Camellia oleifera leaf extract and extraction method thereof

Abstract

The invention belongs to the technical field of camellia oleifera extracts, and particularly relates to camellia oleifera leaf extracts and an extraction method thereof.The camellia oleifera leaf extracts are 3, 5-dimethoxy dihydrostilbene 4 '-O-alpha-L-furan arabinofuranosyl-(1-> 6)-beta-D-glucopyranoside, 3, 5-dimethoxy dihydrostilbene 4'-O-alpha-L-furan arabinofuranosyl-(1-> 6)-beta-D-glucopyranoside, 3, 5- the raw materials comprise 3, 5-dimethoxy dihydrostilbene 4 '-O-alpha-L-arabinopyranosyl-(1-> 6)-beta-D-glucopyranoside and 3, 5-dimethoxy dihydrostilbene 4'-O-beta-D-furan apigenin glycosyl-(1-> 6)-beta-D-glucopyranoside, and the raw materials comprise 3, 5-dimethoxy dihydrostilbene 4 '-O-alpha-L-arabinopyranosyl-(1-> 6)- The anti-inflammatory activity of the new natural products is evaluated by using LPS-stimulated RAW264.7 macrophages, and when the concentration is lower than 40 [mu] M, the tested compound has an inhibiting effect on the generation of nitric oxide, but basically has no cytotoxicity, which indicates that the compound possibly has potential anti-inflammatory activity.

Description

Technical Field

[0001] The present invention belongs to the technical field of camellia oleifera extracts, and particularly relates to camellia oleifera leaf extracts and an extraction method thereof. Background Art

[0002] Camellia oleifera is a plant of the genus Camellia in the family Theaceae, and is widely distributed in Jiangxi, Hunan, Fujian, Guangdong, Guangxi and other places in China. As a traditional Chinese medicine, its dry leaves are officially included in "Chinese Herbal Medicine" and "National Compilation of Chinese Herbal Medicines", and have the effects of clearing heat and detoxifying, astringing and stopping bleeding, refreshing the mind, promoting blood circulation and removing stasis, and relieving pain. It is used as a medicine for treating nosebleeds, skin ulcer itching, ulcerative gangrene, acute pharyngitis, stomachache, sprains and contusions.

[0003] Previous phytochemical studies on this plant have identified triterpenoids and their saponins, flavonoids and their glycosides, dihydrostilbenoids and their glycosides, anthraquinones, lignans, phenolic compounds, organic acids, steroids and fatty acids, and have shown various biological activities, such as anti-tumor, anti-inflammatory, anti-neuroinflammatory, neuroprotective, antioxidant, antibacterial, hypoglycemic and antithrombotic. However, there are many camellia oleifera extracts, and the research on camellia oleifera is still incomplete. At present, further research is still needed. In addition to applying the discovered extracts, continuously discovering new extracts from camellia oleifera also helps to further expand the application of camellia oleifera. For this reason, the present invention provides a camellia oleifera leaf extract and an extraction method thereof. Summary of the Invention

[0004] In order to solve the above technical problems, the present invention provides a camellia oleifera leaf extract and an extraction method thereof. The present invention has studied the methanol extract of camellia oleifera leaves, discovered 3 new natural products, and provided an extraction and separation method, structure determination and anti-inflammatory activity evaluation, providing a basis for further expanding the application of camellia oleifera.

[0005] The present invention is specifically implemented through the following technical solutions.

[0006] The first object of the present invention is to provide a camellia oleifera leaf extract, which is a dihydrostilbenoid glycoside, namely 3,5-dimethoxydihydrostilbene 4'-O-α-L-arabinofuranosyl-(1→6)-β-D-glucopyranoside, 3,5-dimethoxydihydrostilbene 4'-O-α-L-arabinopyranosyl-(1→6)-β-D-glucopyranoside or 3,5-dimethoxydihydrostilbene 4'-O-β-D-apiofuranosyl-(1→6)-β-D-glucopyranoside, specifically having the structures shown in formula (1), formula (2) or formula (3):

[0007]

[0008] The second object of the present invention is to provide a method for extracting the tea leaf extract of Camellia oleifera, comprising the following steps:

[0009] The tea leaves of Camellia oleifera are crushed into powder, extracted with methanol for multiple times, and after combining the extracts, the solvent is removed to obtain a residue; the residue is placed in water and successively extracted with petroleum ether, ethyl acetate and n-butanol to obtain a petroleum ether extract, an ethyl acetate extract and an n-butanol extract respectively; after the ethyl acetate extract is purified, a compound with the structure shown in formula (1), formula (2) or formula (3) is obtained.

[0010] In a preferred embodiment of the present invention, the purification method of the ethyl acetate extract is: separation is carried out by silica gel column chromatography (100-200 mesh), and gradient elution is carried out successively with CH2Cl2-MeOH with a volume ratio of (100:1)-(1:3), each component is collected, and similar components are combined by thin layer chromatography analysis to obtain an elution product Fr.E5 with a volume ratio of (5:1)-(3:1) of CH2Cl2-MeOH;

[0011] Fr.E5 is purified again by silica gel column chromatography, and the elution system is CH2Cl2-MeOH-H2O with a volume ratio of (10:2:0.1)-(1:3:0.2) to obtain an elution product Fr.E5.4 with a volume ratio of 3:1:0.1 of CH2Cl2-MeOH-H2O;

[0012] The Fr.E5.4 is further separated and purified by semi-preparative C 18 high performance liquid chromatography, and gradient elution is carried out with MeOH-H2O with a volume ratio of (30:70)-(70:30) to obtain a compound with the structure shown in formula (1), formula (2) or formula (3).

[0013] In a preferred embodiment of the present invention, HCOOH is further added to the MeOH-H2O elution system, and the addition amount is 0.1% of the total volume of MeOH and H2O. At a retention time t R of 35.73 min and an elution gradient of MeOH-H2O-HCOOH with a volume ratio of 65.73:34.27:0.1, a compound with the structure shown in formula (1) is obtained;

[0014] At a retention time t R of 36.27 min and an elution gradient of MeOH-H2O-HCOOH with a volume ratio of 66.27:33.73:0.1, a compound with the structure shown in formula (2) is obtained;

[0015] At a retention time t RIt was 36.66 min, and the elution gradient was MeOH-H2O-HCOOH with a volume ratio of 66.66:33.34:0.1 to obtain the compound with the structure shown in formula (3).

[0016] In a preferred embodiment of the present invention, when separating and purifying by semi-preparative C 18 high performance liquid chromatography, the flow rate was 3 mL / min.

[0017] In a preferred embodiment of the present invention, when extracting Camellia oleifera leaves, the mass concentration of methanol used was 70%-90%, and the dosage ratio of the total amount of Camellia oleifera leaves and methanol was 1.4 kg:15 L.

[0018] In a preferred embodiment of the present invention, methanol was used for extraction 3 times respectively. The temperature of methanol extraction was 60-70 °C, and each extraction was refluxed for 2-4 h.

[0019] In a preferred embodiment of the present invention, 4.61 mg of the compound with the structure shown in formula (1), 2.96 mg of the compound with the structure shown in formula (2), and 2.61 mg of the compound with the structure shown in formula (3) were obtained from every 1 kg of Camellia oleifera leaves.

[0020] The present invention also provides the application of the extract of Camellia oleifera leaves in the preparation of anti-inflammatory reagents.

[0021] The third object of the present invention is to provide an anti-inflammatory reagent, which includes one or several of the compounds with the structures shown in formula (1), formula (2), and formula (3).

[0022] The present invention has the following beneficial effects compared with the prior art:

[0023] In order to further supplement the types of Camellia oleifera extracts, the present invention conducted extraction research on Camellia oleifera leaves, provided 3 new dihydrostilbene glycoside compounds, and carried out structure identification, which were 3,5-dimethoxydihydrostilbene 4′-O-α-L-arabinofuranosyl-(1→6)-β-D-glucopyranoside, 3,5-dimethoxydihydrostilbene 4′-O-α-L-arabinopyranosyl-(1→6)-β-D-glucopyranoside or 3,5-dimethoxydihydrostilbene 4′-O-β-D-apiofuranosyl-(1→6)-β-D-glucopyranoside respectively.

[0024] The anti-inflammatory activities of these new natural products were evaluated with LPS-stimulated RAW264.7 macrophages. The results showed that when the concentration was lower than 40 μM, the tested compounds 1-3 had an inhibitory effect on the production of nitric oxide, but had no cytotoxicity, indicating that these compounds may have potential anti-inflammatory activities.

[0025] The extraction method of the present invention is simple, providing method support for further research on Camellia oleifera extracts. Description of the Drawings

[0026] Figure 1 1H NMR spectrum of Compound 1 (methanol-d4); 1

[0027] Figure 2 13C NMR spectrum of Compound 1 (methanol-d4); 13

[0028] Figure 3 DEPT spectrum of Compound 1 (methanol-d4);

[0029] Figure 4 HSQC spectrum of Compound 1 (methanol-d4);

[0030] Figure 5 HMBC spectrum of Compound 1 (methanol-d4);

[0031] Figure 6 1H- 1 1 1H COSY spectrum of Compound 1 (methanol-d4);

[0032] Figure 7 NOESY spectrum of Compound 1 (methanol-d4);

[0033] Figure 8 HRESIMS spectrum of Compound 1;

[0034] Figure 9 ESIMS spectrum of Compound 1;

[0035] Figure 10 1H NMR spectrum of Compound 2 (methanol-d4); 1

[0036] Figure 11 13C NMR spectrum of Compound 2 (methanol-d4); 13

[0037] Figure 12 DEPT spectrum of Compound 2 (methanol-d4);

[0038] Figure 13 HSQC spectrum of Compound 2 (methanol-d4);

[0039] Figure 14 HMBC spectrum of Compound 2 (methanol-d4);

[0040] Figure 15 1H- 1 ​​​​​H- 1 1H COSY spectrum (deuterated methanol-d4);

[0041] Figure 16 is the NOESY spectrum (deuterated methanol-d4) of Compound 2;

[0042] Figure 17 is the HRESIMS spectrum of Compound 2;

[0043] Figure 18 is for Compound 3 1 1H NMR spectrum (deuterated methanol-d4);

[0044] Figure 19 is for Compound 3 13 13C NMR spectrum (deuterated methanol-d4);

[0045] Figure 20 is the DEPT spectrum (deuterated methanol-d4) of Compound 3;

[0046] Figure 21 is the HSQC spectrum (deuterated methanol-d4) of Compound 3;

[0047] Figure 22 is the HMBC spectrum (deuterated methanol-d4) of Compound 3;

[0048] Figure 23 is for Compound 3 1 H- 1 1H-1H COSY spectrum (deuterated methanol-d4);

[0049] Figure 24 is the NOESY spectrum (deuterated methanol-d4) of Compound 3;

[0050] Figure 25 is the HRESIMS spectrum of Compound 3;

[0051] Figure 26 Effect of Compounds 1-3 on cell viability in LPS-induced RAW264.7 cells. Values are expressed as the mean ± S.D. of three experiments. Compared with the blank control group, *p<0.05, **p<0.01 and ***p<0.001.

[0052] Figure 27 Inhibitory effect of Compounds 1-3 on NO production in LPS-induced RAW264.7 cells. Butein: positive control. Values are expressed as the mean ± S.D. of three experiments. Detailed implementation method

[0053] To enable those skilled in the art to better understand and implement the technical solution of the present invention, the present invention will be further described below in conjunction with specific embodiments and drawings. However, the exemplified embodiments shall not be construed as limiting the present invention. In the following exemplified embodiments, the experimental methods and detection methods are all conventional methods unless otherwise specified; the reagents and materials are all commercially available unless otherwise specified.

[0054] The present invention provides an extract of Camellia oleifera leaves, which is a dihydrostilbene glycoside compound, namely 3,5-dimethoxydihydrostilbene 4'-O-α-L-arabinofuranosyl-(1→6)-β-D-glucopyranoside, 3,5-dimethoxydihydrostilbene 4'-O-α-L-arabinopyranosyl-(1→6)-β-D-glucopyranoside or 3,5-dimethoxydihydrostilbene 4'-O-β-D-apiofuranosyl-(1→6)-β-D-glucopyranoside, specifically having the structures shown in Formula (1), Formula (2) or Formula (3):

[0055]

[0056] The extraction method of the extract of Camellia oleifera leaves of the present invention comprises the following steps:

[0057] Crush the Camellia oleifera leaves into powder, extract with methanol multiple times, combine the extracts and then remove the solvent to obtain a residue; place the residue in water, and extract successively with petroleum ether, ethyl acetate and n-butanol to obtain a petroleum ether extract, an ethyl acetate extract and an n-butanol extract respectively; after purifying the ethyl acetate extract, the purification method is: separate by silica gel column chromatography (100-200 mesh), and perform gradient elution successively with CH2Cl2-MeOH with a volume ratio of (100:1)-(1:3), collect each component, and combine similar components by thin layer chromatography analysis to obtain a CH2Cl2-MeOH elution product Fr.E5 with a volume ratio of (5:1)-(3:1);

[0058] Purify Fr.E5 again by silica gel column chromatography, and the elution system is CH2Cl2-MeOH-H2O with a volume ratio of (10:2:0.1)-(1:3:0.2) to obtain a CH2Cl2-MeOH-H2O elution product Fr.E5.4 with a volume ratio of 3:1:0.1;

[0059] Further separate and purify Fr.E5.4 by semi-preparative C 18 High performance liquid chromatography, perform gradient elution with MeOH-H2O with a volume ratio of (30:70)-(70:30). The elution method is: HCOOH is also added to the MeOH-H2O elution system, and the addition amount is 0.1% of the total volume of MeOH and H2O. At the retention time tR It is 35.73 min, and the elution gradient is MeOH - H2O - HCOOH with a volume ratio of 65.73:34.27:0.1 to obtain the compound with the structure shown in formula (1);

[0060] At the retention time t R It is 36.27 min, and the elution gradient is MeOH - H2O - HCOOH with a volume ratio of 66.27:33.73:0.1 to obtain the compound with the structure shown in formula (2);

[0061] At the retention time t R It is 36.66 min, and the elution gradient is MeOH - H2O - HCOOH with a volume ratio of 66.66:33.34:0.1 to obtain the compound with the structure shown in formula (3).

[0062] The following is a specific description through the following examples.

[0063] Example 1

[0064] The extraction method of the oil tea leaf extract includes the following steps:

[0065] Crush the dry leaves (2.8 kg) of oil tea into powder, and extract three times with 70% methanol (3 × 10 L) at 60°C for 2 hours each time. Remove the solvent under reduced pressure to obtain a residue, suspend it in distilled water, and extract successively with petroleum ether (60 - 90), ethyl acetate, and n-butanol to obtain petroleum ether extract (36.7 g), ethyl acetate extract (201 g), and n-butanol extract (357.4 g) respectively.

[0066] The ethyl acetate extract (201 g) is separated by silica gel column chromatography, and gradient elution is carried out with CH2Cl2 - MeOH (100:1, 50:1, 30:1, 20:1, 10:1, 5:1, 3:1, 1:1, 1:3, v / v). Collect each component, and combine similar components by thin layer chromatography analysis to obtain 7 main components (Fr.E1 - Fr.E7). Fr.E5 is the elution product of CH2Cl2 - MeOH with a volume ratio of 5:1 - 3:1. Fr.E5 (84.55 g) is purified again by silica gel column chromatography, and the elution system is CH2Cl2 - MeOH - H2O (10:2:0.1 - 1:3:0.2, v / v / v) to obtain 6 sub-components (Fr.E5.1 - Fr.E5.6). Fr.E5.4 is the elution product of CH2Cl2 - MeOH - H2O with a volume ratio of 3:1:0.1. Fr.E5.4 (350 mg) is further purified by semi-preparative C 18Separation and purification were carried out by high performance liquid chromatography, and gradient elution was performed using (MeOH-H2O, 30:70 - 70:30, v / v, containing 0.1% HCOOH by volume, flow rate 3 mL / min), and the compound with the structure shown in formula (1), namely compound 1 (12.9 mg), was obtained at a retention time t R of 35.73 min and an elution gradient of 65.73% MeOH.

[0067]

[0068] The structural characterization was as follows Figures 1 - 9 . The nuclear magnetic resonance and mass spectrometry data of compound 1 were as follows: Compound 1 was a white amorphous powder, 1 1H and 13 13C NMR (400 and 100 MHz, methanol-d4) spectroscopic data are shown in Table 1 in the appendix; the molecular formula was: C 27 H 36 O 12 ; the measured value of HR-ESI-MS was m / z: 575.2092 [M+Na] + (the theoretically calculated value was C 27 H 36 O 12 Na, 575.2099). See Table 1 in detail.

[0069] Example 2

[0070] The extraction method of the Camellia oleifera leaf extract includes the following steps:

[0071] The dry leaves (2.8 kg) of Camellia oleifera were ground into powder and extracted three times with 70% methanol (3×10 L) at 60 °C for 2 hours each time. The solvent was removed under reduced pressure to obtain a residue, which was suspended in distilled water and extracted successively with petroleum ether (60 - 90), ethyl acetate, and n-butanol to obtain petroleum ether extract (36.7 g), ethyl acetate extract (201 g), and n-butanol extract (357.4 g) respectively.

[0072] The ethyl acetate extract (201 g) was separated by silica gel column chromatography, and gradient elution was carried out with CH2Cl2-MeOH (100:1, 50:1, 30:1, 20:1, 10:1, 5:1, 3:1, 1:1, 1:3, v / v). Each fraction was collected, and similar fractions were combined and analyzed by thin layer chromatography to obtain 7 main fractions (Fr.E1-Fr.E7). Fr.E5 was the elution product with CH2Cl2-MeOH at a volume ratio of 5:1-3:1. Fr.E5 (84.55 g) was purified again by silica gel column chromatography, and the elution system was CH2Cl2-MeOH-H2O (10:2:0.1-1:3:0.2, v / v / v) to obtain 6 sub-fractions (Fr.E5.1-Fr.E5.6). Fr.E5.4 was the elution product with CH2Cl2-MeOH-H2O at a volume ratio of 3:1:0.1. Fr.E5.4 (350 mg) was further separated and purified by semi-preparative 18 high performance liquid chromatography. Gradient elution was carried out using (MeOH-H2O, 30:70-70:30, v / v, containing 0.1% HCOOH, flow rate 3 mL / min), and a compound with the structure shown in formula (2), namely compound 2 (8.3 mg), was obtained at a retention time t R of 36.27 min (elution gradient 66.27% MeOH).

[0073]

[0074] The structural characterization was as follows Figures 10 - 17 . The nuclear magnetic resonance and mass spectrometry data of compound 2 were as follows: Compound 2 was a white amorphous powder, 1 1H and 13 13C NMR (400 and 100 MHz, methanol-d4) spectral data are shown in Table 1; the molecular formula was: C 27 H 36 O 12 ; the actual measured value of HR-ESI-MS was m / z: 575.2091 [M+Na] + (the theoretically calculated value for C 27 H 36 O 12 Na was 575.2099). See Table 1 for details.

[0075] Example 3

[0076] The extraction method of the Camellia oleifera leaf extract comprises the following steps:

[0077] The dry leaves of Camellia oleifera (2.8 kg) were ground into powder and extracted three times with 70% methanol (3×10 L) at 60 °C for 2 hours of reflux each time. The solvent was removed under reduced pressure to obtain a residue, which was suspended in distilled water and extracted successively with petroleum ether (60-90), ethyl acetate and n-butanol to obtain petroleum ether extract (36.7 g), ethyl acetate extract (201 g) and n-butanol extract (357.4 g), respectively.

[0078] The ethyl acetate extract (201 g) was separated by silica gel column chromatography and eluted with CH2Cl2-MeOH (100:1, 50:1, 30:1, 20:1, 10:1, 5:1, 3:1, 1:1, 1:3, v / v) in a gradient manner. Each fraction was collected and similar fractions were combined by thin layer chromatography analysis to obtain 7 main fractions (Fr.E1-Fr.E7). Fr.E5 was the elution product with CH2Cl2-MeOH at a volume ratio of 5:1-3:1. Fr.E5 (84.55 g) was purified again by silica gel column chromatography with an elution system of CH2Cl2-MeOH-H2O (10:2:0.1-1:3:0.2, v / v / v) to obtain 6 sub-fractions (Fr.E5.1-Fr.E5.6). Fr.E5.4 was the elution product with CH2Cl2-MeOH-H2O at a volume ratio of 3:1:0.1. Fr.E5.4 (350 mg) was further separated and purified by semi-preparative C 18 high performance liquid chromatography, and gradient elution was carried out with (MeOH-H2O, 30:70-70:30, v / v, containing 0.1% HCOOH, flow rate 3 mL / min). At a retention time t R of 36.66 min (elution gradient 66.66% MeOH), a compound (7.3 mg) with the structure shown in formula (3) was obtained, namely compound 3.

[0079]

[0080] The structural characterization is as Figures 18 - 25 . The nuclear magnetic resonance and mass spectrometry data of compound 3 are as follows: Compound 3 is a white amorphous powder, 1 1H and 13 13C NMR (400 and 100 MHz, methanol-d4) spectral data are shown in Table 1; the molecular formula is: C 27 H 36 O 12 ; the actual measured value of HR-ESI-MS is m / z: 575.2091 [M+Na] + (the theoretically calculated value is C 27 H 36 O 12Na, 575.2099). See Table 1 for details.

[0081] Table 1 of new compounds 1 - 3 1 H (400 MHz) and 13 C NMR (100 MHz) data. δ in ppm, J in Hz.

[0082]

[0083]

[0084] a) Recorded in CD3OD. b) Multiplicities inferred from DEPT and HSQC experiments.

[0085] From the above, it can be seen that three compounds were successfully extracted from the leaves of Camellia oleifera in this invention. Next, a preliminary efficacy test was conducted on the above three compounds. The specific method is as follows:

[0086] In this invention, the anti - inflammatory activities of these compounds 1 - 3 were evaluated using LPS - stimulated RAW264.7 macrophages. An inflammatory model was established by inducing RAW 264.7 cells with LPS at a concentration of 1 μg / mL. A blank control group, an LPS - induced group, an LPS and test group (three different concentrations were set for each compound, and the compound concentrations were 20 μM, 40 μM, and 80 μM respectively), and a positive control group (Butein) were set up. The MTT method was used to detect cell viability, and the Griess reagent was used to detect the production of nitric oxide. The results are as Figure 26 and Figure 27 shown. The results show that when the concentration was 20 μM and 40 μM, the tested compounds 1 - 3 had potential anti - inflammatory activities, and the NO inhibition rates were 32.2%, 27.6%, 18.6% and 63.2%, 46.3%, 51.0% respectively. When the concentration was lower than 40 μM, the tested compounds 1 - 3 had an inhibitory effect on the production of nitric oxide but basically no cytotoxicity, indicating that these compounds may have potential anti - inflammatory activities.

[0087] Obviously, those skilled in the art can make various changes and modifications to this invention without departing from the spirit and scope of this invention. Thus, if these modifications and variations of this invention fall within the scope of the claims of this invention and their equivalent technologies, these changes and variations are also intended to be included.

Claims

1. Tea leaf extract of Camellia oleifera, characterized in that, It is a dihydrostilbene glycoside compound, and its chemical structural formula is shown in Formula (1), Formula (2) or Formula (3):

2. The extraction method of the tea leaf extract of Camellia oleifera according to claim 1, characterized in that, It includes the following steps: Crush the camellia oleifera leaves into powder, extract with methanol multiple times, remove the solvent after combining the extracts to obtain a residue; place the residue in water, and extract successively with petroleum ether, ethyl acetate and n-butanol to obtain a petroleum ether extract, an ethyl acetate extract and an n-butanol extract respectively; after purifying the ethyl acetate extract, obtain the compound shown in Formula (1), Formula (2) or Formula (3).

3. The extraction method according to claim 2, characterized in that, The purification method of the ethyl acetate extract is: separate by silica gel column chromatography, elute with CH2Cl2-MeOH with a volume ratio of (100:1)-(1:3) in gradient, collect each component, and combine similar components by thin layer chromatography analysis to obtain the CH2Cl2-MeOH elution product Fr.E5 with a volume ratio of (5:1)-(3:1). Purify Fr.E5 again by silica gel column chromatography, and the elution system is CH2Cl2-MeOH-H2O with a volume ratio of (10:2:0.1)-(1:3:0.2) to obtain the CH2Cl2-MeOH-H2O elution product Fr.E5.4 with a volume ratio of 3:1:0.

1. The Fr.E5.4 is further separated and purified by semi-preparative C 18 high performance liquid chromatography, and gradient elution is carried out with MeOH-H2O having a volume ratio of (30:70)-(70:30) to obtain a compound having a structure represented by formula (1), formula (2) or formula (3).

4. The extraction method according to claim 3, wherein, HCOOH is also added to the MeOH-H2O elution system, and the addition amount is 0.1% of the total volume of MeOH and H2O. The compound shown in Formula (1) is obtained at the MeOH-H2O-HCOOH with a retention time of 35.73 min and an elution gradient of 65.73:34.27:0.1 by volume. The compound shown in Formula (2) is obtained at the MeOH-H2O-HCOOH with a retention time of 36.27 min and an elution gradient of 66.27:33.73:0.1 by volume. The compound shown in Formula (3) is obtained at the MeOH-H2O-HCOOH with a retention time of 36.66 min and an elution gradient of 66.66:33.34:0.1 by volume.

5. The extraction method according to claim 3, wherein Separation and purification were carried out using semi-preparative C 18 by high performance liquid chromatography at a flow rate of 3 mL / min.

6. The extraction method according to claim 2, wherein When extracting camellia oleifera leaves, the mass concentration of methanol used is 70%-90%, and the dosage ratio of camellia oleifera leaves to methanol is 1.4 kg:15 L.

7. The extraction method according to claim 6, wherein Extract with methanol 3 times respectively, the temperature of methanol extraction is 60-70 °C, and each extraction is refluxed for 2-4 h.

8. The extraction method according to claim 2, characterized in that 4.61 mg of the compound shown in Formula (1), 2.96 mg of the compound shown in Formula (2) and 2.61 mg of the compound shown in Formula (3) are obtained from every 1 kg of camellia oleifera leaves.

9. Use of the camellia oleifera leaf extract according to claim 1 in the preparation of an anti-inflammatory reagent.

10. An anti-inflammatory agent, characterized in that, It includes one or more of the compounds shown in Formula (1), Formula (2) and Formula (3) in claim 1.

Images