Method for separating and purifying active ingredients from Selaginella

Abstract

The invention provides a separation and purification method for multiple active components in Selaginella, and these components have certain anti-tumor activity, which provides the possibility for further research on them.

Description

Technical Field

[0001] The present invention relates to the field of medicinal chemistry. Background Art

[0002] Selaginella (Selaginella tamariscina (P.Beauv.) Spring) is a native or rocky fern of the genus Selaginella in the family Selaginaceae. The roots are bifurcated and densely covered with hairs, forming a tree-like trunk with the stem and branches; the twigs are sparse and regular, the branches are hairless, and the back and abdomen are flat; the leaves are arranged alternately, the leaves are thick and smooth, with white edges, green or brown, and fine teeth on the edges; the megaspores are light yellow, and the microspores are orange-yellow; the flowering period is from July to September; the fruiting period is from September to October. Selaginella is named because its stems and leaves resemble the young branches and leaves of cypress, and the branches and leaves are curled inward.

[0003] Selaginella is native to China and prefers warm, humid and semi-shady environments. It grows mostly on sunny hillside rocks or in dry rock crevices. Selaginella reproduces by spores.

[0004] According to the book "Ben Cao Qiu Zhen", Selaginella has the effects of promoting blood circulation and menstruation, removing blood stasis and stopping bleeding, and can be used to treat amenorrhea, dysmenorrhea, lumps and masses, hematemesis, metrorrhagia, bloody stool, rectal prolapse and other diseases. The branches and leaves of Selaginella are stretched and green. Indoor miniature bonsai are evergreen all year round, shaped like a mountain pine, and are used for rockery and large bonsai cultivation and decoration, with high ornamental value. Selaginella has an evolutionary history of 400 million years and is the oldest group of terrestrial plants to date. Summary of the invention

[0005] The present invention provides a method for separating and purifying active ingredients from Selaginella, comprising the following contents:

[0006] (1) extracting Selaginella with 70-80% ethanol-water solution, concentrating until there is no alcohol taste, and obtaining Selaginella total extract; suspending with water, extracting with petroleum ether, dichloromethane and ethyl acetate in sequence, extracting each organic solvent layer, and recovering the solvent under reduced pressure to obtain an extract;

[0007] (2) The ethyl acetate extract was separated by HP20 macroporous adsorption resin, and eluted with 50%, 60%, 70%, 80%, and 90% methanol-water solution as the elution solvent. After thin layer chromatography, the eluates of the same spot were combined in the order of elution, and concentrated to obtain 5 fractions Fr.1→Fr.5; the Fr.3 fraction was separated by silica gel column chromatography with dichloromethane-methanol 100:0, 50:1, 10:1, 8:1, 5:1, 2:1, and 1:1 v / v gradient elution. After thin layer chromatography, the eluates of the same spot were combined in the order of elution to obtain 7 fractions Fr.3-1→Fr.3-7, among which the Fr.3-5 fraction was eluted with 65% methanol-water by p-HPLC to obtain compound 2;

[0008] The compound structural formula is as follows:

[0009]

[0010] Furthermore, the Selaginella extraction used a 75% ethanol-water solution.

[0011] Furthermore, it also includes the separation and purification method of compounds 1, 3, 4, and 5:

[0012] (3) Taking the dichloromethane layer extract, separating it with HP20 macroporous adsorption resin, using methanol-water solution as the mobile phase, and eluting it with a gradient of 50%, 60%, 70%, 80%, and 90% methanol-water solution in sequence;

[0013] (4) The elution fraction of 60% methanol-water solution was subjected to polyamide column chromatography, and gradient elution was performed with dichloromethane-methanol 100:0, 50:1, 10:1, 5:1, 2:1, and 1:1 v / v in sequence; the elution fraction of dichloromethane-methanol 10:1 was subjected to p-HPLC, and isocratically eluted with 50% methanol-water to obtain compound 5;

[0014] (5) The elution fraction of 80% methanol-water solution was subjected to silica gel column chromatography, and dichloromethane-methanol 100:0, 100:1, 50:1, 30:1, 20:1, 10:1, 8:1, 5:1, 2:1, and 2:3 v / v were used as the mobile phases, and the elution fraction of 8:1 was subjected to p-HPLC isocratic elution with 70% methanol-water to obtain compounds 1, 3, and 4, respectively;

[0015] The compound structural formula is as follows:

[0016]

[0017] The present invention also provides the use of extract A prepared by the following method in the preparation of anticancer drugs; the method comprises the following contents:

[0018] Extract with 70-80% ethanol-water solution of Selaginella, concentrate until there is no alcohol taste, and obtain the total extract of Selaginella; suspend with water, extract with petroleum ether, dichloromethane and ethyl acetate in sequence, extract each organic solvent layer under reduced pressure to recover the solvent to obtain the extract, and take the ethyl acetate layer to obtain extract A.

[0019] The present invention also provides the use of extract B prepared by the following method in the preparation of anticancer drugs; the method comprises the following contents:

[0020] (1) extracting Selaginella with 70-80% ethanol-water solution, concentrating until there is no alcohol taste, and obtaining Selaginella total extract; suspending with water, extracting with petroleum ether, dichloromethane and ethyl acetate in sequence, extracting each organic solvent layer, and recovering the solvent under reduced pressure to obtain an extract;

[0021] (2) Take the ethyl acetate extract and separate it through HP20 macroporous adsorption resin, eluting with 50%, 60%, 70%, 80%, and 90% methanol-water solution as elution solvents. After thin layer chromatography identification, the eluates of the same spots are combined in the order of elution, and concentrated to obtain 5 fractions Fr.1→Fr.5; take the Fr.3 fraction to obtain extract B.

[0022] The present invention also provides the use of extract C prepared by the following method in the preparation of anticancer drugs; the method comprises the following contents:

[0023] (1) extracting Selaginella with 70-80% ethanol-water solution, concentrating until there is no alcohol taste, and obtaining Selaginella total extract; suspending with water, extracting with petroleum ether, dichloromethane and ethyl acetate in sequence, extracting each organic solvent layer, and recovering the solvent under reduced pressure to obtain an extract;

[0024] (2) The ethyl acetate extract was separated by HP20 macroporous adsorption resin, and eluted with 50%, 60%, 70%, 80%, and 90% methanol-water solution. After thin layer chromatography, the eluates of the same spot were combined in the order of elution, and concentrated to obtain 5 fractions Fr.1→Fr.5; the Fr.3 fraction was separated by silica gel column chromatography with dichloromethane-methanol 100:0, 50:1, 10:1, 8:1, 5:1, 2:1, and 1:1 v / v for gradient elution. After thin layer chromatography, the eluates of the same spot were combined in the order of elution to obtain 7 fractions Fr.3-1→Fr.3-7, among which the Fr.3-5 fraction was extract C.

[0025] Furthermore, the breast cancer is triple-negative breast cancer.

[0026] The present invention has found that:

[0027] (1) Compounds 1-3 are new compounds, compounds 4 and 5 are new configurations, and these compounds all have good anticancer activity. The present invention provides methods for extracting and separating these compounds, which makes it possible to further study them.

[0028] (2) Triple-negative breast cancer refers to breast cancer in which the results of immunohistochemical examination of cancer tissue are negative for estrogen receptor (ER), progesterone receptor (PR) and proto-oncogene Her-2. This type of breast cancer accounts for 10.0% to 20.8% of all breast cancer pathological types, has special biological behavior and clinical pathological characteristics, and has a worse prognosis than other types. It is called the "king of breast cancer." According to experimental findings, the drug of the present invention can effectively inhibit the growth of this type of cancer cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 HMBC(→) spectrum of compound 1

[0030] Figure 2 HMBC(→) spectrum of compound 2

[0031] Figure 3 HMBC(→) spectrum of compound 3

[0032] Figure 4 HMBC(→) spectrum of compound 4

[0033] Figure 5 HMBC(→) spectrum of compound 5 DETAILED DESCRIPTION

[0034] Example 1

[0035] 13.0 kg of dried whole herb of Selaginella was extracted by a flash extractor with a liquid-to-solid ratio of 75% ethanol-water solution of 12:1, extracted 3 times, each time for 2 minutes, concentrated by a rotary evaporator under reduced pressure until there was no alcohol taste, and obtained a total extract of Selaginella (1.2 kg), suspended in water, extracted with petroleum ether, dichloromethane, and ethyl acetate, respectively, extracted 3 times, and each fraction extract was taken. The solvent was recovered under reduced pressure to obtain a petroleum ether layer (80.5 g), a dichloromethane layer (160.0 g), and an ethyl acetate layer (180.5 g).

[0036] Take 160.0 g of dichloromethane layer extract, and use HP20 macroporous adsorption resin for separation. The mobile phase is methanol-water, and the gradient elution is 50%, 60%, 70%, 80%, and 90%. Each mobile phase has 7 BV, and the concentrates are combined to obtain five fractions (D1→D5, which means "D1, D2, D3, D4, and D5 in sequence", and the subsequent similar expressions have the same interpretation).

[0037] D2 was subjected to polyamide column chromatography with dichloromethane-methanol (100:0, 50:1, 10:1, 5:1, 2:1, 1:1, v / v) gradient elution to obtain 6 fractions in sequence. The elution portion of dichloromethane-methanol 10:1 was taken and eluted isocratically with 50% methanol-water by p-HPLC to obtain compound 5 (3.0 mg, t R =21.9min); the elution fraction of 80% methanol-water solution was taken and 10 fractions (Fr.1→Fr.10) were obtained by silica gel column chromatography with dichloromethane-methanol 100:0, 100:1, 50:1, 30:1, 20:1, 10:1, 8:1, 5:1, 2:1, 2:3 v / v as mobile phase. Among them, the Fr.7 fraction was eluted isocratically with 70% methanol water by p-HPLC to obtain compound 1 (7.2 mg, t R =23.4min), compound 3 (6.4mg, t R=33.6min), compound 4 (5.2mg, t R =38.2min).

[0038] 178.0 g of ethyl acetate extract was taken and initially separated by HP20 macroporous adsorption resin, with 50%, 60%, 70%, 80%, 90% methanol-water as the elution solvent, gradient elution, 7 BV for each gradient, identification by thin layer chromatography, according to the order of elution, the eluates of the same spot were combined, and concentrated to obtain 5 fractions (Fr.1→Fr.5). The Fr.3 fraction was subjected to silica gel column chromatography with dichloromethane-methanol (100:0, 50:1, 10:1, 8:1, 5:1, 2:1, 1:1) as the solvent system for gradient elution, identification by thin layer chromatography, according to the order of elution, the eluates of the same spot were combined to obtain 7 fractions Fr.3-1→Fr.3-7, of which the Fr.3-5 fraction was subjected to p-HPLC with 65% methanol-water as the elution condition to obtain compound 2 (4.6 mg, t R =26.5min).

[0039] Compound 1

[0040]

[0041] Compound 1 is a red amorphous powder (MeOH), and the quasi-molecular ion peak given by HR-ESI-MS spectrum is m / z 527.1877 [M+H] + , indicating that the molecular weight is 526 and the molecular formula is C 35 H 26 O5, unsaturation is 23. UV spectrum has maximum absorption at 267, 299 and 431nm. 1 H-NMR spectrum data (Table 1) show that δ H 7.73 (1H, d, J = 8.1 Hz) and 7.37 (1H, d, J = 8.1 Hz) are AB spin systems on aromatic rings. H 7.08 and 6.78 (each 2H, d, J = 8.8 Hz), δ H 6.83 and 6.53 (each 2H, d, J = 8.5 Hz), δ H 7.16 and 6.48 (each 4H, d, J = 9.1 Hz) are the proton signals of four para-substituted spin coupling systems, which are respectively the proton signals of the B ring, E ring and the symmetrically substituted C ring and D ring. In addition, 1 H-NMR spectrum suggests the presence of a hydroxymethyl δ H 4.94 (2H, s), one methoxyl group δ H 3.75 (3H, s) hydrogen signal. 13The C-NMR spectrum showed 35 carbon signals, including a carboxyl carbon (δ C 177.5), two alkynyl carbons (δ C 98.6,83.7), an oxygen-containing methylene carbon (δ C 62.0), a methoxy carbon (δ C 54.3) and 30 aromatic carbon signals.

[0042] The structure of compound 1 was further confirmed by HMBC spectrum. H 7.08) and C-27 (δ C 98.6) are related to each other, suggesting that alkynyl (δ C 98.6,83.7) connected to the B ring. H-20 / 24 (δ H 6.83) and C-18 (δ C 142.1) indicates that the E ring is connected to C-18 in the A ring, and the hydroxymethyl group is connected to C-15. H-3, H-5 / C-7 and H-8, H-12 / C-7 are related, suggesting that the connection between the C ring and the D ring is located at C-7 (δ C 166.7).

[0043] Thus, C-19 in the A ring is connected to C-7. In addition, δ H 3.75 and C-30 (δ C 160.0), indicating that the methoxy group is connected to C-30. In addition to the methoxy signal at C-30 [δ H 3.75 (3H, s) and δ C 54.3], compound 1 1 H-NMR and 13 The C-NMR data were similar to those reported for Selaginellin (8).

[0044] In summary, compound 1 is a new compound not reported in the literature and is named Selariscinin H.

[0045] Compound 2

[0046]

[0047] Compound 2 is a red amorphous powder. According to high-resolution mass spectrometry, the quasi-molecular ion peak is m / z 555.1810 [M+H] + , and its molecular formula is deduced to be C 36 H 26 O6, the degree of unsaturation is 24. The UV spectrum shows maximum absorption at 265, 300 and 425 nm, which is the characteristic absorption of acetylphenol in Selaginella.

[0048] Compound 2 1 H-NMR and 13 The C-NMR spectrum showed similar structural features to Selaginellin (8), except that there was an additional acetyl group at the 34-hydroxymethyl position [δ H 2.14(3H,s),δ C 19.4,δ C 171.2], and the rest of the data are basically consistent. In the HMBC spectrum, δ H 2.14 (H-36) and δ C 171.2(C-35),δ H 5.43 (H-34) and δ C The presence of a correlation between 171.2 (C-35) further suggests that -CH2OH at C-15 is replaced by -CH2OOCCH3.

[0049] In summary, compound 2 is a new compound not reported in the literature and is named Selariscinin I.

[0050] Compound 3

[0051]

[0052] Compound 3 is a red amorphous powder (MeOH). In HR-ESI-MS, at m / z 541.1676 [M+H] + The quasi-molecular ion peak is shown at 35 H 24 O6, and showed an unsaturation degree of 24. The UV spectrum showed maximum absorption at 265, 292 and 431 nm. 1 H-NMR and 13 The C-NMR spectrum also showed similar characteristics to compound 1, with the methoxy carbon signal at δ H 3.75 disappears and at δ H 5.92 (2H, s) and δ C A methylenedioxy group appeared at 101.4. The Labat reaction was positive, which further indicated that the structure contained a methylenedioxy group.

[0053] In the HMBC spectrum, the typical 1 H-NMR signal (δ H 5.92) respectively with [δ C 148.4 (C-30) and δ C 147.5(C-31)] 13C-NMR signal correlation indicated that the methylenedioxy group was located at C-30 and C-31. In summary, the compound was a new compound not reported in the literature, and compound 3 was named Selariscinin J.

[0054] Compound 4

[0055]

[0056] Compound 5

[0057]

[0058] Compound 4 and Compound 5 are both red amorphous powders. 1 H-NMR and 13 The C-NMR data are similar to the structures of (S)-selaginellin U and (S)-selaginellin V, except for some differences in the positions of the symmetrical substituents on the C-ring and D-ring. This indicates that compounds 4 and 5 are derivatives of (S)-selaginellin U and (S)-selaginellin V synthesized through tautomerism of the (R) and (S) forms.

[0059] The absolute configuration of compounds 4 and 5 was determined by optical rotation and Cotton Effect (CE) of CD. In the CD spectrum, +CE was shown at 206 and 214 nm, and -CE was shown at 210 and 220 nm. The absolute configuration of compounds 4 and 5 is (R), which is opposite to the (S) configuration reported in the literature (-CE was produced at 206 and 214 nm, and +CE was produced at 210 and 220 nm)

[0060] Table 1 Compounds 1-3 1 H NMR (600MHz), 13 C NMR (150MHz)

[0061]

[0062] Table 2 Compounds 4 and 5 1 H NMR (600 MHz) and 13 C NMR (150MHz)

[0063]

[0064] Example 2 Study on the screening of the anti-tumor activity of Selaginella truncatula in vitro

[0065] 1. Experimental Materials

[0066] The tested monomer compounds were all homemade in the laboratory, with a purity of more than 98%.

[0067] Table 3 Sources of tumor cell lines

[0068]

[0069] 2. CCK-8 assay to detect the inhibitory activity of Selaginella monomer compounds on human triple-negative breast cancer MDA-MB-231 cells and MDA-MB-468 cells

[0070] Human triple-negative breast cancer MDA-MB-231 and MDA-MB-468 cells were seeded in culture flasks and cultured in a 37° C., 0.1% CO 2 incubator using DMEM medium (L-15 medium containing 10% fetal bovine serum, 100 units / mL penicillin and 100 μg / mL streptomycin).

[0071] Take the human triple-negative breast cancer MDA-MB-231 and MDA-MB-468 cells in the logarithmic growth phase, discard the culture medium, add 2mL of PBS buffer to wash twice, discard the PBS, add 1mL of trypsin, gently blow the cells, add 2mL of culture medium to stop digestion, centrifuge at 1500rmp for 5min, discard the supernatant, add an appropriate amount of fresh culture medium, count the cells, mix the cells, add 100μL of cell suspension to each well, inoculate in a 96-well plate (1.5×103 / well), add different concentrations of the monomer compound to be tested or 5-fluorouracil to each well, set 4 replicates for each drug, put it in a CO2 incubator and continue to culture for 48h, and set up a blank control group. After 48 hours, add CCK-8 reagent, put it in the incubator, continue to culture for 1.5h, then take out the cells, turn on the microplate reader, detect at 450nm, record the absorbance (A) value, and calculate the cell proliferation inhibition rate. Observe the inhibition rate at different concentrations and determine its IC50 value.

[0072] 3. Experimental results

[0073] Inhibitory activity of Selaginella monomer compounds on human triple-negative breast cancer MDA-MB-231 cells and MDA-MB-468 cells

[0074] Table 4 Inhibitory activity of compounds 1-5 on triple-negative breast cancer cells

[0075]

[0076] According to the above IC 50 The values ​​show that the compounds of the present invention have certain anti-triple-negative breast cancer activity, among which compound 2 has the best activity, which is similar to the positive control 5-Fu.

Claims

1. A method for separating and purifying active ingredients from Selaginella, characterized in that: It includes the following: (1) extracting Selaginella with 70-80% ethanol-water solution, concentrating until there is no alcohol taste, and obtaining Selaginella total extract; suspending with water, extracting with petroleum ether, dichloromethane and ethyl acetate in sequence, extracting each organic solvent layer, and recovering the solvent under reduced pressure to obtain an extract; (2) The ethyl acetate extract was separated by HP20 macroporous adsorption resin, and eluted with 50%, 60%, 70%, 80%, and 90% methanol-water solution as the elution solvent. After thin layer chromatography, the eluates of the same spot were combined in the order of elution, and concentrated to obtain 5 fractions Fr.1→Fr.5; the Fr.3 fraction was separated by silica gel column chromatography with dichloromethane-methanol 100:0, 50:1, 10:1, 8:1, 5:1, 2:1, and 1:1 v / v gradient elution. After thin layer chromatography, the eluates of the same spot were combined in the order of elution to obtain 7 fractions Fr.3-1→Fr.3-7, among which the Fr.3-5 fraction was eluted with 65% methanol-water by p-HPLC to obtain compound 2; The compound structural formula is as follows:

2. The method according to claim 1, characterized in that Selaginella extraction used 75% ethanol-water solution.

3. The method according to claim 1, characterized in that Also included are separation and purification methods for compounds 1, 3, 4, and 5: (3) Taking the dichloromethane layer extract, separating it with HP20 macroporous adsorption resin, using methanol-water solution as the mobile phase, and eluting it with a gradient of 50%, 60%, 70%, 80%, and 90% methanol-water solution in sequence; (4) The elution fraction of 60% methanol-water solution was subjected to polyamide column chromatography, and gradient elution was performed with dichloromethane-methanol 100:0, 50:1, 10:1, 5:1, 2:1, and 1:1 v / v in sequence; the elution fraction of dichloromethane-methanol 10:1 was subjected to p-HPLC, and isocratically eluted with 50% methanol-water to obtain compound 5; (5) The elution fraction of 80% methanol-water solution was subjected to silica gel column chromatography, and dichloromethane-methanol 100:0, 100:1, 50:1, 30:1, 20:1, 10:1, 8:1, 5:1, 2:1, and 2:3 v / v were used as the mobile phases, and the elution fraction of 8:1 was subjected to p-HPLC isocratic elution with 70% methanol-water to obtain compounds 1, 3, and 4, respectively; The compound structural formula is as follows:

4. Use of extract B prepared by the following method in the preparation of an anti-triple-negative breast cancer drug; the method comprises the following contents: (1) extracting Selaginella with 70-80% ethanol-water solution, concentrating until there is no alcohol taste, and obtaining Selaginella total extract; suspending with water, extracting with petroleum ether, dichloromethane and ethyl acetate in sequence, extracting each organic solvent layer, and recovering the solvent under reduced pressure to obtain an extract; (2) Take the ethyl acetate extract and separate it through HP20 macroporous adsorption resin, eluting with 50%, 60%, 70%, 80%, and 90% methanol-water solution as elution solvents. After thin layer chromatography identification, the eluates of the same spots are combined in the order of elution, and concentrated to obtain 5 fractions Fr.1→Fr.5; take the Fr.3 fraction to obtain extract B.

5. Use of extract C prepared by the following method in the preparation of an anti-triple-negative breast cancer drug; the method comprises the following contents: (1) extracting Selaginella with 70-80% ethanol-water solution, concentrating until there is no alcohol taste, and obtaining Selaginella total extract; suspending with water, extracting with petroleum ether, dichloromethane and ethyl acetate in sequence, extracting each organic solvent layer, and recovering the solvent under reduced pressure to obtain an extract; (2) The ethyl acetate extract was separated by HP20 macroporous adsorption resin, and eluted with 50%, 60%, 70%, 80%, and 90% methanol-water solution. After thin layer chromatography, the eluates of the same spot were combined in the order of elution, and concentrated to obtain 5 fractions Fr.1→Fr.5; the Fr.3 fraction was separated by silica gel column chromatography with dichloromethane-methanol 100:0, 50:1, 10:1, 8:1, 5:1, 2:1, and 1:1 v / v for gradient elution. After thin layer chromatography, the eluates of the same spot were combined in the order of elution to obtain 7 fractions Fr.3-1→Fr.3-7, among which the Fr.3-5 fraction was extract C.

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