阅读说明：本技术 氧化半日花烷型二萜类化合物及其分离方法和应用 (Oxidized labdane diterpenoid compounds and separation method and application thereof ) 是由 余章昕 陈光英 李小宝 韦银约 赵禹恒 曹彦翔 于 2021-08-26 设计创作，主要内容包括：本发明属植物化学技术领域,公开了氧化半日花烷型二萜类化合物及其分离方法和应用,该化合物A环碳-3位被氧化。该化合物通过水提、极性溶剂萃取、有机溶剂梯度洗脱和液相色谱分离等多级分离步骤,得到本发明的化合物I和II。药物活性实验表明,化合物具有明显的抗炎活性,说明其在制备预防或治疗炎症药物方面具有良好的应用前景。(The invention belongs to the technical field of phytochemistry, and discloses an oxidized labdane diterpenoid compound, a separation method and application thereof, wherein the carbon-3 position of the A ring of the compound is oxidized. The compound is subjected to water extraction, polar solvent extraction, organic solvent gradient elution, liquid chromatography separation and other multi-stage separation steps to obtain the compounds I and II. The pharmaceutical activity experiment shows that the compound has obvious anti-inflammatory activity, which shows that the compound has good application prospect in the aspect of preparing medicaments for preventing or treating inflammation.)

1. An oxygenated labdane diterpenoid compound characterized in that the structural formula of the compound is shown as formula I and formula II:

2. the method for isolating oxidized labdane diterpenoids according to claim 1, characterized in that it comprises the following steps:

(1) pulverizing dried folium Callicarpae Formosanae, boiling folium Callicarpae Formosanae powder in water, extracting to obtain extractive solution, and concentrating under reduced pressure to obtain crude extract;

(2) diluting the crude extract in the step (1) with water to prepare a suspension, sequentially extracting with dichloromethane and ethyl acetate, combining organic phases, and concentrating under reduced pressure to obtain an extract;

(3) performing silica gel column chromatography on the extract obtained by extracting the ethyl acetate in the step (2), and performing gradient elution by using a mixed solvent of chloroform and acetone as an eluent, wherein the elution gradient is in a volume ratio of (100:1) to (1:10), and 8 components, namely Fr.1 to Fr.8, are obtained according to the polarity;

(4) performing normal-phase silica gel column chromatography on Fr.2, performing gradient elution by using a mixed solvent of petroleum ether and ethyl acetate as an eluent in a volume ratio of (10:1) - (1:10), performing reduced pressure concentration, performing ODS reverse-phase column separation, performing gradient elution by using a mixed solvent of methanol and water as an eluent in a volume ratio of (10:90) - (90:10), and obtaining 7 components Fr.2a-Fr.2f according to the polarity; and (3) carrying out Sephadex LH-20 gel column chromatography on Fr.2d, eluting 3-6 column volumes by using methanol as an eluent, carrying out reduced pressure concentration, and carrying out HPLC (high performance liquid chromatography) preparation to obtain a compound I and a compound II in sequence.

3. The separation method of the oxidized labdane diterpenoid compounds according to claim 2, wherein in the step (1), the extraction times are more than 2 times, each time for 1-3 h, and the extracting solutions are combined; in the step (2), dichloromethane and ethyl acetate are sequentially used for extraction for more than 3 times.

4. The method for separating labdane diterpenoid compounds according to claim 2, wherein in the step (1), the water is purified water in an amount of 2-3L per kg of callicarpa nudiflora powder; heating to boil, and extracting for 1-2 hr.

5. The method for separating labdane diterpenoid compounds according to claim 2, wherein in the step (2), the amount of water is 300-400 mL per 100 g of crude extract, and the volume of the organic solvent used in each extraction is 1.2-1.3 times of the volume of water.

6. The method for separating labdane diterpenoid compounds according to claim 2, wherein in step (3), the chloroform-acetone mixed solvent is eluted at a gradient of 100:1, 80:1, 50:1, 20:1, 10:1, 5:1, 1:10 by volume, 3 column volumes are collected for each gradient, and each gradient gives one fraction, resulting in 8 fractions, i.e., fr.1 to fr.8.

7. The method for separating labdane diterpenoid compounds according to claim 2 or 6, wherein in step (4), the elution gradient of the mixed solvent of petroleum ether and ethyl acetate is 10:1, 5:1, 1:10 by volume, and each gradient elutes 2-5 column volumes; ODS reversed-phase elution ratios are that methanol and water are in a volume ratio of 10:90, 30:70, 50:50, 60:40, 70:30, 80:20 and 90:10, and each gradient elutes 3-5 column volumes; sephadex LH-20 gel column chromatography, eluting with methanol for 3-6 columnsAccumulating; the conditions of the high performance liquid chromatography are as follows: chromatographic column Waters C₁₈The flow rate is 2mL/min, and the mobile phase is acetonitrile and water in a volume ratio of 60: 40.

8. The method for isolating labdane diterpenoid compounds according to any one of claims 2-7, wherein the callicarpa nudiflora is callicarpa nudiflora harvested from Wuzhishan of Hainan.

9. Use of the labdane diterpenoid of claim 1 for the preparation of a medicament for the prevention or treatment of inflammation.

10. The use of claim 9, wherein the inflammation is systemic inflammatory response syndrome, bronchitis, pneumonia, gastritis, or enteritis.

Technical Field

The invention relates to a natural plant extract, in particular to an oxidized labdane diterpenoid compound and a separation method and application thereof.

Background

Callicarpa nudiflora (Callicarpa nudiflora hook. et Arn.) also known as desmodium and Arthron, is a plant of Callicarpa of Verbenaceae (Verbenacase), is a medicinal material in the province of Hainan province, and is a classic medicinal material in Li nationality. The above-ground parts of beautyberry can be used as medicines, has the effects of diminishing inflammation, removing toxicity, dissipating blood stasis, relieving swelling, resisting bacteria, stopping bleeding and the like, and is mainly used for treating suppurative inflammation, traumatic hemorrhage, digestive tract and respiratory tract infection, burn and scald and the like. The composition is clinically and auxiliarily used for various postoperative hemorrhagic diseases of dermatology, gynecology, ophthalmology and otorhinolaryngology, surgery and the like. Is recorded by newly added Chinese medicine varieties in 'Chinese pharmacopoeia' of 2015 edition.

The research reports on the chemical components of callicarpa nudiflora mainly include various compounds such as flavones (glycosides), phenylpropanoids (glycosides), diterpenes, triterpenes, iridoids, phenolic acids and the like, and all have good biological activity. Some of the labdane diterpenoids reported by Wang Z.H., Xu H.J., Zhai Y.Y., et al, three new labdane-type diterpenoids from Callicarpa macrophylla Vahl, Natural Produvt Research, doi.org/10.1080/14786419.2018.1509336 "have anti-inflammatory effects, but the effects are not good enough. The callicarpa nudiflora contains a large amount of compounds with biological activity, and the invention aims to further separate and research the chemical components of the callicarpa nudiflora so as to obtain the labdane diterpenoid compounds with new structures and remarkable anti-inflammatory effect and fully exert the medicinal value of the callicarpa nudiflora.

Disclosure of Invention

In view of the defects of the prior art, the invention provides an oxidized labdane diterpenoid compound with a novel structure, has a remarkable anti-inflammatory effect, and provides a separation method of a target compound.

The technical scheme of the invention is as follows:

the invention provides an oxidized labdane diterpenoid compound, which has the following structures:

the invention also provides a method for separating the compounds I and II, which comprises the following steps:

(1) pulverizing dried folium Callicarpae Formosanae, boiling folium Callicarpae Formosanae powder with water, extracting under boiling to obtain extractive solution, and concentrating under reduced pressure to obtain crude extract;

(2) diluting the crude extract in the step (1) with water to prepare a suspension, sequentially extracting with dichloromethane and ethyl acetate, combining organic phases, and concentrating under reduced pressure to obtain an extract;

(3) performing silica gel column chromatography on the extract obtained by extracting the ethyl acetate in the step (2), and performing gradient elution by using a mixed solvent of chloroform and acetone as an eluent, wherein the elution gradient is in a volume ratio of (100:1) to (1:10), and 8 components, namely Fr.1 to Fr.8, are obtained according to the polarity;

(4) performing normal-phase silica gel column chromatography on Fr.2, performing gradient elution by using a mixed solvent of petroleum ether and ethyl acetate as an eluent in a volume ratio of (10:1) - (1:10), performing reduced pressure concentration, performing ODS reverse-phase column separation, performing gradient elution by using a mixed solvent of methanol and water as an eluent in a volume ratio of (10:90) - (90:10), and obtaining 7 components Fr.2a-Fr.2f according to the polarity; and (3) carrying out Sephadex LH-20 gel column chromatography on Fr.2d, wherein an eluent is methanol, carrying out reduced pressure concentration, and carrying out High Performance Liquid Chromatography (HPLC) to prepare a compound I and a compound II in sequence.

Preferably, in the step (1), the extraction times are more than 2 times, each time lasts for 1-3 hours, and the extracting solutions are combined; in the step (2), dichloromethane and ethyl acetate are sequentially used for extraction for more than 3 times.

Preferably, in the step (1), the water is purified water, and the dosage of the water is 2-3L per kilogram of callicarpa nudiflora powder.

Preferably, in the step (1), the water is heated to boil, the boiling extraction is kept for 1 to 2 hours, and the extraction times are more than 2 times.

Preferably, in step (2), the extraction is performed 3 or more times by sequentially extracting with dichloromethane and ethyl acetate, and the extracts are combined.

Preferably, in the step (2), the amount of water is 300-400 mL of water per 100 g of crude extract, and the volume of the organic solvent used in each extraction is 1.2-1.3 times of the volume of water.

Preferably, in step (3), the chloroform-acetone mixed solvent has elution gradients of 100:1, 80:1, 50:1, 20:1, 10:1, 5:1, 1:1 and 1:10, 3 column volumes are collected for each gradient, and each gradient obtains one component, and 8 components, namely Fr.1-Fr.8, are obtained.

Preferably, in the step (4), the petroleum ether-ethyl acetate mixed solvent has elution gradients of 10:1, 5:1, 1:1 and 1:10, and each gradient elutes 2-5 column volumes.

Preferably, in the step (4), the ODS reversed-phase elution ratio is methanol to water (V: V)10:90, 30:70, 50:50, 60:40, 70:30, 80:20, 90:10, and each gradient elutes 3-5 column volumes.

Preferably, in the step (4), Sephadex LH-20 gel column chromatography is carried out, an eluent is methanol, and elution is carried out for 3-6 column volumes.

Preferably, in the step (4), the conditions of the high performance liquid chromatography are as follows: chromatographic column Waters C₁₈The flow rate is 2mL/min, and the mobile phase is acetonitrile to water in a volume ratio of 60: 40.

Preferably, the callicarpa nudiflora is callicarpa nudiflora collected from Wuzhishan of Hainan province.

The separated labdane diterpenoid compound is applied to the preparation of medicaments for preventing or treating inflammation. Preferably, the inflammation is systemic inflammatory response syndrome, bronchitis, pneumonia, gastritis or enteritis.

Compared with the prior art, the invention has the beneficial effects that:

the invention extracts and separates the oxidized labdane diterpenoid compounds I and II from the callicarpa nudiflora leaves, and has better anti-inflammatory effect compared with the same type compounds. Specifically, the method obtains the new oxidized labdane diterpenoid compounds from the callicarpa nudiflora extractum by multi-stage separation and extraction methods such as water extraction, distilled water dissolution and dispersion, polar solvent extraction, solvent gradient elution, liquid chromatography separation and the like.

Drawings

FIG. 1: of the Compound I¹H-NMR Spectroscopy (MeOD-d)₄)

FIG. 2: of the Compound I¹³C-NMR Spectroscopy (MeOD-d)₄)

FIG. 3: DEPT (135 ℃) Spectroscopy (MeOD-d) of Compound I₄)

FIG. 4: of the Compound I¹H-¹H COSY spectra (MeOD-d)₄)

FIG. 5: HSQC spectra (MeOD-d) of Compound I₄)

FIG. 6: HMBC Spectroscopy (MeOD-d) of Compound I₄)

FIG. 7: NOESY spectrum (MeOD-d) of Compound I₄)

FIG. 8: HRESIMS spectra of Compound I

FIG. 9: of compounds II¹H-NMR Spectroscopy (MeOD-d)₄)

FIG. 10: of compounds II¹³C-NMR Spectroscopy (MeOD-d)₄)

FIG. 11: DEPT (135 ℃) Spectroscopy (MeOD-d) of Compound II₄)

FIG. 12: of compounds II¹H-¹H COSY spectra (MeOD-d)₄)

FIG. 13: HSQC spectra (MeOD-d) of Compound II₄)

FIG. 14: HMBC Spectroscopy (MeOD-d) of Compound II₄)

FIG. 15: HMBC partial amplification Spectrum (MeOD-d) of Compound II₄)

FIG. 16: NOESY spectrum (MeOD-d) of Compound II₄)

FIG. 17: HRESIMS spectra of Compound II

Detailed Description

In order to better understand the technical content of the invention, specific examples are provided below to further illustrate the invention.

The experimental methods used in the examples of the present invention are all conventional methods unless otherwise specified.

The materials, reagents and the like used in the examples of the present invention can be obtained commercially without specific description.

The experimental material Callicarpa nudiflora is collected from Hainan Wuzhishan, and the used part is leaves.

Example 1 preparation of highly modified labdane-type diterpenoid Compound

The method comprises the following steps:

(1) pulverizing dried folium Callicarpae Formosanae, heating to boil with 2L water per kg of Callicarpa Formosanae, extracting for 2 times (each time for 1.5 hr), mixing extractive solutions, and concentrating under reduced pressure to obtain crude extract (about 300 g);

(2) diluting 100 g of crude extract with 300mL of distilled water to obtain suspension, sequentially extracting with dichloromethane and ethyl acetate for 3 times, mixing organic phases, and concentrating under reduced pressure to obtain extract; the volume of organic solvent used in each extraction was 1.2 times the volume of water.

(3) And (3) performing silica gel column chromatography on the extract (about 10g) obtained by the ethyl acetate extraction in the step (2), performing gradient elution by using a chloroform-acetone mixed solvent, wherein the elution gradient is that the volume ratio is 100:1, 80:1, 50:1, 20:1, 10:1, 5:1, 1:1 and 1:10, 3 column volumes are collected in each gradient, each gradient obtains one component, and 8 components, namely Fr.1-Fr.8, are obtained.

(4) Performing normal-phase silica gel column chromatography on Fr.2, and performing gradient elution by using a petroleum ether-ethyl acetate mixed solvent as an eluent, wherein the elution gradient is 10:1, 5:1, 1:1 and 1:10 in volume ratio, and each gradient elutes 3 column volumes; then carrying out ODS reversed phase separation with the elution ratio of MeOH to H₂O (V: V)10:90, 30:70, 50:50, 60:40, 70:30, 80:20, 90:10, 4 column volumes per gradient elution, giving a total of 7 fractions depending on the polarity size, i.e.Fr.2a to Fr.2g; subjecting Fr.2d to Sephadex LH-20 gel column chromatography with MeOH as eluent, and eluting for 3 column volumes; concentrating under reduced pressure, and preparing by High Performance Liquid Chromatography (HPLC) to obtain compounds I and II; the conditions of the high performance liquid chromatography are as follows: chromatographic column Waters C₁₈The flow rate is 2mL/min, and the mobile phase is MeCN: H with the volume ratio of 60:40₂O。

Example 2 preparation of highly modified labdane-type diterpenoid Compounds

The method comprises the following steps:

(1) pulverizing dried folium Callicarpae Formosanae, heating to boil with 2.5L water per kg of Callicarpa Formosanae powder, extracting for 3 times while maintaining boiling, each time for 1 hr, mixing extractive solutions, and concentrating under reduced pressure to obtain crude extract (about 320 g);

(2) diluting 100 g of crude extract with 400mL of water to obtain suspension, sequentially extracting with dichloromethane and ethyl acetate for 4 times, mixing organic phases, and concentrating under reduced pressure to obtain extract; the volume of organic solvent used in each extraction was 1.3 times the volume of water.

(3) And (3) performing silica gel column chromatography on the extract (about 12g) obtained by the ethyl acetate extraction in the step (2), performing gradient elution by using a chloroform-acetone mixed solvent, wherein the elution gradient is that the volume ratio is 100:1, 80:1, 50:1, 20:1, 10:1, 5:1, 1:1 and 1:10, 3 column volumes are collected in each gradient, each gradient obtains one component, and 8 components are obtained in total, namely Fr.1-Fr.8.

(4) Performing normal phase silica gel column chromatography on Fr.2, performing gradient elution with petroleum ether-ethyl acetate mixed solvent as eluent at volume ratio of 10:1, 5:1, 1:1, and 1:10, wherein each gradient elutes 4 column volumes, performing ODS reverse phase separation at eluting ratio of MeOH: H₂O (V: V)10:90, 30:70, 50:50, 60:40, 70:30, 80:20, 90:10, eluting 4 column volumes per gradient, and obtaining 7 components, namely Fr.2a-Fr.2g, according to the polarity; subjecting Fr.2d to Sephadex LH-20 gel column chromatography with MeOH as eluent, eluting for 6 column volumes, concentrating under reduced pressure, and subjecting to High Performance Liquid Chromatography (HPLC) to obtain compounds I and II; the conditions of the high performance liquid chromatography are as follows: chromatographic column Waters C₁₈The flow rate is 2mL/min, and the mobile phase is MeCN: H with the volume ratio of 60:40₂O。

Example 3 structural identification of highly modified labdane-type diterpenoid Compounds

Application spectrum (b)¹H NMR，¹³C NMR, HSQC, HMBC, NOESY) and MS, etc., to determine the chemical structures of the compounds I and II obtained in examples 1 and 2.

The structure identification data is as follows:

a compound I: it is colorless oil, and is easily dissolved in methanol. High resolution mass spectrum HRESI (-) MS (M/z 357.2032[ M + Na ]]⁺Theoretical value 357.2036) determined to have the molecular formula C₂₀H₃₀O₄(ii) a According to¹H，¹³And C, determining the structure of the material by using two-dimensional nuclear magnetic resonance data, wherein the framework type is labdane diterpene. Slowly volatilizing the methanol to obtain a single crystal of the compound, and determining the absolute configuration as 5R,7S,9R and 10R as shown in the following figure, namely the compound is

3-oxo-7-hydroxy-8(17),13-ent-labdadien-15-oic acid, named callnudoid A¹H and¹³the C NMR data are shown in Table 1. [400MHz ] (¹H)，100MHz(¹³C) The solvent: MeOD-d₄]。

Of the Compound I¹H-NMR Spectroscopy (MeOD-d)₄) As shown in figure 1 of the drawings, in which,

of the Compound I¹³C-NMR Spectroscopy (MeOD-d)₄) As shown in figure 2 of the drawings, in which,

DEPT (135 ℃) Spectroscopy (MeOD-d) of Compound I₄) As shown in figure 3 of the drawings,

of the Compound I¹H-¹H COSY spectra (MeOD-d)₄) As shown in figure 4 of the drawings,

HSQC spectra (MeOD-d) of Compound I₄) As shown in figure 5 of the drawings,

HMBC Spectroscopy (MeOD-d) of Compound I₄) As shown in figure 6 of the drawings,

NOESY spectrum (MeOD-d) of Compound I₄) As shown in figure 7 of the drawings,

the HRESIMS spectrum of Compound I is shown in FIG. 8.

Compound II: it is colorless crystalline and easily soluble in methanol. High resolution mass spectrum HRESI (-) MS (M/z 359.2193[ M + Na ]]⁺Theoretical value 359.2198) determined to have the molecular formula C₁₅H₂₄O₃(ii) a According to¹H，¹³And C, determining the structure of the composite material by using two-dimensional nuclear magnetic resonance data, wherein the framework type is a highly modified labdane diterpene, namely 3-oxo-8-hydroxy-13-ent-labdaidien-15-oic acid, and the composite material is named as callnudoid B. It is composed of¹H and¹³the C NMR data are shown in Table 1. [400MHz ] (¹H)，100MHz(¹³C) The solvent: MeOD-d₄]。

Of compounds II¹H-NMR Spectroscopy (MeOD-d)₄) As shown in figure 9 of the drawings,

of compounds II¹³C-NMR Spectroscopy (MeOD-d)₄) As shown in figure 10 of the drawings,

DEPT (135 ℃) Spectroscopy (MeOD-d) of Compound II₄) As shown in figure 11 of the drawings,

of compounds II¹H-¹H COSY spectra (MeOD-d)₄) As shown in figure 12 of the drawings,

HSQC spectra (MeOD-d) of Compound II₄) As shown in figure 13 of the drawings, in which,

HMBC Spectroscopy (MeOD-d) of Compound II₄) As shown in figure 14 of the drawings,

HMBC partial amplification Spectrum (MeOD-d) of Compound II₄) As shown in figure 15 of the drawings,

NOESY spectrum (MeOD-d) of Compound II₄) As shown in figure 16 of the drawings,

the HRESIMS spectrum of Compound II is shown in FIG. 17.

TABLE 1 of Compounds I and II¹H-NMR and¹³C-NMR (400,100MHz) data

Through the above analysis, the structures of the compounds I and II are determined as follows:

example 4 pharmacological Activity test

Experimental materials:

cell: mouse mononuclear macrophage raw264.7.

Cell culture solution: DMEM medium containing 10% Fetal Bovine Serum (FBS), Lipopolysaccharide (LPS) carbohydrate.

And (3) an NO detection kit: priley (APPLYGEN), cat #: E1030.

the experimental method comprises the following steps:

induction: raw264.7 cells were cultured in DMEM medium containing 10% FBS at 37 ℃ in 5% CO₂Culturing in an incubator by a conventional method. The cells are arranged at 1X 10⁵The cells were inoculated in 96-well plates at 200. mu.L/well in each of a blank control group, an LPS-induced group, and a high, medium, and low (50,25, 12.5. mu.M) dose group of the test drug, and Indomethacin (Indomethacin) was used as a positive control group, and the cells were incubated at 37 ℃ in 5% CO₂And adhering the wall in a cell culture box for 24 hours.

And (3) detection: and (3) sucking 50 mu L of supernatant serving as a liquid to be detected into a 96-well plate, sequentially adding 50 mu L of reagent A and 50 mu L of reagent B according to a detection method of a kit specification, and detecting an OD value at 540 nm by using an enzyme labeling instrument.

The results of the anti-inflammatory activity of the compounds are shown in Table 2, and it is understood from the results that both compounds I and II exhibit significant anti-inflammatory effects, IC₅₀10.8 +/-0.34 mu M and 12.7 +/-0.42 mu M respectively.

Table 2 anti-inflammatory activity of compounds I and II on RAW264.7 cells: (n＝3).

The foregoing is a more detailed description of the present invention that is presented in conjunction with specific embodiments, and the practice of the invention is not to be considered limited to those descriptions. It will be apparent to those skilled in the art that a number of simple derivations or substitutions can be made without departing from the inventive concept.