CN117658811A - Method for separating and purifying active ingredient from selaginella - Google Patents

Abstract

The invention provides a separation and purification method of various active ingredients in selaginella, which have certain anti-tumor activity and provide possibility for further research.

Description

Method for separating and purifying active ingredient from selaginella

Technical Field

The present invention relates to the field of pharmaceutical chemistry.

Background

Herba Selaginellae (Selaginella tamariscina (P.Beauv.) Spring) is a native or stone fern plant of genus Selaginella of family Selagineraceae. The root is multi-forked, the dense fur, the stem and the branches are densely formed into a tree-shaped trunk; branches are sparse and regular, branches have no hair, and the dorsum abdomen is flat; the blades are alternately arranged, the blades are thick and smooth, the edges are provided with white edges, green or brown, and the edges are provided with fine teeth; megaspore is pale yellow and microspore is orange; the flowering period is 7-9 months; the fruit period is 9-10 months. Herba Selaginellae is named because its stem and leaf are similar to the young branches and leaves of cypress, and the branches and leaves are rolled up.

Herba Selaginellae is originally produced in China, and is loved to warm, wet and semi-negative environment, and is mostly grown on sunny hillside rocks or in arid rock joints. The propagation mode of the selaginella tamariscina is spore propagation.

In the book of materia medica, herba Selaginellae has the effects of promoting blood circulation, dredging channels, removing blood stasis and stopping bleeding, and can be used for treating diseases such as amenorrhea, dysmenorrhea, mass, hematemesis, metrorrhagia, hematochezia, rectocele, etc. The tamarisk leaves are stretched, emerald green and lovely, the miniature bonsai in the room is evergreen in four seasons, is like high mountain pine, is used for ornamental cultivation of rockery and large bonsai, and has higher ornamental value. Selaginella tamariscina has an evolution history of up to 4 hundred million years and is by far the oldest group in terrestrial plants.

Disclosure of Invention

The invention provides a method for separating and purifying active ingredients from selaginella, which comprises the following steps:

(1) Extracting herba Selaginellae 70-80% ethanol-water solution, concentrating until no ethanol smell exists, to obtain herba Selaginellae total extract; suspending with water, sequentially extracting with petroleum ether, dichloromethane and ethyl acetate, extracting each organic solvent extraction layer, and recovering solvent under reduced pressure to obtain extract;

(2) Separating ethyl acetate extract with HP20 macroporous adsorbent resin, eluting with 50%, 60%, 70%, 80%, and 90% methanol-water solution as eluting solvent, identifying by thin layer chromatography, mixing the eluates of the same spots in the order of eluting morning and evening, and concentrating to obtain 5 fractions Fr.1-Fr.5; subjecting Fr.3 fractions to gradient elution sequentially with dichloromethane-methanol 100:0, 50:1, 10:1, 8:1, 5:1, 2:1 and 1:1v/v through silica gel column chromatography, identifying by thin layer chromatography, and combining the eluents of the same spots according to the eluting sequence of the morning and evening to obtain 7 fractions Fr.3-1- & gt Fr.3-7, wherein Fr.3-5 fractions are eluted with 65% methanol-water through p-HPLC to obtain a compound 2;

the structural formula of the compound is as follows:

further, herba Selaginellae extraction uses 75% ethanol-water solution.

Further, the method for separating and purifying the compounds 1, 3, 4 and 5 is also included:

(3) Separating dichloromethane layer extract with HP20 macroporous adsorbent resin, and sequentially eluting with 50%, 60%, 70%, 80% and 90% methanol-water solution as mobile phase;

(4) Taking a 60% methanol-water solution elution part, and carrying out gradient elution by polyamide column chromatography sequentially with dichloromethane-methanol 100:0, 50:1, 10:1, 5:1, 2:1 and 1:1 v/v; taking a dichloromethane-methanol 10:1 elution part, and performing isocratic elution with 50% methanol-water by p-HPLC to obtain a compound 5;

(5) Eluting the eluting part of 80% methanol-water solution by silica gel column chromatography sequentially with dichloromethane-methanol 100:0, 100:1, 50:1, 30:1, 20:1, 10:1, 8:1, 5:1, 2:1 and 2:3v/v as mobile phase, eluting the eluting part of 8:1 by p-HPLC with 70% methanol water to obtain compounds 1, 3 and 4 sequentially;

the structural formula of the compound is as follows:

the invention also provides an application of the extract A prepared by the method in preparing anticancer drugs; the method comprises the following steps:

extracting herba Selaginellae 70-80% ethanol-water solution, concentrating until no ethanol smell exists, to obtain herba Selaginellae total extract; suspending with water, sequentially extracting with petroleum ether, dichloromethane and ethyl acetate, extracting each organic solvent layer, recovering solvent under reduced pressure to obtain extract, and collecting ethyl acetate layer to obtain extract A.

The invention also provides an application of the extract B prepared by the method in preparing anticancer drugs; the method comprises the following steps:

(1) Extracting herba Selaginellae 70-80% ethanol-water solution, concentrating until no ethanol smell exists, to obtain herba Selaginellae total extract; suspending with water, sequentially extracting with petroleum ether, dichloromethane and ethyl acetate, extracting each organic solvent extraction layer, and recovering solvent under reduced pressure to obtain extract;

(2) Separating ethyl acetate extract with HP20 macroporous adsorbent resin, eluting with 50%, 60%, 70%, 80%, and 90% methanol-water solution as eluting solvent, identifying by thin layer chromatography, mixing the eluates of the same spots in the order of eluting morning and evening, and concentrating to obtain 5 fractions Fr.1-Fr.5; taking Fr.3 fraction to obtain extract B.

The invention also provides an application of the extract C prepared by the method in preparing anticancer drugs; the method comprises the following steps:

(1) Extracting herba Selaginellae 70-80% ethanol-water solution, concentrating until no ethanol smell exists, to obtain herba Selaginellae total extract; suspending with water, sequentially extracting with petroleum ether, dichloromethane and ethyl acetate, extracting each organic solvent extraction layer, and recovering solvent under reduced pressure to obtain extract;

(2) Separating ethyl acetate extract with HP20 macroporous adsorbent resin, eluting with 50%, 60%, 70%, 80%, and 90% methanol-water solution as eluting solvent, identifying by thin layer chromatography, mixing the eluates of the same spots in the order of eluting morning and evening, and concentrating to obtain 5 fractions Fr.1-Fr.5; taking Fr.3 fractions, sequentially carrying out gradient elution with dichloromethane-methanol 100:0, 50:1, 10:1, 8:1, 5:1, 2:1 and 1:1v/v through silica gel column chromatography, identifying through thin layer chromatography, and combining the eluents of the same spots according to the eluting sequence of the morning and evening to obtain 7 fractions Fr.3-1- & gt Fr.3-7, wherein Fr.3-5 fractions are the extract C.

Further, the breast cancer is triple negative breast cancer.

The research of the invention finds that:

(1) The compounds 1-3 are novel compounds, the compounds 4 and 5 are novel configurations, and the compounds have good anticancer activity. The invention provides an extraction and separation method of the compounds, which provides possibility for further research.

(2) Triple negative breast cancer refers to breast cancer that is negative for both Estrogen Receptor (ER), progestogen Receptor (PR) and protooncogene Her-2 as a result of cancer tissue immunohistochemical examination. The breast cancer accounts for 10.0% -20.8% of all pathological types of breast cancer, has special biological behaviors and clinical pathological characteristics, and is inferior to other types in prognosis, and is called as breast cancer king. According to experiments, the medicine provided by the invention can effectively inhibit the growth of the cancer cells.

Drawings

FIG. 1 HMBC (→) spectra of Compound 1

FIG. 2 HMBC (→) spectra of Compound 2

FIG. 3 HMBC (→) spectra of Compound 3

FIG. 4 HMBC (→) spectra of Compound 4

FIG. 5 HMBC (→) spectra of Compound 5

Detailed Description

Example 1

Extracting herba Selaginellae dry whole plant 13.0kg with flash extractor at a ratio of 75% ethanol-water solution 12:1 for 3 times and 2min each time, concentrating under reduced pressure by rotary evaporator until no ethanol smell, to obtain herba Selaginellae total extract (1.2 kg), suspending with water, extracting with petroleum ether, dichloromethane and ethyl acetate for 3 times, and collecting each fraction extract. The solvent was recovered under reduced pressure to give a petroleum ether layer (80.5 g), a methylene chloride layer (160.0 g) and an ethyl acetate layer (180.5 g).

160.0g of methylene dichloride extract is taken, the methylene dichloride extract is separated by adopting HP20 macroporous adsorption resin, the mobile phase is methanol-water, the gradient elution is carried out by 50%, 60%, 70%, 80% and 90%, 7 BV are carried out on each mobile phase, the concentrated solution is combined to obtain five fractions (D1-D5, which means "D1, D2, D3, D4 and D5 in turn", and the similar expression is carried out later.

D2 was eluted gradient-wise by polyamide column chromatography with dichloromethane-methanol (100:0, 50:1, 10:1, 5:1, 2:1, 1:1, v/v) to give 6 fractions in sequence. The dichloromethane-methanol 10:1 eluate was taken and eluted with 50% methanol-water isocratically by p-HPLC to give compound 5 (3.0 mg, t _R =21.9 min); the elution fraction of 80% methanol-water solution was subjected to silica gel column chromatography with methylene chloride-methanol 100:0, 100:1, 50:1, 30:1, 20:1, 10:1, 8:1, 5:1, 2:1, 2:3v/v as mobile phase to obtain 10 fractions (Fr.1. Fwdarw. Fr.10). Wherein Fr.7 fraction was eluted with 70% methanol in water isocratically by p-HPLC to give compound 1 (7.2 mg, t _R =23.4 min), compound 3 (6.4 mg, t _R =33.6 min), compound 4 (5.2 mg, t _R ＝38.2min)。

Taking 178.0g of ethyl acetate extract, carrying out primary separation by using HP20 macroporous adsorption resin, taking 50%, 60%, 70%, 80% and 90% gradient methanol-water as an eluting solvent, carrying out gradient elution, carrying out thin layer chromatography identification on 7 BV of each gradient, combining the eluents of the same spots according to the eluting order, and concentrating to obtain 5 fractions (Fr.1-Fr.5). The Fr.3 fractions were subjected to gradient elution by silica gel column chromatography using dichloromethane-methanol (100:0, 50:1, 10:1, 8:1, 5:1, 2:1, 1:1) as solvent system, and identified by thin layer chromatography, and the eluents of the same spots were combined in the order of the elution morning and evening to obtain 7 fractions Fr.3-1→Fr.3-7, wherein the Fr.3-5 fractions were subjected to p-HPLC using 65% methanol water as eluting condition to obtain compound 2 (4.6 mg, t _R ＝26.5min)。

Compound 1

Compound 1 is a red amorphous powder (MeOH), and HR-ESI-MS spectrum gives an excimer ion peak of M/z527.1877[ M+H ]] ⁺ Molecular weight 526, molecular formula C ₃₅ H ₂₆ O ₅ The unsaturation was 23. The UV spectra 267,299 and 431 have maximum absorption. ¹ The H-NMR spectrum data (Table 1) shows that delta _H 7.73 (1 h, d, j=8.1 Hz) and 7.37 (1 h, d, j=8.1 Hz) are AB spin systems on the aromatic ring. From delta _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 four para-substituted spin coupling systems, which are proton signals on the B-ring, E-ring, and symmetrically substituted C-and D-rings, respectively. In addition, in the case of the optical fiber, ¹ H-NMR spectra indicated the presence of a hydroxymethyl delta _H 4.94 (2H, s), a methoxy group delta _H 3.75 (3H, s) hydrogen signal. ¹³ The C-NMR spectrum showed a 35 carbon signal comprising a carboxyl carbon (delta _C 177.5 Two alkynyl carbons (delta) _C 98.6,83.7), an oxygen-containing methylene carbon (delta) _C 62.0 A methoxy carbon (. Delta.) _C 54.3 And 30 aromatic carbon signals.

Further confirmation of the structure of Compound 1 by HMBC Spectrometry, H-28/32 (delta) _H 7.08 And C-27 (delta) _C 98.6 With related hint alkynyl (delta) _C 98.6,83.7) is attached to the B ring. H-20/24 (delta) _H 6.83 And C-18 (delta) _C 142.1 The correlation between ring E attached to C-18 in ring A and hydroxymethyl attached 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-and D-rings is located at C-7 (delta) _C 166.7)。

Thus, C-19 in ring A is linked to C-7. In addition, delta _H 3.75 and C-30 (delta) _C 160.0 Methoxy proton signal at), indicating that methoxy is attached to C-30. Except that the C-30 position is more than methoxy signal [ delta ] _H 3.75 (3H, s) and delta _C 54.3]In addition, compound 1 ¹ H-NMR ¹³ The data for C-NMR are similar to those reported for Selaginellin (8).

In summary, compound 1 is a novel compound not reported in the literature and is named as selarisine H.

Compound 2

Compound 2 was a red amorphous powder. Gives an excimer ion peak M/z555.1810[ M+H ] according to high resolution mass spectrometry] ⁺ Deducing that its molecular formula is C ₃₆ H ₂₆ O ₆ The unsaturation was 24. The UV spectrum shows maximum absorption at 265, 300 and 425nm, which is a characteristic absorption of propargyl in Selaginella tamariscina.

Compound 2 ¹ H-NMR ¹³ C-NMR spectra showed structural features similar to those of Selaginellin (8), except that there was one more acetyl [ delta ] at the hydroxymethyl group 34 _H 2.14(3H,s),δ _C 19.4,δ _C 171.2]The remaining data is substantially identical. In HMBC spectra, delta was observed _H 2.14 (H-36) and delta _C 171.2(C-35)，δ _H 5.43 (H-34) and delta _C 171.2 There is a correlation between (C-35), further indicating-CH at C-15 ₂ OH is-CH ₂ OOCCH ₃ And (3) substitution.

In summary, compound 2 is a novel compound which is not reported in the literature and is named as selarisine I.

Compound 3

Compound 3 is a red amorphous powder (MeOH). In HR-ESI-MS, at m/z 541.1676[ M+H ]] ⁺ The excimer ion peak is shown, which shows the molecular formula C ₃₅ H ₂₄ O ₆ And shows an unsaturation degree of 24. The ultraviolet spectrum shows maximum absorption at 265, 292 and 431 nm. Compound 3 ¹ H-NMR ¹³ C-NMR spectra also showed similar characteristics to Compound 1, methoxy carbon signal at delta _H Vanishing at 3.75, at delta _H 5.92 (2H, s) and delta _C One methylenedioxy group appears at 101.4. Labat reaction was positive, further indicating that the structure contained a methylenedioxy structure.

In HMBC spectra, the methylenedioxy group is typical ¹ H-NMR Signal (delta) _H 5.92 Respectively with [ delta ] _C 148.4 (C-30) and delta _C 147.5(C-31)]A kind of electronic device ¹³ The C-NMR signals correlate indicating that methylenedioxy is located at C-30 and C-31. In summary, the compound is a novel compound which is not reported in the literature, and the compound 3 is named as selarisine J.

Compound 4

Compound 5

Compound 4 and compound 5 were both red amorphous powders. Compounds 4 and 5 ¹ H-NMR ¹³ The C-NMR data were similar to the structures of (S) -selaginellin U and (S) -selaginellin V, except that there were some differences in the symmetrical substituent positions of the C-and D-rings. Compounds 4 and 5 are illustrated as derivatives of (S) -selaginellin U and (S) -selaginellin V which are tautomerically synthesized by the (R) and (S) forms.

The absolute configuration of compounds 4 and 5 was determined by the optical rotation and the Compact Effect (CE) of CD. In the CD spectra, + CEs are shown at 206 and 214nm, and-CEs are shown at 210 and 220 nm. The absolute configuration of compounds 4 and 5 is (R), contrary to the (S) configuration reported in the literature (production of-CE at 206 and 214nm, and +CE at 210 and 220 nm)

TABLE 1 Compounds 1-3 ¹ H NMR(600MHz)、 ¹³ C NMR(150MHz)

TABLE 2 Compounds 4 and 5 ¹ H NMR (600 MHz) and ¹³ C NMR(150MHz)

EXAMPLE 2 screening study of in vitro anti-tumor Activity of Chinese herba Selaginellae

1. Experimental materials

The tested monomer compounds are prepared by a laboratory, and the purity of the tested monomer compounds reaches more than 98 percent.

TABLE 3 tumor cell line sources

2. CCK-8 method for detecting inhibition activity of selaginella monomer compound on human triple negative breast cancer MDA-MB-231 cells and MDA-MB-468 cells

Human triple negative breast cancer MDA-MB-231 and MDA-MB-468 cells were inoculated into culture flasks and incubated in DMEM medium (medium containing L-15 with 10% fetal bovine serum, 100units/mL penicillin and 100. Mu.g/mL streptomycin) at 37℃in a 0.1% CO2 incubator.

Taking human triple negative breast cancer MDA-MB-231 and MDA-MB-468 cells in logarithmic growth phase, discarding culture solution, adding 2mL of PBS buffer solution for washing twice, discarding PBS, adding 1mL of pancreatin, gently blowing the cells, adding 2mL of culture solution for stopping digestion, centrifuging 1500rmp for 5min, discarding supernatant, adding a proper amount of fresh culture solution, performing cell counting, mixing the cells uniformly, adding 100 mu L of cell suspension into each hole, inoculating to 96-well plates (1.5X103/hole), adding to-be-detected monomer compounds or 5-fluorouracil with different concentrations into each hole, setting 4 compound holes for each medicine, putting into a CO2 incubator for continuous culture for 48h, and setting a blank control group. After 48 hours, CCK-8 reagent was added, placed in an incubator for further cultivation for 1.5 hours, then the cells were taken out, the microplate reader was turned on, absorbance (A) was recorded for detection at 450nm, and the cell proliferation inhibition rate was calculated. The inhibition ratios at different concentrations were observed and the IC50 values were measured.

3. Experimental results

Selaginella tamariscina monomer compound has inhibitory activity on human triple negative breast cancer MDA-MB-231 cells and MDA-MB-468 cells

TABLE 4 inhibitory Activity of Compounds 1-5 on triple negative breast cancer cells

According to the IC ₅₀ The compound of the invention has a certain activity of resisting triple negative breast cancer, wherein the activity of the compound 2 is optimal and is similar to that of positive control 5-Fu.

Claims (7)

1. A method for separating and purifying active ingredients from selaginella tamariscina, which is characterized by comprising the following steps:

(1) Extracting herba Selaginellae 70-80% ethanol-water solution, concentrating until no ethanol smell exists, to obtain herba Selaginellae total extract; suspending with water, sequentially extracting with petroleum ether, dichloromethane and ethyl acetate, extracting each organic solvent extraction layer, and recovering solvent under reduced pressure to obtain extract;

(2) Separating ethyl acetate extract with HP20 macroporous adsorbent resin, eluting with 50%, 60%, 70%, 80%, and 90% methanol-water solution as eluting solvent, identifying by thin layer chromatography, mixing the eluates of the same spots in the order of eluting morning and evening, and concentrating to obtain 5 fractions Fr.1-Fr.5; subjecting Fr.3 fractions to gradient elution sequentially with dichloromethane-methanol 100:0, 50:1, 10:1, 8:1, 5:1, 2:1 and 1:1v/v through silica gel column chromatography, identifying by thin layer chromatography, and combining the eluents of the same spots according to the eluting sequence of the morning and evening to obtain 7 fractions Fr.3-1- & gt Fr.3-7, wherein Fr.3-5 fractions are eluted with 65% methanol-water through p-HPLC to obtain a compound 2;

the structural formula of the compound is as follows:

2. the method according to claim 1, wherein the selaginella extract is extracted with 75% ethanol-water solution.

3. The method according to claim 1, further comprising a method for separating and purifying compounds 1, 3, 4, 5:

(3) Separating dichloromethane layer extract with HP20 macroporous adsorbent resin, and sequentially eluting with 50%, 60%, 70%, 80% and 90% methanol-water solution as mobile phase;

(4) Taking a 60% methanol-water solution elution part, and carrying out gradient elution by polyamide column chromatography sequentially with dichloromethane-methanol 100:0, 50:1, 10:1, 5:1, 2:1 and 1:1 v/v; taking a dichloromethane-methanol 10:1 elution part, and performing isocratic elution with 50% methanol-water by p-HPLC to obtain a compound 5;

(5) Eluting the eluting part of 80% methanol-water solution by silica gel column chromatography sequentially with dichloromethane-methanol 100:0, 100:1, 50:1, 30:1, 20:1, 10:1, 8:1, 5:1, 2:1 and 2:3v/v as mobile phase, eluting the eluting part of 8:1 by p-HPLC with 70% methanol water to obtain compounds 1, 3 and 4 sequentially;

the structural formula of the compound is as follows:

4. the extract A prepared by the following method is applied to the preparation of anticancer drugs; the method comprises the following steps:

extracting herba Selaginellae 70-80% ethanol-water solution, concentrating until no ethanol smell exists, to obtain herba Selaginellae total extract; suspending with water, sequentially extracting with petroleum ether, dichloromethane and ethyl acetate, extracting each organic solvent layer, recovering solvent under reduced pressure to obtain extract, and collecting ethyl acetate layer to obtain extract A.

5. The extract B prepared by the following method is applied to the preparation of anticancer drugs; the method comprises the following steps:

(1) Extracting herba Selaginellae 70-80% ethanol-water solution, concentrating until no ethanol smell exists, to obtain herba Selaginellae total extract; suspending with water, sequentially extracting with petroleum ether, dichloromethane and ethyl acetate, extracting each organic solvent extraction layer, and recovering solvent under reduced pressure to obtain extract;

(2) Separating ethyl acetate extract with HP20 macroporous adsorbent resin, eluting with 50%, 60%, 70%, 80%, and 90% methanol-water solution as eluting solvent, identifying by thin layer chromatography, mixing the eluates of the same spots in the order of eluting morning and evening, and concentrating to obtain 5 fractions Fr.1-Fr.5; taking Fr.3 fraction to obtain extract B.

6. The extract C prepared by the following method is applied to the preparation of anticancer drugs; the method comprises the following steps:

(1) Extracting herba Selaginellae 70-80% ethanol-water solution, concentrating until no ethanol smell exists, to obtain herba Selaginellae total extract; suspending with water, sequentially extracting with petroleum ether, dichloromethane and ethyl acetate, extracting each organic solvent extraction layer, and recovering solvent under reduced pressure to obtain extract;

(2) Separating ethyl acetate extract with HP20 macroporous adsorbent resin, eluting with 50%, 60%, 70%, 80%, and 90% methanol-water solution as eluting solvent, identifying by thin layer chromatography, mixing the eluates of the same spots in the order of eluting morning and evening, and concentrating to obtain 5 fractions Fr.1-Fr.5; taking Fr.3 fractions, sequentially carrying out gradient elution with dichloromethane-methanol 100:0, 50:1, 10:1, 8:1, 5:1, 2:1 and 1:1v/v through silica gel column chromatography, identifying through thin layer chromatography, and combining the eluents of the same spots according to the eluting sequence of the morning and evening to obtain 7 fractions Fr.3-1- & gt Fr.3-7, wherein Fr.3-5 fractions are the extract C.

7. The use according to any one of claims 5-6, wherein the breast cancer is a triple negative breast cancer.