A polyhydroxy cembrane diterpenoid compound, its preparation method and application

Abstract

The present invention belongs to the technical field of biomedicine, relates to the extraction of active ingredients from traditional Chinese medicine, and relates to a polyhydroxy cembrane diterpenoid compound, its preparation method and application. The compound is compound 1 or compound 2, and the chemical structural formulas of compound 1 or compound 2 are shown as follows: The present invention extracts polyhydroxy cembrane diterpenoid compounds from frankincense, and through research, it is found that the polyhydroxy cembrane diterpenoid compounds have strong activity against liver injury caused by anti-tuberculosis drugs.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, relates to the extraction of active ingredients from traditional Chinese medicine, and relates to a polyhydroxy cembrane diterpenoid compound, a preparation method thereof and an application thereof. Background Art

[0002] Disclosing the information of this background art section is only intended to enhance the understanding of the overall background of the present invention, and is not necessarily to be regarded as an admission or an implication in any form that this information constitutes the prior art already known to those of ordinary skill in the art.

[0003] Frankincense is the resin exuded from the bark of Boswellia carterii Birdw. and its congeners Boswellia bhawdajiana Birdw. of the Burseraceae family, which are mainly produced in Somalia, Ethiopia and other places, and are divided into Somali frankincense and Ethiopian frankincense. Frankincense is pungent, bitter and warm, and has the effects of promoting blood circulation to relieve pain and detumescence and promoting granulation. The chemical components of frankincense mainly include diterpenoids and triterpenoids. The triterpenoids are mainly pentacyclic triterpenoids and tetracyclic triterpenoids, while the diterpenoids are mainly cembrane diterpenoids and aromadendrane diterpenoids, and the cembrane diterpenoids are the main type. The structural characteristics of cembrane diterpenoids are that they mostly have an oxygen ring in the ring, a three-membered oxygen ring, and an isopropyl group or an isopropyl group-substituted fourteen-membered ring skeleton, and the parent nucleus mostly has acyl oxygen groups, carbonyl groups, epoxy groups, hydroxyl groups and other substituents. Summary of the Invention

[0004] The inventors' research shows that the cembrane diterpenoid compounds in frankincense contain at most 1-2 hydroxyl substitutions. Through further research, the present invention unexpectedly extracts polyhydroxy (trihydroxy, tetrahydroxy) cembrane diterpenoid compounds from frankincense, and conducts activity research on the polyhydroxy cembrane diterpenoid compounds, and finds that the polyhydroxy cembrane diterpenoid compounds have strong activity against liver injury caused by antituberculosis drugs, so the present invention is proposed.

[0005] Based on the above research results, the technical solution of the present invention is as follows:

[0006] On the one hand, a polyhydroxy cembrane diterpenoid compound, which is compound 1 or compound 2, and the chemical structural formulas of compound 1 or compound 2 are shown as follows:

[0007]

[0008] On the other hand, a preparation method of the above polyhydroxy cembrane diterpenoid compound, comprising the following steps:

[0009] Using an ethanol solution with a volume fraction of 75-85% to extract frankincense to obtain a frankincense extract;

[0010] The frankincense extract was extracted with petroleum ether to obtain the petroleum ether extraction part;

[0011] The petroleum ether extraction part was subjected to one - step gradient elution using silica gel column chromatography to obtain the first elution part; in the one - step gradient elution, petroleum ether, a mixed solvent with a volume ratio of petroleum ether to ethyl acetate of 49 - 51:1, and a mixed solvent with a volume ratio of petroleum ether to ethyl acetate of 24 - 26:1 were successively used for gradient elution, and the elution part with a volume ratio of petroleum ether to ethyl acetate of 24 - 26:1 was the first elution part;

[0012] The first elution part was subjected to two - step gradient elution using silica gel column chromatography to obtain the second elution part; in the two - step gradient elution, a mixed solvent with a volume ratio of petroleum ether to ethyl acetate of 49 - 51:1, a mixed solvent with a volume ratio of petroleum ether to ethyl acetate of 24 - 26:1, a mixed solvent with a volume ratio of petroleum ether to ethyl acetate of 9 - 11:1, and a mixed solvent with a volume ratio of petroleum ether to ethyl acetate of 4 - 6:1 were successively used for gradient elution, and the elution part with a volume ratio of petroleum ether to ethyl acetate of 4 - 6:1 was the second elution part;

[0013] The second elution part was subjected to three - step gradient elution using silica gel column chromatography to obtain the elution part of Product 1; in the three - step gradient elution, a mixed solvent with a volume ratio of petroleum ether to ethyl acetate of 6.9 - 7.1:1 and a mixed solvent with a volume ratio of petroleum ether to ethyl acetate of 4.9 - 5.1:1 were successively used for gradient elution, and the elution part with a volume ratio of petroleum ether to ethyl acetate of 4.9 - 5.1:1 was the elution part of Product 1;

[0014] Using a mixed solution with a volume ratio of acetonitrile to water of 64.5 - 65.5:35.5 - 34.5 as the mobile phase, the elution part of Product 1 was separated using a semi - preparative chromatographic column to obtain Compound 1.

[0015] In the third aspect, a preparation method of the above - mentioned polyhydroxy cembrane diterpenoid compound comprises the following steps:

[0016] Frankincense was extracted with an ethanol solution with a volume fraction of 75 - 85% to obtain a frankincense extract;

[0017] The frankincense extract was extracted with petroleum ether to obtain the petroleum ether extraction part;

[0018] The petroleum ether extraction part was subjected to one - step gradient elution using silica gel column chromatography to obtain the first - eluted part; in the one - step gradient elution, petroleum ether, a mixed solvent with a volume ratio of petroleum ether to ethyl acetate of 49 - 51:1, and a mixed solvent with a volume ratio of petroleum ether to ethyl acetate of 24 - 26:1 were successively used for gradient elution, and the eluted part with a volume ratio of petroleum ether to ethyl acetate of 24 - 26:1 was the first - eluted part;

[0019] The first - eluted part was subjected to two - step gradient elution using silica gel column chromatography to obtain the second - eluted part; in the two - step gradient elution, a mixed solvent with a volume ratio of petroleum ether to ethyl acetate of 49 - 51:1, a mixed solvent with a volume ratio of petroleum ether to ethyl acetate of 24 - 26:1, a mixed solvent with a volume ratio of petroleum ether to ethyl acetate of 9 - 11:1, and a mixed solvent with a volume ratio of petroleum ether to ethyl acetate of 4 - 6:1 were successively used for gradient elution, and the eluted part with a volume ratio of petroleum ether to ethyl acetate of 4 - 6:1 was the second - eluted part;

[0020] The second - eluted part was subjected to three - step gradient elution using silica gel column chromatography to obtain the eluted part of product 2; in the three - step gradient elution, a mixed solvent with a volume ratio of petroleum ether to ethyl acetate of 6.9 - 7.1:1, a mixed solvent with a volume ratio of petroleum ether to ethyl acetate of 4.9 - 5.1:1, and a mixed solvent with a volume ratio of petroleum ether to ethyl acetate of 3.9 - 4.1:1 were successively used for gradient elution, and the eluted part with a volume ratio of petroleum ether to ethyl acetate of 3.9 - 4.1:1 was the eluted part of product 2;

[0021] A mixed solution with a volume ratio of acetonitrile to water of 67.5 - 68.5:32.5 - 31.5 was used as the mobile phase, and a semi - preparative chromatographic column was used to separate the eluted part of product 2 to obtain compound 2.

[0022] Fourthly, a composition includes the above - mentioned polyhydroxy cembrane diterpenoid compounds and pharmaceutical excipients.

[0023] Fifthly, an application of the above - mentioned polyhydroxy cembrane diterpenoid compounds or composition in the preparation of drugs for preventing liver injury caused by anti - tuberculosis drugs.

[0024] The beneficial effects of the present invention are as follows:

[0025] One 5,7,8,11 - tetrahydroxy cembrane - type diterpenoid and one 7,8,11 - trihydroxy cembrane - type diterpenoid compound isolated and purified from frankincense by the present invention have more hydroxyl substituents, and the two obtained new polyhydroxy cembrane - type diterpenoids have good activities against liver injury caused by anti - tuberculosis drugs, and have low toxic and side effects, showing good medicinal prospects. Description of the Drawings

[0026] The accompanying drawings forming a part of this invention are used to provide a further understanding of the invention. The schematic embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0027] Figure 1 It is the HRESIMS spectrum of Compound 1 in the embodiments of the present invention;

[0028] Figure 2 It is of Compound 1 in the embodiments of the present invention 1 H NMR spectrum;

[0029] Figure 3 It is of Compound 1 in the embodiments of the present invention 13 C NMR spectrum;

[0030] Figure 4 It is the HMQC spectrum of Compound 1 in the embodiments of the present invention;

[0031] Figure 5 It is the HMBC spectrum of Compound 1 in the embodiments of the present invention;

[0032] Figure 6 It is the NOESY spectrum of Compound 1 in the embodiments of the present invention;

[0033] Figure 7 It is the ECD spectrum of Compound 1 in the embodiments of the present invention;

[0034] Figure 8 It is the HRESIMS spectrum of Compound 2 in the embodiments of the present invention;

[0035] Figure 9 It is of Compound 2 in the embodiments of the present invention 1 H NMR spectrum;

[0036] Figure 10 It is of Compound 2 in the embodiments of the present invention 13 C NMR spectrum;

[0037] Figure 11 It is the HMQC spectrum of Compound 2 in the embodiments of the present invention;

[0038] Figure 12 It is the HMBC spectrum of Compound 2 in the embodiments of the present invention;

[0039] Figure 13 It is the NOESY spectrum of Compound 2 in the embodiments of the present invention;

[0040] Figure 14 It is the ECD spectrum of Compound 2 in the embodiments of the present invention. Detailed implementation manners

[0041] It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs.

[0042] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0043] In view of the fact that the present invention has first extracted polyhydroxycembraane diterpenoids with strong anti-tuberculosis drug-induced liver injury activity from frankincense, the present invention provides a polyhydroxycembraane diterpenoid, its preparation method and application.

[0044] A typical embodiment of the present invention provides a polyhydroxycembraane diterpenoid, which is Compound 1 or Compound 2. The chemical structural formulas of Compound 1 or Compound 2 are shown as follows:

[0045]

[0046] Another embodiment of the present invention provides a preparation method of the above polyhydroxycembraane diterpenoid, comprising the following steps:

[0047] Extract frankincense with an ethanol solution having a volume fraction of 75-85% to obtain a frankincense extract;

[0048] Extract the frankincense extract with petroleum ether to obtain a petroleum ether extraction part;

[0049] Perform a one-step gradient elution on the petroleum ether extraction part by silica gel column chromatography to obtain a one-step elution part; in the one-step gradient elution, successively use petroleum ether, a mixed solvent of petroleum ether and ethyl acetate with a volume ratio of 49-51:1, and a mixed solvent of petroleum ether and ethyl acetate with a volume ratio of 24-26:1 for gradient elution. The elution part with a volume ratio of petroleum ether to ethyl acetate of 24-26:1 is the one-step elution part;

[0050] The first elution fraction was subjected to secondary gradient elution using silica gel column chromatography to obtain the secondary elution fraction; in the secondary gradient elution, a mixed solvent with a volume ratio of petroleum ether to ethyl acetate of 49 - 51:1, a mixed solvent with a volume ratio of petroleum ether to ethyl acetate of 24 - 26:1, a mixed solvent with a volume ratio of petroleum ether to ethyl acetate of 9 - 11:1, and a mixed solvent with a volume ratio of petroleum ether to ethyl acetate of 4 - 6:1 were successively used for gradient elution, and the elution fraction with a volume ratio of petroleum ether to ethyl acetate of 4 - 6:1 was the secondary elution fraction;

[0051] The secondary elution fraction was subjected to tertiary gradient elution using silica gel column chromatography to obtain the elution fraction of Product 1; in the tertiary gradient elution, a mixed solvent with a volume ratio of petroleum ether to ethyl acetate of 6.9 - 7.1:1 and a mixed solvent with a volume ratio of petroleum ether to ethyl acetate of 4.9 - 5.1:1 were successively used for gradient elution, and the elution fraction with a volume ratio of petroleum ether to ethyl acetate of 4.9 - 5.1:1 was the elution fraction of Product 1;

[0052] A mixture of acetonitrile and water with a volume ratio of 64.5 - 65.5:35.5 - 34.5 was used as the mobile phase, and Compound 1 was obtained by separating the elution fraction of Product 1 using a semi - preparative chromatographic column.

[0053] In some embodiments, heating under reflux was carried out during the extraction of frankincense with an ethanol solution.

[0054] In some embodiments, when extracting frankincense with an ethanol solution, the solid - liquid ratio was 1:2.5 - 3.5.

[0055] In some embodiments, when separating the elution fraction of Product 1 using a semi - preparative chromatographic column, the detection wavelength was 204 - 206 nm and the flow rate was 4 - 6 mL / min -1 。

[0056] The third embodiment of the present invention provides a method for preparing the above - mentioned polyhydroxy cembrane diterpenoids, comprising the following steps:

[0057] Frankincense was extracted with an ethanol solution having a volume fraction of 75 - 85% to obtain a frankincense extract;

[0058] The frankincense extract was extracted with petroleum ether to obtain a petroleum ether extraction fraction;

[0059] The petroleum ether extraction part was subjected to one - step gradient elution using silica gel column chromatography to obtain the first elution part; in the one - step gradient elution, petroleum ether, a mixed solvent with a volume ratio of petroleum ether to ethyl acetate of 49 - 51:1, and a mixed solvent with a volume ratio of petroleum ether to ethyl acetate of 24 - 26:1 were successively used for gradient elution, and the elution part with a volume ratio of petroleum ether to ethyl acetate of 24 - 26:1 was the first elution part;

[0060] The first elution part was subjected to two - step gradient elution using silica gel column chromatography to obtain the second elution part; in the two - step gradient elution, a mixed solvent with a volume ratio of petroleum ether to ethyl acetate of 49 - 51:1, a mixed solvent with a volume ratio of petroleum ether to ethyl acetate of 24 - 26:1, a mixed solvent with a volume ratio of petroleum ether to ethyl acetate of 9 - 11:1, and a mixed solvent with a volume ratio of petroleum ether to ethyl acetate of 4 - 6:1 were successively used for gradient elution, and the elution part with a volume ratio of petroleum ether to ethyl acetate of 4 - 6:1 was the second elution part;

[0061] The second elution part was subjected to three - step gradient elution using silica gel column chromatography to obtain the elution part of product 2; in the three - step gradient elution, a mixed solvent with a volume ratio of petroleum ether to ethyl acetate of 6.9 - 7.1:1, a mixed solvent with a volume ratio of petroleum ether to ethyl acetate of 4.9 - 5.1:1, and a mixed solvent with a volume ratio of petroleum ether to ethyl acetate of 3.9 - 4.1:1 were successively used for gradient elution, and the elution part with a volume ratio of petroleum ether to ethyl acetate of 3.9 - 4.1:1 was the elution part of product 2;

[0062] A mixed solution with a volume ratio of acetonitrile to water of 67.5 - 68.5:32.5 - 31.5 was used as the mobile phase, and a semi - preparative chromatographic column was used to separate the elution part of product 2 to obtain compound 2.

[0063] In some embodiments, heating under reflux was carried out during the extraction of frankincense with an ethanol solution.

[0064] In some embodiments, when extracting frankincense with an ethanol solution, the solid - liquid ratio was 1:2.5 - 3.5.

[0065] In some embodiments, when separating the elution part of product 2 using a semi - preparative chromatographic column, the detection wavelength was 204 - 206 nm and the flow rate was 4 - 6 mL / min -1 。

[0066] The fourth embodiment of the present invention provides a composition, comprising the above - mentioned polyhydroxy cembrane diterpenoids and pharmaceutical excipients.

[0067] Specifically, the pharmaceutical excipients are pharmaceutical carriers and / or excipients. The pharmaceutical carriers include, but are not limited to, aluminum stearate, serum albumin, alumina, phosphate buffer solution, polyvinylpyrrolidone, polyethylene glycol, etc. The content of the pharmaceutical carrier in the composition can be 1-98% by weight, usually about 80% by weight. The excipients include, but are not limited to, binders, fillers, lubricants, disintegrants, wetting agents, emulsifiers, preservatives, etc.

[0068] The fifth embodiment of the present invention provides an application of the above-mentioned polyhydroxy cembrane diterpenoids or composition in the preparation of drugs for preventing liver injury caused by anti-tuberculosis drugs.

[0069] In order to enable those skilled in the art to more clearly understand the technical solutions of the present invention, the technical solutions of the present invention will be described in detail below with reference to specific examples.

[0070] Examples

[0071] The method for extracting polyhydroxy cembrane diterpenoids (5,7,8,11-tetrahydroxy cembrane diterpenoids (Compound 1) and 7,8,11-trihydroxy cembrane diterpenoids (Compound 2)) from frankincense is as follows:

[0072] (1) Take the frankincense medicinal material, extract it by heating under reflux with 80% ethanol three times, the solid-liquid ratio is 1:3 (g / mL), 2 hours each time, combine and filter the extract, and concentrate it until the alcohol smell disappears to obtain the 80% ethanol extract of frankincense.

[0073] (2) Extract the obtained 80% ethanol extract with petroleum ether and ethyl acetate 5 times respectively to obtain the petroleum ether fraction and the ethyl acetate fraction. The petroleum ether fraction is preliminarily separated by silica gel column chromatography, and gradient elution is carried out with a petroleum ether-ethyl acetate mixed solvent. The gradient is: 100:0 (v:v) → 50:1 (v:v) → 25:1 (v:v) → 10:1 (v:v) → 5:1 (v:v) → 1:1 (v:v), the elution volume for each gradient is 2 L, and after combining the elution parts, they are concentrated under reduced pressure to obtain the elution parts of each gradient.

[0074] (3) Continue to separate the petroleum ether-ethyl acetate 25:1 (v:v) elution fraction obtained in step 2 by silica gel column chromatography, and carry out gradient elution with a petroleum ether-ethyl acetate mixed solvent. The gradient is: 50:1 (v:v) → 25:1 (v:v) → 10:1 (v:v) → 5:1 (v:v), the elution volume for each gradient is 2 L, and after combining the elution parts, they are concentrated under reduced pressure to obtain the elution parts of each gradient.

[0075] (4) The fraction eluted with petroleum ether - ethyl acetate (5:1, v:v) obtained in step 3 was further separated by silica gel column chromatography, and gradient elution was carried out using a mixed solvent of petroleum ether - ethyl acetate. The gradient was: 7:1 (v:v) → 5:1 (v:v) → 4:1 (v:v) → 3:1 (v:v) → 1:1 (v:v). The elution volume for each gradient was 1 L. After combining the eluted fractions, they were concentrated under reduced pressure to obtain the eluted fractions for each gradient.

[0076] (5) The fraction eluted with petroleum ether - ethyl acetate (5:1, v:v) obtained in step 4 was separated by semi - preparative column chromatography. The separation conditions included a mobile phase of CH3CN - H2O (65:35, v / v), a detection wavelength of 205 nm, and a flow rate of 5 mL min -1 , and compound 1 (10 mg) was obtained.

[0077] (6) The fraction eluted with petroleum ether - ethyl acetate (4:1, v:v) obtained in step 4 was separated by semi - preparative column chromatography. The separation conditions included a mobile phase of CH3CN - H2O (68:32, v / v), a detection wavelength of 205 nm, and a flow rate of 5 mL min -1 , and compound 2 (11 mg) was obtained.

[0078] Structure identification: The monomeric components obtained by separation were measured by HR - ESI - MS and NMR spectra using a Bruker Impact II mass spectrometer and a Burker 400 MHz nuclear magnetic resonance spectrometer, respectively. The obtained nuclear magnetic data are shown in Table 1, and the structures of a 5,7,8,11 - tetrahydroxy cembranoid diterpene and a 7,8,11 - trihydroxy cembranoid diterpene were identified.

[0079] (1S,5S,7R,8S,11R,12R,3E)-1:12 - epoxy - 3 - cembranene - 5,7,8,11 - tetraol (1): Light yellow oily substance; HR - ESI - MS: m / z 355.2472 [M - H] - (Theoretical value: 355.2479, C 20 H 35 O5), as Figure 1 shown, its molecular formula was determined to be: C 20 H 36 O5, and the degree of unsaturation was 3. The infrared spectrum showed an absorption at 3441 cm -1 indicating the presence of a hydroxyl functional group.

[0080] 1 The 1H - NMR spectrum showed a set of isopropyl signals at δ H1.04(3H, d, J = 6.8Hz, Me-16), 0.90(3H, d, J = 6.8Hz, Me-17), 1.77(1H, overlapped, H-15), three methyl singlet signals δ H 1.07(3H, s, Me-20), 1.16(3H, s, Me-19), 1.74(3H, s, Me-18), three oxygen - linked methylene signals δ H 4.44(1H, d, J = 10.8Hz, H-5), 3.48(1H, m, H-7), 4.21(1H, dd, J = 2.8, 9.6Hz, H-11), one vinylic hydrogen signal δ H 6.29(1H, t, J = 8.0Hz, H-3), as Figure 2 shown. 13 The C - NMR spectrum shows the presence of 20 carbon signals, including one quaternary carbon double - bond signal δ C 130.4(C-4), three oxygen - linked quaternary carbon signals δ C 88.6(C-1), 85.9(C-8), 86.6(C-12), one CH vinylic carbon signal δ C 129.6(C-3), 3 oxygen - linked CH signals δ C 89.9(C-5), 77.1(C-7), 83.1(C-11), one isopropyl CH signal δ C 41.9(C-15), 6 CH2 signals δ C 31.7(C-2), δ C 34.6(C-6), δ C 36.6(C-9), δ C 28.3(C-10), δ C 31.0(C-13), δ C 35.6(C-14), 5 methyl signals δ C 17.7(C-16), δ C 18.6(C-17), δ C 14.9(C-18), δ C 18.7(C-19), δ C 25.3(C-20), as Figure 3 shown.

[0081] Through 1 1H - NMR and 13 C - NMR spectra, it is inferred that compound 1 contains one isopropyl group, one double bond, one 1:12 peroxy bridge, and four hydroxyl groups. This inference is further confirmed by two - dimensional spectra, and the results are as Figures 4 - 5As shown, in the HMBC spectrum, H-3 is correlated with C-1 / C-4 / C-5 / Me-18, H-5 is correlated with C-3 / C-4 / C-6 / C-7 / Me-18, H-7 is correlated with C-5 / C-6 / C-8 / C-9 / Me-19, H-11 is correlated with C-9 / C-13 / C-12 / Me-20, and Me-20 is correlated with C-12 / C-1 / C-13 / C-11, further determining the above functional groups. Finally, the planar structure of compound 1 is determined to be 1:12-epoxy-3-cembranene-5,7,8,11-tetraiol.

[0082] The relative and absolute configurations of compound 1 were obtained from the NOESY spectrum and the calculated ECD spectrum. In the NOESY spectrum, the correlation signal between H-3 and Me-18 was not observed, demonstrating that the double bond at the C3=C4 position has an E configuration; H-5 is correlated with H-3, H-7, Me-16, and Me-19, H-7 is correlated with H-3 and H-5, and H-11 is correlated with H-7, Me-16, and Me-20, demonstrating that H-5, H-7, H-11, and Me-19 are in the β configuration. Correspondingly, the OH groups at C-5, C-7, C-8, and C-11 positions are in the α configuration, as Figure 6 shown. By comparing the calculated ECD with the experimentally measured ECD spectrum, the absolute configuration of compound 1 was determined to be 1S,5S,7R,8S,11R,12R, as Figure 7 shown. In summary, the structure of compound 1 was determined to be (1S,5S,7R,8S,11R,12R,3E)-1:12-epoxy-3-cembranene-5,7,8,11-tetraol.

[0083] (1S,7R,8R,11R,12R,3E)-1:12-epoxy-3-cembranene-5,7,8,11-tetraol (2): light yellow oil; HR-ESI-MS: m / z 339.2499 [M-H] - (theoretical value: 339.2529, C 20 H 35 O4), as Figure 8 shown, and its molecular formula was determined to be: C 20 H 36 O4, with an unsaturation degree of 3. The infrared spectrum shows absorption at 3432 cm -1 indicating the presence of a hydroxyl functional group. Comparing with compound 1, it was found that the mass spectrometry data of compound 2 is 16 less, that is, 1 O atom less. By comparing the 1 H-NMR and 13 C-NMR spectral data of compounds 1 and 2, as Figures 2 - 3, as shown in Figures 9 - 10, it was found that the structures of the two were similar, with the difference being that the oxygen - connected CH signal at the C - 5 position in Compound 1 was replaced by the CH2 signal in Compound 2. This difference was further confirmed by the relevant signals in the two - dimensional nuclear magnetic resonance, as Figures 11 - 12 shown. In the HMBC spectrum, H - 3 was correlated with C - 1 / C - 4 / C - 5 / Me - 18, H2 - 5 was correlated with C - 3 / C - 4 / C - 6 / C - 7 / Me - 18, H - 7 was correlated with C - 5 / C - 6 / C - 8 / C - 9 / Me - 19, and Me - 18 was correlated with C - 4 / C - 3 / C - 5, further determining the above - mentioned difference. Finally, the planar structure of Compound 2 was determined to be 1:12 - epoxy - 3 - cembranene - 7,8,11 - triol.

[0084] The relative configuration and stereoconfiguration of Compound 2 were obtained through the NOESY spectrum and the calculated ECD spectrum. In the NOESY spectrum, the correlation signal between H - 3 and Me - 18 was not observed, proving that the double bond at the C3 = C4 position was in the E configuration; H - 7 was correlated with H - 3, H - 11, and Me - 19, and H - 11 was correlated with H - 7 and Me - 20, proving that H - 7 and H - 11 were in the β configuration and Me - 19 was in the α configuration. Correspondingly, the OH groups at C - 7 and C - 11 were in the α configuration, and the OH group at C - 8 was in the β configuration, as Figure 13 shown. By comparing the calculated ECD with the experimentally measured actual ECD spectrum, the absolute configuration of Compound 2 was determined to be 1S,7R,8R,11R,12R,3E, as Figure 14 shown. In summary, the structure of Compound 2 was determined to be (1S,7R,8R,11R,12R,3E)-1:12 - epoxy - 3 - cembranene - 7,8,11 - triol.

[0085] Table 1 1 1H NMR (400 MHz, CDCl3) and 13 13C NMR data (100 MHz, CDCl3) of Compounds 1 and 2

[0086]

[0087]

[0088] Pharmacological experiments:

[0089] I. Study on the anti - tuberculosis drug - induced liver injury activities of 5,7,8,11 - tetrahydroxy cembranoid diterpenoids and 7,8,11 - trihydroxy cembranoid diterpenoids in frankincense

[0090] 1. Establishment of the tuberculosis drug - induced liver injury model

[0091] The HepG2 cell line was incubated in a cell culture incubator with 5% CO2 at 37°C and cultured using RPMI-1640 medium, 100 U / mL penicillin, 100 mg / mL streptomycin, and 10% fetal bovine serum (FBS) until logarithmic cell proliferation occurred for subculture. Take a 96-well cell plate and add 100 μL of cell suspension with a concentration of 1×10 5 cells / mL to each well, and culture adherently for 24 h. Set up a blank group and an HRZ model group for model construction. The blank group was added with 100 μL of RPMI-1640 medium without fetal bovine serum, and the HRZ model group was given 100 μL of RPMI-1640 medium without fetal bovine serum containing 100 mg / mL isoniazid, 100 mg / mL rifampicin, and 100 mg / mL pyrazinamide to construct a cell model of liver injury induced by antituberculosis drugs.

[0092] 2. Screening for the activities of compounds 1 and 2 against liver injury induced by antituberculosis drugs

[0093] (1) The MTT method was used to detect the survival rates of compounds 1 and 2 on HepG2 cells. Take a 96-well cell plate and add 100 μL of cell suspension with a concentration of 1×10 5 cells / mL to each well, and culture adherently for 24 h. Set up a blank group, a compound 1 group, and a compound 2 group. The blank group was added with 100 μL of RPMI-1640 medium without fetal bovine serum, and the compound 1 group and the compound 2 group were respectively added with 100 μL of RPMI-1640 medium without fetal bovine serum containing 10 μM of compound 1 and 2. After continued incubation for 24 h, 10 μL of MTT (5 mg·mL -1 ) solution was added to each well. After placing in the dark for 4 h, the supernatant was aspirated, 100 μL of DMSO was added to each well, shaken evenly, and the A value of each well was measured at 490 nm with an enzyme-linked immunosorbent assay (ELISA) reader. The experiment was repeated 3 times to calculate the cell survival rate.

[0094] (2) Using the cell model established in (1), a blank group, an HRZ model group, a positive control group, and a drug administration group were set up for activity screening. The blank group was added with 100 μL of RPMI-1640 medium without fetal bovine serum. The HRZ model group was given 100 μL of RPMI-1640 medium without fetal bovine serum containing 100 mg / mL isoniazid, 100 mg / mL rifampicin, and 100 mg / mL pyrazinamide. The positive control group was given 100 μL of medium containing 10 μM bicyclol (containing 100 mg / mL isoniazid, 100 mg / mL rifampicin, and 100 mg / mL pyrazinamide). The drug administration group was given 100 μL of medium containing 10 μM compounds 1 and 2 (containing 100 mg / mL isoniazid, 100 mg / mL rifampicin, and 100 mg / mL pyrazinamide). Three replicate wells were set for each compound. After culturing for 48 h, 15 μL of 15 mg / mL MTT solution was added to each well. After placing in the dark for 4 h, the supernatant was aspirated. 100 μL of DMSO was added to each well and shaken. The absorbance (A) value of each well was measured at 490 nm using a multifunctional microplate reader, and the anti-tuberculosis drug-induced liver injury activities of compounds 1 and 2 were calculated. The results are shown in Table 2.

[0095] Table 2 Screening results of the anti-tuberculosis drug-induced liver injury activities of polyhydroxycembraane diterpenoids 1 and 2 in Boswellia

[0096]

[0097] The MTT method was used to determine the survival rates of compounds 1 and 2 on HepG2 cells. The data in Table 2 showed that neither of them had a significant inhibitory effect on the growth of HepG2 cells, and the cell survival rate was above 85%. Through the constructed tuberculosis drug-induced liver injury model, the hepatoprotective activities of compounds 1 and 2 were determined. From the data in Table 2, it can be seen that compound 2 had good inhibitory activity against liver injury with an inhibition rate of 69.1%, and compound 1 had moderate inhibitory activity against liver injury with an inhibition rate of 50.7%. In addition, the hepatoprotective activities of compounds 1 and 2 were both higher than those of the positive control drug.

[0098] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A polyhydroxy cembrane diterpenoid compound, characterized in that it is Compound 1 or Compound 2, the chemical structural formulas of Compound 1 or Compound 2 are shown as follows:

2. A preparation method of the polyhydroxy siphonane diterpenoid compound according to claim 1, characterized in that, Comprising the following steps: Using an ethanol solution with a volume fraction of 75 - 85% to extract frankincense to obtain a frankincense extract; Using petroleum ether to extract the frankincense extract to obtain a petroleum ether extraction fraction; Performing a first gradient elution on the petroleum ether extraction fraction using silica gel column chromatography to obtain a first elution fraction; in the first gradient elution, successively using petroleum ether, a mixed solvent with a volume ratio of petroleum ether to ethyl acetate of 49 - 51:1, and a mixed solvent with a volume ratio of petroleum ether to ethyl acetate of 24 - 26:1 for gradient elution, and the elution fraction with a volume ratio of petroleum ether to ethyl acetate of 24 - 26:1 is the first elution fraction; Performing a second gradient elution on the first elution fraction using silica gel column chromatography to obtain a second elution fraction; in the second gradient elution, successively using a mixed solvent with a volume ratio of petroleum ether to ethyl acetate of 49 - 51:1, a mixed solvent with a volume ratio of petroleum ether to ethyl acetate of 24 - 26:1, a mixed solvent with a volume ratio of petroleum ether to ethyl acetate of 9 - 11:1, and a mixed solvent with a volume ratio of petroleum ether to ethyl acetate of 4 - 6:1 for gradient elution, and the elution fraction with a volume ratio of petroleum ether to ethyl acetate of 4 - 6:1 is the second elution fraction; Performing a third gradient elution on the second elution fraction using silica gel column chromatography to obtain an elution fraction of Product 1; in the third gradient elution, successively using a mixed solvent with a volume ratio of petroleum ether to ethyl acetate of 6.9 - 7.1:1 and a mixed solvent with a volume ratio of petroleum ether to ethyl acetate of 4.9 - 5.1:1 for gradient elution, and the elution fraction with a volume ratio of petroleum ether to ethyl acetate of 4.9 - 5.1:1 is the elution fraction of Product 1; Using a mixed solution with a volume ratio of acetonitrile to water of 64.5 - 65.5:35.5 - 34.5 as the mobile phase, and separating the elution fraction of Product 1 using a semi - preparative chromatographic column to obtain Compound 1.

3. The preparation method of the polyhydroxy cembrane diterpenoid compound according to claim 2, characterized in that, When separating the elution fraction of Product 1 using a semi-preparative chromatographic column, the detection wavelength is 204 - 206 nm and the flow rate is 4 - 6 mL / min -1 .

4. A method for preparing the polyhydroxy cembrane diterpenoid compound according to claim 1, characterized in that, Comprising the following steps: Using an ethanol solution with a volume fraction of 75 - 85% to extract frankincense to obtain a frankincense extract; Using petroleum ether to extract the frankincense extract to obtain a petroleum ether extraction fraction; Performing a first gradient elution on the petroleum ether extraction fraction using silica gel column chromatography to obtain a first elution fraction; in the first gradient elution, successively using petroleum ether, a mixed solvent with a volume ratio of petroleum ether to ethyl acetate of 49 - 51:1, and a mixed solvent with a volume ratio of petroleum ether to ethyl acetate of 24 - 26:1 for gradient elution, and the elution fraction with a volume ratio of petroleum ether to ethyl acetate of 24 - 26:1 is the first elution fraction; The primary elution fraction was subjected to secondary gradient elution using silica gel column chromatography to obtain the secondary elution fraction; in the secondary gradient elution, a mixed solvent with a volume ratio of petroleum ether to ethyl acetate of 49 - 51:1, a mixed solvent with a volume ratio of petroleum ether to ethyl acetate of 24 - 26:1, a mixed solvent with a volume ratio of petroleum ether to ethyl acetate of 9 - 11:1, and a mixed solvent with a volume ratio of petroleum ether to ethyl acetate of 4 - 6:1 were successively used for gradient elution, and the elution fraction with a volume ratio of petroleum ether to ethyl acetate of 4 - 6:1 was the secondary elution fraction; The secondary elution fraction was subjected to tertiary gradient elution using silica gel column chromatography to obtain the product 2 elution fraction; in the tertiary gradient elution, a mixed solvent with a volume ratio of petroleum ether to ethyl acetate of 6.9 - 7.1:1, a mixed solvent with a volume ratio of petroleum ether to ethyl acetate of 4.9 - 5.1:1, and a mixed solvent with a volume ratio of petroleum ether to ethyl acetate of 3.9 - 4.1:1 were successively used for gradient elution, and the elution fraction with a volume ratio of petroleum ether to ethyl acetate of 3.9 - 4.1:1 was the product 2 elution fraction; Using a mixed solution with a volume ratio of acetonitrile to water of 67.5 - 68.5:32.5 - 31.5 as the mobile phase, compound 2 was obtained by separating the product 2 elution fraction using a semi - preparative chromatographic column.

5. The preparation method of the polyhydroxy cembrane diterpenoid compound according to claim 2 or 4, characterized in that, When extracting frankincense with an ethanol solution, heating under reflux was carried out.

6. The preparation method of the polyhydroxy cembrane diterpenoid compound according to claim 2 or 4, characterized in that, When extracting frankincense with an ethanol solution, the solid - liquid ratio was 1:2.5 - 3.

5.

7. The preparation method of the polyhydroxy cembrane diterpenoid compound according to claim 4, characterized in that, When separating the elution part of product 2 using a semi-preparative chromatographic column, the detection wavelength is 204 - 206 nm and the flow rate is 4 - 6 mL min -1 .

8. A composition, characterized in that, It includes the polyhydroxy sesterterpene diterpenoid compound described in claim 1 and a pharmaceutical excipient.

9. The composition according to claim 8, characterized in that, The pharmaceutical excipient is a pharmaceutical carrier and / or excipient.

10. Use of the polyhydroxy sesterterpene diterpenoid compound described in claim 1 or the composition described in claim 8 or 9 in the preparation of a drug for treating liver injury.

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