Chenane diterpenoid compound as well as preparation method and application thereof

Abstract

The invention discloses a cembrane diterpenoid compound as well as a preparation method and application thereof, and relates to the technical field of natural compounds. The cembrane diterpenoids have a structural formula as shown in formulas 1-9, and the preparation method comprises the following steps: firstly extracting and coarsely separating cigar tobacco leaves, and then finely separating and purifying to obtain the cembrane diterpenoids. The cembrane diterpenoids which are not reported are extracted and separated from cigar tobacco leaves, and the compounds 1-9 are proved to be capable of promoting proliferation of human oral mucosa fibroblasts and have a certain effect on healing of oral soft tissue defects.

Description

Technical Field

[0001] The present invention relates to the technical field of natural cembranoid compounds, and in particular to a cembranoid diterpenoid compound, a preparation method and an application thereof. Background Art

[0002] Cembranoids are a class of naturally occurring diterpenoids whose structural characteristics and diverse activities (including anti-tumor, anti-inflammatory, and neuroprotective) make them of great value in the pharmaceutical field. Cembranoids are primarily derived from marine organisms, but the extraction and isolation of cembranoids from tobacco has been less studied. Summary of the Invention

[0003] The present invention successfully extracted and separated unreported cembranoid diterpenoid compounds from cigar tobacco leaves, and found that the compounds can promote the proliferation of human oral mucosal fibroblasts and have a certain effect on the healing of oral soft tissue defects.

[0004] To this end, in a first aspect, the present invention provides a cembranoid diterpenoid compound, including Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8 and / or Compound 9, whose structural formulas are:

[0005]

[0006] The second aspect of the present invention provides a method for preparing the above-mentioned cembranoid diterpenoid compound, comprising:

[0007] Extraction and crude fractionation: cigar tobacco leaves were crushed and then extracted, and the extract was concentrated under reduced pressure to obtain an extract. The extract was appropriately diluted and extracted, and the extract was concentrated under reduced pressure. The extract was subjected to silica gel column chromatography, and gradient elution was performed with petroleum ether and acetone in a volume ratio of 1:0 to 0:1. The extracts were numbered according to the order of collection, and then detected and developed using silica gel thin layer plate. Similar fractions were combined to finally obtain 9 fractions, which were labeled as fractions I to IX.

[0008] Fine separation and purification: taking the third, fourth and fifth fractions obtained by the extraction and crude fractionation, and performing fine separation and purification; wherein,

[0009] The third part, 18 Separation was performed on a silica gel preparative column with a gradient elution of methanol-water at a volume ratio of 50% to 100%. 14 fractions were collected and labeled as fractions III-1 to III-14.

[0010] Take the third part of C 18Separation by a semi-preparative column in silica gel, gradient elution with methanol-water in a volume ratio of 60% - 100%, 10 fractions were collected successively, labeled as fraction III-3-1 to fraction III-3-10; Take fraction III-3-6 and separate it by a Sephadex LH-20 gel column, elute with methanol, 4 fractions were collected successively, labeled as fraction III-3-6-1 to fraction III-3-6-4; Take fraction III-3-6-2 and purify it by an HPLC preparative column, gradient elution with acetonitrile-water in a volume ratio of 45% - 55%, the compound 1 was obtained, with a retention time of 26.3 min;

[0011] Take fraction III-4, and pass through C 18 Separation by a semi-preparative column in silica gel, gradient elution with methanol-water in a volume ratio of 60% - 100%, 18 fractions were collected successively, labeled as fraction III-4-1 to fraction III-4-18; Take fraction III-4-13 and purify it by an HPLC preparative column, gradient elution with acetonitrile-water in a volume ratio of 60% - 75%, the compound 4 was obtained, with a retention time of 41.3 min;

[0012] Take fraction III-6, separate it by a silica gel column, gradient elution with petroleum ether-ethyl acetate in a volume ratio of 20:1 - 0:1, 8 fractions were collected successively, labeled as III-6-1 to III-6-8; Take fraction III-6-7 and separate it by a gel column, elute with methanol, and then purify it by an HPLC preparative column, gradient elution with acetonitrile-water in a volume ratio of 80% - 90%, the compound 8 with a retention time of 41.0 min and the compound 6 with a retention time of 44.1 min were obtained successively; Take fraction III-6-8 and purify it by an HPLC preparative column, gradient elution with acetonitrile-water in a volume ratio of 50% - 65%, the compound 7 was obtained, with a retention time of 24.7 min;

[0013] Take fraction IV, and pass through C 18 Separation by a semi-preparative column in silica gel, gradient elution with methanol-water in a volume ratio of 60% - 100%, 8 fractions were collected successively, labeled as fraction IV-1 to fraction IV-8; Take fraction IV-3 and separate it by a gel column, elute with methanol, 9 fractions were collected successively, labeled as fraction IV-3-1 to fraction IV-3-9; Take fraction IV-3-4 and purify it by an HPLC preparative column, gradient elution with acetonitrile-water in a volume ratio of 50% - 65%, the compound 5 with a retention time of 31.5 min and the compound 2 with a retention time of 33.1 min were obtained successively; Take fraction IV-7 and separate it by a gel column, elute with 90% methanol by volume, and then purify it by an HPLC preparative column, gradient elution with acetonitrile-water in a volume ratio of 80% - 92%, the compound 9 was obtained, with a retention time of 35.9 min;

[0014] Take fraction V, and pass through C 18Separated by a preparative column in silica gel, eluted with a gradient of methanol-water at a volume ratio of 40% - 100%, and 10 fractions were collected successively, labeled as fraction V-1 to fraction V-10; fraction V-5 was taken and separated by silica gel column chromatography, eluted with a gradient of petroleum ether-ethyl acetate at a volume ratio of 20:1 - 1:1, and 7 fractions were collected successively, labeled as fraction V-5-1 to fraction V-5-7; fraction V-5-4 was taken and separated by a Sephadex LH-20 gel column, eluted with methanol, and 5 fractions were collected successively, labeled as fraction V-5-4-1 to fraction V-5-4-5; fraction V-5-4-3 was taken and purified by an HPLC preparative column, eluted with a gradient of acetonitrile-water at a volume ratio of 55% - 70%, and the compound 3 was obtained, with a retention time of 24.0 min.

[0015] Furthermore, the extraction and crude separation specifically include:

[0016] The cigar tobacco leaves were crushed to 20 mesh, extracted with methanol solution at room temperature for 3 times, the extraction solutions were combined, and concentrated under reduced pressure to obtain the extract. The extract was diluted with a 0.5% hydrochloric acid solution by volume and partitioned 3 times between equal volumes of ethyl acetate. The ethyl acetate layer was combined and concentrated under reduced pressure to obtain the extract. The extract was separated by silica gel column chromatography and eluted with a gradient of petroleum ether and acetone at a volume ratio of 1:0 - 0:1, numbered in the order of collection, then detected using a silica gel thin layer plate and developed by heating with 5% sulfuric acid ethanol, and similar components were combined. Finally, 9 fractions were obtained, labeled as fraction I to fraction IX.

[0017] Furthermore, the chromatographic conditions for purifying the compound 1-9 by an HPLC preparative column also include: using an HPLC XBridge column, with a mobile phase flow rate of 8 mL / min.

[0018] In the third aspect of the present invention, an application of the above cembrane diterpenoid compounds is provided, and the cembrane diterpenoid compounds are used in drugs or daily care products for protecting oral ulcers.

[0019] Furthermore, the cembrane diterpenoid compounds are used to prepare drugs or daily care products for promoting the proliferation of human oral mucosal fibroblasts, and / or for preparing drugs or daily care products for promoting the healing of oral soft tissue defects.

[0020] Compared with the prior art, the present invention at least includes the following beneficial effects:

[0021] The present invention provides a cembrane diterpenoid compound, its preparation method and application, extracts and separates cembrane diterpenoid compounds 1-9 that have not been reported from cigar tobacco leaves, and compounds 1-9 can all promote the proliferation of human oral mucosal fibroblasts and have a certain effect on the healing of oral soft tissue defects. Description of the Drawings

[0022] Figure 1 The activity of compounds 1-9 provided by the embodiments of the present invention in promoting the proliferation of human oral mucosal fibroblasts; (*p<0.05, **p<0.01, ***p<0.001 vs control group). Detailed implementation manners

[0023] In order to fully understand the purpose, features and effects of the present invention, the present invention will be described in detail through the following specific implementation manners. Except for the following content, the process methods of the present invention all adopt conventional methods or devices in the art. Unless otherwise specified, the following noun terms have the meanings commonly understood by those skilled in the art.

[0024] Example 1 A cembrane diterpenoid compound, its preparation method and application

[0025] (1) Cembrane diterpenoid compounds[[ID=E15]]

[0026] (1) The structural formulas of compounds 1-9 are as follows:

[0027]

[0028] (2) The physicochemical data of compounds 1-9 are as follows

[0029] Cigarcembrane A(1): C 20 H 34 O3; (c 0.20, CH3OH); UV(CH3OH) λ max (logε)202.8(3.17), 219.0(2.69), 273.8(1.96) nm; 1 1H(600 MHz) and 13 13C(150 MHz) NMR data (chloroform-d) are shown in Tables 1 and 2; HREIMS(m / z 322.2504[M] + , C 20 H 34 O3 calculated value 322.2502). CD(MeOH, Δε) λ max : 200(5.85) nm.

[0030] Cigarcembrane B(2): C 20 H 34 O2; (c 0.12, CH3OH); UV(CH3OH) λ max (logε)204.2(3.47), 227.8(2.40), 273.0(1.81) nm;1 H (600 MHz) and 13 C (150 MHz) NMR data (chloroform-d) are shown in Tables 1 and 2; HREIMS (m / z 306.2553 [M] + , C 20 H 34 O2 calcd 306.2553).

[0031] Cigarcembrane C(3): C 20 H 34 O3; (c 0.11, CH3OH); UV(CH3OH) λ max (logε) 204.5(3.37), 219.0(2.96), 274.5(2.06) nm; 1 H (500 MHz) and 13 C (125 MHz) NMR data (chloroform-d) are shown in Tables 1 and 2; HREIMS (m / z 322.2500 [M] + , C 20 H 34 O3 calcd 306.2502).

[0032] Cigarcembrane D(4): C 21 H 36 O3; (c 0.14, CH3OH); UV(CH3OH) λ max (logε) 203.0(3.44), 221.5(3.05), 273.0(2.30) nm; 1 H (800 MHz) and 13 C (200 MHz) NMR data (chloroform-d) are shown in Tables 1 and 2; HREIMS (m / z 336.2656 [M] + , C 21 H 36 O3 calcd 336.2659).

[0033] Cigarcembrane E(5): C 20 H 34 O2; (c 0.14, CH3OH); UV(CH3OH) λ max (logε) 204.5(3.55), 229.5(2.61), 268.0(1.95), 285.0(1.84) nm; 1 H (600 MHz) and13 13C(150 MHz) NMR data (in chloroform-d) are shown in Tables 2 and 3; HREIMS (m / z 306.2553 [M] + , C 20 1H 34 2O2, calcd. 306.2553).

[0034] Cigarcembrane F(6): C 20 1H 32 O; (c 0.09, CH3OH); UV (in CH3OH) λ max (log ε) 204.5 (3.47), 245.5 (2.94) nm; 1 1H(500 MHz) and 13 13C(125 MHz) NMR data (in chloroform-d) are shown in Tables 2 and 3; HREIMS (m / z 288.2449 [M] + , C 20 1H 32 O, calcd. 288.2448).

[0035] Cigarcembrane G(7): C 20 1H 32 O2; (c 0.16, CH3OH); UV (in CH3OH) λ max (log ε) 203.5 (3.08), 272.0 (2.51) nm; 1 1H(600 MHz) and 13 13C(150 MHz) NMR data (in chloroform-d) are shown in Tables 2 and 3; HREIMS (m / z 304.2397 [M] + , C 20 1H 32 O2, calcd. 304.2399).

[0036] Cigarcembrane H(8): C 20 1H 32 O; (c 0.14, CH3OH); UV (in CH3OH) λ max (log ε) 204.2 (3.73), 238.0 (2.98) nm; 1 1H(500 MHz) and 13 13C(125 MHz) NMR data (in chloroform-d) are shown in Table 4; HREIMS (m / z 288.2449 [M] +, C 20 H 32 O calculated value 288.2448). CD(MeOH, Δε) λ max : 212(-1.73) nm.

[0037] Cigarcembrane I(9): C 20 H 32 O; (c 0.11, CH3OH); UV(CH3OH) λ max (logε) 203.6(3.42), 235.2(2.72) nm; 1 1H(500 MHz) and 13 13C(125 MHz) NMR and (chloroform-d) are shown in Table 4; HRESIMS (m / z 289.2526 [M + H] + , calculated value C 20 H 33 O 289.2520). CD(MeOH, Δε) λ max : 214(-0.30), 235(0.16) nm.

[0038] Table 1 1H NMR data of compounds 1 - 4 (chloroform-d) (δ in ppm, J in Hz).

[0039]

[0040]

[0041] a recorded at 600 MHz; b at 500 MHz; c at 800 MHz Table Ⅱ 13C NMR data of compounds 1 - 7 (chloroform-d) (δ in ppm).

[0042]

[0043]

[0044] a recorded at 150 MHz; b at 125 MHz; c at 200 MHz

[0045] Table 3 1H NMR data of compounds 5 - 7 (chloroform-d) (δ in ppm and J in Hz).

[0046]

[0047] a Recorded at 600MHz; b At 500MHz

[0048] Table 4 1H and 13C NMR data of compounds 8 - 9 (chloroform - d) (δ in ppm and J in Hz).

[0049]

[0050] (II) Preparation method

[0051] (1) Extraction and rough separation: Take shredded cigar tobacco leaves (20 kg), pulverize them, and extract with methanol solution 3 times at room temperature. Combine the extracts and concentrate under reduced pressure to obtain an extract. Dilute the extract with an appropriate volume of 0.5% hydrochloric acid solution and partition it 3 times between ethyl acetate. Combine the ethyl acetate layers and concentrate under reduced pressure to obtain 400 g of a crude extract. Subject the crude extract to silica gel column chromatography and elute with petroleum ether and acetone (1:0 - 0:1, v / v). The volume ratios of petroleum ether to acetone are 1:0, 9:1, 8:2, 7:3, 1:1, 0:1 in sequence. Number them in the order of collection, detect using silica gel thin - layer plates, and develop color by heating with 5% sulfuric acid - ethanol. Combine the similar ones to obtain 9 fractions (labeled as Fraction I - Fraction IX).

[0052] (2) Fine separation and purification: Take Fraction III, Fraction IV, and Fraction V obtained from extraction and rough separation for fine separation and purification; among them,

[0053] Fraction III (30 g) is separated by a semi - preparative column in silica gel, eluted with methanol - water in a gradient of 50% - 100% by volume, and 14 fractions are collected successively, labeled as Fraction III - 1 to Fraction III - 14. Among them, take Fraction III - 3 (1.62 g) and separate it by a semi - preparative column in silica gel, elute with methanol - water in a gradient of 60% - 100% by volume, and 10 fractions are collected successively, labeled as Fraction III - 3 - 1 to Fraction III - 3 - 10; take Fraction III - 3 - 6 and separate it by a gel column (Sephadex LH - 20), elute with methanol, and 4 fractions are collected successively, labeled as Fraction III - 3 - 6 - 1 to Fraction III - 3 - 6 - 4; take Fraction III - 3 - 6 - 2 and purify it by an HPLC Xbridge preparative chromatographic column, elute with acetonitrile - water in a gradient of 45% - 55% by volume for 45 min to obtain the compound 1 (t 18 18 26.3 min, 3.2 mg). R

[0054] Take Fraction III - 4 (1.68 g) and subject it to C​​18 Separation was carried out on a sub-preparative column in silica gel, and elution was performed with a gradient of methanol-water in a volume ratio of 60% to 100%. A total of 18 fractions were collected successively and labeled as fraction III-4-1 to fraction III-4-18. The 13th fraction of III-4 was purified on an HPLC preparative column, and elution was carried out with a gradient of acetonitrile-water in a volume ratio of 60% to 75% for 55 min to obtain compound 4 (t R 41.3 min, 2.0 mg).

[0055] The 6th fraction of III (1.26 g) was separated on a silica gel column, and elution was carried out with a gradient of petroleum ether-ethyl acetate in a volume ratio of 20:1 to 0:1. A total of 8 fractions were collected successively and labeled as III-6-1 to III-6-8. The 7th fraction of III-6 was separated on a gel column, eluted with methanol, and then purified on an HPLC preparative column. Elution was carried out with a gradient of acetonitrile-water in a volume ratio of 80% to 90% for 45 min to obtain compound 8 (t R 41.0 min, 17.2 mg) and compound 6 (t R 44.1 min, 10.6 mg); The 8th fraction of III-6 was purified on an HPLC preparative column, and elution was carried out with a gradient of acetonitrile-water in a volume ratio of 50% to 65% for 45 min to obtain compound 7 (t R 24.7 min, 2.1 mg).

[0056] The 4th fraction (27 g) was passed through C 18 Separation was carried out on a sub-preparative column in silica gel, and elution was performed with a gradient of methanol-water in a volume ratio of 60% to 100%. A total of 8 fractions were collected successively and labeled as fraction IV-1 to fraction IV-8. The 3rd fraction of IV (2.11 g) was separated on a gel column, eluted with methanol, and a total of 9 fractions were collected successively and labeled as fraction IV-3-1 to fraction IV-3-9. The 4th fraction of IV-3 was purified on an HPLC preparative column, and elution was carried out with a gradient of acetonitrile-water in a volume ratio of 50% to 65% for 40 min to obtain compound 5 (t R 31.5 min, 2.5 mg) and compound 2 (t R 33.1 min, 3.6 mg); The 7th fraction of IV (1.33 g) was separated on a gel column, eluted with methanol at a volume concentration of 90%, and then purified on an HPLC preparative column. Elution was carried out with a gradient of acetonitrile-water in a volume ratio of 80% to 92% for 50 min to obtain compound 9 (t R 35.9 min, 4.1 mg).

[0057] The 5th fraction (16 g) was passed through C 18Separation by a preparative column in silica gel, gradient elution with methanol-water in a volume ratio of 40% - 100%, 10 fractions were collected successively and labeled as fraction V-1 to fraction V-10; Take fraction V-5 (1.72 g) and perform silica gel column chromatography, gradient elution with petroleum ether - ethyl acetate in a volume ratio of 20:1 - 1:1, 7 fractions were collected successively with petroleum ether - ethyl acetate and labeled as fraction V-5-1 to fraction V-5-7; Take fraction V-5-4 and separate it by a Sephadex LH-20 gel column, elute with methanol, 5 fractions were collected successively and labeled as fraction V-5-4-1 - fraction V-5-4-5; Take fraction V-5-4-3 and purify it by an HPLC preparative column, gradient elution with acetonitrile - water in a volume ratio of 55% - 70% for 55 min to obtain compound 3 (t R 24.0 min, 7.9 mg).

[0058] (III) Application

[0059] Evaluation of the cell proliferation activity of cembrane diterpenoids 1 - 9 on human oral mucosal fibroblasts (h-OMF) is as follows:

[0060] Inoculate cells: Take cells in the logarithmic growth phase, digest them with trypsin, and make a cell suspension with FM-2 fibroblast special medium. h-OMF cells are inoculated into a 96-well plate at a density of 4000 cells / well, with a volume of 100 μL per well, and placed in an incubator for 12 - 16 hours. Add the test compound: The compound is dissolved in DMSO to prepare a 10 mM stock solution, diluted to the required concentration with complete medium, 100 μL is added to each well, and the final concentration is 50 μM. Each treatment is set with 3 replicates, and cultured at 37°C and 5% CO2 for 48 hours. Color development: Discard the old culture medium in the wells, add a mixed solution of medium and MTS at a ratio of 4:1, 100 μL per well; at the same time, set 3 blank replicates, continue to incubate for 2 - 4 hours, and measure the light absorption value. Data processing: Survival rate % = 100 - (OD value of the negative group - OD value of the experimental group) / (OD value of the negative group - OD value of the blank group) × 100%. Use EXCEL software for data statistical analysis, and use the two independent samples t-test for measurement data. A P < 0.05 is considered statistically significant for the difference.

[0061] At a concentration of 50 μM, as Figure 1 shown, all compounds can promote the proliferation of human oral mucosal fibroblasts and have a certain effect on the healing of oral soft tissue defects. Among them, compound 1 has a new 6 / 8 ring system and is the most potential lead compound in oral care.

[0062] It is easily understandable to those skilled in the art that, on the premise of no conflict, the above-mentioned advantageous ways can be freely combined and superimposed. The above are only the preferred embodiments of the present application and are not intended to limit the present application. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present application shall be included within the protection scope of the present application. The above is only the preferred implementation manner of the present application. It should be noted that for those of ordinary skill in the art, without departing from the technical principle of the present application, several improvements and modifications can still be made, and these improvements and modifications should also be regarded as within the protection scope of the present application.

Claims

1. A cembranoid diterpenoid compound, characterized in that: Including compound 1, compound 2, compound 3, compound 4, compound 5, compound 6, compound 7, compound 8 and / or compound 9, wherein the structural formulas of compounds 1-9 are:

2. A method for preparing a cembranoid diterpenoid compound, characterized in that: The method for preparing the cembranoid diterpenoid compound according to claim 1, comprising: Extraction and crude fractionation: cigar tobacco leaves were crushed and then extracted, and the extract was concentrated under reduced pressure to obtain an extract. The extract was appropriately diluted and extracted, and the extract was concentrated under reduced pressure. The extract was subjected to silica gel column chromatography, and gradient elution was performed with petroleum ether and acetone in a volume ratio of 1:0 to 0:

1. The extracts were numbered according to the order of collection, and then detected and developed using silica gel thin layer plate. Similar fractions were combined to finally obtain 9 fractions, which were labeled as fractions I to IX. Fine separation and purification: taking the third, fourth and fifth fractions obtained by the extraction and crude fractionation, and performing fine separation and purification; wherein, The third part, 18 Separation was performed on a silica gel preparative column with a gradient elution of methanol-water at a volume ratio of 50% to 100%. 14 fractions were collected and labeled as fractions III-1 to III-14. Take the third part of C 18 The product was separated by a silica gel preparative column and eluted with methanol-water at a volume ratio of 60% to 100%, and 10 fractions were collected, labeled as fractions III-3-1 to III-3-10; fraction III-3-6 was separated by a Sephadex LH-20 gel column and eluted with methanol, and 4 fractions were collected, labeled as fractions III-3-6-1 to III-3-6-4; fraction III-3-6-2 was purified by HPLC preparative column chromatography and eluted with acetonitrile-water at a volume ratio of 45% to 55%, to obtain compound 1 with a retention time of 26.3 min. Take the third-4th part, and pass through C 18 The product was separated by a silica gel preparative column and eluted with methanol-water at a volume ratio of 60% to 100%. 18 fractions were collected and labeled as fractions III-4-1 to III-4-18. Fraction III-4-13 was purified by HPLC preparative column chromatography and eluted with acetonitrile-water at a volume ratio of 60% to 75% to obtain compound 4 with a retention time of 41.3 min. Fraction III-6 was separated by silica gel column, and petroleum ether-ethyl acetate was gradient eluted at a volume ratio of 20:1 to 0:

1. Eight fractions were collected and labeled as fractions III-6-1 to III-6-8. Fraction III-6-7 was separated by gel column, eluted with methanol, and then purified by HPLC preparative column chromatography, acetonitrile-water was gradient eluted at a volume ratio of 80% to 90%, and compound 8 was obtained, with a retention time of 41.0 min and compound 6, with a retention time of 44.1 min. Fraction III-6-8 was purified by HPLC preparative column chromatography, acetonitrile-water was gradient eluted at a volume ratio of 50% to 65%, and compound 7 was obtained, with a retention time of 24.7 min. Take the fourth part, pass through C 18 The product was separated by a silica gel column and eluted with methanol-water at a volume ratio of 60% to 100%, and 8 fractions were collected, labeled as fractions IV-1 to IV-8; fraction IV-3 was separated by a gel column and eluted with methanol, and 9 fractions were collected, labeled as fractions IV-3-1 to IV-3-9; fraction IV-3-4 was purified by HPLC preparative column and eluted with acetonitrile-water at a volume ratio of 50% to 65%, to obtain compound 5 with a retention time of 31.5 min and compound 2 with a retention time of 33.1 min; fraction IV-7 was separated by a gel column and eluted with methanol at a volume concentration of 90%, and then purified by HPLC preparative column and eluted with acetonitrile-water at a volume ratio of 80% to 92% to obtain compound 9 with a retention time of 35.9 min; Take the fifth part, pass through C 18 The product was separated by a silica gel preparative column and eluted with methanol-water in a gradient ratio of 40% to 100% by volume, and 10 fractions were collected, labeled as fraction V-1 to fraction V-10; fraction V-5 was subjected to silica gel column chromatography and gradient elution with petroleum ether-ethyl acetate in a volume ratio of 20:1 to 1:1, and 7 fractions were collected, labeled as fraction V-5-1 to fraction V-5-7; fraction V-5-4 was separated by a Sephadex LH-20 gel column and eluted with methanol, and 5 fractions were collected, labeled as fraction V-5-4-1 to fraction V-5-4-5; fraction V-5-4-3 was purified by HPLC preparative column chromatography and eluted with acetonitrile-water in a gradient ratio of 55% to 70% by volume to obtain compound 3 with a retention time of 24.0 min.

3. The method for preparing cembranoid diterpenoids according to claim 2, wherein: The extraction and rough separation specifically include: Cigar tobacco leaves were crushed into 20 meshes, extracted three times with a methanol solution at room temperature, the extracts were combined, and concentrated under reduced pressure to obtain the extract. The extract was diluted with a 0.5% by volume hydrochloric acid solution and distributed three times with an equal volume of ethyl acetate. The ethyl acetate layers were combined and concentrated under reduced pressure to obtain the extract. The extract was chromatographed on a silica gel column, and gradient eluted with petroleum ether and acetone in a volume ratio of 1:0 to 0:

1. The extracts were numbered in the order of collection, and then detected using a silica gel thin layer plate and heated with 5% sulfuric acid ethanol for color development. Similar components were combined to finally obtain 9 parts, which were marked as parts I to IX.

4. The method for preparing cembranoid diterpenoids according to claim 2 or 3, wherein: The chromatographic conditions for purifying the compound 1-9 by HPLC preparative column also include: using an HPLC XBridge column and a mobile phase flow rate of 8 mL / min.

5. An application of a cembranoid diterpenoid compound, characterized in that: Use of the cembranoid diterpenoid compound according to claim 1 in a medicine or daily care product for protecting oral ulcers.

6. The use of the cembranoid diterpenoid compound according to claim 5, characterized in that: The cembranoid diterpenoid compound is used to prepare a medicine or daily care product for promoting the proliferation of human oral mucosal fibroblasts, and / or for preparing a medicine or daily care product for promoting the healing of oral soft tissue defects.

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