Tobacco source compound composition and application

Abstract

The invention discloses a tobacco source compound composition and application, and relates to the technical field of skin care. The composition is prepared from any one or more of telangiol, sclareol, 2-oxo telangiol, 2 [alpha]-hydroxy telangiol, 2 [beta]-hydroxy telangiol and lavandan enediol. The monomeric compound composition disclosed by the invention has relatively high synergistic activity for inhibiting propionibacterium acnes. Within a certain concentration range, the diterpenoid monomeric compounds play a synergistic antibacterial effect with high efficiency and low dosage, and the purposes of synergism and toxicity reduction can be achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of skin care, and more particularly to a tobacco-derived compound composition and application thereof. Background Art

[0002] Acne is a chronic inflammatory skin disease that is common in adolescents. It affects the hair follicles and sebaceous glands of the skin, causing scars and pigmentation, and causing skin damage. When the ducts of the hair follicles and sebaceous glands are blocked, an anaerobic environment rich in lipids will be generated, which is conducive to the proliferation of Propionibacterium acnes, a skin flora colonized in the sebaceous follicles, producing a lipase that decomposes triglycerides in sebum and produces free fatty acids, changing the skin environment and destroying the balance of the flora. It also secretes hydrolases, hyaluronidases, etc., further destroying the skin barrier and causing acne inflammation. It is the main microorganism that affects the onset of acne. Therefore, inhibiting the growth and reproduction of Propionibacterium acnes is an effective means of treating acne. At present, the common way to treat acne is oral or topical antibiotics, such as erythromycin, tetracycline, retinoic acid, clindamycin, nitroimidazole, etc., but there are still problems such as skin irritation, obvious drug resistance and cross-resistance. Natural products have the advantages of rich pharmacological effects, safety, high efficiency and few side effects. Therefore, exploring and developing natural products with good antibacterial effects, few adverse reactions and low drug resistance has good application prospects.

[0003] Studies have found that diterpenoids have the characteristics of broad antibacterial spectrum, low toxicity, multiple targets, and high antibacterial activity, and have been proven to have the effect of inhibiting Propionibacterium acnes. Some scholars have found that farnesol, a sesquiterpenoid, has the effect of inhibiting acne, eliminating body odor and sweat odor, and exhibits a mild antibacterial effect; and Xin Xiaoen's research found that sclareol in Salvia miltiorrhiza is a potential effective ingredient in the Xiaocuo Decoction for the treatment of acne. When the cyclic monoterpenoid compound terpinene-4-ol in plant essential oils and α-bisabolol, one of the main active ingredients in chamomile, are used in combination, they can inhibit the growth of Propionibacterium acnes, with an inhibitory concentration index FICI < 0.5, showing a synergistic effect. The minimum inhibitory concentration (MIC) of terpinene-4-ol is 0.31 g / L, and the MIC of α-bisabolol is reduced to 0.39-1.56 g / L.

[0004] Nicotiana glutinosa is a wild species of tobacco, native to Peru, Bolivia and Ecuador, and its surface is rich in lybendazim diterpenoids, mainly including sclareol, 2-ketoepimanool, labdenediol, manool and other compounds. Currently, there are few reports on drug combination formulas of lybendazim diterpenoids specifically used to inhibit Propionibacterium acnes and with high synergistic antibacterial activity.

[0005] Therefore, providing new uses for tobacco-derived compounds, optimizing the combination ratio of the compounds, improving the antibacterial effect of drugs while reducing the drug concentration, reducing side effects, and reducing the risk of drug resistance development are issues that need to be urgently addressed by those skilled in the art. Summary of the invention

[0006] In view of this, the present invention provides a tobacco-derived compound composition and application.

[0007] In order to achieve the above object, the present invention adopts the following technical solution:

[0008] A tobacco-derived compound composition comprises any one or more of dacryotaxol, sclareol, 2-oxodacryotaxol, 2α-hydroxydacryotaxol, 2β-hydroxydacryotaxol and lisdandiol.

[0009] Furthermore, the composition comprises at least any two of sclareol, 2β-hydroxylactotaxel and lisdendioic acid.

[0010] Furthermore, the mass ratio of the composition includes at least one of the following three ratios:

[0011] The ratio of sclareol to 2β-hydroxylactotaxol is 2 to 5:1;

[0012] The ratio of sclareol to lysine glycol is 1-20:1-20;

[0013] The ratio of 2β-hydroxy-lactotaxel to lisdendiolide is 1 to 5:1.

[0014] The invention discloses an application of a tobacco-derived compound composition in preparing an active drug for inhibiting Propionibacterium acnes.

[0015] It can be seen from the above technical solution that, compared with the prior art, the beneficial effects of the present invention are:

[0016] The monomer compounds extracted by the present invention, such as dacryotaxol, sclareol, 2-oxodacryotaxol, 2α-hydroxydacryotaxol, 2β-hydroxydacryotaxol and lisdendioic acid, have a higher inhibition rate on Propionibacterium acnes than tetracycline at 50 μg / mL.

[0017] The monomer compound composition of the present invention has a high synergistic inhibition activity of Propionibacterium acnes. When the concentration of sclareol is 20-25 μg / mL, the concentration of 2β-hydroxy-dacryl is 10-5 μg / mL, and the ratio of the two is 2-5:1, a synergistic antibacterial effect is achieved. When the concentration of sclareol is 20-1 μg / mL, the concentration of lysine glycol is 1-20 μg / mL, and the two are compounded in a ratio of 1-20:1-20, a synergistic antibacterial effect can be achieved. When the concentration of 2β-hydroxy-dacryl is 20-10 μg / mL, and the concentration of lysine glycol is 4-10 μg / mL, the two are compounded in a ratio of 1-5:1, a synergistic antibacterial effect can be achieved. Within a certain concentration range, diterpenoid monomer compounds exert synergistic antibacterial effects with high efficiency and low doses, which can achieve the purpose of synergism and toxicity reduction. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0019] Figure 1 The accompanying drawing is a diagram of monomer compounds separated and identified from the glandular hair secretions of the tobacco of the present invention;

[0020] Figure 2 The accompanying drawing is a graph showing the inhibitory effect of the primary separation product of tobacco of the present invention on Propionibacterium acnes;

[0021] Figure 3 The accompanying drawing is a diagram showing the inhibitory effect of the tobacco sticky monomer compound of the present invention on Propionibacterium acnes. DETAILED DESCRIPTION

[0022] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0023] Source of test materials:

[0024] Nicotiana glutinosa resources: Nicotiana glutinosa seeds were provided by the National Tobacco Germplasm Resource Medium-term Bank. They were planted at the Jimo Experimental Base of the Tobacco Research Institute of the Chinese Academy of Agricultural Sciences (Jimo, Shandong). Conventional methods were used for sowing, transplanting and transplanting.

[0025] Test bacteria: Propionibacterium acnes freeze-dried powder, purchased from Beina Biotechnology, product number BNCC330605.

[0026] Test reagents:

[0027] Table 1 Main test reagents

[0028]

[0029] Test equipment:

[0030] Table 2 Main test instruments

[0031]

[0032]

[0033] Example 1

[0034] Preparation of crude extract of Nicotiana tabacum

[0035] Collect the inflorescence of Nicotiana tabacum, extract it with ethanol (95%) at room temperature, the solid-liquid ratio is 1:5, the extraction time is 1-2min, and the extraction is continued for 2-3 times. Then spray and rinse the surface of the tobacco leaves with anhydrous ethanol twice the mass of the raw material, and combine the extracts. The above extracts are concentrated under reduced pressure at 50°C, and the solvent is removed to obtain a crude extract of Nicotiana tabacum glandular hairs. Then, the crude extract of Nicotiana tabacum glandular hairs is redissolved with dichloromethane: methanol (1:1, v / v), and the solvent is removed from the dissolved part to obtain a crude extract of glandular hairs.

[0036] Primary separation of crude extracts from tobacco

[0037] The crude extract of glandular hair components of Nicotiana tabacum was separated by reduced pressure silica gel column chromatography (TLC).

[0038] Mixing samples: Use the obtained glandular hair crude extract as raw material, gradually add it into 100-200 mesh silica gel of equal mass, stir thoroughly, and place in a dry place to dry.

[0039] Column packing: Use 300g of 100-200 mesh silica gel for dry column packing. Gently tap the column to make the column surface flat, turn on the vacuum pump to pump the bottom of the column for 20 minutes, and set aside. Carefully add the dried sample to the column surface, and make the sample surface flat, turn on the vacuum pump and continue to pump for 20 minutes.

[0040] Elution: According to the principle of increasing polarity, ethyl acetate / petroleum ether system (1:50→1:1.v) was first used, followed by methanol / dichloromethane system (1:20→1:1.v), and finally pure methanol was used for elution under reduced pressure, and 12 groups of eluents with different polarities were obtained. After decompression and concentration to remove the solvent, the primary separation products (Fr.1-T~Fr.6) were obtained. GC / MS was used to detect and analyze and merge similar components, among which Fr.1-T was mainly weakly polar higher alkanes and alkanols, and there were no diterpenoid compounds, which were not used for subsequent separation.

[0041] Separation of monomeric compounds

[0042] Based on the component composition, Fr.2 to Fr.5 rich in diterpenes were further separated and purified.

[0043] Specific separation process:

[0044] Fr.2 was eluted through a forward silica gel column with an elution system of petroleum ether: ethyl acetate (50:1, v / v) (Note: the elution system is v / v, the same below) to obtain Fr.2.1 and Fr.2.2. The eluate of Fr.2.1 was enriched and concentrated by TLC, and then eluted through a gel column with an elution system of dichloromethane: methanol (1:1) to obtain compound 1. Fr.2.2 was subjected to preparative thin layer chromatography (Prep-TLC) with a developing system of petroleum ether: ethyl acetate (50:1) to obtain compound 2.

[0045] Fr.3 was separated using a C-18 reverse phase silica gel column and analyzed by TLC. A mixture of 50% methanol concentration and 60% methanol concentration was mixed and analyzed by Prep-TLC under the development conditions of dichloromethane:methanol (30:1) to obtain compound 3.

[0046] After Fr.4 was chromatographed on a C-18 reverse phase silica gel column, combined with TLC and GC / MS analysis, the sample eluted at 80% methanol concentration was eluted through a gel column with an elution system of dichloromethane: methanol (1:1) to obtain compound 4. The samples eluted at 60%-70% methanol concentration were combined and eluted through a forward silica gel column with an elution system of dichloromethane: methanol (80:1→50:1→30:1), and separated into Fr.4.1 and Fr.4.2 by TLC. Fr.4.1 was eluted through a forward silica gel column with an elution system of petroleum ether: ethyl acetate (7:1) to obtain compounds 5 and 6. Fr.4.2 was eluted through a forward silica gel column with an elution system of petroleum ether: ethyl acetate (4:1) to obtain Fr.4.2.1 and compound 7. Fr.4.2.1 was analyzed by TLC, HPLC and other means, and Fr.4.2.1 was purified by Prep-TLC under the development conditions of petroleum ether:acetone (5:1) to obtain compound 8.

[0047] Fr.5 was chromatographed on a gel column with methanol as the eluent at a flow rate of 0.4 mL / min. A C-18 reverse phase column was then used for separation. Combined with TLC analysis, the samples eluted at 60% methanol concentration and 70% methanol concentration were combined. Prep-HPLC was used with acetonitrile: water (65:35) as the mobile phase. Fr.5.1 was divided into two sections according to the peak time: Fr.5.1.1 and Fr.5.1.2. According to TLC and HPLC analysis, Fr.5.1.1 and compound 7 are the same substance. Fr.5.1.2 was eluted with a dichloromethane: methanol (50:1) elution system through a forward silica gel column, and after reduced pressure concentration, it was purified again using preparative liquid chromatography (Prep-HPLC), and the chromatographic conditions were the same as above. The sample after reduced pressure concentration will be used again using Prep-TLC with a developing system of petroleum ether: ethyl acetate (3:1) to obtain compound 9.

[0048] Identification of diterpenoid monomer compounds

[0049] A total of 9 compounds were isolated and identified, including 8 lisdactyl diterpenoids and 1 sesquiterpenoid ( Figure 1 ).

[0050] Compound 1 is a colorless colloid with no fluorescence under 254nm ultraviolet light. It turns purple-red after heating with anisaldehyde-sulfuric acid color developer. Its chemical structure was obtained by NMR nuclear magnetic resonance spectroscopy analysis. HNMR (500Hz, CDCl3), δH 1.52(4H,m,H-1),0.99(1H,td,J=10.5,3.5Hz,H-2α),1.52(4H,m,H-2β),1. 45(1H,m,H-3α),1.67(1H,dt,J=10.5,2.0Hz,H-3β),1.72(1H,d,J=11.0Hz,H -5),0.77(3H,s,H-6),1.92(1H,td,J=10.5,4.0Hz,H-7α),2.32(1H,dq,J=10 .5Hz,1.5,H-7β),3.17(1H,dd,H-9),1.07(1H,dd,J=10.5Hz,2.0,H-11α),1. 35(1H,dd,J=10.5Hz,2.0,H-11β),1.52(4H,m,H-12α),4.35(1H,s,H-12β),5 .82(1H,dd,J=14.5,9.0Hz,H-14),4.93(1H,s,H-15α),5.11(1H,dd,J=14.5H z,2.0,H-15β),1.22(3H,m,H-16),4.48(1H,s,H-17α),4.77(1H,s,H-17β),0 .85(3H,s,H-18),1.11(3H,s,H-19),0.62(3H,s,H-20),1.52(4H,m,OH-13). 13C NMR(125Hz,CDCl3)δC 39.2(C-1,CH2),19.5(C-2,CH2),42.3(C-3,CH2),39.9(C-4,C),55.7(C-5.CH),24. 5(C-6,CH2),38.5(C-7,CH),148.9(C-8,C),57.3(C-9,CH),33.6(C-10,C),17.7(C-1 1, CH2), 41.4 (C-12, CH2), 73.8 (C-13, C), 145.2 (C-14, CH), 111.7 (C-15, CH2), 33.7 (C-16, CH3), 106.4 (C-17, CH2), 28.1 (C-18, CH3), 21.8 (C-19, CH3), 14.5 (C-20, CH3). After literature search, its NMR data is basically consistent with the report in the literature (BURDEN et al., 1975), so compound 1 is identified as Manool.

[0051] Compound 2 is in the form of white powder, has no fluorescence under 254nm ultraviolet light, and turns purple-red after heating with anisaldehyde-sulfuric acid colorimetric agent. Its chemical structure was obtained by NMR nuclear magnetic resonance spectrum analysis. 1HNMR (500Hz, CDCl3), δH 1.22 (4H, s, H-1), 1.37 (2H, dd, (10.5, 3.0), H-2), 1.46 (5H, m, H-3), 0.85 (4H, s, H-5), 0.94 (1H, dd, J = 10.5, 2.5Hz, H-6α), 1.77 (1H, dt, J = 10, 3.0Hz, H-6β), 1.46 (5H, m, H-7α), 1.62 (1H, m, H-7β), 2.21 (1H, m, H-9), 0.85 (4H, s, H-11), 1.46(5H,m,H-12),4.97(1H,d,J=15Hz,H-14),4.91(1H,d,J=9.5Hz,H-15α),6.01(1H,dd,J=10.5,3.0Hz,H-15β),1 .13(4H,s,H-16),1.22(4H,s,H-16),1.13(4H,s,H-17),0.85(4H,s,H-18),0.783(4H,s,H-19),0.723(4H,s,H-20). 13C NMR(125Hz,CDCl3)δC 34.8(C-1,CH2),15.9(C-2,CH2),39.3(C-3,CH2),33.3(C-4,C),56.5(C-5,CH),19. 9(C-6,CH2),42.2(C-7,CH2),76.1(C-8,C),58.5(C-9,CH),36.8(C-10,C),18.6(C-1 1, CH2), 43.1 (C-12, CH2), 73.3 (C-13, C), 147.7 (C-14, CH), 109.5 (C-15, CH2), 33.2 (C-16, CH3), 32.7 (C-17, CH3), 23.9 (C-18, CH3), 21.2 (C-19, CH3), 15.8 (C-20, CH3). After literature search, its NMR data was basically consistent with that reported in the literature (JEONG et al., 2022), so compound 2 was identified as Manoyl oxide.

[0052] Compound 3 is in the form of white powder, and has fluorescence under 254nm ultraviolet light. Anisaldehyde-sulfuric acid color developer turns green after heating. Its chemical structure was obtained by NMR nuclear magnetic resonance spectrum analysis. 1HNMR (500Hz, CDCl3), δH 5.92 (1H, s, H-1), 3.8 (1H, d, J = 10.0Hz, H-3), 1.25 (4H, d, J = 5.5Hz, H-4), 1.65 (1H, m, H-5), 2.23 (4H, m, H-6α), 2.57 (1H, dt, J = 12.5, 3.0Hz, H-6β), 2.23 (4H, m, H-7), 1.4 (1H, qd, J = 11.0, 3.5Hz,H-8α),1.93(1H,dq,J=9.0,2.5Hz,H-8β),1.05(1H,q,J=10.5Hz,H-9α),2.23(4H,m,H -9β), 4.47 (2H, d, H-12), 1.74 (3H, s, H-13), 1.25 (4H, d, J = 5.5 Hz, H-14), 2.23 (4H, m, OH-3). 13C NMR (125Hz,CDCl3)δC 121(C-1,CH),199.6(C-2,C),76.3(C-3,CH),29.7(C-4,CH),42.5(C-5,CH),35.4(C-6,CH2),44.5(C-7,CH),31.4(C-8,CH2),36.9(C-9,CH2),148.6(C-10,C),166.9(C-11,C),109.5(C-12,CH2),20.7(C-13,CH3),15.6(C-14,CH3). According to literature search, its NMR data is basically consistent with that reported in the literature (BOHLMANN et al.,1982), so compound 3 is identified as Glutinosone.

[0053] Compound 4 is a white powder with no fluorescence under 254nm ultraviolet light. It turns purple-red after heating with anisaldehyde-sulfuric acid color developer. Its chemical structure was obtained by NMR nuclear magnetic resonance spectrum analysis. 1HNMR (500Hz, CDCl3), δH 1.4 (5H, m, H-1α), 1.6 (5H, m, H-1β), 1.4 (5H, m, H-2), 0.8 (4H, S, H-3α), 1.6 (5H, m, H-3β), 3.3 (2H, d, J = 2.25Hz, H-5), 1.1 (4H, s, H-6 / 7), 4.1 (1H, q, J = 6.0, 3.0Hz, H-9), 1.4 (5H, m, H-11), 3.2 (3H, d, J = 1.5Hz, H-12), 4.9 ( 1H,d,J=3.0Hz,H-14),5.1(1H,d,J=10.5Hz,H-15α),5.8(1H,dd.J=10.0,6.0Hz,H-15β),1.6(5H,m,H-16),1. 0(3H,s,H-17),0.8(4H,s,H-18),0.7(3H,s,H-19),3.2(3H,d,J=1.5Hz,OH-8),3.3(2H,d,J=2.25Hz,OH-13). 13C NMR(125Hz,CDCl3)δC 38.8(C-1,CH2),18.0(C-2,CH2),44.1(C-3,CH2),33.3(C-4,C),55.8(C-5,CH),20. 1(C-6,CH2),45.5(C-7,CH2),72.3(C-8,C),61.1(C-9,CH),41.7(C-10,C),19.0(C-1 1, CH2), 48.6 (C-12, CH2), 72.1 (C-13, C), 146.6 (C-14, CH), 110.6 (C-15, CH2), 32.9 (C-16, CH3), 27.7 (C-17, CH3), 23.9 (C-18, CH3), 21.4 (C-19, CH3), 15.2 (C-20, CH3). According to literature search, its NMR data is consistent with the literature ( et al., 2010) reported that compound 4 was identified as Sclareol.

[0054] Compound 5 is a colorless, granular crystal with no fluorescence under 254nm ultraviolet light. It turns blue-purple after heating with anisaldehyde-sulfuric acid color developer. Its chemical structure was obtained by NMR nuclear magnetic resonance spectroscopy analysis. 1HNMR (500Hz, CDCl3), δH 1.63(2H,m,H-1α),2.17(1H,d,J=10.5Hz,H-1β),1.79(2H,m,H-3α),2.28(1H,d,J=10.5Hz,H-3β),2.4(2,d,J=10.0Hz,H-5),1.32(4H, m,H-6α),1.79(2H,m,H-6β),2.0(1H,td,J=10.5,4.0Hz,7-Hα),2.4(2H,d,J=10.0Hz,H-7β),2.1(1H,dd,J=11.0,2.0Hz,H-9),1.32(4H, m,H-12α),1.63(2H,m,H-12β),5.84(1H,dd,J=14.5,9.0Hz,H-14),5.01(1H,d,J=9.0Hz,H-15α),5.16(1H,d,J=14.5,9.0Hz,H-15β),1. 01(3H,s,H-16),4.51(1H,S,H-17α),4.84(1H,S,H-17β),1.22(3H,s,H-18),0.79(2H,s,H-19),0.64(3H,s,H-20),5.25(1H,s,OH-13). 13CNMR(125Hz,CDCl3)δC 54(C-1,CH2),211.9(C-2,C),56.1(C-3,CH),39(C-4,C),54.7(C-5,CH2),24.3(C -6,CH2),37.5(C-7,CH2),146.9(C-8,C),56.6(C-9,CH),45(C-10,C),18(C-11,C H2),40.8(C-12,CH2),73.3(C-13,C),144.8(C-14,CH),111.8(C-15,CH2),33.3( C-16,CH3),107.7(C-17,CH2),27.9(C-18,CH3),23(C-19,CH3),15.1(C-20,CH3). After literature search, its NMR data was basically consistent with the report in the literature (COHEN et al., 1983), and compound 5 was identified as 2-ketoepimanool.

[0055] Compound 6 is a colorless, granular crystal with no fluorescence under 254nm ultraviolet light. It turns purple-red when heated with anisaldehyde-sulfuric acid color developer. Its chemical structure was obtained by NMR nuclear magnetic resonance spectroscopy analysis. 1HNMR (500Hz, CDCl3), δH 1.55(3H,m,H-1α),1.79(1H,dd,J=12.5,4.5Hz,H-1β),4.16(1H,m,H-2),1.55(3H,m,H-3α),1.65(1H,m,H-3β),1.15(1H,dd,J=10.0,2.5 Hz,H-5),1.41(5H,m,H-6α),1.71(2H,m,H-6β),1.95(1H,td,J=10.5,4.5Hz,H-7α),2.38(1H,dt,J=10.5Hz,3.0Hz,H-7β),1.55(3H,m,H- 9),1.41(5H,m,H-11),1.28(4H,s,H-12α),1.71(2H,m,H-12β),5.91(1H,dd,J=14.5Hz,9.0,H-14),5.21(1H,d,J=14.5Hz,H-15),5.38(1 H,d,J=8.5Hz,H-15),0.92(6H,s,H-16),4.51(1H,s,H-17),4.84(1H,s,H-17),1.18(4H,s,H-18),1.00(3H,s,H-19),0.98(6H,s,H-20). 13C NMR(125Hz,CDCl3)δC 45.8(C-1,CH2),67.8(C-2,CH),46.6(C-3,CH2),33(C-4,C),53.2(C-5,CH),24.5( C-6,CH2),38.1(C-7,CH2),148(C-8,C),57.7(C-9,CH),39.8(C-10,C),18.2(C-11 ,CH2),41.4(C-12,CH2),73.6(C-13,C),145(C-14,CH),111.7(C-15,CH2),33.3(C -16,CH3),107.1(C-17,CH2),28.0(C-18,CH3),24.6(C-19,CH3),17.3(C-20,CH3). After literature search, its NMR data was basically consistent with the report in the literature (CUTLER et al., 1977), so compound 6 was identified as 2α-hydroxylacrimal taxol (2α-Hydroxymanool).

[0056] Compound 7 is a colorless, granular crystal with no fluorescence under 254nm ultraviolet light. It turns purple when heated with anisaldehyde-sulfuric acid color developer. Its chemical structure was obtained by NMR nuclear magnetic resonance spectroscopy analysis. 1HNMR (500Hz, CDCl3), δH 0.98(1H,t,J=10.0Hz,H-1α),2.12(1H,t,J=10.0Hz,H-1β),3.87(1H,d,J=9 .5Hz,H-2α),1.14(1H,t,J=10.0Hz,H-2β),1.76(1H,m,H-3),1.06(1H,dd,J =18.0,2.5Hz,H-5),1.26(4H,s,h-6),1.71(2H,m,H-6),1.96(1H,td,J=10. 5,3.0Hz,H-7),2.38(1H,dt,J=10.5,3.0Hz,H-7),1.62(4H,m,H-9),1.32(2H ,m,H-9),1.62(4H,m,H-11),1.32(2H,m,H-12α),1.71(2H,m,H-12β),5.89( 1H,dd,J=14.5,9.0Hz,H-14),5.1(1H,d,J=9.0Hz,H-15α),5.2(1H,d,J=14. 5Hz,H-15β),0.93(3H,s,H-16),4.51(1H,s,H-17α),4.83(1H,s,H-17β),1. 26(4H,s,H-18),0.83(3H,s,H-19),0.7(3H,s,H-20),1.62(4H,m,OH-2 / 13). 13C NMR(125Hz,CDCl3)δC48.2(C-1,CH2),65.6(C-2,CH),51(C-3,CH2),33.4(C-4,C),54.9 (C-5,CH),23.9(C-5,CH2),38(C-7,CH2),147.8(C-8,C),57(C-9,CH),35(C-10,C),17.9 (C-11, CH2), 41.2 (C-12, CH2), 73.6 (C-13, C), 145.1 (C-14, CH), 111.7 (C-15, CH2), 33.7 (C-16, CH3), 107.2 (C-17, CH2), 27.9 (C-18, CH3), 22.6 (C-19, CH3), 15.3 (C-20, 15.3). After literature search, its NMR data is basically consistent with the report in the literature (ABDELFATTAH et al., 2010), so compound 7 is identified as 2β-hydroxylacrimal taxol (2β-Hydroxymanool).

[0057] Compound 8 is in the form of a colorless colloid, has no fluorescence under 254nm ultraviolet light, and turns purple when heated with anisaldehyde-sulfuric acid color developer. Its chemical structure was obtained by NMR nuclear magnetic resonance spectrum analysis, and the molecular formula of the compound is C20H34O. 1HNMR (500Hz, CDCl3), δH 1.36 (5H, m, H-1α), 1.59 (5H, m, H-1β), 1.36 (5H, m, H-2), 1.59 (5H, m, H-3), 3.77 (1H, s, H-5), 1.11 (4H, d, J=2.5Hz, H-6 / 7), 4.33 (1H, d, J=16.5Hz, H-9), 1.36 (5H, m, H-11), 3.32 (2H, s, H- 12),4.91(1H,d,J=8.5Hz,3.0Hz,H-14),5.11(1H,ddd,J=14,8.5,2.0Hz,H-15),1.59(5H,m, H-16),0.97(3H,d,J=5.5Hz,H-17),0.83(4H,s,H-18),0.75(3H,s,H-19),0.72(3H,s,H-20). 13C NMR(125Hz,CDCl3)δC 38.8(C-1,CH2),18.0(C-2,CH2),44.1(C-3,CH2),33.3(C-4,C),55.8(C-5,CH),20. 1(C-6,CH2),45.5(C-7,CH2),72.3(C-8,C),61.1(C-9,CH),41.7(C-10,C),19.0(C-1 1, CH2), 48.6 (C-12, CH2), 72.1 (C-13, C), 146.6 (C-14, CH), 110.6 (C-15, CH2), 32.9 (C-16, CH3), 27.7 (C-17, CH3), 23.9 (C-18, CH3), 21.4 (C-19, CH3), 15.2 (C-20, CH3). According to literature search, its NMR data is consistent with the literature ( et al., 1975), the compound 8 was identified as 13-Epimanoyl oxide.

[0058] Compound 9 is in the form of white powder, has no fluorescence under 254nm ultraviolet light, and turns purple when heated by anisaldehyde-sulfuric acid color developer. Its chemical structure was obtained by NMR nuclear magnetic resonance spectroscopy analysis. 1HNMR (500Hz, CDCl3), δH 0.97(2H,m,H-1),1.62(2H,d,J=11.0Hz,H-1),1.38(4H,m,H-2α),1.54(4H,m,H-2β),0.93(2H,m,H-3α),1.12(4H,s,H-3β),1.54(4H,m,H -5),1.24(1H,m,H-6),1.64(2H,d,J=11.0Hz,H-6),1.38(4H,m,H-7α),2.09(1H,t,J=6.5Hz,H-7β),1.05(1H,t,J=4.0Hz,H-9),0.78(6H, d,J=4.5Hz,H-18),1.54(4H,m,H-12α),1.38(4H,m,H-12β),1.85(1H,dt,J=7.5,3.0Hz,H-12),5.43(1H,t,J=6.0Hz,H-14),4.14(2H,m,H -15),1.69(3H,s,H-16),0.86(3H,s,H-17),1.12(4H,s,H-19α),1.38(4H,m,H-19β),0.78(6H,d,J=4.5Hz,H-20),1.54(4H,m,OH-8 / 15). 13CNMR(125Hz,CDCl3)δC 39.7(C-1,CH2),18.4(C-2,CH2),41.9(C-3,CH2),33.2(C-4,C),56.1(C-5,CH),20. 5(C-6,CH2),42.9(C-7,CH2),74.1(C-8,C),61.2(C-9,CH),39.2(C-10,C),23.5(C-1 1, CH2), 44.5 (C-12, CH2), 140.9 (C-13, C), 123.2 (C-14, CH), 59.2 (C-15, CH2), 16.4 (C-16, CH3), 33.4 (C-17, CH3), 21.5 (C-18, CH3), 23.9 (C-19, CH3), 15.5 (C-20, CH3). According to literature search, its NMR data is consistent with the literature ( et al., 1975), so compound 9 was identified as Labda-13(E)-en-8α,15-diol.

[0059] Inhibitory activity of crude extracts, primary separation products and monomeric compounds of Nicotiana tabacum against Propionibacterium acnes

[0060] Solution preparation

[0061] The test extracts (Nicotiana tabacum glandular hair crude extract, primary separation product, diterpenoid monomers) were dissolved in 95% ethanol and prepared into solutions of required concentrations for use.

[0062] Bacteria activation

[0063] Take 0.2g of freeze-dried powder and add it to 10mL of NB medium, dissolve and mix thoroughly. Pipette 200μL of bacterial suspension into Columbia blood plate, spread evenly, repeat three times. Place the plate in an anaerobic culture box and culture at 37℃ for 48h.

[0064] Antibacterial test

[0065] Pick a single colony and dissolve it in 1 mL of NB medium. Dissolve and mix thoroughly, dilute the bacterial suspension to 10-4 times, and set aside.

[0066] Mix 100 μL of diluted bacterial suspension, 100 μL of test agent (blank control is 95% ethanol), and 800 μL of NB medium to make 1 mL of drug-containing bacterial suspension. Take 100 μL of drug-containing bacterial suspension in Columbia blood plate and spread it evenly. Repeat 3 times for each treatment. Place the plate in an anaerobic culture box and culture at 37°C. When the colonies on the blank control bacterial plate grow, the culture is terminated. The antibacterial rate of the test agent is calculated based on the number of colonies in the treatment group and the control group. The calculation formula for the colony inhibition rate is as follows:

[0067] Inhibition rate = [(CFUc-CFUt) / CFUc] × 100%;

[0068] Among them, CFUc is the number of colonies in the control group, and CFUt is the number of colonies in the treatment group.

[0069] Minimum inhibitory concentration (MIC) determination

[0070] The monomer compound was dissolved in 95% ethanol, and the final concentration of the monomer compound was diluted to an appropriate gradient range using bacterial suspension and culture medium according to the results of the antibacterial test. Each treatment was repeated 3 times, and the plate was placed in an anaerobic culture box and cultured at 37°C. The culture was terminated when the colonies of the blank control bacterial plate grew, and the MIC of the monomer compound was determined according to the number of colonies. Each treatment was repeated 3 times.

[0071] Fractional Inhibitory Concentration Index (FICI) determination

[0072] FICI was used to determine the combined effect between monomer compounds. The drug combination ratios of different concentrations of monomer compounds were designed and the antibacterial rate was determined. The interaction between compounds was determined according to the following formula (1): FIC ≤ 0.5 was a synergistic effect, 0.5 < FICI ≤ 1 was an additive effect, 1 < FIC ≤ 2 was no correlation, and FICI > 2 was an antagonistic effect.

[0073]

[0074] Inhibitory effects of crude extracts and primary separation products on Propionibacterium acnes

[0075] The crude extract showed significant inhibition against Propionibacterium acnes at a concentration of 1 mg / mL, with an inhibition rate of 89.45%. Among the primary separation products, Fr.2 to Fr.7 all showed inhibitory activity against Propionibacterium acnes, and the inhibitory effects are shown in Table 3 and Figure 2 As shown. At 200 μg / mL, the inhibitory activity of Fr.2 to Fr.7 was high, with an inhibition rate of 83.17% to 100%. At 50 μg / mL, the inhibition rates of Fr.2 to Fr.7 were 52.27% to 91.10%, all higher than the positive control tetracycline (15.53%), especially Fr.4, which had the highest inhibition rate of 91.10%, followed by Fr.6, which had an inhibition rate of 78.40%.

[0076] Table 3 Inhibition rate of primary separation products on Propionibacterium acnes

[0077]

[0078] Inhibitory effects of monomeric compounds

[0079] At 50 μg / mL, the compounds that showed inhibitory effects on Propionibacterium acnes are shown in Tables 4 and Figure 3 As shown. Among them, compound 9 was the best, with an inhibitory activity of 94.23%, significantly higher than other compounds, followed by compound 4, with an inhibition rate of 52.56%. The antibacterial activity of all monomer compounds was higher than that of the positive control tetracycline.

[0080] Table 4 Inhibition rate of monomer compounds against Propionibacterium acnes at 50 μg / mL

[0081]

[0082] Combined antibacterial effect of monomer compounds

[0083] Table 3, Table 4, Figure 2 , Figure 3 The results showed that there was a high possibility of synergistic effects between monomeric compounds, so Fr.4, which had the best activity and contained the most monomeric compounds among the primary separation products, was selected to study the synergistic effects between diterpenes.

[0084] The MICs of compound 4 (sclareol), compound 7 (2β-hydroxy-dacryl) and compound 9 (lisdandiol) were 60, 75 and 52 μg / mL, respectively. When the concentration of sclareol was 10-20 μg / mL and the concentration of 2β-hydroxy-dacryl was 50-20 μg / mL, the combination effect was additive when the two were compounded in a ratio of 1:1-5; when the proportion of sclareol was increased and the two were compounded in a ratio of 2-5:1, the concentration of sclareol was 20-25 μg / mL and the concentration of 2β-hydroxy-dacryl was 10-5 μg / mL, and the two achieved a synergistic antibacterial effect.

[0085] When the concentration of sclareol is between 20 and 1 μg / mL and the concentration of lisdendol is between 1 and 20 μg / mL, a synergistic antibacterial effect can be achieved when the two are compounded in a ratio of 1 to 20:1 to 20.

[0086] When the concentration of 2β-hydroxylacrimal taxol is between 20 and 10 μg / mL, and the concentration of lisdendol is between 4 and 10 μg / mL, and the two are compounded in a ratio of 1 to 5:1, a synergistic antibacterial effect can be achieved.

[0087] Within a certain concentration range, diterpenoid monomer compounds exert synergistic antibacterial effects with high efficiency and low doses, achieving the purpose of enhancing efficacy and reducing toxicity.

[0088] Table 5 Concentration and antibacterial effect of the combined use of compounds

[0089]

[0090] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A tobacco-derived compound composition, characterized in that: The composition comprises any one or more of dacryotaxol, sclareol, 2-oxodacryotaxol, 2α-hydroxydacryotaxol, 2β-hydroxydacryotaxol and lisdandiol.

2. The tobacco-derived compound composition according to claim 1, characterized in that: The composition comprises at least any two of sclareol, 2β-hydroxylactotaxol and lisdendioic acid.

3. The tobacco-derived compound composition according to claim 2, characterized in that: The mass ratio of the composition includes at least one of the following three ratios: The ratio of sclareol to 2β-hydroxylactotaxol is 2 to 5:1; The ratio of sclareol to lysine glycol is 1-20:1-20; The ratio of 2β-hydroxy-lactotaxel to lisdendiolide is 1 to 5:

1.

4. Use of the tobacco-derived compound composition according to claim 1 in the preparation of an active drug for inhibiting Propionibacterium acnes.

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