Novel compound separated from pogonatum insignis as well as preparation method and application of novel compound

Abstract

The invention discloses a novel compound separated from pogonatum insignis as well as a preparation method and application thereof, and belongs to the technical field of natural medicines. The preparation method comprises the following steps: step 1, taking a pogonatum insignis sun-dried plant, crushing, carrying out heating reflux extraction with 95% ethanol for at least two times (at least 2 hours each time), filtering, combining extracting solutions, and carrying out reduced pressure concentration to obtain a thick extract, so as to obtain a crude extract; 2, dispersing the crude extract with hot water, and sequentially extracting with petroleum ether, dichloromethane, acetic ether and n-butyl alcohol; performing vacuum concentration on the extraction liquid of each polar part to obtain an extract; step 3, loading the dichloromethane extract on an MCI column, and carrying out gradient elution by using a methanol-water solvent system; and 4, concentrating the 50% methanol eluent, preparing a liquid phase, and carrying out isocratic elution by using an acetonitrile-water solvent. According to the invention, the new flavanol compound is separated from pogonatum indarum, and the new compound has good anti-hepatoma activity, and lays a foundation for development and utilization of pogonatum indarum plant resources.

Description

Technical Field

[0001] The invention relates to a new compound separated from golden moss and a preparation method and application thereof, belonging to the technical field of natural medicines. Background Art

[0002] Polytrichum commune Hedw. belongs to the Polytrichum genus of the Polytrichum family. It usually grows in acidic and humid coniferous forests, mostly in large patches, and often mixes with plants of the genus Hedw or Hedw. It is sturdy in appearance and looks like a pine or fir seedling. The plant body is several centimeters to tens of centimeters high. The stem has a differentiated central axis, and there are a large number of reddish-brown adventitious roots at the base. The leaves are relatively stiff, with multiple layers of cells, and the ventral surface is covered with many green single-layer cell pectens; the leaf margins have coarse teeth; the midrib is wide, almost occupying the entire leaf surface. The capsule stalk is reddish-brown and several centimeters long. The spore capsule is a four-sided ellipse with a prominent platform. There are 64 capsule teeth. The capsule cap is oblate-conical, and the capsule cap is covered with many golden cilia. In acidic and humid coniferous forests, this species often grows in large patches, and often mixes with plants of the genus Hedw or Hedw.

[0003] In the prior art, no flavanol derivative compounds have been obtained from the golden moss, and the flavanol derivative compounds have been widely used in the fields of medicine, pesticides, plastics, dyes, electronic chemicals, and daily chemicals. Obtaining flavanol derivative compounds from the natural drug golden moss by extraction and separation can not only enrich the flavanol derivative natural drugs, but also provide a certain reference for the classification of golden moss plants from the perspective of chemical taxonomy. Summary of the invention

[0004] In view of the above problems, the object of the present invention is to provide a new compound separated from golden moss, which is a new flavanol derivative natural medicine.

[0005] Meanwhile, the invention provides a preparation method of a new compound separated from golden moss.

[0006] At the same time, the present invention provides an application of a new compound separated from golden moss in the preparation of an anti-liver cancer drug.

[0007] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0008] A new compound isolated from golden moss has the following structure:

[0009]

[0010] A method for preparing a new compound isolated from golden moss comprises the following steps:

[0011] Step 1, taking dried plants of Polytrichum commune Hedw., crushing, sieving, and extracting at least twice with 95% ethanol under heating and refluxing, with the amount of solvent used each time being 8 to 10 times the amount of the feed, each time for at least 2 hours, filtering and combining the extracts, and concentrating under reduced pressure into a thick extract to obtain a crude extract;

[0012] Step 2: After the crude extract is dispersed with hot water, it is extracted with petroleum ether, dichloromethane, ethyl acetate and n-butanol in sequence; the extracts of each polar part are then concentrated under reduced pressure to obtain extracts of different polar parts;

[0013] Step 3, the dichloromethane extract is loaded onto an MCI column, gradient eluted with a methanol-water solvent system, the 50% methanol elution portion is collected, and concentrated under reduced pressure to obtain an extract;

[0014] Step 4, the extract is separated by a reverse phase chromatography column method, with 30% acetonitrile-water solution as the mobile phase and a detection wavelength of 262 nm, and the chromatographic peak with a retention time of 23 min is collected to obtain a crude compound;

[0015] Step 5: The crude product is purified by Sephadex LH-20 gel column and eluted with methanol to obtain compound (I).

[0016] In step 1, the powder is crushed and passed through a 20-30 mesh sieve.

[0017] In step 2, the boiling range of petroleum ether is 30-60°C.

[0018] In step 2, the amount of hot water used is 10 to 12 times the weight of the crude extract; the amount of petroleum ether, dichloromethane, ethyl acetate and n-butanol used in each extraction is the same volume as the hot water, and each extractant is extracted at least 3 times; the temperature of the hot water is 80 to 95°C.

[0019] In step 3, the methanol-water solvent system uses water, 30%, 50%, 70% and 90% MeOH / H2O as eluents in sequence, and each gradient elution is 3 to 5 column volumes.

[0020] In step 4, the reverse phase C18 column is a Kromasil C18 preparative column, the injection volume is 50-80 mg, and the flow rate is 10 mL / min.

[0021] Application of a new compound isolated from golden moss in the preparation of anti-hepatocarcinoma drugs.

[0022] An anti-liver cancer drug is prepared by using a new compound separated from golden moss of the present invention.

[0023] Compared with the prior art, the present invention has the following advantages:

[0024] The invention separates and obtains new flavanol compounds from golden moss, and the new compound has good anti-liver cancer activity. The invention lays a foundation for the development and utilization of golden moss plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 is the H spectrum of compound (I) of the present invention;

[0026] Figure 2 is the C spectrum of compound (I) of the present invention;

[0027] Figure 3 is the HMBC spectrum of compound (I) of the present invention;

[0028] Figure 4 is the HMBC spectrum of compound (I) of the present invention;

[0029] Figure 5 It is the HH-COSY spectrum of compound (I) of the present invention. DETAILED DESCRIPTION

[0030] The present invention will be further described in detail below in conjunction with specific examples. The following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0031] Source of moss: The sample of Polytrichum commune Hedw. was collected in Gaopo Yunding Miao Village, Gaopo Township, Huaxi District, Guiyang City in October 2019. The specimen was identified as the whole herb of Polytrichum commune Hedw. of the Polytrichumaceae family by Professor Zhao Jiancheng of the School of Life Sciences of Hebei Normal University. The voucher specimen (HBSD2019110501) is preserved in the Laboratory of Systematics and Evolution of the School of Life Sciences of Hebei Normal University.

[0032] Example 1

[0033] A new compound isolated from golden moss has the following structure:

[0034]

[0035] A preparation method of a new compound isolated from golden moss comprises the following steps: 1.5 kg of golden moss plants are dried in the sun, crushed into coarse powder, and then passed through a 30-mesh sieve, and extracted twice with 95% ethanol under hot reflux (12 kg of solvent each time, time 2 hours), filtered and combined, and concentrated under reduced pressure at 70°C to form a thick extract (41 g) to obtain a crude extract. The crude extract is dispersed with 400 g (i.e., 400 mL) of 90°C hot water, and then extracted with petroleum ether (30-60°C), dichloromethane, ethyl acetate and n-butanol in sequence. The amount of petroleum ether, dichloromethane, ethyl acetate and n-butanol used for each extraction is 400 mL, and each extractant is extracted 4 times. The extracts of each polar part are concentrated under reduced pressure at 60°C and dried at 50°C to obtain an extract. The dichloromethane extract (8.5 g) was dissolved in 10 mL of methanol and filtered. The filtrate was loaded onto an MCI gel column with a diameter-to-height ratio of 1:8 and an MCI gel filler of 160 g. The column was gradient eluted using a methanol-water solvent system. The methanol-water solvent system used water, 30%, 50%, 70% and 90% MeOH / H2O as eluents in sequence, and each gradient elution lasted for 3 column volumes. The 50% methanol elution portion was concentrated under reduced pressure at 60°C and dried at 50°C to obtain an extract (0.7 g). The extract was dissolved in 5 mL of methanol and subjected to reverse phase chromatography using a 30% acetonitrile-water solution as the mobile phase. The reverse phase C18 column was Kromasil C18 4.6×250mm, each injection volume is 1mL, the ultraviolet detection wavelength is 262nm, the flow rate is 10mL / min, and preparative liquid separation is performed. The chromatographic peak with a retention time of 23min is collected. After enrichment and merging, it is concentrated under reduced pressure at 60°C and dried at 50°C to obtain a crude compound. The crude product is dissolved in 2mL of methanol, filtered, loaded, and purified on a Sephadex LH-20 column. The diameter-to-height ratio of the Sephadex LH-20 column is 1:30, and the amount of Sephadex LH-20 filler is 100g. After loading, methanol is eluted for 6 column volumes, and the eluate is collected, concentrated under reduced pressure at 60°C, and dried at 50°C to obtain compound (I) (9.1mg).

[0036] English name of compound (I):

[0037] brainin E

[0038] Chinese name of compound (I):

[0039] Flavone E

[0040] like Figure 1 to Figure 5 As shown, the NMR signals of the structure of compound (I) are assigned as shown in Table 1.

[0041] Table 1 Attribution of NMR signals of compound (I)

[0042]

[0043]

[0044] The application of the new compound isolated from golden moss obtained in this example in the preparation of anti-liver cancer drugs.

[0045] Pharmacological activity: This compound has good anti-hepatocarcinoma activity.

[0046] Cell viability culture:

[0047] Human liver cancer cell lines HepG2, Huh7, Hep3B and SMMC7721 were provided by the cell bank of Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. The above cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum, 100U / mL penicillin, and 100μg / mL streptomycin in a saturated humidity, 37°C, and 5% CO2 cell culture incubator. The next day, the growth of the cells was observed and the culture medium was replaced. When the cells covered 80% of the bottom of the culture bottle, they could be subcultured. The cell culture medium was discarded, an appropriate amount of PBS was taken to rinse the cells twice, 0.25% trypsin was added, and the cells were shaken and digested. When the cells became round, the cell culture medium was added to terminate the digestion, and the cell suspension was collected and centrifuged at 900r / min for 4min. The supernatant was discarded and the cells were resuspended in the culture medium for subculture.

[0048] MTT assay:

[0049] Cancer cells in the logarithmic growth phase were prepared into 5×10 4 / mL cell suspension. The cells were seeded into a 96-well plate and 100μL of cell suspension was added to each well. The compound was dissolved in fresh culture medium at four concentrations of 0μM, 5μM, 25μM, 125μM and 625μM, respectively, and then added to the 96-well plate in sequence, with each concentration repeated 3 times. After culturing in the incubator for 48 hours, the culture medium of each well was aspirated and 100μL of 0.5mg / mL MTT solution was added. After culturing for 1 hour, the MTT solution was aspirated and 200μL of DMSO was added to each well to dissolve the purple crystals of MTT. The UV absorbance value of each well was measured at a wavelength of 570nm by enzyme-linked immunosorbent assay. The results of the MTT experiment are shown in Table 2 below.

[0050] Table 2 MTT test results

[0051]

[0052] The above experimental results show that compared with the blank control group, the compound has a certain inhibitory effect on liver cancer cell lines HepG2, Huh7, Hep3B and SMMC7721 cells, among which the inhibitory effect on Hep3B cell line is better.

[0053] An anti-liver cancer drug is prepared by using a new compound isolated from golden moss in this example.

[0054] Example 2

[0055] The difference between this embodiment and embodiment 1 is only that:

[0056] A preparation method of a new compound isolated from golden moss comprises the following steps: 1.5 kg of golden moss plants are dried in the sun, crushed into coarse powder, and then passed through a 20-mesh sieve, and extracted with 95% ethanol under hot reflux for 3 times (15 kg of solvent is used each time, and the time for each time is 2.5 hours), and the extracts are combined after filtration, and concentrated under reduced pressure at 70°C to form a thick extract (45 g) to obtain a crude extract. The crude extract is dispersed with 540 g (i.e., 540 mL) of 80°C hot water, and then extracted with petroleum ether (30-60°C), dichloromethane, ethyl acetate and n-butanol in sequence. The amount of petroleum ether, dichloromethane, ethyl acetate and n-butanol used for each extraction is 540 mL, and each extractant is extracted 3 times. The extracts of each polar part are concentrated under reduced pressure at 60°C and dried at 50°C to obtain an extract. The dichloromethane extract (9.2 g) was dissolved in 10 mL of methanol and filtered. The filtrate was loaded onto an MCI gel column with a diameter-to-height ratio of 1:10 and an MCI gel filler of 180 g. The column was gradient eluted using a methanol-water solvent system. The methanol-water solvent system used water, 30%, 50%, 70% and 90% MeOH / H2O as eluents in sequence, and each gradient elution lasted for 5 column volumes. The 50% methanol elution portion was concentrated under reduced pressure at 60°C and dried at 50°C to obtain an extract (0.82 g). The extract was dissolved in 10 mL of methanol and subjected to reverse phase chromatography using a 30% acetonitrile-water solution as the mobile phase. The reverse phase C18 column was Kromasil C18 4.6×250mm, each injection volume is 1mL, the ultraviolet detection wavelength is 262nm, the flow rate is 10mL / min, and preparative liquid separation is performed. The chromatographic peak with a retention time of 23min is collected. After enrichment and merging, it is concentrated under reduced pressure at 60°C and dried at 50°C to obtain a crude compound. The crude product is dissolved in 2mL of methanol, filtered, loaded, and purified on a Sephadex LH-20 column. The diameter-to-height ratio of the Sephadex LH-20 column is 1:40, and the amount of Sephadex LH-20 filler is 100g. After loading, methanol is eluted for 7 column volumes, and the eluate is collected, concentrated under reduced pressure at 60°C, and dried at 50°C to obtain compound (I) (9.5mg).

[0057] Example 3

[0058] The difference between this embodiment and embodiment 1 is only that:

[0059] A preparation method of a new compound isolated from golden moss comprises the following steps: 1.5 kg of golden moss is dried in the sun, crushed into coarse powder, and then passed through a 30-mesh sieve, and extracted twice with 95% ethanol under hot reflux (13.5 kg of solvent is used each time, and the extraction time is 3 hours each time), and the extracts are combined after filtration, and concentrated under reduced pressure at 75°C to form a thick extract (47 g) to obtain a crude extract. The crude extract is dispersed with 517 g (i.e., 517 mL) of 95°C hot water, and then extracted with petroleum ether (30-60°C), dichloromethane, ethyl acetate and n-butanol in sequence. The amount of petroleum ether, dichloromethane, ethyl acetate and n-butanol used for each extraction is 517 mL, and each extractant is extracted 4 times. The extracts of each polar part are concentrated under reduced pressure at 70°C and dried at 60°C to obtain an extract. The dichloromethane extract (8.8 g) was dissolved in 10 mL of methanol and filtered. The filtrate was loaded onto an MCI gel column with a diameter-to-height ratio of 1:9 and an MCI gel filler of 160 g. The column was gradient eluted using a methanol-water solvent system. The methanol-water solvent system used water, 30%, 50%, 70% and 90% MeOH / H2O as eluents in sequence, and each gradient elution had 4 column volumes. The 50% methanol elution portion was concentrated under reduced pressure at 70°C and dried at 60°C to obtain an extract (0.75 g). The extract was dissolved in 6 mL of methanol and subjected to reverse phase chromatography using a 30% acetonitrile-water solution as the mobile phase. The reverse phase C18 column was Kromasil C18 4.6×250mm, each injection volume is 1mL, the ultraviolet detection wavelength is 262nm, the flow rate is 10mL / min, and preparative liquid separation is performed. The chromatographic peak with a retention time of 23min is collected. After enrichment and merging, it is concentrated under reduced pressure at 70°C and dried at 60°C to obtain a crude compound. The crude product is dissolved in 2mL of methanol, filtered, loaded, and purified on a Sephadex LH-20 column. The diameter-to-height ratio of the Sephadex LH-20 column is 1:35, and the amount of Sephadex LH-20 filler is 100g. After loading, methanol is eluted for 6 column volumes, and the eluate is collected, concentrated under reduced pressure at 70°C, and dried at 60°C to obtain compound (I) (9.13mg).

[0060] It should be understood that in order to streamline the present disclosure and aid in understanding one or more of the various inventive aspects, in the above description of exemplary embodiments of the present invention, various features of the present invention are sometimes grouped together into a single embodiment, or description thereof. However, this disclosed method should not be interpreted as reflecting the intention that the claimed invention requires more features than those expressly recited in each claim. Rather, as reflected in the claims, inventive aspects lie in less than all of the features of the previously disclosed embodiments. Therefore, the claims that follow the detailed description are hereby expressly incorporated into the detailed description, with each claim itself serving as a separate embodiment of the present invention.

[0061] Although the present invention has been described according to a limited number of embodiments, it will be apparent to those skilled in the art, with the benefit of the above description, that other embodiments may be envisioned within the scope of the invention thus described. In addition, it should be noted that the language used in this specification is selected primarily for readability and teaching purposes, rather than for explaining or defining the subject matter of the present invention. Therefore, many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the appended claims. The disclosure of the present invention is illustrative, not restrictive, with respect to the scope of the present invention, which is defined by the appended claims.

[0062] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A new compound isolated from golden moss, characterized in that Its structure is as follows:

2. The method for preparing a new compound isolated from golden moss according to claim 1, characterized in that: The following steps are involved: Step 1, taking dried plants of Polytrichum commune Hedw., crushing, sieving, and extracting at least twice with 95% ethanol under heating and refluxing, with the amount of solvent used each time being 8 to 10 times the amount of the feed, each time for at least 2 hours, filtering and combining the extracts, and concentrating under reduced pressure into a thick extract to obtain a crude extract; Step 2: After the crude extract is dispersed with hot water, it is extracted with petroleum ether, dichloromethane, ethyl acetate and n-butanol in sequence; the extracts of each polar part are then concentrated under reduced pressure to obtain extracts of different polar parts; Step 3, the dichloromethane extract is loaded onto an MCI column, gradient eluted with a methanol-water solvent system, the 50% methanol elution portion is collected, and concentrated under reduced pressure to obtain an extract; Step 4, the extract is separated by a reverse phase chromatography column method, with 30% acetonitrile-water solution as the mobile phase and a detection wavelength of 262 nm, and the chromatographic peak with a retention time of 23 min is collected to obtain a crude compound; Step 5: The crude product is purified by Sephadex LH-20 gel column and eluted with methanol to obtain compound (I).

3. The preparation method according to claim 2, characterized in that: In step 1, the powder is crushed and passed through a 20-30 mesh sieve.

4. The preparation method according to claim 2, characterized in that: In step 2, the boiling range of petroleum ether is 30-60°C.

5. The preparation method according to claim 2, characterized in that: In step 2, the amount of hot water used is 10 to 12 times the weight of the crude extract; the amount of petroleum ether, dichloromethane, ethyl acetate and n-butanol used in each extraction is the same volume as the hot water, and each extractant is extracted at least 3 times; the temperature of the hot water is 80 to 95°C.

6. The preparation method according to claim 2, characterized in that: In step 3, the methanol-water solvent system uses water, 30%, 50%, 70% and 90% MeOH / H2O as eluents in sequence, and each gradient elution is 3 to 5 column volumes; the diameter-to-height ratio of the MCI column is 1:(8 to 10).

7. The preparation method according to claim 2, characterized in that: In step 4, the reverse phase C18 column is a Kromasil C18 preparative column, the injection volume is 82-140 mg, and the flow rate is 10 mL / min.

8. The preparation method according to claim 2, characterized in that: In step 5, the diameter-to-height ratio of the Sephadex LH-20 gel column is 1:(30-40). After loading the sample, the column is eluted with methanol for at least 6 column volumes. The methanol eluate is collected, concentrated and dried to obtain compound (I).

9. Use of the new compound isolated from golden moss according to claim 1 in the preparation of anti-liver cancer drugs.

10. An anti-liver cancer drug, characterized in that: The method is prepared by using the new compound isolated from golden moss as claimed in claim 1.

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