Sesquiterpenoids in glabrous sarcandra herb as well as extraction and separation method and application of sesquiterpenoids

Abstract

The invention discloses sesquiterpenoids in glabrous sarcandra herb as well as an extraction and separation method and application of the sesquiterpenoids, and relates to the technical field of phytochemical extraction and separation. The invention provides a sesquiterpenoids compound in sarcandra glabra, which is characterized by comprising at least one of sarcanlactone F, sarcanlactone G and sarcanlactone H. The invention also provides a preparation method of the sesquiterpenoids compound in sarcandra glabra. The invention provides an extraction method of sesquiterpenoids in sarcandra glabra. Three sesquiterpenoids obtained by the extraction and separation method provided by the invention are new compounds which are not reported. The sarcandra glabra sesquiterpenoids provided by the invention have antibacterial and anti-inflammatory activity, and have potential development value on diseases such as fungal infection, pharyngitis, pneumonia and the like. The sarcandra glabra sesquiterpenoids provided by the invention also have the effects of repairing skin injury and resisting aging, and can also be applied to the field of cosmetics.

Description

Technical Field

[0001] The invention relates to the technical field of plant chemical extraction and separation, in particular to a sesquiterpenoid compound in Camellia sinensis and an extraction and separation method and application thereof. Background Art

[0002] Sarcandra glabra (Thunb.) Nakai, also known as Sarcandra glabra, ...

[0003] Sesquiterpenoids are the most abundant component type in Jiujie Tea, which are distributed in the entire Chrysanthemiaceae family and have multiple activities such as antibacterial, anti-inflammatory, anti-tumor, and liver protection. Li et al. found that chloranthalactone B, a sesquiterpenoid component in Jiujie Tea, can significantly inhibit the production of pro-inflammatory mediators in RAW264.7 macrophages induced by LPS. The mechanism of action is to inhibit the production of inflammatory mediators by inhibiting the AP-1 and p38 MAPK pathways. Yaemaimaiti et al. reported that sarglabolide L, a sesquiterpenoid component in Jiujie Tea, has an inhibitory effect on NO production in RAW 264.7 macrophages induced by LPS, with an IC50 value of 30.65±3.94M. The sesquiterpenoid components 4α,7α-epoxyguaiane-10α,11-diol and nerolidol in Jiujie Tea also inhibited the proliferation of human lung adenocarcinoma cell line A-549, with IC50 values ​​of 7.2 and 4.7 μmol·L, respectively. -1 Shizukaol B, a dimer sesquiterpenoid, can inhibit vascular cell adhesion, especially intracellular cell adhesion molecule-1 (ICAM-1), suggesting that it can be used for arteriosclerosis and inflammatory diseases (rheumatoid arthritis, allergic asthma, osteoporosis, etc.). Sarglaperoxide A has an inhibitory effect on the growth of Staphylococcus aureus. At 25 μg·mL -1The inhibition rate was 64.5%. Oral administration of crude extract of Jiujiecha tea to mice had a significant protective effect on the increase of serum alanine aminotransferase caused by injection of concanavalin A. In addition, sarcaglaboside A-E, chloranoside A and chloranthalactone E-8-O-β-D-glucopyranoside of sesquiterpenoids had a significant protective effect on the toxicity of WB-F344 rat liver epithelial stem cell-like cells induced by D-galactosamine.

[0004] At present, there is no report on sarcandralactone FH (sarcandralactone F-H), nor on its pharmaceutical composition as an active ingredient, nor on its use in the preparation or treatment of fungal infections, anti-inflammatory and analgesic, or tumor drugs, nor on its use in functional cosmetics for skin damage repair and anti-aging. Summary of the invention

[0005] Based on this, the purpose of the present invention is to overcome the shortcomings of the above-mentioned prior art and provide a sesquiterpenoid compound in Jiujiecha and a method for extracting and separating the same and an application thereof.

[0006] To achieve the above object, the technical scheme adopted by the present invention is: a sesquiterpenoid compound in chrysanthemum officinale, including at least one of chrysanthemum officinale lactone F, chrysanthemum officinale lactone G and chrysanthemum officinale lactone H, wherein the structural formula of chrysanthemum officinale lactone F is shown in formula (I), the structural formula of chrysanthemum officinale lactone G is shown in formula (II), and the structural formula of chrysanthemum officinale lactone H is shown in formula (III).

[0007]

[0008] Preferably, the method for extracting sesquiterpenoid compounds from the tea of ​​the Chinese wolfberry leaf comprises the following steps:

[0009] (1) using 10-20 times the amount of water to decoct the tea, reducing the pressure to concentrate, and cooling to obtain an extract;

[0010] (2) extracting the extract with an equal volume of ethyl acetate and collecting the extract;

[0011] (3) subjecting the extract to normal phase silica gel column chromatography, using dichloromethane and / or methanol in different volume ratios as mobile phases, to fractionate the extract to obtain 8 component segments Fr.1→Fr.8;

[0012] Among them, using the volume ratio of dichloromethane to methanol as 100:1 as the mobile phase, component segment Fr.1 is obtained; using the volume ratio of dichloromethane to methanol as 50:1 as the mobile phase, component segment Fr.2 is obtained; using the volume ratio of dichloromethane to methanol as 20:1 as the mobile phase, component segment Fr.3 is obtained; using the volume ratio of dichloromethane to methanol as 10:1 as the mobile phase, component segment Fr.4 is obtained; using the volume ratio of dichloromethane to methanol as 5:1 as the mobile phase, component segment Fr.5 is obtained; using the volume ratio of dichloromethane to methanol as 2:1 as the mobile phase, component segment Fr.6 is obtained; using the volume ratio of dichloromethane to methanol as 1:1 as the mobile phase, component segment Fr.7 is obtained; using methanol as the mobile phase, component segment Fr.8 is obtained;

[0013] (4) Purifying the Fr.1 part using a C18 reverse phase chromatography column with water and methanol to obtain a monomer compound of chamomile lactone F; the volume ratio of water to methanol is 85:15-75:25;

[0014] And / or, purifying the Fr.4 part by using a C18 reverse phase chromatography column with water and methanol to obtain a monomer compound of chamomile lactone G; the volume ratio of water to methanol is 75:25-65:35;

[0015] and / or, fractionating the Fr.7 portion using a reverse phase silica gel column, and gradient eluting using a methanol-water system, with each increase of 10% of the methanol ratio forming a gradient, to obtain 10 component segments Fr.7-1→Fr.7-10;

[0016] Using methanol and water volume ratio of 10:90 as mobile phase, component segment Fr.7-1 is obtained; using methanol and water volume ratio of 20:80 as mobile phase, component segment Fr.7-2 is obtained; using methanol and water volume ratio of 30:70 as mobile phase, component segment Fr.7-3 is obtained; using methanol and water volume ratio of 40:60 as mobile phase, component segment Fr.7-4 is obtained; using methanol and water volume ratio of 50:50 as mobile phase, component segment Fr.7-5 is obtained; using methanol and water volume ratio of 60:40 as mobile phase, component segment Fr.7-6 is obtained; using methanol and water volume ratio of 70:30 as mobile phase, component segment Fr.7-7 is obtained; using methanol and water volume ratio of 80:20 as mobile phase, component segment Fr.7-8 is obtained; using methanol and water volume ratio of 90:10 as mobile phase, component segment Fr.7-9 is obtained; using methanol as mobile phase, component segment Fr.7-10 is obtained;

[0017] The Fr.7-1 part was purified using a C18 reverse phase column with water, acetonitrile and methanol to obtain the monomer compound chamomile lactone H; the volume ratio of water, acetonitrile and methanol was water: acetonitrile: methanol = 80: (1-10): (10-19).

[0018] In addition, the present invention provides the use of the sesquiterpenoid compounds in the nine-section tea in the preparation of antifungal infection, anti-inflammatory and analgesic, and anti-tumor drugs. The present invention also provides the use of the sesquiterpenoid compounds in the nine-section tea in the preparation of skin damage repair and anti-aging cosmetics.

[0019] Furthermore, the present invention provides an antifungal, anti-inflammatory, analgesic and anti-tumor drug, comprising the sesquiterpenoid compounds in the nine-section tea and a pharmaceutically acceptable carrier. The present invention also provides a cosmetic, comprising the sesquiterpenoid compounds in the nine-section tea and a cosmetic matrix.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows: the three sesquiterpenes obtained by the extraction and separation method provided by the present invention are all new compounds that have not been reported, and are respectively named sarcandralactone FH (sarcandralactone F-H). The sarcandra sesquiterpenoid compounds provided by the present invention have antibacterial and anti-inflammatory activities, and have potential development value for fungal infections, pharyngitis, pneumonia and other diseases. The sarcandra sesquiterpenoid compounds provided by the present invention also have the effects of skin damage repair and anti-aging, and can also be applied to the field of cosmetics. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 is the high-resolution mass spectrum of compound 1;

[0022] Figure 2 is the hydrogen spectrum of compound 1;

[0023] Figure 3 is the carbon spectrum of compound 1;

[0024] Figure 4 For compound 1 1 H- 1 H COSY spectrum and key correlations

[0025] Figure 5 HMBC spectrum and key related information of compound 1

[0026] Figure 6 NOESY spectrum and key correlations of compound 1

[0027] Figure 7 is a comparison of the measured and calculated ECD spectra of compound 1;

[0028] Figure 8 is the high-resolution mass spectrum of compound 2;

[0029] Fig. 9 is the hydrogen spectrum of compound 2;

[0030] Fig.10 is the carbon spectrum of compound 2;

[0031] Fig.11 For compound 2 1 H- 1 H COSY spectrum and key correlations

[0032] Fig.12 The HMBC spectrum and key correlations of compound 2

[0033] Fig.13 NOESY spectrum and key correlations of compound 2

[0034] Fig.14 is a comparison diagram of the measured and calculated ECD spectra of compound 2;

[0035] Fig.15 is the high-resolution mass spectrum of compound 3;

[0036] Fig.16 is the hydrogen spectrum of compound 3;

[0037] Fig.17 is the carbon spectrum of compound 3;

[0038] Fig.18 For compound 3 1 H- 1 H COSY spectrum and key correlations

[0039] Fig.19 The HMBC spectrum and key correlations of compound 3

[0040] Fig. 20 NOESY spectrum and key correlations of compound 3

[0041] Fig.21 is a comparison diagram of the measured and calculated ECD spectra of compound 3;

[0042] Fig. 22 Figure 2 is the ON content diagram of the blank control group, model group and drug-added group. DETAILED DESCRIPTION

[0043] In order to better illustrate the purpose, technical scheme and advantages of the present invention, the present invention will be further described below in conjunction with the accompanying drawings and specific embodiments. Its purpose is to understand the content of the present invention in detail, rather than to limit the present invention. All other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. Unless otherwise specified, the experimental reagents and instruments designed for the implementation and comparative examples of the present invention are all commonly used ordinary reagents and instruments, which can be obtained from commercial channels. In the implementation and comparative examples, the experimental methods used are conventional methods unless otherwise specified; and unless otherwise specified, the raw materials used in parallel experiments are the same batch of raw materials.

[0044] The raw materials and instruments used in the experiment are shown in Table 1 below:

[0045] Table 1

[0046]

[0047]

[0048] Example 1

[0049] The present invention provides a method for extracting sesquiterpenoid compounds from Jiujie Tea, and the method for extracting sesquiterpenoid compounds from Jiujie Tea comprises the following steps:

[0050] (1) using 10 times the amount of water to decoct 10 kg of Jiujiecha for 2 hours, decocting twice, concentrating under reduced pressure, and cooling to obtain an extract;

[0051] (2) dissolving the extract powder in water, extracting with equal volumes of ethyl acetate three times, and collecting the extracts;

[0052] (3) subjecting the extract to normal phase silica gel column chromatography, using dichloromethane and / or methanol in different volume ratios as mobile phases, to fractionate the extract to obtain 8 component segments Fr.1→Fr.8;

[0053] Among them, using the volume ratio of dichloromethane to methanol as 100:1 as the mobile phase, component segment Fr.1 is obtained; using the volume ratio of dichloromethane to methanol as 50:1 as the mobile phase, component segment Fr.2 is obtained; using the volume ratio of dichloromethane to methanol as 20:1 as the mobile phase, component segment Fr.3 is obtained; using the volume ratio of dichloromethane to methanol as 10:1 as the mobile phase, component segment Fr.4 is obtained; using the volume ratio of dichloromethane to methanol as 5:1 as the mobile phase, component segment Fr.5 is obtained; using the volume ratio of dichloromethane to methanol as 2:1 as the mobile phase, component segment Fr.6 is obtained; using the volume ratio of dichloromethane to methanol as 1:1 as the mobile phase, component segment Fr.7 is obtained; using methanol as the mobile phase, component segment Fr.8 is obtained;

[0054] (4) The Fr.1 fraction was purified using a C18 reverse phase column with a water / methanol ratio of 75:25 to obtain the monomer compound sarcandralactone F;

[0055] The Fr.4 fraction was purified by C18 reverse phase chromatography with 70:30 water / methanol to obtain the monomer compound sarcandralactone G.

[0056] The Fr.7 part was fractionated using a reverse phase silica gel column and gradient eluted using a methanol-water system. Each increase of 10% methanol ratio constituted a gradient, and 10 component segments Fr.7-1→Fr.7-10 were obtained.

[0057] Using methanol and water volume ratio of 10:90 as mobile phase, component segment Fr.7-1 is obtained; using methanol and water volume ratio of 20:80 as mobile phase, component segment Fr.7-2 is obtained; using methanol and water volume ratio of 30:70 as mobile phase, component segment Fr.7-3 is obtained; using methanol and water volume ratio of 40:60 as mobile phase, component segment Fr.7-4 is obtained; using methanol and water volume ratio of 50:50 as mobile phase, component segment Fr.7-5 is obtained; using methanol and water volume ratio of 60:40 as mobile phase, component segment Fr.7-6 is obtained; using methanol and water volume ratio of 70:30 as mobile phase, component segment Fr.7-7 is obtained; using methanol and water volume ratio of 80:20 as mobile phase, component segment Fr.7-8 is obtained; using methanol and water volume ratio of 90:10 as mobile phase, component segment Fr.7-9 is obtained; using methanol as mobile phase, component segment Fr.7-10 is obtained;

[0058] The Fr.7-1 fraction was purified using a C18 reverse phase column with a ratio of 80:2:18 of water / acetonitrile / methanol to obtain the monomer compound sarcandralactone H.

[0059] Wherein, the structural formula of chamomile lactone F is shown in formula (I), the structural formula of chamomile lactone G is shown in formula (II), and the structural formula of chamomile lactone H is shown in formula (III).

[0060]

[0061] Structural identification and derivation of new compounds extracted and separated by the present invention

[0062] (1) Compound 1: 5β,6β-epoxy-8βH-lidan-7(11)-en-8α,12-olide.

[0063]

[0064] Yellow oil (methanol), +56.7(c 0.1,MeOH). HR-ESI-MS( Figure 1 ) gives the quasi-molecular ion peak [M+Na] + m / z:269.0785(calcd.269.0790for C 14 H 14 O4Na). Combined 1 H-NMR and 13 C-NMR confirmed that its molecular formula is C 14 H 14 O4, unsaturation is 8.

[0065] 1 H NMR (600 MHz, DMSO-d6) Figure 2 ), the high field region gives two methyl hydrogen signals: δ H 1.93(3H,d,J=1.8Hz,C H 3-13),1.10(3H,s,C H 3-14); a group of three-membered ring hydrogen signals: δ H 1.98 (1H, m, H-1), 1.38 (1H, m, H-2α), 1.18 (1H, m, H-2β), 2.14 (1H, m, H-3); a group of methylene hydrogen signals: δ H 2.38 (1H, dd, J = 11.8, 4.3 Hz, H-9α), 1.60 (1H, t, J = 12.2 Hz, H-9β); 2 oxygen-linked methylene hydrogen signals: δ H 4.09(1H,s,H-6),4.95(1H,m,H-8).

[0066] 13 C NMR (150 MHz, DMSO-d6) Figure 3 ), a total of 14 carbon signals are displayed, and 2 methyl carbon signals are visible in the high field region: δ C 8.7( C H3-13),20.1( C H3-14); 1 group of three-membered ring carbon signals: δ C 27.7 (C-1), 11.1 (C-2), 26.6 (C-3); 3 oxygen-linked carbon signals: δ C 56.3 (C-6), 70.1 (C-5), 75.8 (C-8). A group of double bond carbon signals can be seen in the low field region: δ C 128.1 (C-11), 155.6 (C-7); 2 carbonyl carbon signals: δ C 172.7 (C-12), 205.2 (C-4).C 155.6 (C-7), 75.8 (C-8), 128.1 (C-11), 172.7 (C-12), and 8.7 (C-13) indicate that compound 1 has a 3-methyl-5-hydrofuran-2-one structure.

[0067] 1 H- 1 H COSY( Figure 4 ) spectrum, δ H 1.98(H-1),2.14(H-3) and δ H 1.38 (H-2α) is related, suggesting that C-1, C-2, and C-3 are connected in the form of a three-membered ring; δ H 4.95(H-8) and δ H 1.60 (H-9α) and 2.38 (H-9β) were correlated, suggesting the connection of the C8-C9 structural fragment.

[0068] HMBC( Figure 5 ) spectrum, δ H 1.11(C H 3-14) and δ C 27.7 (C-1), 70.1 (C-5), 43.6 (C-9), 34.3 (C-10) long-range correlation, confirm C H 3-14 connected to C-10; δ H 1.93(C H 3-13) and δ C 56.3 (C-6), 155.6 (C-7), 75.8 (C-8), 128.1 (C-11), 172.7 (C-12) long-range correlation, confirm C H 3-13 connected to C-11; δ H 4.95(H-8) and δ C 155.6(C-7),128.1(C-11) telecorrelation;δ H 4.09(H-6) and δ C 205.2 (C-4), 70.1 (C-5), 155.6 (C-7) long-range correlation, through the above long-range correlation, it is further determined that the parent nucleus of the compound is an α, β unsaturated γ-lactone five-membered ring linderane-type sesquiterpenoid. Through literature comparison, it is found that the NMR data of compound 1 and chloranthalactone F are basically consistent, the difference lies in the position of the epoxide ring and the carbon signal of the 4-position carbon. It is inferred that C-4 of compound 1 is a keto carbon. By comparing the carbon-hydrogen signals of chloranthalactone F and compound 1, it is known that C-5 and C-6 of compound 1 are connected to an ethylene oxide. Combining HSQC and 1 H- 1The H COSY spectrum confirmed the planar structure of the compound.

[0069] See literature: Takeda Y, Yamashita H, Matsumoto T, et al.Chloranthalactone F,asesquiterpenoid from the leaves of chloranthus glaber[J].Phytochemistry 1993(33):713-715.

[0070] The relative configuration of compound 1 was determined by NOESY spectroscopy ( Figure 6 ) identification. H 1.11(C H 3-14) and δ H 4.95(H-8),2.38(H-9β)correlation;δ H 1.60(H-9α) and δ H 1.98(H-1),2.14(H-3) related, suggesting C H 3-14, H-8 are located on the same side, H-6, H-1 and H-3 are located on the same side. It is determined that the compound has two possible configurations: 1R, 3S, 6R, 8S, 10R and 1S, 3R, 6S, 8R, 10S. By further testing the ECD spectrum of compound 1, it is compared with the calculated ECD spectrum ( Figure 7 ) were compared and analyzed, and the absolute configuration of compound 1 was determined to be 1R, 3S, 6R, 8S, 10R. According to Sci-finder search, compound 1 is a new α, β-unsaturated γ-lactone five-membered ring linderane-type sesquiterpenoid compound that has not been reported in the literature, named 5β, 6β-epoxy-8βH-lidan-7(11)-en-8α, 12-olide. Its hydrogen and carbon spectrum data are shown in Table 2.

[0071] Table 2 Hydrogen and carbon spectrum data of compound 1 (DMSO-d6)

[0072]

[0073] (2) Compound 2: 4α,15-dihydroxy-5α,8βH-eudesman-1,7(11)-en-8α,12-olide

[0074]

[0075] Yellow oil (methanol), +60.0(c 0.1,MeOH). HR-ESI-MS( Figure 8 ) gives the quasi-molecular ion peak [M+H]+ m / z:265.1440(calcd.265.1440for C 15 H 21 O4). Combine 1 H-NMR and 13 C-NMR confirmed that its molecular formula is C 15 H 20 O4, unsaturation is 6.

[0076] 1 H NMR (600 MHz, DMSO-d6) Fig. 9 ), the high field region gives two methyl hydrogen signals: δ H 1.72(3H,s,C H 3-13),1.11(3H,s,C H 3-14); three groups of methylene hydrogen signals: δ H 0.95 (1H, t, J = 11.4 Hz, H-9α), 2.23 (1H, dd, J = 11.4, 7.8 Hz, H-9β), 1.84 (1H, m, H-3α), 2.25 (1H, m, H-3β), 3.16 (1H, dd, J = 14.0, 3.0 Hz, H-6α), 2.46 (1H, d, J = 13.9 Hz, H-6β); a group of oxymethylene hydrogen signals: δ H 3.44 (1H, d, J = 11.2 Hz, H-15α), 3.37 (1H, d, J = 11.2 Hz, H-15β). The low field region gives a signal of 1 oxygen-linked methylene hydrogen: δ H 5.00 (1H, m, H-8); a group of olefin hydrogen signals: δ H 5.40(1H,ddd,J=10.0,5.1,2.2Hz,H-2),5.45(1H,dd,J=10.0,2.6Hz,H-1).

[0077] 13 C NMR (151 MHz, DMSO-d6) Fig.10 ), a total of 15 carbon signals are displayed, and 2 methyl carbon signals are visible in the high field region: δ C 8.0( C H3-13),20.2( C H3-14); 1 oxymethylene carbon signal: δ C 64.6 (C-15); 1 oxygen-linked quaternary carbon signal: δ C 71.3 (C-4); 1 oxygen-linked methylene carbon signal: δ C 78.0 (C-8). The low field region includes two groups of double bond carbon signals: δ C135.6 (C-1), 122.6 (C-2), 164.7 (C-7), 118.0 (C-11); 1 acyl carbon signal: δ C 174.1(C-12). C 164.7 (C-7), 78.0 (C-8), 118.0 (C-11), 174.1 (C-12), and 8.0 (C-13) indicate that compound 2 has a 3-methyl-5-hydrofuran-2-one structure.

[0078] 1 H- 1 H COSY( Fig.11 ), δ H 5.45(H-1) and δ H 1.84 (H-3α), δ H 5.40 (H-2) is correlated with 2.25 (H-3β), suggesting the connection of C1-C2-C3 structural fragments; δ H 1.56(H-5) and δ H 3.16 (H-6α) and 2.46 (H-6β) are related, suggesting the connection of C5-C6 structural fragments; δ H 5.00(H-8) and δ H There is a correlation between 2.23 (H-9β) and 0.95 (H-9α), suggesting the connection of the C8-C9 structural fragment.

[0079] HMBC( Fig.12 ), δ H 1.11(CH3-14) and δ C 135.6 (C-1), 52.3 (C-5), 47.6 (C-9), 37.3 (C-10) long-range correlation, confirming that CH3-14 is connected to C-10; δ H 1.56(H-5) and δ C 71.3(C-4),47.6(C-9),37.3(C-10),64.6(C-15) long-distance correlation;δ H 1.72(CH3-13) and δ C 165.7 (C-7), 118.0 (C-11), 174.1 (C-12) long-range correlation, confirming that CH3-13 is connected to C-11; δ H 3.44 (H-15β) and 3.37 (H-15α) are respectively C 37.8 (C-3), 71.3 (C-4), 52.2 (C-5) long-range correlation, inferred that the hydroxymethyl fragment is connected to C-4. The above long-range correlation further confirmed that the parent nucleus of the compound is eucalyptane-type sesquiterpenoid. The molecular formula C15 H 20 O4, indicating that there is an extra oxygen atom in its structure, indicating the presence of hydroxyl. Through literature comparison, it was found that the NMR data of compound 2 and atractylenolide IV are basically consistent, and the only difference is the peak shape, coupling constant and carbon signal data of hydrogen at positions 1 and 2 on atractylenolide IV. This shows that the hydroxyl group is connected to C-4, and compound 2 has one more double bond than atractylenolide IV. By comparing the carbon-hydrogen signals at positions 1 and 2 of atractylenolide IV and compound 2, it is known that the double bond is located at positions 1 and 2. Combining HSQC and 1 H- 1 The H COSY spectrum confirmed the planar structure of the compound.

[0080] See the literature: Hu XR, Wu HF, Zhang XP, et al. A new sesquiterpene lactone from Sarcandra glabra [J]. Natural Product Research, 2013, 27(13): 1-5.

[0081] The relative configuration of compound 2 was determined by NOESY spectroscopy ( Fig.13 ) identification. H 1.11(C H 3-14) and δ H 2.46 (H-6β), 5.00 (H-8), 3.45 (H-15) correlation; δ H 1.56(H-5) and δ H 3.16 (H-6α) related, suggesting C H 3-14, H-8, and H-15 are located on the same side, and H-5 and 4-OH are located on the same side. It is determined that the compound has two possible configurations: 4R, 5R, 8S, 10R and 4S, 5S, 8R, 10S. By further testing the ECD spectrum of compound 2, it is compared with the calculated ECD spectrum ( Fig.14 ) were compared and analyzed to determine that the absolute configuration of compound 2 was 4R, 5R, 8S, 10R. According to Sci-finder, compound 2 is a new eudesmanolide-type sesquiterpenoid compound that has not been reported in the literature and is named 4α,15-dihydroxy-5α,8βH-eudesman-1,7(11)-en-8α,12-olide. Its hydrogen and carbon spectrum data are shown in Table 3.

[0082] Table 3 Hydrogen and carbon spectrum data of compound 2 (DMSO-d6)

[0083]

[0084] (3) Compound 3: 4α-hydroxy-5αH-1(2),7(11)-en,8α,12-olide-15-O-β-D-glucopyranoside

[0085]

[0086] Yellow oil (methanol), +50.9(c 0.1, MeOH). HR-ESI-MS( Fig.15 ) gives the quasi-ion peak [M+Na] + m / z:449.1796(calcd.449.1788for C 21 H 30 O9Na). Combined 1 H-NMR and 13 C-NMR confirmed that its molecular formula is C 21 H 30 O9, unsaturation is 7.

[0087] 1 H NMR (600 MHz, DMSO-d6) Fig.16 ), the high field region gives two methyl hydrogen signals: δ H 1.72(3H,s,C H 3-13),1.13(3H,s,C H 3-14); a group of oxymethylene hydrogen signals: δ H 3.37 (1H, d, J = 10.7 Hz, H-15α), 3.97 (1H, d, J = 10.7 Hz, H-15β); 1 sugar terminal hydrogen signal: δ H 4.13 (1H, d, J = 7.7 Hz, H-1'). The low field region gives a signal of a hydrogen bonded to oxygen: δ H 5.01 (1H, m, H-8); a group of olefin hydrogen signals: δ H 5.40(1H,ddd,J=10.0,5.1,2.2Hz,H-2),5.46(1H,dd,J=10.1,2.6Hz,H-1).

[0088] 13 C NMR (151 MHz, DMSO-d6) Fig.17 ), a total of 21 carbon signals are displayed, and 2 methyl carbon signals are visible in the high field region: δ C 8.1 (CH3-13), 20.0 (CH3-14); 8 oxygen-linked carbon signals: δ C70.8 (C-4), 78.0 (C-8), 73.7 (C-15), 73.9 (C-2'), 76.4 (C-3'), 70.1 (C-4'), 77.0 (C-5'), 61.1 (C-6'). The low field region includes a sugar terminal carbon signal: δ C 104.1 (C-1'); two groups of double bond carbon signals: δ C 135.7 (C-1), 122.2 (C-2), 164.7 (C-7), 118.0 (C-11); 1 acyl carbon signal: δ C 174.1 (C-12). The carbon-hydrogen signals suggest that compound 8 is a β-D-glycoside eudesmanolide-type sesquiterpenoid.

[0089] 1 H- 1 H COSY( Fig.18 ), δ H 5.40(H-2) and δ H 2.24 (H-3β), δ H 5.46(H-1) and δ H 1.93 (H-3α) is related, suggesting the connection of C1-C2-C3 structural fragments; δ H 1.54(H-5) and δ H 3.17(H-6α),δ H 2.49 (H-6β) is related, suggesting the connection of C5-C6 structural fragments; δ H 5.01(H-8) and δ H There is a correlation between 2.21 (H-9α) and 0.95 (H-9β), suggesting the connection of the C8-C9 structural fragment.

[0090] HMBC( Fig.19 ), δ H 1.13(C H 3-14) and δ C 135.7 (C-1), 52.4 (C-5), 47.6 (C-9), 37.3 (C-10) long-range correlation, confirming that CH3-14 is connected to C-10; δ H 1.72(CH3-13) and δ C 164.7 (C-7), 78.0 (C-8), 118.0 (C-11), 174.2 (C-12) long-range correlation, confirming that CH3-13 is connected to C-11; δ H 3.97(H-15β), 3.37(H-15α) and δ C38.0 (C-3), 70.8 (C-4), 52.4 (C-5), 104.1 (C-1') long-range correlation, determined that 15-CH2-O-β-D-Glu is connected to C-4. The above long-range correlation further determined that the parent nucleus of the compound is a eucalyptane-type sesquiterpene glycoside. The molecular formula C was given by high-resolution mass spectrometry. 21 H 30 O9, through literature comparison, it was found that the NMR data of compound 3 and compound 3 were basically consistent, and the only difference was the peak shape, coupling constant and carbon signal data of hydrogen at position 1 and 2. In addition, the molecular formula of compound 3 had two fewer hydrogens than compound 3, indicating that compound 3 had one more double bond than compound 3. By comparing the carbon-hydrogen signals at positions 1 and 2 of compound 3 and compound 3, combined with HSQC and 1 H- 1 The H COSY spectrum confirmed the planar structure of the compound.

[0091] See the literature: Hu XR, Yang JS, Xu

[0092] The relative configuration of compound 3 was determined by NOESY spectroscopy ( Fig. 20 ) to determine the relative configuration. H 1.13(C H 3-14) and δ H 5.01 (H-8), 3.96 (H-15β), 0.95 (H-9β) correlation; δ H 1.54(H-5) and δ H 3.17 (H-6α), suggesting that CH3-14, H-8, H-15 are located on the same side, and H-5 and 4-OH are located on the same side. It is determined that the compound has two possible configurations: 4R, 5R, 8S, 10R and 4S, 5S, 8R, 10R. By further testing the ECD spectrum of compound 3, it is consistent with the calculated ECD spectrum ( Fig.21 ) were compared and analyzed to determine that the absolute configuration of compound 3 was 4R, 5R, 8S, 10R. According to Sci-finder search, compound 3 is a new eudesmanolide-type sesquiterpene glycoside compound that has not been reported in the literature and is named 4α-hydroxy-5αH-1(2),7(11)-en-8α,12-olide-15-O-β-D-glucopyranoside. Its hydrogen and carbon spectrum data are shown in Table 4.

[0093] Table 4 Hydrogen spectrum and carbon spectrum data of compound 8 (DMSO-d6)

[0094]

[0095]

[0096] Performance Test-1 Anti-inflammatory Activity Test

[0097] In this experiment, a lipopolysaccharide (LPS)-induced mouse macrophage (RAW264.7 cell) inflammation screening model was used. The anti-inflammatory activity of the monomeric compounds was evaluated by detecting the amount of NO produced by RAW264.7 cells under the induction of LPS as an inducer using the Griess method.

[0098] RAW264.7 cells cultured to the logarithmic growth phase were digested with trypsin and added to the culture medium to adjust the concentration to 1×10 5 500 μL of cell suspension with 5×10 cells / mL was pipetted. 4 The cells were inoculated with LPS at a concentration of 1 g / mL in each well of a 24-well plate and placed in a 5% CO2 incubator at 37°C for 24 hours. Three groups of experiments were set up in the plate, namely, a blank control group (culture solution), a model group (culture solution + LPS) and a drug-added group (culture solution + LPS + different concentrations of compounds). The blank control group and the model group each had 2 wells. The drug-added group added the required concentration of compounds. After the drug-acting cells were incubated in the incubator for 4 hours, LPS with a final concentration of 1 g / mL was added to each well except the blank control group for induction stimulation, and the culture was continued for 24 hours. 50 μL of supernatant from each well was transferred to a 96-well plate, with 2 replicates per well, and then the steps in the NO kit instructions were followed. After sufficient shaking, the absorbance (OD) value was detected at 540 nm using an enzyme reader. 5 μmol / L of parthenolide was set as the positive control. The content and inhibition rate of NO were calculated, and the half inhibition concentration (IC50) was calculated using SPSS software. The experiment was repeated 3 times and the average value was taken. The results are shown in Table 5.

[0099] Table 5 Inhibition rate and IC50 of compounds on NO

[0100]

[0101] In this experiment, the Griess method was used to detect the effects of three compounds at concentrations of 10μmol / L and 20μmol / L on the release of NO from RAW 264.7 cells after LPS induction. The results are shown in Table 5. The inhibition rate of the positive control was 98.49% at a concentration of 5μmol / L. The experimental results showed that the three compounds had varying degrees of inhibitory effects on the secretion of NO in RAW264.7 cells after LPS induction at concentrations of 10μmol / L and 20μmol / L. At a concentration of 20μmol / L, the inhibition rates all reached more than 50%, which were 62.21%, 51.64% and 69.18%, respectively, indicating that the three sesquiterpenoid compounds have good anti-inflammatory activity. The concentrations of the compounds were set at 2.5μmol / L, 5μmol / L, 10μmol / L, 20μmol / L and 40μmol / L to calculate the content and IC50 of NO produced by RAW264.7 cells after LPS induction. The results are shown in Table 5. Fig. 22 As shown in Table 5. Observation and analysis of the chart shows that the NO content in the model group is significantly different from that in the blank control group, and the content has increased significantly, indicating that the anti-inflammatory activity model has been successfully established, while the NO content in the drug-added group at different concentrations has decreased to varying degrees compared with the model group, indicating that the compound has a significant inhibitory effect on the release of NO in the cell model. The IC50 results of each compound show that the compound has good anti-inflammatory activity, and the IC50 are 14.61 1.03μmol / L, 6.00 2.13μmol / L, and 12.08 1.12μmol / L, respectively.

[0102] Performance Test-2 Antimicrobial Activity Test

[0103] Test strains: Two types of test bacteria were selected in this study, including Gram-positive bacteria (G + ) Staphylococcus aureus (S. aureus, ATCC 43300) and Gram-negative bacteria (G - )Escherichia coli (E. coli).

[0104] Preparation of Luria-Bertani (LB) liquid medium: First, weigh 10 g of tryptone, 10 g of sodium chloride, and 5 g of yeast extract, respectively, and dissolve them evenly in 900 mL of deionized water. Then, adjust the pH value to 7.0 with 5 M sodium hydroxide solution and 5 M hydrochloric acid solution, and make up to 1 L with deionized water. Finally, place it at 121 ° C for high temperature sterilization.

[0105] Activation of test bacteria: Take out the bacteria stored in the glycerol tube from the -80℃ refrigerator, take a small amount of the original bacterial solution and activate it twice in fresh sterile LB medium before use.

[0106] In vitro antibacterial activity test: In this study, the isolated compounds were tested for in vitro antibacterial activity using the two-fold dilution method, and the lowest drug concentration corresponding to the well that can be observed by the naked eye was used as its minimum inhibitory concentration (MIC) by visual inspection. At the same time, the bacterial solution can be regarded as a solution, and its bacteria can be idealized into a spherical shape and uniformly distributed. This solution will absorb light of a certain wavelength. Under a certain linear range and a specific wavelength (600nm), its optical density (OD) is proportional to the concentration of the bacterial solution, that is, the larger the OD600 value, the higher the concentration of the bacterial solution, and the smaller the OD600 value, the lower the concentration of the bacterial solution (the higher the inhibition rate). Therefore, the change in optical density (OD600) measured by an enzyme marker can reflect its inhibitory effect and verify the accuracy of the MIC value. In the experiment, 0.5% solvent DMSO was used as a negative control, ciprofloxacin and ampicillin sodium were used as positive controls (25μM), and LB culture medium was used as a blank control. Inhibition rate (I) / % = (OD600 of negative control well - OD600 of test well) / (OD600 of negative control well - OD600 of blank control well) × 100. The specific operation of the experiment is as follows:

[0107] First, the bacteria were inoculated into LB culture medium and cultured to the logarithmic phase. Then the cultured bacterial solution was diluted with culture medium to 2×10 6 CFU / mL (When OD600 is 1, the bacterial density is about 1×10 9 CFU / mL) and inoculated in a 96-well plate. After the test compound is dissolved in DMSO, it is diluted twice continuously with LB culture medium to obtain a series of test solutions with a concentration gradient (100-3.12μM). The test solution of different concentrations is mixed with the bacterial solution in the well in equal volumes (the final concentration of DMSO should be less than 0.5%), placed in a 37°C constant temperature and humidity incubator for 14-16 hours, and then the growth of bacteria in the well plate is carefully observed. The wells with bacterial growth will become turbid, and the wells with sterile growth will be clear (the corresponding concentration is the MIC value of the compound). Each drug concentration set in the experiment is parallel to three wells, and all operations are carried out in a clean bench. The experimental tools, culture medium, etc. are sterilized by ultraviolet or high temperature.

[0108] The minimum inhibitory concentration (MIC) and corresponding inhibition rate (I) of compounds 1, 2 and 3 against Staphylococcus aureus and Escherichia coli are shown in Table 6. It can be seen from the table that these three compounds have obvious inhibitory effects on these four bacteria at their respective MIC concentrations.

[0109] Table 6

[0110]

[0111] Performance test-3 Anti-light damage and anti-aging test

[0112] Cell culture: Skin keratinocytes were cultured in MEM containing 10% fetal bovine serum at 37°C and 5% CO2 saturated humidity.

[0113] Establishment of cell photoaging model: Based on the calculation of ultraviolet radiation dose (J / cm 2 ) = irradiation intensity (W / cm 2 )× irradiation time (s), and the irradiation intensity was measured by an ultraviolet radiation meter.

[0114] Cells in the logarithmic growth phase were inoculated into 96-well plates according to the literature method. After the cells were attached to the wall, the culture medium was discarded and 200 μl PBS was added to each well. 64 mJ / cm 2 UVB irradiation, the blank group was covered with aluminum foil.

[0115] See the literature: Li Wei. Effects of long-wave ultraviolet rays on the morphology, number and expression of inducible nitric oxide synthase in human keratinocytes and fibroblasts [D]. Changsha: Central South University, 2007: 8.

[0116] Experimental grouping: When the cells grew to 80% confluence, they were digested and passaged, inoculated into 96-well plates, and cultured at 37°C, 5% CO2 for 24 hours, and then divided into blank group, model group, low-dose group of compound 1-3, medium-dose group of compound 1-3, and high-dose group of compound 1-3. The concentration of the low-dose group was 10 μmol / L, the concentration of the medium-dose group was 20 μmol / L, and the concentration of the high-dose group was 40 μmol / L. The model group and the drug-treated group were irradiated with ultraviolet light at the above doses.

[0117] MTT detection of cell viability: 48 hours after administration, add 20μl MTT (5mg / ml) to each well, continue to culture for 4 hours, discard the supernatant, add 150μl DMSO to each well to dissolve, shake at room temperature for 10 minutes to dissolve the crystals, and use a microplate reader to detect the light absorption (OD) value at 492nm of each well, and then calculate the cell viability according to the formula. Cell viability = light absorption value of experimental group / light absorption value of control group × 100%

[0118] Detection of SOD, GSH-Px, CAT activity and MDA content in cells: At 48h, collect cells in 6-well plates, wash twice with cold PBS, disrupt cells by ultrasound (power 400W, action 3s, interval 5s, 30 cycles) in an ice bath, centrifuge at 4°C, 12000×g for 15min, collect supernatant in EP tubes precooled on ice. Strictly follow the instructions of the kit to detect SOD, GSH-Px, CAT activity and MDA content.

[0119] The effects of compounds 1-3 on cell viability after UVB irradiation (x±s) are shown in Table 7, and the effects of compounds 1-3 on SOD, GSH-Px, CAT activities and MDA content in HaCaT cells.

[0120] Table 7

[0121]

[0122]

[0123] Compared with the model control group, *P<0.05, **P<0.01

[0124] UVB has a significant inhibitory effect on the growth of keratinocytes cultured in vitro. Different doses of compounds can alleviate the inhibition of keratinocyte growth caused by ultraviolet irradiation. Medium and high doses of compounds 1-3 can significantly increase cell viability after UVB irradiation, among which the high dose of compound 3 has the most obvious effect.

[0125] Table 8

[0126]

[0127] Compared with the model control group, *P<0.05, **P<0.01.

[0128] The effects of UVB irradiation and different doses of compounds 1-3 on the activities of SOD, GSH-Px, CAT and MDA content in HaCaT cells are shown in Table 8. -2 UVB irradiation of HaCaT cells caused oxidative damage, resulting in a significant decrease in the activities of SOD, GSH-Px, and CAT in the model control group, and a significant increase in the content of MDA (P < 0.01); while the medium and high dose groups of compounds 1-3 were able to increase the activities of SOD, GSH-Px, and CAT, and reduce the content of MDA, and the difference was statistically significant compared with the model control group (P < 0.05 or P < 0.01), among which the high dose of compound 3 had the most obvious effect. This indicates that compounds 1-3 can inhibit skin damage caused by ultraviolet rays, and can effectively eliminate free radicals, playing an anti-aging role.

[0129] Performance Test-4 Cell Proliferation and Cell Scratch Test

[0130] Cell grouping: The cells were divided into control group, model group and compound 1-3 intervention group. The control group used complete medium, and the model group and compound 1-3 intervention group were cultured in complete medium with human TNF-α (40 μg / L) for 24 hours. After that, the compound 1-3 group was added with the corresponding compound at a concentration of 40 mol / L. Each group was cultured for 24 hours.

[0131] CCK8 method for detecting cell proliferation: Take each group of keratinocytes in a 96-well plate, set up 3 replicate wells for each group, and put them in an incubator for 48 hours; add CCK-8 reagent (concentration 10ml / well) to each group of cell culture wells. -1 ) and then continue to culture for 5 h. The absorbance (OD) at 450 nm was measured by an enzyme-labeled instrument and the measurement was repeated 3 times to evaluate the cell proliferation activity of each group.

[0132] Cell migration assay: HaCaT cells in the logarithmic growth phase were digested and resuspended at 6×10 3 pc.ml -1 The cells were inoculated in a 96-well plate at a density of 100 μL per well, and cultured in a cell culture incubator at 37°C and 5% CO2 for 24 hours. The culture medium was discarded, and the cells were treated according to the experimental groups. Each group had 3 replicate wells and was cultured in the incubator for another 24 hours. A scratch instrument was used to scratch the 96-well plate vertically, and the culture medium PBS was discarded for washing. Serum-free DMEM was added, and the scratch width was observed at 0 hours, and the healing condition was observed and photographed after 24 hours. ImageJ software was used to estimate the cell migration distance.

[0133] The effects of the compounds on the proliferation and migration of keratinocytes induced by TNF-α are shown in Table 9.

[0134] Table 9

[0135]

[0136] Compared with TNF-α group, *P<0.05; compared with TNF-α group, **P<0.01

[0137] The effects of compounds 1-3 on the proliferation and migration of keratinocytes induced by TNF-α are shown in Table 9. HaCaT cells showed dysregulation of proliferation under the induction of TNF-α, the healing rate of cell scratches decreased, and the number of migrating cells decreased (P<0.01). Compounds 1-3 can significantly inhibit the proliferation of HaCaT cells induced by TNF-α, increase the healing rate of cell scratches, and increase the number of migrating cells (P<0.05), among which compound 1 and compound 3 have the most significant effects (P<0.01). This shows that compounds 1-3 can inhibit cell proliferation caused by inflammatory response and can be used to resist skin damage caused by skin inflammation.

[0138] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A sesquiterpenoid compound in tea of ​​nine sections, characterized in that: It includes at least one of chamomile lactone F, chamomile lactone G and chamomile lactone H, wherein the structural formula of chamomile lactone F is shown in formula (I), the structural formula of chamomile lactone G is shown in formula (II), and the structural formula of chamomile lactone H is shown in formula (III).

2. The method for extracting sesquiterpenoid compounds from Rhizoma Cibotii as claimed in claim 1, characterized in that: The steps include: (1) using 10-20 times the amount of water to decoct the tea, reducing the pressure to concentrate, and cooling to obtain an extract; (2) extracting the extract with an equal volume of ethyl acetate and collecting the extract; (3) subjecting the extract to normal phase silica gel column chromatography, using dichloromethane and / or methanol in different volume ratios as mobile phases, to fractionate the extract to obtain 8 component segments Fr.1→Fr.8; Among them, using the volume ratio of dichloromethane to methanol as 100:1 as the mobile phase, component segment Fr.1 is obtained; using the volume ratio of dichloromethane to methanol as 50:1 as the mobile phase, component segment Fr.2 is obtained; using the volume ratio of dichloromethane to methanol as 20:1 as the mobile phase, component segment Fr.3 is obtained; using the volume ratio of dichloromethane to methanol as 10:1 as the mobile phase, component segment Fr.4 is obtained; using the volume ratio of dichloromethane to methanol as 5:1 as the mobile phase, component segment Fr.5 is obtained; using the volume ratio of dichloromethane to methanol as 2:1 as the mobile phase, component segment Fr.6 is obtained; using the volume ratio of dichloromethane to methanol as 1:1 as the mobile phase, component segment Fr.7 is obtained; using methanol as the mobile phase, component segment Fr.8 is obtained; (4) Purifying the Fr.1 part using a C18 reverse phase chromatography column with water and methanol to obtain a monomer compound of chamomile lactone F; the volume ratio of water to methanol is 85:15-75:25; And / or, purifying the Fr.4 part by using a C18 reverse phase chromatography column with water and methanol to obtain a monomer compound of chamomile lactone G; the volume ratio of water to methanol is 75:25-65:35; and / or, fractionating the Fr.7 portion using a reverse phase silica gel column, and gradient eluting using a methanol-water system, with each increase of 10% of the methanol ratio forming a gradient, to obtain 10 component segments Fr.7-1→Fr.7-10; Using methanol and water volume ratio of 10:90 as mobile phase, component segment Fr.7-1 is obtained; using methanol and water volume ratio of 20:80 as mobile phase, component segment Fr.7-2 is obtained; using methanol and water volume ratio of 30:70 as mobile phase, component segment Fr.7-3 is obtained; using methanol and water volume ratio of 40:60 as mobile phase, component segment Fr.7-4 is obtained; using methanol and water volume ratio of 50:50 as mobile phase, component segment Fr.7-5 is obtained; using methanol and water volume ratio of 60:40 as mobile phase, component segment Fr.7-6 is obtained; using methanol and water volume ratio of 70:30 as mobile phase, component segment Fr.7-7 is obtained; using methanol and water volume ratio of 80:20 as mobile phase, component segment Fr.7-8 is obtained; using methanol and water volume ratio of 90:10 as mobile phase, component segment Fr.7-9 is obtained; using methanol as mobile phase, component segment Fr.7-10 is obtained; The Fr.7-1 part was purified using a C18 reverse phase column with water, acetonitrile and methanol to obtain the monomer compound chamomile lactone H; the volume ratio of water, acetonitrile and methanol was water: acetonitrile: methanol = 80: (1-10): (10-19).

3. A use of the sesquiterpenoid compounds in the Herba Lycopodii of claim 1 in the preparation of antifungal, anti-inflammatory, analgesic and antitumor drugs.

4. A use of the sesquiterpenoid compounds in the Herba Lycopodii of claim 1 in the preparation of skin damage repair and anti-aging cosmetics.

5. An antifungal, anti-inflammatory, analgesic, and anti-tumor drug, characterized in that: The invention comprises the sesquiterpenoid compounds in the tea of ​​claim 1 and a pharmaceutically acceptable carrier.

6. A cosmetic, characterized in that: The invention comprises the sesquiterpenoid compounds in the tea of ​​claim 1 and a cosmetic matrix.

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