus species as inhibitors of the NF-κB signaling pathway[J]. European Journal of Pharmacology, 2011, 657: 26-34.). Its NMR data were carefully compared with those of pleuropyrone A (Reference: Min BS, Lee JP, Na MK, et al. A New Naphthopyrone from the Root of Pleuropterus ciliinervis[J]. Chemical & Pharmaceutical Bulletin, 2003, 51(11): 1322-1324.). They were found to be very similar. The main difference is that compound 4 has a signal of a set of 1,2,3-trisubstituted benzene rings, while pleuropyrone A has a pair of meta-coupled aromatic protons. This inference can be further confirmed by the correlation points of HMBC and the H-7 / H-8 / H-9 correlations suggested by 1H-1H COSY. Therefore, the structure of compound 4 was identified and named bercheminol D.

[0042] Compound 5bercheminol E is a white amorphous powder, [α]20D-1.1 (c 0.05, MeOH); UV (MeOH)λmax (logε) 235 (2.36), 270 (2.49), 350 (1.90) nm; 1H NMR (500 MHz, CD3OD) and 13C NMR (125 MHz, CD3OD); HRESIMS m / z 433.14941 [M+H]+ (calcd for C22H25O9, 433.14931). The white amorphous powder, when analyzed by HR-ESI-MS, revealed that the molecular ion peak of compound 5 was [M+H]+ at m / z 433.14941 (calculated value 433.14931), thus its molecular formula is C22H24O9, with 11 degrees of unsaturation, 14 more mass units than pleuropyrone A. NMR signals indicated that compound 5 also contains a β-configuration glucose, which was confirmed to be D-form glucose through acid hydrolysis and HPLC analysis. According to 1H-NMR (500MHz, CD3OD), the aglycone of compound 5 also possesses an isolated aromatic proton signal [δH 7.40 (1H, s, H-6)] and a set of meta-coupled aromatic proton signals [δH 6.95 (1H, d, J = 2.5Hz, H-7), 6.95 (1H, d, J = 2.5Hz, H-9)], which is very similar to pleuropyrone A. Careful comparison of the NMR data of compound 5 and pleuropyrone A revealed the only difference: the former contains an additional methoxy group signal [δH 3.93 (3H, s)], indicating that compound 5 is a product of C-8 methylation of pleuropyrone A. This is further confirmed by the correlation between methoxy groups and C-8 on the HMBC spectrum, leading to the determination of the structure of compound 5 and its naming as bercheminol E.

[0043] Compound 6bercheminol F is a brown amorphous powder, [α]20D -65.0 (c 0.02, MeOH); UV (MeOH)λmax (logε) 230 (3.84), 290 (3.79) nm; ECD (c 0.5, MeOH)λ (θ) 207 (+8.93), 220 (-16.13), 245 (+0.41), 260 (-2.24), 294 (+10.27), 329 (-3.36) nm; 1H NMR (500MHz, CD3OD) and 13C NMR (125MHz, CD3OD); HRESIMS m / z 493.13515 [MH]- (calcd for C23H26O12, 493.13515). Compounds 6 and 7 are both brown amorphous powders. HR-ESI-MS in negative ion mode showed their quasi-molecular ion peaks at m / z: 493.13760 and 493.13589 [MH]- (calculated value: 493.13515), respectively. Therefore, it is inferred that the molecular formula of both compounds is C23H26O12, with 11 degrees of unsaturation. Detailed analysis of their NMR data revealed that compounds 6 and 7 have the same planar structure and belong to dihydroflavonol glycosides containing L-type rhamnose. These conclusions were further confirmed by sugar hydrolysis and HPLC analysis. Comparing their data with those of 6,6-bisastilbi (Reference: Shang XY, Li S, Wang SJ, et al. Chemical constituents of Bauhinia aurea[J]. Journal of Asian Natural Products Research, 2012, 14(10): 966-972.), it was found that they are very similar to the individual structures of this symmetrical structure, except for the substitution mode of the C ring. Specifically, the p-hydroxyphenyl in the C ring of 6,6-bisastilbi is replaced by 3,5-dimethoxy-4-hydroxyphenyl. The correlation between H-2 and 6 with C-2, C-1 and C-4, and the correlation between methoxy and C-3 and C-5 in the HMBC spectrum further confirms this view. Careful analysis of the NMR data of 6 and 7 revealed that the difference between them lies in the absolute configuration of C-2 and C-3, which is very similar to the relationship between neoastilbin and astilbin in the literature.Compared to the (2R,3R) configuration, in the (2S,3S) configuration, H-1 and H-2 at the C-3 position shift to a lower field, while H-5 and CH3-6 shift to a higher field (Reference: DeBritto J, Manickam VS, Gopalakrishnan S, et al. Determination of Aglycone Chirality in Dihydroflavonol 3-O-α-L-Rhamnosides by 1H-NMR Spectroscopy[J]. Chemical & Pharmaceutical Bulletin, 1995, 43, 338-339.). Based on this pattern, we hypothesize that compounds 6 and 7 have (2S,3S) and (2R,3R) configurations at the C-2 and C-3 positions, respectively. The presence of negative and positive Cotton effects at 330 nm in the CD spectra of compounds 6 and 7 further confirms this. Meanwhile, we performed ECD calculations on compounds 6 and 7, and the results supported the above view. Therefore, they were identified and named bercheminol F and bercheminol G, respectively.

[0044] Compound 7bercheminol G is a brown amorphous powder, [α]20D+17.8 (c 0.02, MeOH); UV (MeOH)λmax(logε)230(3.81),295(3.70)nm; ECD (c 0.5, MeOH)λ(θ)208(-7.38),229(+9.17),295(-4.61),321(+2.54)nm; 1H NMR (500MHz, CD3OD) and 13C NMR (125MHz, CD3OD); HRESIMS m / z 493.13515 [MH]- (calcd for C23H26O12, 493.13515).

[0045] Compound 8bercheminol H is a yellow amorphous powder with [α]20D+ 6.0 (c 0.05, MeOH); UV(MeOH)λmax(logε) 210 (3.04), 275 (2.19) nm; 1H NMR (500MHz, CD3OD) and 13C NMR (125MHz, CD3OD); HRESIMS m / z 407.17007 [M+H]+ (calcd for C21H27O8, 407.17004). As an amorphous yellow powder, according to HR-ESI-MS, the quasi-molecular ion peak m / z of compound 8 is 407.17007 [M+H]+, with a calculated value of 407.17004. Therefore, its molecular formula is deduced to be C21H26O8, with 9 degrees of unsaturation. Its NMR spectrum also showed signals belonging to glucose [δC 102.4 (C-1”), 75.0 (C-2”), 78.2 (C-3”), 71.5 (C-4”), 78.2 (C-5”), 62.6 (C-6”)], and terminal proton signals [δH 4.78 (1H,d,J=7.5Hz,H-1”)]. Based on the coupling constant of its terminal protons, it was determined to be β-glucose. Using acid hydrolysis and HPLC analysis, it was identified as D-type β-glucose. Excluding the glucose signal, 1H-NMR (500MHz, CD3OD) showed that compound 8 had two sets of multiplet methylene signals [δH 2.76 (2H,m,H-α), 2.76 (2H,m,H-β)] and seven aromatic proton signals, including [δH 6.39 (1H,br]]. The values ​​[s,H-2), 6.37 (1H,t,J=2.0Hz,H-4), 6.29 (1H,br s,H-6)] suggest a 1,3,5-substituted benzene ring. [δH 7.06 (2H,d,J=8.5Hz,H-2',6'), 6.79 (2H,d,J=8.5Hz,H-3',5')] belong to AA'BB', indicating a para-substituted benzene ring. Based on this information, it is inferred that it has a bibenzyl skeleton. Literature review revealed that the aglycone data are largely consistent with the NMR data of vitarin A, thus it is speculated that compound 8 is a 3-O-glucosylated derivative of vitarin A. The correlation between the anoproton of glucose and C-3 in the HMBC spectrum also supports this inference. Therefore, the structure of compound 8 was determined and named bercheminol H.

[0046] Compound 9, Vittarin-B, is a red amorphous powder with the molecular formula C15H16O3. Its 1H-NMR (500MHz, CD3OD) and 13C-NMR (125MHz, CD3OD) spectra were obtained. A review of the literature (Wu PL, Hsu YL, Zao CW, et al. Constituents of Vittaria anguste-elongata and Their Biological Activities[J]. Journal of Natural Products, 2005, 68(08):1180-1184.) revealed that its spectral data were basically consistent with those of Vittarin-B. Therefore, compound 9 was identified as Vittarin-B.

[0047] Compound 10, Demethylflavasperone-10-O-β-D–glucopyranoside, is a yellow needle-like crystal with the molecular formula C21H22O10. It was analyzed by 1H-NMR (500MHz, DMSO-d6) and 13C-NMR (125MHz, DMSO-d6). By comparing with the literature (Shen JW, Jiang JS, Zhang XF, et al. Two new benzochromone glycosides from thestem of Berchemia racemosa[J]. Journal of Asian Natural Products Research, 2007, 9(6-8):499-503.), compound 10 was finally identified as Demethylflavasperone-10-O-β-D-glucopyranoside.

[0048] Compound 11, Rubrofusarin-6-O-β-D-glucopyranoside, is a yellowish-brown amorphous powder with the molecular formula C24H28O7. It was determined by 1H-NMR (500 MHz, DMSO-d6) and 13C-NMR (125 MHz, DMSO-d6). Comparison with literature confirmed that compound 11 is Rubrofusarin-6-O-β-D–glucopyranoside.

[0049] Compound 12, Rubrofusarin-6-O-β-D-glucopyranoside, is a yellow powder with the molecular formula C27H32O14. It was detected by 1H-NMR (500MHz, DMSO-d6) and 13C-NMR (125MHz, DMSO-d6). Comparison with literature data confirmed that compound 12 is Rubrofusarin-6-O-α-L-rhamnosyl-(1→6)-O-β-D-glucopyranside.

[0050] Compound 13Kaempferol-3-O-α-L-rhamnopyranosyl-(1→6)-β-D-glucopyranoside is a yellow amorphous powder with the molecular formula C27H30O15. ¹H-NMR (600MHz, DMSO-d6) and ¹³C-NMR (150MHz, DMSO-d6) were used. Its spectral data were compared with those in the literature (Sang S, Cheng X, Zhu N, et al. Flavonol glycosides and novel iridoid glycoside from the leaves of Morinda citrifolia[J]. Journal of Agricultural & Food Chemistry, 2001, 49(09):4478-4481.), and it was finally identified as Kaempferol-3-O-α-L-rhamnopyranosyl-(1→6)-β-D-glucopyranoside.

[0051] Compound 14, quercetin, is a yellow powder with the molecular formula C15H10O7. ¹H-NMR (600MHz, DMSO-d6) and ¹³C-NMR (150MHz, DMSO-d6) were used. After careful comparison with data reported in the literature (Xiong Yan, Du Caixia, Duan Yushu, et al. Study on chemical constituents and pharmacological activities of Sedum aizoon from Guizhou [J]. Chinese Traditional and Herbal Drugs, 2019, 50(22):5404-5410.), it was finally identified as quercetin.

[0052] Compound 15, kaempferol, is a pale yellow powder with the molecular formula C15H10O6. Its NMR spectra were obtained using 1H-NMR (600MHz, DMSO-d6) and 13C-NMR (150MHz, DMSO-d6). Careful analysis of the NMR data revealed a strong similarity to the aglycone of compound 13. Further comparison with the literature (Li J, Deng GR, Cheng W, et al. Chemical Constituents of Berchemia Lineata [C]. Medicine and Biopharmaceuticals, Proceedings of the International Conference, 2016, 1140-1148.) confirmed its identity as kaempferol.

[0053] Compound 16 naringenin is a yellow powder with the molecular formula C15H12O5. Its 1H-NMR (500MHz, CD3OD) and 13C-NMR (125MHz, CD3OD) spectra were carefully compared with those in the literature (Gao S, Guang-Miao FU, Fan LH, et al. Flavonoids from Lysidice rhodostegia Hance[J]. Acta Botanica Sinica, 2005, 47(06):759-763.), and it was finally identified as naringenin.

[0054] Compound 17dihydrokaempferol is a yellow powder with the molecular formula C15H12O6. 1H-NMR (500MHz, CD3OD) and 13C-NMR (125MHz, CD3OD). The spectral data of compound 17 and 16 are very similar. The only difference is that the doublet signal has become a single doublet signal [δH 4.52 (1H,d,J=11.5Hz,H-3)]. Therefore, it is considered to be the product of the C-3 position of compound 16 being replaced by a hydroxyl group. This conclusion was confirmed by consulting the literature (Lu CL, Zhu W, Wang DM, et al. Inhibitory Effects of Chemical Compounds Isolated from the Rhizome of Smilax glabra on Nitric Oxide and Tumor Necrosis Factor-α Production in Lipopolysaccharide-Induced RAW264.7 Cell[J]. Evidence-based Complementary and Alternative Medicine, 2015.). Compound 17 was identified as dihydrokaempferol.

[0055] Compound 18, 2-hydroxyemodin 1-methyl ether, is a yellow powder with the molecular formula C16H12O6. ¹H-NMR (500MHz, CD3OD) and ¹³C-NMR (125MHz, CD3OD) were performed. Comparison with literature (…)

[0056] Lin LC, Chou CJ, and Kuo YC. Cytotoxic Principles from Ventilagoleiocarpa[J]. Journal of Natural Products, 2001, 64(05): 674-676.) After comparison, it was finally determined to be 2-hydroxyemodin 1-methyl ether.

[0057] Compound 19, emodin, is a yellow powder with the molecular formula C15H10O5. It was identified as emodin by 1H-NMR (600MHz, DMSO-d6) and 13C-NMR (150MHz, DMSO-d6). Comparison with literature (Cohen PA, Towers GH N. The anthraquinones of Heterodermia obscurata[J]. Phytochemistry, 1995, 40, 911-915.)

[0058] Compound 20 trans-cinnamic acid is a brown powder with the molecular formula C9H8O2. ¹H-NMR (600MHz, CDCl3): 7.54 (¹H, m, H-2), 7.40 (¹H, m, H-3), 7.40 (¹H, m, H-4), 7.40 (¹H, m, H-5), 7.54 (¹H, m, H-6), 6.45 (¹H, d, J=15.6Hz, H-β), 7. 79 (1H, d, J = 15.6 Hz, H-γ); 13C-NMR (150 MHz, CDCl3): 134.2 (C-1), 128.6 (C-2), 129.2 (C-3), 130.9 (C-4), 129.2 (C-5), 128.6 (C-6), 172.6 (C-α), 117.5 (C-β), 147.3 (C-γ). Careful comparison of the 1H-NMR (600 MHz, CDCl3) and 13C-NMR (150 MHz, CDCl3) data of compound 20 revealed that it was essentially consistent with the trans–cinnamicacid reported in the literature (Yang Hui, Hou Aijun, Jiang Bei, et al. A novel compound in *Serratumia serratum*—Serratumin A[J]. *Journal of Plant Taxonomy and Resources*, 2000, 22: 75-80.).

[0059] Compound 21, 7-hydroxy-2,5-dimethylchromon, is a white needle-like crystal with the molecular formula C11H10O3. ¹H-NMR (500MHz, CD3OD): 6.00 (¹H, s, H-3), 6.62 (¹H, d, J=2.5, H-6), 6.63 (¹H, d, J=2.5, H-8), 2.32 (³H, s, 2-Me), 2.71 (³H, br... s,5-Me); 13C-NMR (125MHz, CD3OD): 166.8 (C-2), 118.1 (C-3), 182.1 (C-4), 143.8 (C-5), 111.5 (C-6), 163.3 (C-7), 101.8 (C-8), 161.6 (C-9), 115.7 (C-10), 19.9 (2-Me), 23.3 (5-Me). Its spectral data were compared with those in the literature (Ayer WA, Racok J S. The metabolites of Talaromycesflavus: Part 1. Metabolites of the organic extracts[J]. Cheminform, 2010, 22(15): 2085-2094.), and it was identified as 7-hydroxy-2,5-dimethylchromon.

[0060] The compound 22-methyl 3,4-dihydroxybenzoate is a light brown amorphous powder with the molecular formula C8H8O4. ¹H-NMR (500MHz, CD3OD): 7.22 (¹H, d, J = 3.0 Hz, H-2), 6.79 (¹H, d, J = 9.0 Hz, H-5), 6.97 (¹H, dd, J = 3.0, 9.0 Hz, H-6), 3.92 (³H, s, H-OMe), 119.2 (C-1), 115.5 (C-2), 150.9 (C-3), 156.3 (C-4), 113.4 (C-5), 125.2 (C-6), 171.8 (C=O), 52.9 (OMe). Its spectral data were compared with those in the literature (Tsuda T, Watanabe M, Ohshima K, et al. Antioxidative Components Isolated from the Seed of Tamarind (Tamarindus indica L.)[J]. Journal of Agricultural and Food Chemistry, 1994, 42(12): 2671-2674.), and it was identified as methyl 3,4-dihydroxybenzoate.

[0061] Compound 23Isoarborinol is a white powder with the molecular formula C30H50. ¹H-NMR (600MHz, Pyridine-d5): 3.49 (¹H, m, H-3), 5.33 (¹H, d, J = 6.0Hz, H-11); ¹³C-NMR (150MHz, Pyridine-d5): 36.61 (C-1), 28.49 (C-2), 78.07 (C-3), 40.04 (C-4), 52.84 (C-5), 21.82 (C-6), 27.06 (C-7), 41.16 (C-8), 149.51 (C-9), 39.72 (C-10), 1 14.32(C-11),36.23(C-12),36.96(C-13),38.40(C-14),29.78(C-15), 36.21(C-16),43.01(C-17),52.24(C-18),20.38(C-19),28.73(C-20), 59.68(C-21),30.97(C-22),28.93(C-23),16.59(C-24),22.46(C-25), 17.26(C-26),15.54(C-27),14.15(C-28),23.18(C-29),22.28(C-30). Based on its spectral characteristics, it was inferred to be a terpene compound, and by comparing it with the literature (Sun XB, Zhao PH, Xu YJ, et al. Chemical constituents from the roots of Polygonum bistorta[J]. Chemistry of Natural Compounds, 2007, 43(05): 563-566.), it was identified as Isoarborinol.

[0062] 4. Summary

[0063] This experiment investigated the chemical constituents of *Tea japonica*, extracting and isolating 23 monomeric compounds. Structural identification revealed that compound 1 is a novel skeleton compound; compounds 2 and 3 are naphthoquinones (α-pyranones); compounds 4, 5, 10, 11, and 12 are naphthoquinones (γ-pyranones); compounds 6 and 7 are flavonoids; compounds 8 and 9 are bibenzyl groups; compounds 13, 14, 15, 16, and 17 are flavonoids and dihydroflavonoids; compounds 18 and 19 are anthraquinones; compounds 20, 21, and 22 are simple phenols; and compound 23 is a terpene. Compounds 1-8 are new compounds, and compounds 9-23 are known compounds. Compounds 9-13, 16-18, and 20-23 are compounds isolated from this plant for the first time. The names and molecular formulas of the 23 isolated monomeric compounds are shown in Table 1; the classification and structural formulas of the compounds are shown in the appendix. Figure 1 .

[0064] Table 1

[0065]

[0066] II. Experimental Study on the Anti-inflammatory Activity of Monomer Compounds in *Tea japonica*

[0067] The 23 monomeric compounds extracted and separated using the method described above were screened for anti-inflammatory activity, as follows:

[0068] 1. The specific steps for screening the anti-inflammatory activity of 23 monomeric compounds from *Tea japonica* in this experiment are as follows:

[0069] (1) RAW264.7 cells were cultured in a humid incubator at 37°C and 5% CO2 using complete culture medium (DMEM + 10% FBS + 1% P / S). The cells were passaged every 24 hours, and cells in the logarithmic growth phase were selected for experiments.

[0070] (2) The drug toxicity of the compound was detected by the CCK-8 method;

[0071] (3) RAW264.7 cells were evenly seeded into 96T plates, 100 μL per well. After the cells adhered, the supernatant was discarded. A blank control group and a drug group were set up. Cell culture medium and 100 μL of 50 μM monomer compound were added to the blank control group and the drug group, respectively. After incubation for 24 h, the supernatant was discarded.

[0072] (4) Strictly follow the CCK-8 instructions and measure the absorbance at a wavelength of 450 nm to calculate the cell viability. Calculation formula: Cell viability (%) = [(As-Ab) / (Ac-Ab)] × 100%, Ac = absorbance of experimental wells (absorbance of wells containing cells, culture medium, CCK-8 and the test compound), As = absorbance of blank wells (absorbance of wells containing culture medium and CCK-8), Ab = absorbance of control wells (absorbance of wells containing cells, culture medium and CCK-8);

[0073] (5) Statistical analysis: GraphPad Prism 9.0 software was used to analyze the data. This indicates that comparisons among multiple groups were performed using one-way ANOVA, and pairwise comparisons were performed using the Scheffe method. A p-value < 0.05 or p-value < 0.01 indicates that the data differences are statistically significant.

[0074] (6) The experimental results are shown in Table 2.

[0075] Table 2 Cell viability ( n=3)

[0076]

[0077] 2. The release of NO was detected using the Griess method. The specific steps are as follows:

[0078] (1) A classic inflammatory response model was established by inducing mouse macrophages RAW264.7 with LPS;

[0079] (2) Set up blank control (Control), model group (LPS), positive control group (DXM) and drug group;

[0080] (3) The blank control group was added to DMEM medium, the model group was added to LPS (0.5 μg / mL), the positive control group was added to LPS (0.5 μg / mL) and 10 μM dexamethasone (DXM) and co-incubated, and the drug group was added to LPS (0.5 μg / mL) and 50 μM drug and co-incubated; incubated at 37℃ in an incubator with a CO2 concentration of 5% for 12 h; 50 μL of cell supernatant was taken, and the NO content in the cell supernatant was detected by the Griess method: a new 96-well plate was taken, and 50 μL of cell supernatant, 50 μL of room temperature GriessReagent I, and 50 μL of GriessReagent II were added to each well in sequence, and the absorbance was measured at 540 nm using an ELISA reader. The NO release amount was calculated according to the standard curve, and the NO inhibition rate was calculated according to the OD value;

[0081] (4) Statistical analysis is the same as above;

[0082] (5) The experimental results are shown in Table 3.

[0083] Table 3 NO inhibition rate

[0084]

[0085] Note: Compounds 14, 15, and 19 are known compounds and will not be analyzed.

[0086] 3. Anti-inflammatory activity gradient detection, the specific steps are as follows:

[0087] (1) Set up a blank control group, a model group, a positive drug group, and a drug group;

[0088] (2) Spread RAW264.7 cells in a 96-well plate, wait for the cells to adhere to the wall, and discard the cell supernatant;

[0089] (3) The blank control group was given DMEM medium, and all groups except the normal group were given 0.5 μg / mL LPS. The positive drug group was given DXM with a final concentration of 10 μM. The drug groups were given different concentrations of the test drug (6.25, 12.5, 25, 75 μM).

[0090] (4) The statistical methods are the same as above;

[0091] (5) The experimental results are shown in Table 4.

[0092] Table 4. Effects of compounds on LPS-induced NO release in RAW264.7 cells ( n=3)

[0093]

[0094] Note: **P < 0.01 compared to the control group; **P < 0.01 compared to the LPS model group.

[0095] 4. Experimental results showed that (compounds 14, 15, and 19 were known and not analyzed), after 12 hours of administration, the NO inhibition rate of the novel skeleton compound 01 was 70.81%, and the novel naphthoquinone compounds 02 and 03 exhibited certain anti-inflammatory effects. The NO inhibition rates of the novel bibenzyl compound 08, along with the newly isolated bibenzyl compound 09, flavonoid compounds 06 and 07, the newly isolated anthraquinone compound 18, and the newly isolated dihydroflavonoid compounds 16 and 17, were all above 50%, demonstrating good anti-inflammatory effects. Among these, the NO inhibition rate of the novel skeleton compound was 70.81%, and the NO inhibition rates of the bibenzyl compound 09, dihydroflavonoid compound 17, and anthraquinone compound 19 were all above 80%, indicating good anti-inflammatory activity. The newly isolated simple phenolic compounds 20 and 21 also exhibited some anti-inflammatory activity, but the newly isolated naphthopyranone compound 11 and terpene compound 23 showed poor anti-inflammatory activity. New skeleton compounds, bibenzyl groups, anthraquinones, and dihydroflavonoids all exhibited good anti-inflammatory activity, suggesting that these compounds may be the pharmacodynamic basis for the anti-inflammatory effects of *Gnaphalium affine*.

[0096] The new skeletal compounds and compounds 02, 16, 18, and 21 can all inhibit NO production in a dose-dependent manner. In summary, the new skeletal compounds 1, 2-8, and the monomeric compounds 9, 16-18, 20-21, and 23 isolated from *Gnaphalium affine* can inhibit NO release to varying degrees and have certain anti-inflammatory activity.

Claims

1. A method for extracting multiple monomeric compounds from *Tea japonica*, characterized in that, Includes the following steps: (1) The aerial parts of the fine-leafed tea were dried and crushed, and extracted three times with 70% ethanol-water for 24 hours each time to obtain ethanol extract; then extracted three times with equal volumes of petroleum ether, ethyl acetate and n-butanol respectively, and dried completely to obtain petroleum ether extract, ethyl acetate extract and n-butanol extract. (2) After the n-butanol extract was dissolved in water, it was separated by adsorption of D-101 macroporous resin. The gradient eluents were 30%, 50%, 70%, and 95% ethanol-water. After drying, the extracts were obtained as 30%, 50%, and 70% ethanol extracts. (3) The 50% ethanol extract was subjected to normal phase silica gel column chromatography with dichloromethane / methanol at volume ratios of 20:1, 9:1, 8:2, 7:3, and 1:

1. After TCL detection, similar fractions were combined to obtain fractions A to L in sequence. (4) Component C was repeatedly prepared by HPLC. The ratio of CH3CN:H2O = 32:68 was used to obtain compound 9. Compound 12 was obtained by recrystallizing component H. Component F was subjected to reversed-phase silica gel column chromatography with 30%, 50%, 70%, and 90% methanol-water as gradient eluents. After TCL detection and merging, components F.1 to F.11 were obtained. (5) Component F.2 was repeatedly prepared by HPLC under the condition of CH3CN:H2O=19:81 to obtain compound 6 and compound 7; After recrystallization of fraction F.3, compound 10 was obtained. The mother liquor after filtration was separated by gel column chromatography with methanol as the isocratic eluent, and then repeatedly prepared by HPLC with CH3CN:H2O=19:81 to obtain three compounds: compounds 4, 5 and 8. After recrystallization of fraction F.4 with methanol as solvent, compound 11 was precipitated. (6) The J component was prepared by reversed-phase silica gel column chromatography with 30%, 50%, and 70% methanol-water as gradient eluents, and the results were combined by TCL detection and then repeatedly prepared by HPLC with CH3CN:H2O=18:82 to obtain compound 13. (7) The ethyl acetate extract was separated by normal-phase silica gel column chromatography with petroleum ether / ethyl acetate volume ratios of 15:1, 10:1, 9:1, 8:2, 7:3, 6:4, and 1:1 as the mobile phase. The fractions Fr.1 to Fr.16 were obtained by TCL detection and combination. Fr.6 fraction was recrystallized to obtain white crystals, which was compound 23. Fr.15 fraction was recrystallized to obtain yellow powder, which was compound 14. (8) The Fr.9 fraction was separated by normal-phase silica gel column chromatography with petroleum ether / ethyl acetate volume ratios of 15:1, 10:1, 9:1, 8:2, 7:3, 6:4, and 1:1 as mobile phases. After TCL detection and merging, the fractions were repeatedly prepared by HPLC. Compound 19 was obtained under the condition of CH3CN:H2O = 50:50, and compound 22 was obtained under the condition of CH3CN:H2O = 22:

78. The Fr.14 fraction was separated by reverse-phase silica gel column chromatography with 30%, 50%, 70%, and 90% methanol-water as mobile phases. The fractions were then merged by TCL detection to obtain the Fr.14.1 to Fr.14.14 fractions. (9) Fr.14.7 was repeatedly prepared by HPLC using CH3CN:H2O = 25:75 to obtain three compounds, namely compounds 17, 20 and 21; Compound 16 was obtained by repeated HPLC preparation of Fr.14.10 with CH3CN:H2O = 35:65; compound 1 was obtained by repeated HPLC preparation of Fr.14.11 with CH3CN:H2O = 30:70; and compounds 2, 3, 15 and 18 were obtained by repeated HPLC preparation of Fr.14.14 with CH3CN:H2O = 40:

60. (10) The 23 monomeric compounds extracted above, and the classification and structural formulas of compounds 1-23 are as follows: 。 2. The application of compound 1 obtained by the method for extracting multiple monomeric compounds from *Tea japonica* as described in claim 1 in the preparation of anti-inflammatory drugs.

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