A compound extracted and separated from chloranthus kirkii and application thereof

Abstract

The application discloses a compound extracted and separated from Chloranthus glaucescens and application thereof, relates to the technical field of medicinal compounds, and provides a structural formula, a preparation method and application. A novel compound is separated from Chloranthus glaucescens, the compound has a good protective effect on lipopolysaccharide-induced acute lung injury, and thus can be used for preparing an acute lung injury improvement drug.

Description

Technical Field

[0001] The present invention relates to the technical field of medicinal compounds, and more particularly to a compound extracted and isolated from Chloranthus fortunei and its applications. Background Art

[0002] Chloranthus fortunei, a plant of the genus Chloranthus in the family Chloranthaceae, is produced in Shandong, Jiangsu, Anhui and other places. Chloranthus fortunei is one of the sources of the traditional Chinese medicine Sikuaiwa. The whole herb is used medicinally, which can antibacterial and anti-inflammatory, promote blood circulation and remove stasis; treat women's dry blood tuberculosis, traumatic injury, stomachache or internal injury pain, and scabies, etc. [[ID=I0]]

[0003] Acute lung injury (ALI) is a pathological feature characterized by pulmonary edema and atelectasis caused by extensive damage to diffuse alveolar capillary endothelial cells and alveolar epithelial cells after the body is struck by severe infection, trauma, shock, etc. Its severe stage is acute respiratory distress syndrome (ARDS). The clinical manifestations are progressive respiratory distress and refractory hypoxemia. It is a common clinical critical illness with a high incidence and mortality rate. At present, there is no specific treatment method, and mainly mechanical ventilation and drugs are comprehensively used for treatment. Therefore, it is of great significance to invent a drug for treating acute lung injury.

[0004] Therefore, whether a drug for treating acute lung injury can be extracted from Chloranthus fortunei is an urgent problem to be solved by those skilled in the art. Summary of the Invention

[0005] In view of this, the present invention provides a compound extracted and isolated from Chloranthus fortunei and its applications. The structure of the monomer compound was identified by comprehensively using high-resolution mass spectrometry, spectroscopic analysis, and ECD calculation. The results of the bioactivity test show that the compound has a good protective effect on acute lung injury, indicating that this compound has a good anti-inflammatory effect and has a good application prospect in the treatment of acute lung injury.

[0006] In order to achieve the above object, the present invention adopts the following technical scheme:

[0007] A compound extracted and isolated from Chloranthus fortunei, the structural formula of the compound is as follows:

[0008]

[0009] The present invention also provides a preparation method of the above compound, including the following steps:

[0010] (1) Take the roots of Chloranthus fortunei, crush and sieve them, extract with 95% ethanol, combine the extraction solutions, and concentrate to a fluid extract state to obtain the total extract;

[0011] (2) Extract with petroleum ether and ethyl acetate in sequence, and keep the ethyl acetate dissolved portion for later use;

[0012] (3) The ethyl acetate dissolved fraction was sequentially purified by silica gel column chromatography gradient elution, Sephadex LH-20 gel chromatography, and preparative high performance liquid chromatography to obtain the compound.

[0013] Preferred: Step (1) Mass-volume ratio of *Chloranthus sylvestris* root and ethanol: 15 kg: 200 L; Sieving: 20 mesh sieve; Extraction conditions: Extracted 3 times at 25°C with 95% ethanol for 48 hours each time.

[0014] Preferred: Step (2) specifically involves: suspending the total extract in 5.0L of water, and extracting it sequentially with petroleum ether and ethyl acetate, 5.0L each time, and extracting it 3 times each time, to obtain the petroleum ether dissolved portion and the ethyl acetate dissolved portion.

[0015] Preferred: Step (3) specifically includes:

[0016] The ethyl acetate dissolved fraction was separated by silica gel column chromatography, and eluted sequentially with dichloromethane-methanol at volume ratios of 100:1, 50:1, 25:1, 10:1, 5:1, and 0:1; the Fr.EB fraction was obtained, concentrated under reduced pressure and dried to a fluid extract state for later use.

[0017] The obtained fraction Fr.EB was separated by silica gel column chromatography and eluted sequentially with dichloromethane-methanol at volume ratios of 100:1, 50:1, 25:1, 10:1, 5:1, and 0:1. The fraction Fr.EB6 obtained at a dichloromethane-methanol volume ratio of 5:1 was reserved for later use.

[0018] The component Fr.EB6 was purified by Sephadex LH-20 gel chromatography, eluted isocratically with 100% methanol, and then isocratically eluted by preparative high performance liquid chromatography with a methanol / water ratio of 60:40, a flow rate of 10.0 ml / min, and a retention time of 9.630 min. The chromatographic peak with a retention time of 9.630 min was collected to obtain the purified compound.

[0019] The present invention also provides the application of the above-mentioned compounds in pharmaceutical manufacturing.

[0020] Preferred: Used in the preparation of drugs for treating acute lung injury.

[0021] Preferred: Used in the preparation of anti-inflammatory drugs.

[0022] As can be seen from the above technical solution, compared with the prior art, the present invention discloses a compound extracted and isolated from Chloranthus spicatus and its application. The technical effect achieved is that the present invention isolates a novel compound from Chloranthus spicatus, which has a good protective effect against lipopolysaccharide-induced acute lung injury, and can thus be used to improve the preparation of drugs for acute lung injury. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0024] Figure 1 The attached figure shows the HRESIMS spectrum of the compound provided by this invention.

[0025] Figure 2 The attached figure shows the 1H-NMR spectrum of the compound provided by this invention.

[0026] Figure 3 The attached figure shows the compound provided by this invention. 13 C-NMR spectrum.

[0027] Figure 4 The attached figure shows the HSQC spectrum of the compound provided by this invention.

[0028] Figure 5 The attached figure shows the HMBC spectrum of the compound provided by this invention.

[0029] Figure 6 The attached figure shows the NOESY spectrum of the compound provided by this invention.

[0030] Figure 7 The attached figure shows the ECD spectrum of the compound provided by this invention.

[0031] Figure 8 The attached figure is a schematic diagram of key correlation signals in the HMBC and NOESY spectra of the compounds provided in this invention.

[0032] Figure 9 The attached figure shows a detection graph of the protective effect of the compound provided by the present invention. Detailed Implementation

[0033] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0034] This invention discloses a compound extracted and isolated from Chloranthus spicatus and its application.

[0035] Example 1

[0036] Preparation of new compounds

[0037] Step 1: Take 15.0 kg of Chloranthus sylvestris root, crush it through a 20-mesh sieve, and extract it three times with 200 L of 95 v / v ethanol at room temperature (about 25°C) for 48 hours each time. Combine the extracts and concentrate them under reduced pressure using a rotary evaporator to obtain a fluid extract of 696.8 g.

[0038] Step 2: The total extract obtained in Step 1 was suspended in 5.0L of water and extracted with petroleum ether and ethyl acetate in sequence, 5.0L each time, for 3 extractions each time, to obtain the petroleum ether dissolved fraction (99.7g) and the ethyl acetate dissolved fraction (476.9g).

[0039] Step 3: The ethyl acetate dissolved fraction (476.9 g) obtained in Step 2 was separated by silica gel column chromatography, using a gradient elution of dichloromethane-methanol at volume ratios of 100:1, 50:1, 25:1, 10:1, 5:1, and 0:1. TLC was performed to combine similar fractions, yielding seven fractions from Fr.EA to EG. These fractions were then concentrated under reduced pressure using a rotary evaporator and dried to a fluid extract state for later use.

[0040] The obtained fraction Fr.EB (47.17 g) was separated by silica gel column chromatography, using a gradient elution of dichloromethane-methanol at volume ratios of 100:1, 50:1, 25:1, 10:1, 5:1, and 0:1. TLC was performed, and similar fractions were combined to obtain fractions Fr.EB1 to EB77. Fr.EB6 was the fraction eluted at a dichloromethane-methanol volume ratio of 5:1.

[0041] The fraction Fr.EB6 was purified by Sephadex LH-20 gel chromatography, eluted isocratically with 100% methanol at a flow rate of one drop every seven seconds until all sample was elute. Preparative high-performance liquid chromatography was then performed, eluted isocratically with methanol-water (CH3OH / H2O, 60:40 v / v, flow rate 10.0 ml / min, retention time 9.630 min). The chromatographic peak with a retention time of 9.630 min was collected, yielding the purified new compound Fortuneilide C (10.4 mg).

[0042] Structural identification: High-resolution mass spectrometry (HRESIMS) and nuclear magnetic resonance spectroscopy were used. 1 HNMR, 13 CNMR, HMQC, and HMBC, as well as calculated ECD, were used to identify the structure of the new compound obtained in step 5. The results are shown in [Figure 1]. Figures 1 to 7 ;

[0043] Upon identification, the quasi-molecular ion peak of the new compound obtained in step 5 was found to be 635.2849 [M+H] at m / z. + (The calculated value is C) 36 H 43 O 10 (635.2850), the molecular formula is presumed to be C 36 H 42 O 10 The NMR spectrum data are shown in Table 1.

[0044] Table 1: Compounds of Formula (1) 1 HNMR and 13 CNMR data ( 1 H 500MHz, 13 C 125MHz, in CDCl3, δ: ppm, JinHz.)

[0045]

[0046]

[0047] The relevant signals of the HMBC spectrum of the obtained compound are as follows: Figure 8 As shown

[0048] The structural formula of the obtained compound is shown below:

[0049]

[0050] The chemical formula is C 36 H 42 O 10 It is named Fortuneilide C.

[0051] Example 2

[0052] Detection of the protective effect of compound Fortuneilide C against acute lung injury

[0053] Step 1: MLE-12 lung epithelial cell line was cultured at 1×10⁻⁶ cells / year. 6 Cells were seeded at a density of 100 cells / well in 6-well plates for 24 hours. Cells were then divided into the following groups: control, LPS (model, 50 μM), and LPS + Fortuneilide C (1.0, 10.0, 100.0 μM). MLE-12 cells were stimulated with LPS (50 mM) for 24 hours, and then incubated with the drugs from each group for another 24 hours. The control group was stimulated with the same PBS. Cell supernatant was then collected and frozen with MLE-12 cells at -20°C for later use.

[0054] Step 2: MLE-12 lung epithelial cell line was cultured at 1×10⁻⁶ cells / year. 6 Cells were seeded at a density of 100 cells / well in 6-well plates for 24 hours. Cells were then divided into the following groups: control, LPS (model, 50 μM), and LPS + Fortuneilide C (1.0, 10.0, 100.0 μM). MLE-12 cells were stimulated with LPS (50 mM) for 24 hours, followed by incubation with the respective drugs for another 24 hours. The control group was stimulated with the same PBS. The culture medium was then removed, and 200 μL of MTT (0.5 mg / ml) was added to each well for 4 hours of incubation. The supernatant was discarded, and 150 μL of DMSO was added to each well. The absorbance of each well was measured at 490 nm using a microplate spectrophotometer.

[0055] The test results for the new compound Fortuneilide C are as follows: Figure 9 As shown, the results indicated that this compound improved the survival rate of the lipopolysaccharide (LPS)-induced lung epithelial cell line MLE-12 in a dose-dependent manner. After administration of the new compound at concentrations of 1.0, 10.0, and 100.0 μM, the cell survival rate was significantly higher than that of the model group. However, at a concentration of 100 μM, the cell survival rate was comparable to that of the control group. Therefore, this compound possesses a protective effect against acute lung injury.

[0056] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0057] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A compound extracted and isolated from Chloranthus spicatus, characterized in that, The structural formula of the compound is as follows: 。 2. The method for preparing the compound according to claim 1, characterized in that, Includes the following steps: (1) Take the root of Chloranthus sylvestris, crush and sieve it, extract it with 95% ethanol, combine the extracts, concentrate it into a fluid extract, and obtain the total extract. (2) Extraction was performed sequentially using petroleum ether and ethyl acetate, and the dissolved portion of ethyl acetate was reserved for later use; (3) The ethyl acetate dissolved fraction was separated by silica gel column chromatography, and dichloromethane-methanol was used for gradient elution at volume ratios of 100:1, 50:1, 25:1, 10:1, 5:1, and 0:1 to obtain the Fr.EB fraction, which was concentrated and dried under reduced pressure to a fluid extract state for later use. The obtained fraction Fr.EB was separated by silica gel column chromatography and eluted sequentially with dichloromethane-methanol at volume ratios of 100:1, 50:1, 25:1, 10:1, 5:1, and 0:

1. The fraction Fr.EB6 obtained at a dichloromethane-methanol volume ratio of 5:1 was reserved for later use. The component Fr. EB6 was purified by Sephadex LH-20 gel chromatography, eluted isocratically with 100% methanol, and then isocratically eluted by preparative high performance liquid chromatography with a methanol / water ratio of 60:40, a flow rate of 10.0 ml / min, and a retention time of 9.630 min. The chromatographic peak with a retention time of 9.630 min was collected to obtain the compound.

3. The preparation method according to claim 2, characterized in that, The mass-to-volume ratio of the *Chloranthus sylvestris* root and 95% ethanol in step (1) is 15 kg: 200 L; the sieving method is a 20-mesh sieve; the extraction conditions are: extraction at 25°C for 3 times with 95% ethanol, each time for 48 hours.

4. The preparation method according to claim 3, characterized in that, Step (2) is as follows: The total extract is suspended in 5.0 L of water and extracted sequentially with petroleum ether and ethyl acetate, 5.0 L each time, and each extraction is performed 3 times to obtain the petroleum ether dissolved portion and the ethyl acetate dissolved portion.

5. The use of the compound of claim 1 in the preparation of a medicament for treating acute lung injury.

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