A new compound isolated from pucciniastrum schweinitzii and a preparation method and application thereof

Abstract

The application discloses a new compound separated from haircap moss and a preparation method and application thereof, and belongs to the technical field of natural medicines. The preparation method comprises the following steps: step one, taking the dried haircap moss plants, crushing, extracting with 95% ethanol by heating and refluxing for at least 2 times, each time for at least 2 hours, filtering, combining the extraction liquid, and concentrating into thick extract under reduced pressure to obtain a crude extract; step two, dispersing the crude extract with hot water, and sequentially extracting with petroleum ether, dichloromethane, ethyl acetate and n-butanol; each polar part extraction liquid is concentrated under reduced pressure to obtain an extract; step three, gradient elution of the dichloromethane extract on an MCI column with a methanol-water solvent system; and step four, concentration of the 50% methanol eluent, and then preparation liquid phase, acetonitrile-water solvent isocratic elution. The new flavanol compound is separated from the haircap moss, has good anti-liver cancer activity, and lays a foundation for the development and utilization of the haircap moss plant resources.

Description

TECHNICAL FIELD

[0001] The present application relates to a new compound isolated from Polytrichum commune Hedw. and a preparation method and application thereof, and belongs to the technical field of natural medicine. BACKGROUND

[0002] Polytrichum commune Hedw. belongs to Polytrichaceae and Polytrichum. It usually grows in acid and humid coniferous forest land, and is often mixed with Hypnum or Polytrichum. The plant body is thick and looks like a coniferous seedling, with a height of several centimeters to several tens of centimeters. The stem has a central differentiation, and the base has a large number of red-brown false roots. The leaf is relatively hard, has multiple layers of cells, and the ventral surface is attached to a large number of green single-layer cells. The leaf edge has coarse teeth. The middle rib is wide and almost occupies the entire leaf surface. The peristome is red-brown, with a length of several centimeters. The capsule is ellipsoidal with four edges, and the platform is obvious. The peristome has 64 teeth. The operculum is conical, and the cap is covered with a large number of golden yellow hairs. In acid and humid coniferous forest land, the species often grows in large pieces, and is often mixed with Hypnum or Polytrichum.

[0003] The flavanol derivative compound has been widely used in medicine, pesticide, plastic, dye, electronic chemical industry and daily chemical industry. The flavanol derivative compound obtained by extraction and separation from the natural medicine Polytrichum commune Hedw. can not only enrich the flavanol derivative natural medicine, but also provide a certain reference for the classification of Polytrichaceae plants from the perspective of chemical taxonomy. SUMMARY

[0004] In view of the above problems, the present application provides a new compound isolated from Polytrichum commune Hedw., which is a new flavanol derivative natural medicine.

[0005] Meanwhile, the present application provides a preparation method of the new compound isolated from Polytrichum commune Hedw.

[0006] Meanwhile, the present application provides an application of the new compound isolated from Polytrichum commune Hedw. in preparing an anti-liver cancer drug.

[0007] To solve the above technical problems, the technical scheme adopted by the present application is as follows:

[0008] A new compound isolated from Polytrichum commune Hedw. has the following structure:

[0009]

[0010] A preparation method of the new compound isolated from Polytrichum commune Hedw. comprises the following steps:

[0011] Step one, take Polytrichum commune Hedw. dried plants, crush, sieve, 95% ethanol reflux extraction at least 2 times, each solvent dosage is equivalent to 8-10 times the amount of feed, each at least 2 hours, filter and combine the extract, concentrated under reduced pressure into thick extract, obtain the crude extract;

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

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

[0014] Step four, the extract is subjected to preparative liquid chromatography with 30% acetonitrile-water solution as the mobile phase, and the detection wavelength is 262 nm, and the compound crude product is obtained by collecting the chromatographic peak with a retention time of 23 min;

[0015] Step five, the crude product is purified by Sephadex LH-20 gel column, and the compound (I) is obtained after elution with methanol.

[0016] In step one, the crushed product is sieved through a 20-30 mesh sieve.

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

[0018] In step two, the amount of hot water is 10-12 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 as that of hot water, and each extraction agent is extracted at least 3 times; the temperature of hot water is 80-95°C.

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

[0020] In step four, the reverse phase C18 column is a Kromasil C18 preparation column, the sample amount is 50-80 mg, and the flow rate is 10 mL / min.

[0021] The application of a new compound isolated from Polytrichum commune Hedw. in the preparation of an anti-liver cancer drug.

[0022] An anti-liver cancer drug is prepared from a new compound isolated from Polytrichum commune Hedw.

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

[0024] The present application separates a new flavanol compound from Polytrichum commune Hedw., the new compound has good anti-liver cancer activity, and the present application lays a foundation for the development and utilization of Polytrichum commune Hedw. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 The H spectrum of the compound (I) of the present application is as follows:

[0026] Figure 2 The C spectrum of the compound (I) of the present application is as follows:

[0027] Figure 3 The HMBC spectrum of the compound (I) of the present application is as follows:

[0028] Figure 4 The HMBC spectrum of the compound (I) of the present application is as follows:

[0029] Figure 5 The H-H-COSY spectrum of the compound (I) of the present application is as follows. DETAILED DESCRIPTION

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

[0031] The moss source: Polytrichum commune Hedw. sample was collected in Gaopo Yungtopia Miao Village, Gaopo Township, Huaxi District, Guiyang City in October 2019, and the specimen was identified by Professor Zhao Jiancheng of the School of Life Sciences of Hebei Normal University as the whole grass of Polytrichum commune Hedw. of Polytrichaceae and Polytrichum commune Hedw.. The voucher specimen (HSBD2019110501) is preserved in the System and Evolution Laboratory of the School of Life Sciences of Hebei Normal University.

[0032] Example 1

[0033] A new compound isolated from Polytrichum commune Hedw. has the following structure:

[0034]

[0035] A preparation method of a new compound isolated from Pogonatum japonicum Steph, comprising the following steps: drying 1.5 kg of Pogonatum japonicum Steph, crushing into coarse powder, and then screening through a 30-mesh sieve; and extracting the coarse powder twice by hot reflux extraction with 95% ethanol (12 kg of solvent is used each time, and the extraction time is 2 hours); filtering and then combining the extracted solutions; and concentrating the combined extracted solutions into thick extract (41 g) at 70 DEG C under reduced pressure to obtain a crude extract. After dispersing the crude extract with 400 g (400 mL) of hot water at 90 DEG C, the crude extract is extracted with petroleum ether (30-60 DEG C), dichloromethane, ethyl acetate and n-butanol in sequence. The amount of petroleum ether, dichloromethane, ethyl acetate and n-butanol used in each extraction is 400 mL, and each extraction agent is used for 4 times. After each polar part extraction solution is concentrated at 60 DEG C under reduced pressure, it is dried at 50 DEG C to obtain an extract. The dichloromethane extract (8.5 g) is dissolved in 10 mL of methanol, filtered, and then the filtrate is subjected to MCI gel column chromatography. The diameter-height ratio of the MCI gel column is 1:8, and the amount of MCI gel filler used is 160 g. The MCI gel filler is eluted with a methanol-water solvent system in gradient, and the methanol-water solvent system is water, 30%, 50%, 70% and 90% MeOH / H2O used in sequence as eluent, and each gradient elution is 3 column volumes. The 50% methanol elution part is concentrated at 60 DEG C under reduced pressure, and then dried at 50 DEG C to obtain extract (0.7 g). The extract is dissolved in 5 mL of methanol, and then subjected to preparative liquid chromatography with 30% acetonitrile-water solution as mobile phase, reverse phase C18 column as Kromasil C18 4.6*250 mm, 1 mL of sample each time, ultraviolet detection wavelength as 262 nm, and flow rate as 10 mL / min. Compound (I) is obtained by collecting the chromatographic peak with retention time of 23 min, and then drying the collected and combined product at 60 DEG C under reduced pressure and at 50 DEG C. The crude product is dissolved in 2 mL of methanol, filtered, and then subjected to Sephadex LH-20 column chromatography. The diameter-height ratio of the Sephadex LH-20 column is 1:30, and the amount of Sephadex LH-20 filler used is 100 g. After the sample is loaded, the Sephadex LH-20 filler is eluted with methanol for 6 column volumes, and then the eluate is collected. Compound (I) (9.1 mg) is obtained by concentrating the collected eluate at 60 DEG C under reduced pressure and drying the concentrated eluate at 50 DEG C.

[0036] The English name of compound (I) is:

[0037] brainin E

[0038] The Chinese name of compound (I) is:

[0039] Pogonatum flavanol E

[0040] As shown in Table 1, the nuclear magnetic signal attribution of the structure of compound (I) is as follows. Figures 1-5

[0041] Table 1 Nuclear magnetic signal attribution of compound (I)

[0042] ​

[0043]

[0044] The application of the new compound separated from the Pogonatum japonicum in preparing anti-hepatoma drugs.

[0045] Pharmacological activity: the compound has good anti-hepatoma activity.

[0046] Cell activity culture:

[0047] Human hepatoma cell lines HepG2, Huh7, Hep3B and SMMC7721 are provided by the Cell Bank of Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. The above cells are cultured in DMEM high-sugar medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin in a saturated humidity, 37°C, 5% CO2 cell incubator. The next day, the cell growth is observed and the culture medium is replaced. When the cells grow to 80% of the bottom of the culture bottle, they can be subcultured. Discard the cell culture medium, rinse the cells with PBS twice, add 0.25% trypsin, shake well, and then digest. When the cells are rounded, add cell culture medium to stop digestion, collect the cell suspension, centrifuge at 900 r / min for 4 min, discard the supernatant, and resuspend with culture medium for subculture.

[0048] MTT experiment:

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

[0050] Table 2 MTT experiment results

[0051]

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

[0053] The application discloses an anti-hepatoma drug prepared from a new compound separated from a haircap moss.

[0054] Example 2

[0055] The difference between the example and the example 1 is only that:

[0056] A preparation method of a new compound separated from a haircap moss, comprising the following steps: 1.5 kg of dried haircap moss is crushed into coarse powder and then sieved through a 20-mesh sieve, and then the haircap moss is extracted by hot refluxing with 95% ethanol for three times (15 kg of solvent is used for each time, and each time lasts for 2.5 hours), and then the extracted solution is filtered and combined, and then the combined solution is concentrated into thick extract (45 g) at 70 DEG C under reduced pressure to obtain a crude extract. After the crude extract is dispersed with 540 g (namely 540 mL) of hot water at 80 DEG C, the crude extract is sequentially extracted with petroleum ether (30-60 DEG C), dichloromethane, ethyl acetate and n-butanol. The petroleum ether, dichloromethane, ethyl acetate and n-butanol used for each extraction are all 540 mL, and each extraction agent is used for three times. After each polar part extraction solution is concentrated at 60 DEG C under reduced pressure, the solution is dried at 50 DEG C to obtain an extract. The dichloromethane extract (9.2 g) is dissolved in 10 mL of methanol, filtered, and then the filtrate is subjected to MCI gel column chromatography. The diameter-height ratio of the MCI gel column is 1:10, the amount of MCI gel filler is 180 g, the methanol-water solvent system is used for gradient elution, the methanol-water solvent system is water, 30%, 50%, 70% and 90% MeOH / H2O used as eluents in sequence, and each gradient elution is performed for 5 column volumes; the 50% methanol elution part is concentrated at 60 DEG C under reduced pressure, and then dried at 50 DEG C to obtain extract (0.82 g). The extract is dissolved in 10 mL of methanol, and then subjected to preparative liquid chromatography. The mobile phase is 30% acetonitrile-water solution, the reverse phase C18 column is Kromasil C18 4.6*250 mm, the sample injection amount is 1 mL each time, the ultraviolet detection wavelength is 262 nm, the flow rate is 10 mL / min, and the compound is separated by preparative liquid chromatography. The chromatographic peak with a retention time of 23 min is collected, and then the collected peak is concentrated at 60 DEG C under reduced pressure and dried at 50 DEG C to obtain a crude compound. The crude compound is dissolved in 2 mL of methanol, filtered, and then subjected to Sephadex LH-20 column chromatography. The diameter-height ratio of the Sephadex LH-20 column is 1:40, the amount of Sephadex LH-20 filler is 100 g, the compound is eluted with methanol for 7 column volumes after being subjected to the Sephadex LH-20 column, and then the eluent is collected, concentrated at 60 DEG C under reduced pressure and dried at 50 DEG C to obtain the compound (I) (9.5 mg).

[0057] Example 3

[0058] The difference between the example and the example 1 is only that:

[0059] A method for preparing a new compound isolated from the haircap moss, comprising the following steps: 1.5 kg of the haircap moss is dried, crushed into coarse powder, and then sieved through a 30-mesh sieve; the powder is extracted twice by hot refluxing with 95% ethanol (13.5 kg of solvent is used each time, and each extraction lasts for 3 hours); the extracted solution is filtered and combined; and the combined solution is concentrated into thick extract (47 g) at 75°C under reduced pressure to obtain a crude extract. The crude extract is dispersed with 517 g (517 mL) of hot water at 95°C, and then sequentially extracted with petroleum ether (30-60°C), dichloromethane, ethyl acetate, and n-butanol. The amount of petroleum ether, dichloromethane, ethyl acetate, and n-butanol used in each extraction is 517 mL, and each extraction agent is used for 4 times. Each polar part of the extracted solution is concentrated at 70°C under reduced pressure, and then dried at 60°C to obtain an extract. The dichloromethane extract (8.8 g) is dissolved in 10 mL of methanol, filtered, and then subjected to MCI gel column chromatography. The diameter-height ratio of the MCI gel column is 1:9, and the amount of MCI gel filler used is 160 g. The MCI gel column is eluted with a methanol-water solvent system in gradient, and the methanol-water solvent system is water, 30% MeOH / H2O, 50% MeOH / H2O, 70% MeOH / H2O, and 90% MeOH / H2O used in sequence as eluent, and each gradient elution is performed for 4 column volumes. The 50% methanol eluted part is concentrated at 70°C under reduced pressure, and then dried at 60°C to obtain an extract (0.75 g). The extract is dissolved in 6 mL of methanol, and subjected to preparative liquid chromatography with a reverse phase column. The mobile phase is 30% acetonitrile-water solution, the reverse phase C18 column is Kromasil C18 4.6x250 mm, the sample injection amount is 1 mL each time, the ultraviolet detection wavelength is 262 nm, the flow rate is 10 mL / min, and the retention time of the collected chromatographic peak is 23 min. The collected chromatographic peak is combined, concentrated at 70°C under reduced pressure, and then dried at 60°C to obtain a crude compound. The crude compound is dissolved in 2 mL of methanol, filtered, and then subjected to Sephadex LH-20 column chromatography. The diameter-height ratio of the Sephadex LH-20 column is 1:35, and the amount of Sephadex LH-20 filler used is 100 g. The eluent is methanol, and the column is eluted for 6 column volumes. The eluent is collected, concentrated at 70°C under reduced pressure, and then dried at 60°C to obtain compound (I) (9.13 mg).

[0060] It should be understood that, in order to concisely illustrate the present disclosure and to help understand one or more of the various inventive aspects, various features of the present application are sometimes grouped together in a single embodiment or described in conjunction with one another in the above description of the exemplary embodiments of the present application. However, the method of the present disclosure should not be interpreted as reflecting an intention that the claimed present application requires more features than those explicitly recited in each claim. Rather, as reflected by the claims, the inventive aspects are in less than all the features of the previously disclosed embodiments. Therefore, the claims following the detailed description are hereby expressly incorporated into the detailed description, with each claim itself being a separate embodiment of the present application.

[0061] While the application has been described in accordance with a limited number of embodiments, these are merely aimed to provide an overview of the application and are not intended to limit the scope of the application in any way. Furthermore, it should be noted that the language used is merely used for readability and instructional purposes and should not be construed as limiting, in any way, the scope of the application. Accordingly, many modifications and variations of the application can be made without departing from its spirit or scope, which is defined by the scope of the appended claims.

[0062] The above description is merely illustrative of the preferred embodiments of the application and is not intended to limit the scope of the application in any way. Rather, the scope of the application is to be limited solely by the claims appended hereto and equivalents thereof.

Claims

1. A method for isolating compound (I) from *Moss hygrophorae*, characterized in that, Includes the following steps: Step 1, take golden moss Polytrichum commune Hedw. Dry the plant, crush it, sieve it, and extract it with 95% ethanol under reflux at least twice, each time using 8 to 10 times the amount of the plant material, for at least 2 hours each time. After filtration, combine the extracts and concentrate them under reduced pressure to obtain a thick extract. Step 2: After dispersing the crude extract with hot water, it is extracted sequentially with petroleum ether, dichloromethane, ethyl acetate and n-butanol; the extracts of each polar fraction are then concentrated under reduced pressure to obtain extracts of different polar fractions. Step 3: Load the dichloromethane extract onto an MCI column and elute with a gradient using a methanol-water solvent system. Collect the 50% methanol eluent and concentrate under reduced pressure to obtain the extract. In step three, the methanol-water solvent system uses water, 30%, 50%, 70%, and 90% MeOH / H2O as eluents sequentially, eluting 3-5 column volumes for each gradient; the diameter-to-height ratio of the MCI column is 1:(8-10). Step 4: The extract was separated by reversed-phase chromatography with 30% acetonitrile-water solution as the mobile phase and a detection wavelength of 262 nm. The chromatographic peak with a retention time of 23 min was collected to obtain crude compound (I). In step four, the reversed-phase column method uses a reversed-phase C18 column, specifically a Kromasil C18 preparative column, with an injection volume of 82–140 mg and a flow rate of 10 mL / min. Step 5: The crude product was purified by Sephadex LH-20 gel column chromatography, and the elution with methanol yielded compound (I). 。 2. The method according to claim 1, characterized in that, In step one, the powder is crushed and then passed through a 20-30 mesh sieve.

3. The method according to claim 1, characterized in that, In step two, the boiling range of petroleum ether is 30–60°C.

4. The method according to claim 1, characterized in that, In step two, 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.

5. The method according to claim 1, characterized in that, In step five, the diameter-to-height ratio of the Sephadex LH-20 gel column is 1:(30~40). After loading the sample, elute with methanol for at least 6 column volumes, collect the methanol eluent, concentrate and dry it to obtain compound (I).

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