CN108434180B

CN108434180B - Herba Saussureae Involueratae extract, diterpenoid compounds and application thereof in preparation of anti-pulmonary fibrosis drugs

Info

Publication number: CN108434180B
Application number: CN201810346526.3A
Authority: CN
Inventors: 陆金健; 林理根; 李傲; 张乐乐
Original assignee: University of Macau
Current assignee: University of Macau
Priority date: 2018-04-17
Filing date: 2018-04-17
Publication date: 2024-08-20
Anticipated expiration: 2038-04-17
Also published as: CN108434180A

Abstract

The invention discloses a herba phellodendri extract, a diterpenoid compound and application thereof in preparing medicines for resisting pulmonary fibrosis, and relates to the field of medicines. The cypress extract disclosed by the invention is selected from the total cypress extract, ethyl acetate extract and n-butanol extract; the diterpene compound has a structure as shown in formula (I), (II) or (III). The research of the invention shows that the total extract of the bamboo and cypress, the ethyl acetate extract, the n-butyl alcohol extract and the diterpene compound with the structure shown in the formula (I), (II) or (III) have remarkable anti-pulmonary fibrosis activity, can be used for preparing anti-pulmonary fibrosis medicines, and can be used as active substances or active ingredients to provide a new thought or means for preventing or treating pulmonary fibrosis diseases.

Description

Herba Saussureae Involueratae extract, diterpenoid compounds and application thereof in preparation of anti-pulmonary fibrosis drugs

Technical Field

The invention relates to the field of medicines, in particular to a herba phellodendri extract and a diterpene-reducing compound and application thereof in preparing medicines for resisting pulmonary fibrosis.

Background

Pulmonary fibrosis is a common outcome of pulmonary interstitial disease caused by various pathogenic factors, and is extremely harmful to human health. Pulmonary fibrosis is a relatively complex physiological and pathological process, and currently, safe and effective therapeutic drugs and means are still lacking clinically. In recent years, the search for novel compounds with anti-pulmonary fibrosis activity from natural medicinal plants is focused, and the further development of safe and effective anti-pulmonary fibrosis drugs on the basis is a hot spot of current medical research.

Disclosure of Invention

The invention aims to provide a kind of cypress extract, a diterpenoid compound or a pharmaceutically acceptable salt or ester thereof, which has anti-pulmonary fibrosis activity.

The invention also aims to provide application of the thuja extract, the norditerpenoid compound or the pharmaceutically acceptable salt or ester thereof.

It is another object of the present invention to provide a class of anti-pulmonary fibrosis drugs. The medicine has anti-pulmonary fibrosis activity.

Another object of the present invention is to provide a method for preparing the above-mentioned anti-pulmonary fibrosis drug.

The invention is realized in the following way:

a herba Saussureae Involueratae extract is selected from the group consisting of total extract, ethyl acetate extract and n-butanol extract of herba Saussureae Involueratae (Podocarpus nagi).

The preparation method of the cypress extract is referred as follows:

the method comprises pulverizing herba Selaginellae Doederleinii seed, reflux-extracting with 80-95% ethanol for three times, concentrating to obtain extract (i.e. the above total extract), suspending the extract with distilled water, sequentially extracting with petroleum ether for three times, ethyl acetate for three times (to obtain the above ethyl acetate extract), and n-butanol for three times (to obtain the above n-butanol extract), and sequentially collecting petroleum ether, ethyl acetate, n-butanol and the rest water-soluble extract.

A class of diterpene compounds or pharmaceutically acceptable salts or esters thereof, having the structure shown in the following general formula (I), (II) or (III):

In the general formulae (I) and (II):

R1 to R3 are each independently a hydrogen atom, a hydroxyl group or a hydroxyl group protected by a protecting group, or any adjacent two groups of R1 to R3 form an oxirane ring together with the carbon atom to which they are attached, the remaining one being a hydrogen atom or a hydroxyl group;

r5 is isopropyl, ethyl, 1-hydroxyethyl, 2-hydroxyisopropyl or hydroxyl protected by a protecting group;

in the general formula (III):

R1 to R4 are each independently a hydrogen atom, a hydroxyl group or a hydroxyl group protected by a protecting group, or any adjacent two groups of R1 to R3 form together with the carbon atom to which they are attached an oxirane ring;

r5 is isopropyl, ethyl, 1-hydroxyethyl, 2-hydroxyisopropyl or hydroxyl protected by a protecting group.

Further, in some embodiments of the invention, the diterpene-lowering compound is selected from the group consisting of bamboo Bai Nazhi D, bamboo Bai Nazhi E, bamboo Bai Nazhi G, 3β -hydroxy bamboo Bai Nazhi F, and bambusa lactone B.

Wherein, the structure of bamboo Bai Nazhi D is shown as the following formula (IV):

the structure of the bambusa lactone E is shown as the following formula (V):

the structure of bamboo Bai Nazhi G is shown in the following formula (VI):

the structure of 3 beta-hydroxy bamboo Bai Nazhi F is shown in the following formula (VII):

the structure of the bamboo Bai Nazhi B is shown as the following formula (VIII):

diterpenoid-lowering compounds are widely present in the arhat pine family of plants. The compound has a certain antitumor activity, and can inhibit proliferation of tumor cells such as colorectal cancer. A series of diterpenoid compounds are separated from ethyl acetate and n-butanol extracts of the cypress seeds in the early stage, and the compounds are found to have obvious anti-tumor activity, and part of the compounds can induce tumor cells to autophagy. In addition, it has been reported that the diterpenoid compound bamboo Bai Nazhi B has an anti-atherosclerosis effect and can inhibit atherosclerosis of mice with ApoE gene defects. But there are no reports of its anti-pulmonary fibrosis activity.

Transforming growth factor-beta 1 (TGF-beta 1) is widely recognized as one of key molecules causing fibrosis, plays an important role in the occurrence and development of pulmonary fibrosis, and is also widely used as a modeling medicament for an in vitro cell model of pulmonary fibrosis. The injection of bleomycin into the annulus of the tracheal cartilage is currently the most commonly used method for replicating animal models of pulmonary fibrosis. The research of the invention carries out drug screening after the TGF-beta 1 is used for inducing the alveolar epithelial cell strains A549 and RLE-6TN to generate the myofibroblast-like transdifferentiation change, discovers that the herba phellodendri extract and the diterpenoid compounds have remarkable anti-pulmonary fibrosis activity, and uses a bleomycin-induced pulmonary fibrosis animal model to carry out in vivo experimental verification.

Based on the above, the herba cypress extract, the diterpene-reducing compound or the pharmaceutically acceptable salt or ester thereof can be used for preparing medicines for preventing or resisting pulmonary fibrosis, or can be used for preparing inhibitors for inhibiting the expression of protein related to cell fibrosis and other fields, thereby providing a new thought or means for preventing or treating pulmonary fibrosis diseases.

The application of the herba Selaginellae extract, the diterpene compound or the pharmaceutically acceptable salt or ester thereof in preparing medicines for treating pulmonary fibrosis is provided.

Further, in some embodiments of the invention, the pulmonary fibrosis is caused by transforming growth factor- β1 or a pharmaceutical factor.

Use of a thuja extract, a norditerpenoid compound or a pharmaceutically acceptable salt or ester thereof as described above for the preparation of an inhibitor for inhibiting the expression of a protein associated with cellular fibrosis.

Further, in some embodiments of the invention, the cellular fibrosis-associated protein is selected from one or more of type I collagen, fibronectin, connective tissue growth factor, vimentin, and alpha-smooth muscle actin.

An anti-pulmonary fibrosis medicine contains the extract of herba Saussureae Involueratae, nor diterpenoid compound or pharmaceutically acceptable salt or ester thereof.

Further, in some embodiments of the invention, the anti-pulmonary fibrosis drug further comprises a pharmaceutically acceptable adjuvant.

Further, in some embodiments of the invention, the pharmaceutically acceptable excipients are selected from one or more of a cosolvent, excipient, emulsifier, preservative, suspending agent, preservative, filler, binder, disintegrant, lubricant, surfactant, or flavoring agent.

Further, in some embodiments of the invention, the anti-pulmonary fibrosis drug is an injection, aerosol, paste, patch, tablet, capsule, granule, oral liquid, syrup, pill.

A method of preparing an anti-pulmonary fibrosis drug as described above comprising: mixing the herba Saussureae Involueratae extract, diterpene compounds or pharmaceutically acceptable salts or esters thereof with pharmaceutically acceptable adjuvants.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 shows the results of detection of the total extract, ethyl acetate extract and n-butanol extract of herba Saussureae Involueratae in example 1 of the present invention, which significantly inhibit TGF- β1 induced morphology changes of A549 and RLE-6TN cell interstitium samples;

FIG. 2 shows the results of the detection of the total extract, ethyl acetate extract and n-butanol extract of the herba Saussureae Involueratae in example 2 of the present invention, which significantly inhibit TGF- β1 induced changes in A549 and RLE-6TN cell fibrosis related proteins;

FIG. 3 shows the results of detection of the diterpene compounds bamboo Bai Nazhi D, bamboo Bai Nazhi E, bamboo Bai Nazhi G, 3 beta-hydroxy bamboo Bai Nazhi F, and bambusa lactone B in example 3 of the present invention significantly inhibiting TGF-beta 1 induced A549, RLE-6TN cell interstitial-like morphological changes;

FIG. 4 shows the results of detection of the changes in the diterpene compounds bamboo Bai Nazhi D, bamboo Bai Nazhi E, bamboo Bai Nazhi G, 3β -hydroxy bamboo Bai Nazhi F, and hinokitiol B in example 4 significantly inhibiting TGF- β1 induced changes in A549, RLE-6TN cell fibrosis associated proteins;

FIG. 5 is a view of the results of the observation of HE and Masson's trichrome staining of bleomycin-induced pulmonary fibrosis mouse pulmonary tissue with bamboo Bai Nazhi D under light microscope at 200 Xmagnification in example 5 of the present invention;

FIG. 6 is a graph showing the effect of bamboo Bai Nazhi D in example 6 of the present invention on hydroxyproline content in bleomycin-induced pulmonary fibrosis mouse lung tissue; in the figure: * Representing: p <0.05 compared to sham surgery group; # represents: p <0.05 compared to bleomycin model control;

FIG. 7 shows the effect of bamboo Bai Nazhi D in example 7 of the present invention on the expression of Collagen I and Collagen III proteins in the lung tissue of bleomycin-induced pulmonary fibrosis mice; in the figure: * Representing: p <0.05 compared to sham surgery group; # represents: p <0.05 compared to bleomycin model control.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

The features and capabilities of the present invention are described in further detail below in connection with the examples.

Example 1

The herba Saussureae Involueratae extract can significantly improve TGF-beta 1 induced morphological changes of lung fibrosis cell model

A549 cells and RLE-6TN cells were inoculated in 6-well plates for wall-adhering culture overnight, and a control group, a model group, and an extract group were set respectively. Wherein the control group was further cultured with the medium for 48 hours, the model group was treated with 5ng/mL of TGF-. Beta.1 for 48 hours, the extract group was treated with 5 ng/mLTGF-. Beta.1 together with each extract (total extract of Sabina chinensis, ethyl acetate extract, n-butanol extract) for 48 hours, and the extract was set at a concentration of 10. Mu.g/mL. After 48 hours, each group of cells was morphologically photographed under an inverted light microscope in a random field of view, respectively. The results are shown in FIG. 1.

As can be seen from fig. 1, TGF- β1-induced model groups a549 and RLE-6TN are fusiform in morphology and present in a mesenchymal morphology, and the synchronous treatment of the total extract of the bambusa, the ethyl acetate extract and the n-butanol extract can significantly improve the mesenchymal morphology change of the cells.

Example 2

The herba Saussureae Involueratae extract can significantly improve TGF-beta 1 induced change of lung fibrosis index of cell model

A549 cells and RLE-6TN cells were inoculated in 6-well plates for wall-adhering culture overnight, and a control group, a model group, and an extract group were set respectively. Wherein the control group is cultured with culture medium for 48 hours, the model group is treated with 5ng/mL TGF-beta 1 for 48 hours, the compound treatment group is treated with 5ng/mL TGF-beta 1 together with each extract (total extract of herba Lophatheri, ethyl acetate extract, n-butanol extract) for 48 hours, and the extracts are respectively set to 5 μg/mL and 10 μg/mL. After 48 hours, the cells were scraped off with a cell scraper, lysed with cell lysate on ice for 20 minutes, centrifuged at 4℃at 15 000 rpm for 20 minutes, the supernatant was aspirated, the protein concentration was determined with BCA ^TM kit, and the corresponding amount of protein loading buffer was added for denaturation at 95℃for 5 minutes. Protein expression was determined by Western Blot at 20. Mu.g loading. The results are shown in FIG. 2.

TGF- β1 induces changes in a range of fibrotic marker proteins, including upregulation of Collagen type I (Collagen I), fibronectin (Fibronectin), connective Tissue Growth Factor (CTGF), vimentin (Vimentin), alpha-smooth muscle actin (alpha-SMA), and the like, during cellular fibrosis. As can be seen from fig. 2, treatment of the total extract of the phellodendron, the ethyl acetate extract and the n-butanol extract can significantly inhibit the change of TGF-beta 1 induced fibrosis related proteins of model groups A549 and RLE-6TN cells.

Example 3

Diterpenoid-lowering compounds significantly improve TGF-beta 1-induced morphological changes in lung fibrosis cell models

A549 cells and RLE-6TN cells were inoculated in 6-well plates for adherent culture overnight, and a control group, a model group, and a compound treatment group were set, respectively. Wherein, the control group is cultured for 48 hours with the culture medium, the model group is treated with 5ng/mL TGF-beta 1 for 48 hours, the compound treatment group is treated with 5ng/mL TGF-beta 1 together with each compound (bamboo Bai Nazhi D, bamboo Bai Nazhi E, bamboo Bai Nazhi G, 3 beta-hydroxy bamboo Bai Nazhi F, bamboo Bai Nazhi B) for 48 hours, and the compounds are all set at 4 mu M concentration. After 48 hours, each group of cells was morphologically photographed under an inverted light microscope in a random field of view, respectively. The results are shown in FIG. 3.

As can be seen from fig. 3, TGF- β1-induced model groups a549 and RLE-6TN are fusiform in cell morphology, and present a mesenchymal cell-like morphology, and the synchronous treatment of the diterpene compounds bamboo Bai Nazhi D, bamboo Bai Nazhi E, bamboo Bai Nazhi G, 3 β -hydroxy bamboo Bai Nazhi F and bamboo Bai Nazhi B can significantly improve the mesenchymal morphology change of the cells.

Example 4

Reducing diterpenoid compounds significantly improves TGF-beta 1 induced changes in lung fibrosis index of cell model

A549 cells and RLE-6TN cells were inoculated in 6-well plates for adherent culture overnight, and a control group, a model group, and a compound treatment group were set, respectively. Wherein the control group was cultured with the culture medium for 48 hours, the model group was treated with 5ng/mL TGF-. Beta.1 for 48 hours, the compound treatment group was treated with 5ng/mL TGF-. Beta.1 together with each compound (bamboo Bai Nazhi D, bamboo Bai Nazhi E, bamboo Bai Nazhi G, 3-. Beta. -hydroxy bamboo Bai Nazhi F, bamboo Bai Nazhi B) for 48 hours, and the compounds were set to 2. Mu.M and 4. Mu.M concentrations, respectively. After 48 hours, the cells were scraped off with a cell scraper, lysed with cell lysate on ice for 20 minutes, centrifuged at 4℃at 15 000 rpm for 20 minutes, the supernatant was aspirated, the protein concentration was determined with BCA ^TM kit, and the corresponding amount of protein loading buffer was added for denaturation at 95℃for 5 minutes. Protein expression was determined by Western Blot at 20. Mu.g loading. The results are shown in FIG. 4.

As can be seen from fig. 4, the treatment of the diterpene compounds bamboo Bai Nazhi D, bamboo Bai Nazhi E, bamboo Bai Nazhi G, 3 beta-hydroxy bamboo Bai Nazhi F and bamboo Bai Nazhi B can significantly inhibit the TGF-beta 1 induced changes of the protein associated with fibrosis of the model group a549 and RLE-6TN cells.

Example 5

Bambusolide D significantly improves lung histopathological changes of lung fibrosis model mice

The mice were randomly divided into 3 groups according to body weight, 10 mice each, respectively, a sham operation group, a model control group, and a bambusa lactone D group. After the mice in each group were anesthetized by intraperitoneal injection of phenobarbital sodium (50 mg/kg), the neck skin was sterilized with ethanol, the middle of the neck was subjected to blunt separation, the trachea was exposed, and 50 μl (3.5 mg/kg) of bleomycin was slowly injected into the tracheal cartilage annulus between the model group and the bambusactone group D, and the control group was injected with the same volume of physiological saline. Immediately after injection, the mice were rotated upright, the fluid was evenly distributed across the lungs, and the neck skin was sutured. From the 7 th day of molding, bamboo Bai Nazhi D (10 mg/kg) was administered by i 0mL/kg volume of intraperitoneal injection, 1 time a day, 15 times continuously; sham-operated and model control mice were intraperitoneally injected daily with the same volume of physiological saline. The experimental animals were sacrificed after 21 days of molding, the lower lobe tissue of the right lung was taken and fixed in 4% paraformaldehyde solution, the lung tissue was verified, cell degeneration and fibrosis were observed by hematoxylin-eosin (H & E) staining, and collagen hyperplasia in the lung tissue was observed by Masson's trichrome staining.

The results of HE staining of the lung tissue sections of mice are shown in FIG. 5, the lung tissue structures of mice in the sham operation group are clear, alveoli are polygonal saccular bodies, secretion does not exist in alveolar cavities, the alveolar epithelium has complete structure, the alveolar walls are not thickened, inflammatory cells are not found in alveolar compartments, and capillary vessels are enriched. The bleomycin model group mice have disordered alveolar structure, the alveolar walls are obviously thickened, and macrophages and lymphocytes in the alveolar septum are obviously infiltrated, and more fibrous tissue hyperplasia is visible. Compared with the model group, the inflammatory cell infiltration and the lung parenchymal lesions in the lung tissue of the mice of the administration group of the bambusa lactone D are obviously reduced, the alveolar collagen-isolated fiber hyperplasia focus is obviously reduced, and the alveolar structure is basically normal.

The Masson's trichrome staining results showed that only a small amount of blue collagen fibrils were deposited in the bronchiole and alveolar septum of the sham mice (fig. 5). A large amount of blue-stained collagen fibers can be seen on the bronchiole wall and surrounding, alveolar wall and space of the model group mice. The alveolar walls of mice in the treatment group with hinokitiol D had slight thickening, and only a small amount of inflammatory cells had infiltrated and blue-stained collagen fiber deposited.

Example 6

The bambusa lactone D obviously reduces the content of hydroxyproline in lung tissues of lung fibrosis model mice

The mice were randomly divided into 3 groups according to body weight, 10 mice each, respectively, a sham operation group, a model control group, and a bambusa lactone D group. After the mice in each group were anesthetized by intraperitoneal injection of phenobarbital sodium (50 mg/kg), the neck skin was sterilized with ethanol, the middle of the neck was subjected to blunt separation, the trachea was exposed, and 50 μl (3.5 mg/kg) of bleomycin was slowly injected into the tracheal cartilage annulus between the model group and the bambusactone group D, and the control group was injected with the same volume of physiological saline. Immediately after injection, the mice were rotated upright, the fluid was evenly distributed across the lungs, and the neck skin was sutured. From the 7 th day of molding, bamboo Bai Nazhi D (10 mg/kg) was administered by i 0mL/kg volume of intraperitoneal injection, 1 time a day, 15 times continuously; sham-operated and model control mice were intraperitoneally injected daily with the same volume of physiological saline. The experimental animals are sacrificed after 21 days of molding, 50mg of upper lobe tissue of the right lung is accurately weighed, and the hydroxyproline content is detected by the alkaline hydrolysis according to the instruction of the kit.

Further examination of the hydroxyproline content of collagen fiber I characteristic component in lung tissue revealed that the hydroxyproline content in lung tissue was significantly increased in the bleomycin model group mice compared to the sham operation group (P <0.05; FIG. 6). Compared with the model group, the bambusa lactone D can obviously reduce the hydroxyproline content (P < 0.05) in lung tissues of mice.

Example 7

The bambusolide D obviously reduces I, III type collagen expression in lung tissue of lung fibrosis model mice

The mice were randomly divided into 3 groups according to body weight, 10 mice each, respectively, a sham operation group, a model control group, and a bambusa lactone D group. After the mice in each group were anesthetized by intraperitoneal injection of phenobarbital sodium (50 mg/kg), the neck skin was sterilized with ethanol, the middle of the neck was subjected to blunt separation, the trachea was exposed, and 50 μl (3.5 mg/kg) of bleomycin was slowly injected into the tracheal cartilage annulus between the model group and the bambusactone group D, and the control group was injected with the same volume of physiological saline. Immediately after injection, the mice were rotated upright, the fluid was evenly distributed across the lungs, and the neck skin was sutured. From the 7 th day of molding, bamboo Bai Nazhi D (10 mg/kg) was administered by i 0mL/kg volume of intraperitoneal injection, 1 time a day, 15 times continuously; sham-operated and model control mice were intraperitoneally injected daily with the same volume of physiological saline. The experimental animals were sacrificed 21 days after molding, and part of the lung tissue was snap frozen with liquid nitrogen, and the extracellular matrix component I, III-type collagen expression level was measured by Western Blot.

As shown in fig. 7, the sham-operated group expressed only a small amount of Collagen type I, III (Collagen I, collagen III), and the bleomycin-induced pulmonary fibrosis model group had a significantly up-regulated Collagen type I, III expression; compared with the model group, the expression level of Collagen I and CollagenIII of the bambusicolin D group is obviously lower than that of the bleomycin model group (P < 0.05), which indicates that the bambusicolin D can obviously reduce the deposition of Collagen components in lung tissues and improve the degree of pulmonary fibrosis.

Example 8

This example provides the preparation of the extract and the different parts of the cypress used in examples 1-7, and the separation of bamboo Bai Nazhi D, bamboo Bai Nazhi E, bamboo Bai Nazhi G, 3 beta-hydroxy bamboo Bai Nazhi F, and bamboo Bai Nazhi B.

15Kg of seeds of a medicinal plant, namely, phellodendron amurense (Podocarpus nagi), are taken as a raw material, crushed, extracted with 40L of 80-95% ethanol under reflux for three times, concentrated to obtain an extract, the extract is suspended with distilled water, extracted with petroleum ether for three times, ethyl acetate for three times and n-butanol for three times in sequence, 174.6g of the extract obtained by using petroleum ether is collected, 138.7g of the extract obtained by using ethyl acetate is collected, 78.3g of the extract obtained by using n-butanol is collected, and 878.2g of the residual water-soluble extract is collected.

Separating the extract obtained by extraction with ethyl acetate with MCI resin, gradient eluting with 0-100% methanol water, separating out crystals at 20% methanol position and 40% methanol position, and identifying as bamboo Bai Nazhi B and bamboo Bai Nazhi D; the mobile phase chloroform methanol 19 is subjected to silica gel column chromatography in a 70% methanol elution section: eluting and separating out crystals to obtain compounds of the bambusa lactone E and the bambusa lactone G.

Subjecting n-butanol fraction to MCI column chromatography, eluting with methanol-water ratio 19:1 to 1:19, combining MCI column chromatography 10:1 eluting sections and eluting with silica gel column chromatography chloroform methanol 60:1 to 5:1, eluting with 15: and (3) combining the elution sections, and eluting by using high-efficiency preparation liquid phase 45% acetonitrile to obtain the compound 3 beta-hydroxy bambusa lactone F.

Bamboo Bai Nazhi B: crystallizing in acetone, and white needle-like crystal ,mp 320℃.MF:C₁₉H₂₄O₇.EI-MS:m/z 364(M⁺),347(M⁺-OH),346(M⁺-H₂O).¹H NMR(600MHz,C₅D₅N)δ4.32(d,J＝6.1Hz,1H,H-1),4.28(ddd,J＝4.6,6.1,13.1Hz,1H,H-2),2.10(dd,J＝13.1,4.5Hz,1H,H-3α),2.74(dd,J＝13.1,13.1Hz,1H,H-3β),1.89(d,J＝6.6Hz,1H,H-5),5.16(dd,J＝6.6,8.4Hz,1H,H-6),5.64(d,J＝8.4Hz,1H,H-7),7.01(s,1H,H-11),3.47(m,J＝6.7Hz,1H,H-15),1.27,1.24(d,J＝6.8Hz,6H,Me-16,Me-17),2.01(s,3H,Me-18),1.45(s,3H,Me-20).

Bamboo Bai Nazhi D: crystallizing in acetone, and obtaining colorless needle-like crystals ,mp 265-266℃.MF:C₁₈H₂₀O₆.ESI-MS:m/z 333[M+H]⁺,355[M+Na]⁺,687[2M+Na]⁺.¹HNMR(400MHz,CDCl₃)δ3.63(d,J＝4.3Hz,1H,H-l),3.48(dd,J＝4.3,6.7Hz,1H,H-2),4.48(d,J＝6.7Hz,1H,H-3),1.91(d,J＝6.5Hz,1H,H-5),5.0(m,H-6),3.46(dd,J＝10.4,16.4Hz,1H,H-7α),2.80(dd,J＝7.1,16.4Hz,1H,H-7β),6.35(s,1H,H-11),2.64(q,J＝7.5Hz,1H,H-15),1.18(t,J＝7.5Hz,3H,Me-16),1.43(s,3H,Me-18),1.26(s,3H,Me-20).¹³C NMR(100MHz,CDCl₃):57.1(C-1),50.5(C-2),67.5(C-3),48.6(C-4),50.5(C-5),73.2(C-6),25.5(C-7),106.7(C-8),163.6(C-9),37.6(C-10),106.2(C-11),161.9(C-12),162.9(C-14),24.6(C-15),11.7(C-16),25.8(C-18),178.2(C-19),17.5(C-20).

Bambusa lactone E: crystallizing in acetone, and obtaining colorless needle-like crystals ,mp 295℃.MF:C₁₉H₂₄O₆.EI-MS:m/z 348(M⁺).¹H NMR(600MHz,DMSO)δ3.50(m,1H,H-3),1.82(d,J＝4.6Hz,1H,H-5),15.00(dd,J＝4.1,1.3Hz,1H,H-6),4.20(d,J＝1.3Hz,1H,H-7),5.96(s,H-11),4.63(d,J＝2.9Hz,1H,H-14),1.97(m,H-15),0.97(d,J＝6.8Hz,3H,Me-16),0.94(d,J＝6.7Hz,3H,Me-17),1.32(s,3H,Me-18),1.07(s,3H,Me-20).¹³CNMR(125MHz,DMSO)36.1(C-1),44.8(C-2),71.3(C-3),44.2(C-4),28.8(C-5),71.7(C-6),53.5(C-7),58.3(C-8),159.0(C-9),27.8(C-10),116.2(C-11),163.8(C-12),82.1(C-14),25.9(C-15),16.3(C-16),23.7(C-17),21.3(C-18),178.5(C-19),21.1(C-20).

Bamboo Bai Nazhi G: crystallizing in acetone, and obtaining colorless needle-like crystals ,mp 296-298℃.MF:C₁₉H₂₄O₅ESI-MS:m/z 333[M+H]⁺,355[M+Na]⁺,687[2M+Na]⁺.^lHNMR(400MHz,CDCl₃)1.85(d,J＝5Hz,1H,H-5),4.91(dd,J＝2.6,5.9Hz,1H,H-6),3.95(d,J＝2.6Hz,1H,H-7),4.40(d,J＝5.1Hz,1H,H-14),δ5.95(s,1H,H-11),1.10(d,J＝7Hz,6H,Me-16,Me-17),1.28(s,3H,Me-18),1.15(s,3H,Me-20);¹³C NMR(100Hz,CDCl₃)29.7(C-1),17.5(C-2),26.8(C-3),41.9(C-4),43.8(C-5),72.4(C-6),53.7(C-7),58.1(C-8),159.0(C-9),36.0(C-10),117.0(C-11),163.5(C-12),82.9(C-14),28.4(C-15),16.4(C-16),21.3(C-17),25.1(C-18),180.0(C-19),24.1(C-20).

3Β -hydroxy bamboo Bai Nazhi F: crystallizing in methanol, and making yellow granular crystal .MF:C₁₉H₂₄O_5.ESI-MS:m/z 333[M+H]⁺.¹H NMR(600MHz,DMSO)2.02(d,J＝4.2Hz,1H,H-5),5.16(dd,J＝5.2,2.6Hz,1H,H-6),6.32(d,J＝2.6Hz,1H,H-7),5.02(m,H-14),δ5.73(s,1H,H-11),1.09(d,J＝7.2Hz,6H,Me-16,Me-17),0.89(s,3H,Me-18),0.87(s,3H,Me-20).

The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. The application of the diterpene compound or the pharmaceutically acceptable salt thereof in preparing the anti-pulmonary fibrosis medicine is characterized in that the diterpene compound is selected from at least one of bamboo Bai Nazhi D, bamboo Bai Nazhi E, bamboo Bai Nazhi G, 3 beta-hydroxy bamboo Bai Nazhi F and bambusa lactone B.

2. The use according to claim 1, wherein the diterpene-reducing compound is selected from the group consisting of bamboo Bai Nazhi D, bamboo Bai Nazhi E, bamboo Bai Nazhi G, 3β -hydroxy bambusa lactone F and bambusa lactone B.

3. The use according to claim 1 or 2, wherein the pulmonary fibrosis is caused by transforming growth factor- β1 or a pharmaceutical factor.