CN109851644B

CN109851644B - Diterpene alcohol glycoside compound, preparation method and application thereof

Info

Publication number: CN109851644B
Application number: CN201910163337.7A
Authority: CN
Inventors: 李建晨; 田蜜; 史利瑶; 潘晶环
Original assignee: Hebei University of Science and Technology
Current assignee: Hebei University of Science and Technology
Priority date: 2019-03-05
Filing date: 2019-03-05
Publication date: 2020-10-20
Anticipated expiration: 2039-03-05
Also published as: CN109851644A

Abstract

The invention relates to a diterpene alcohol glycoside compound with a molecular formula of C₂₆H₄₀O₇The preparation method of the diterpene alcohol glycoside compound is to extract the diterpene alcohol glycoside compound from the silverweed cinquefoil root. The diterpene alcohol glycoside compounds have application in preparing antitumor drugs. The invention extracts, separates and purifies chemical components from the silvery meadow fern by using a natural product chemical means, and has simple and controllable operation. The diterpene alcohol glycoside compound obtained by the preparation method is a compound type which is never reported in the Thymus chinensis (lour.) Merr, and provides a new research direction and thought for the future chemical component research of the Stenoloma japonicum; the diterpene alcohol glycoside compound is subjected to anti-tumor cell detection, and the result shows that the compound can obviously inhibit the proliferation and survival of various tumor cells, has certain tumor inhibition activity, and has potential value as an anticancer lead compound. Has wide application in many fields.

Description

Diterpene alcohol glycoside compound, preparation method and application thereof

Technical Field

The invention relates to a diterpene alcohol glycoside compound, a preparation method and application thereof, and particularly belongs to the field of natural product chemistry.

Background

All-grass of whiteflower StenolomaAleuritopteris argentea(Gmel.) Fee, a plant of Stenoloma of Pteridaceae, also called TONGJINGCAO, has effects of promoting blood circulation, regulating menstruation, tonifying deficiency and relieving cough, and can be used for treating menoxenia, amenorrhea and abdominal pain, pulmonary tuberculosis cough, and hemoptysis. At present, no report is available on the extraction of diterpene alcohol glycoside compounds with biological activity from silverweed cinquefoil.

Disclosure of Invention

The invention provides a diterpene alcohol glycoside compound named as drynaria fortunei C, and provides an extraction method from drynaria fortunei and application thereof in the anti-tumor field.

The molecular formula of the novel diterpene alcohol glycoside compound is C₂₆H₄₀O₇The structure is shown in formula 1.

Formula 1

Further, a preparation method of diterpene alcohol glycoside compounds, which is extracted from the silvery meadow fern.

Further, the specific method for extracting diterpene alcohol glycoside compounds from the silvery meadow fern comprises the following steps:

(1) weighing the entire plant of the Tongdong creeper, performing reflux extraction twice by using 80% ethanol, filtering an extracting solution, combining filtrates, performing reduced pressure concentration to 2-5 times of volume, centrifuging (4000 rpm) for 5-10 minutes, sequentially performing partition extraction on an upper layer of solution by using cyclohexane, chloroform, ethyl acetate and n-butyl alcohol with equal volume, and recovering a solvent to respectively obtain extracts of cyclohexane, chloroform, ethyl acetate and n-butyl alcohol;

(2) dissolving the ethyl acetate extract by adding a proper amount of methanol, adding 1-2 times of silica gel, stirring uniformly, volatilizing the solvent, loading on a silica gel chromatographic column (the dosage is 50-100 times, 200-300 meshes), eluting by using a mixed solvent of petroleum ether-ethyl acetate (5: 1 → 4:1 → 3:1 → 2:1 → 1:1 → 0: 1) → ethyl acetate-methanol (9: 1 → 8:1 → 7:1 → 6:1 → 5: 1), and collecting the fractions by tracing and identifying through thin layer chromatography; the mixed solvent of ethyl acetate and methanol (9: 1 → 8:1 → 7:1 → 6:1 → 5: 1) is used for eluting firstly with a mixed solution of ethyl acetate and methanol in a volume ratio of 9:1, then with a mixed solution of ethyl acetate and methanol in a volume ratio of 8:1, then with a mixed solution of ethyl acetate and methanol in a volume ratio of 7:1, then with a mixed solution of ethyl acetate and methanol in a volume ratio of 6:1, and finally with a mixed solution of ethyl acetate and methanol in a volume ratio of 5:1, and the other steps are carried out in the same way.

(3) Collecting the eluted part (2-5 times of column volume) of the mixed solvent of ethyl acetate-methanol (9: 1), recovering solvent under reduced pressure, concentrating, adding silica gel, and volatilizing solvent. Taking blank silica gel (300-400 meshes) in an amount which is 50-100 times that of the sample-mixed silica gel, filling the mixture into a column, eluting the mixture by using a mixed solvent of cyclohexane-chloroform-methanol (10: 1:0.1 → 10:2:1 → 5:1:1 → 5:2:1 → 5:3: 2), collecting an elution part (2-3 times of the column volume) of the cyclohexane-chloroform-methanol (5: 2: 1), and recovering the solvent under reduced pressure to obtain an extract;

(4) adding 50% methanol into the extract to form a suspension, adding the suspension onto a reverse phase silica gel column, performing gradient elution with 30% -65% methanol (30% -40% -50% -60% -65%) with the amount of the reverse phase silica gel being 150 times that of the suspension, collecting the 50% methanol elution part, recovering the solvent, and concentrating under reduced pressure to obtain the extract;

(5) continuously loading the obtained extract on a Sephadex LH-20 gel column, performing gradient elution (35% -40% -45% -50% -55%) by using 35% -55% methanol, collecting the 50% methanol elution part, recovering the solvent, and performing reduced pressure concentration to obtain the extract;

(6) and (5) drying the extract to obtain a white amorphous powdery monomer compound.

Further, in the step (1), reflux extraction is carried out, 15 times of ethanol in volume of the tongjincao is added for the first time, heating and refluxing are carried out for 2 hours, 10 times of ethanol in volume of the tongjincao is added for the second time, heating and refluxing are carried out for 1 hour, and ethanol used for refluxing is 80% ethanol in volume fraction; vacuum degree of decompression concentration is 0.08-0.1 MPa; the rotation speed during centrifugation was 4000 rpm.

Further, in the step (2), methanol is added to dissolve the ethyl acetate extract, and the adding amount of the methanol is 2-3 times of the volume of the ethyl acetate extract.

Further, in the step (4), the drying temperature is 45-50 ℃, and the drying time is 3-4 hours.

The compound obtained by the method is identified as a novel compound by the structure identification through the modern spectrum technology, and is named as the sildenafil C.

The invention also provides application of the diterpene alcohol glycoside compounds in preparation of antitumor drugs.

The multiples of the present invention are all volume multiples.

Compared with the modern technology, the invention has the beneficial effects that:

(1) the invention extracts, separates and purifies chemical components from the silvery meadow fern by using a natural product chemical means, and has simple and controllable operation. The diterpene alcohol glycoside compound obtained by the preparation method is a compound type which is never reported in the Thymus chinensis (lour.) Merr, and provides a new research direction and thought for the future chemical component research of the Stenoloma japonicum;

(2) the diterpene alcohol glycoside compound is subjected to anti-tumor cell detection, and the result shows that the compound can obviously inhibit the proliferation and survival of various tumor cells (MCF-7 cells, SKOV3 cells and HL-60 cells), has certain tumor inhibition activity, and has potential value as an anti-cancer lead compound. Has wide application in many fields.

Drawings

FIG. 1 is a drawing of a compound of the present invention¹³C-NMR spectrum;

FIG. 2 is a drawing of a compound of the present invention¹H-NMR spectrum;

FIG. 3 is a DEPT spectrum of a compound of the present invention;

FIG. 4 is a drawing of a compound of the present invention¹H-¹H COSY spectrum;

FIG. 5 is an HMQC spectrum of a compound of the invention;

FIG. 6 is an HMBC spectrum of a compound of the present invention;

FIG. 7 is a NOESY spectrum of a compound of the present invention.

Detailed Description

Preparation of the Compound of example 1

Weighing 2.5 kg of the entire plant of the Tongdong herb, performing reflux extraction twice by using 80% ethanol, adding 15 times of ethanol of the volume of the Tongdong herb for heating and refluxing for 2h for the first time, adding 10 times of ethanol of the volume of the Tongdong herb for heating and refluxing for 1h for the second time, filtering the extracting solution, combining the filtrates, performing reduced pressure concentration to 5 times of the volume, centrifuging (4000 rpm) for 10 min, sequentially performing partition extraction on the supernatant by using cyclohexane, chloroform, ethyl acetate and n-butanol of equal volume, and recovering the solvent to respectively obtain extracts of cyclohexane, chloroform, ethyl acetate and n-butanol.

The ethyl acetate extract was dissolved in 50mL of methanol 25.5 g, and then 41g of silica gel was added and stirred uniformly, the solvent was evaporated, and the mixture was eluted by a silica gel column (100 times the amount used, 300 mesh), and eluted with a mixed solvent of petroleum ether-ethyl acetate (5: 1 → 4:1 → 3:1 → 2:1 → 1:1 → 0: 1) → ethyl acetate-methanol (9: 1 → 8:1 → 7:1 → 6:1 → 5: 1), followed by thin layer chromatography to collect fractions.

Collecting the eluted part (5 times of column volume) of the above mixed solvent of ethyl acetate-methanol (9: 1), recovering solvent under reduced pressure, concentrating, adding 10 g of silica gel, and volatilizing the solvent. Another blank silica gel (400 mesh) in an amount which is 100 times that of the mixed silica gel is taken, the mixture is filled into a column, the mixture is eluted by a mixed solvent of cyclohexane-chloroform-methanol (10: 1:0.1 → 10:2:1 → 5:1:1 → 5:2:1 → 5:3: 2), the eluted part (3 times of the column volume) of the cyclohexane-chloroform-methanol (5: 2: 1) is collected, and the solvent is recovered under reduced pressure to obtain 2.4 g of extract.

Adding 50% methanol into the extract to form a suspension, adding the suspension onto a reverse phase silica gel column, wherein the dosage of the reverse phase silica gel is 150 times of that of the suspension, performing gradient elution by using 30% -65% methanol (30% -40% -50% -60% -65%), collecting the elution part of the 50% methanol, recovering the solvent, and performing reduced pressure concentration to obtain 1.7 g of the extract.

And continuously loading the obtained extract on a Sephadex LH-20 gel column, performing gradient elution by using 35-55% methanol (35-40-45-50-55%), collecting the 50% methanol elution part, recovering the solvent, and performing reduced pressure concentration to obtain 1.3 g of extract.

The extract thus obtained was dried to obtain 1.3 g of a white amorphous powdery monomeric compound.

White amorphous powder, HR-ESI-MS showing the excimer ion peak [ M + H ]]⁺Is composed ofm/z465.28562 (calculation: 465.28536, C)₂₆H₄₀O₇）。

¹³In the C-NMR (DMSO) spectrum, 26 carbon signal peaks, including 3 primary carbons, 7 secondary carbons, 12 tertiary carbons and 4 quaternary carbons, appeared together in the d 11.1-149.4 range, suggesting the possibility of one diterpene glycoside compound. Of these, d 108.2 and d 149.4, d141.9 and d 129.3, d 127.3 and d 125.6 are considered signals for double bonds.

In that¹The above hypothesis is also supported by a total of 40 hydrogen signal peaks in the H-NMR (DMSO) spectrum between d 0.77 and 6.00. After subtracting the signal of a six-carbon sugar, the unsaturation degree is 6, and the carbon spectrum analysis is combined, 3 double bonds and 3 cyclic structures are included. d 0.77 (3H, s), 0.91 (3H, s), 1.72 (3H, s) are the three methyl signals.

The HMQC spectrum shows that d 4.77 (1H, s), 5.06 (1H, s) and d 108.2, d 5.48 (1H, dd, J =1, 12 Hz) and d 125.6, d 5.46 (1H, m) and d 127.3, d 6.00 (1H, d, J =1.5 Hz) and d141.9 have a correlation, suggesting that there are 1 exocyclic double bond and 2 endocyclic double bonds. Whereas the correlation of d 3.85 (1H, s), 4.11 (1Hs) with d 71.6, d 3.54 (1H, br d, J =4 Hz) with d 71.4 indicates the presence of 1 hydroxymethyl group and 1 hydroxyl group. There is a correlation between d 3.45 (1H, dd, J =6, 12 Hz), d 3.67 (1H, dd, J =4, 12 Hz) and d 61.0, d 3.06(1H, m) and d 70.1, d 3.01 (1H, dd, J =8, 8Hz) and d 73.6, d 3.14 (1H, dd, J =8, 8Hz) and d 76.8, d 3.07 (1H, m) and d 76.9, and d 4.14 (1H, d, J = 8Hz) and d 102.6, suggesting the presence of a six-carbon sugar structure.

From the HMBC spectra, it can be seen that d 149.4 has a correlation with d 5.06 (1H, s), 2.62 (1H, m), 1.45(1H, m), suggesting that d 39.2 and d 51.6 are in their vicinities, respectively.¹H-¹In the H COSY spectrum, the connection of C-4 'and C-5' can be deduced through the coupling between d 2.62 and d 1.45(1H, m), 2.10 (1H, m), d 2.10 and d 1.24 (1H, o) and 1.49 (1H, m), and the structure of an independent five-membered ring can be deduced by combining the correlation between d 1.49 and d 51.6 in the HMBC spectrum. In addition, the methyl signals d 0.77 and d 37.4, 45.5, 71.4, 141.9 all show clear correlations suggesting the possibility of direct connection of C-3a, C-1, C-8 to C-8a, and the coupling between d 6.00 (1H, d, J =1.5Hz H-8) and d 45.5, 71.4, 37.4 also supports this inference. In combination with the number of carbon atoms, the presence of an azulene skeleton can be considered. According to this idea, by HMBC spectral sum¹H-¹Analysis of the H COSY spectrum confirms the basic skeleton of the compound and the connection positions of each double bond, hydroxyl, methyl and five-membered ring. The coupling between d 2.62 and d 71.6 and d 102.6 suggests that the sugar group is attached to the hydroxymethyl group at the C-3' position.

To confirm the three-dimensional structures of C-1 and C-3', we performed experiments using MTPA (α -methoxy- α -trifluoromethylphenyl acetate) method. Collecting the separated compound 45 mg, adding 4% H₂SO₄The solution (3 ml) was refluxed for 1h and purified by MCI resin column. 27 mg of the resulting aglycone fraction was taken, and 50mg of R- (-) MTPA-Cl reagent, 1 ml DMAC and 0.1 ml triethylamine were added thereto, followed by stirring at room temperature for 4 hours, followed by addition of CH₂Cl₂Extraction and chromatography on silica gel (eluent C)₆H₁₂:CH₂Cl₂EtOAc =5:2: 2) separation to yield two products F₁(16 mg) and F₂(10 mg) were measured, respectively¹H nuclear magnetic spectrum. As a result: f₁Is/are as follows¹The H-NMR spectrum showed that the chemical shifts of the two hydrogens on C-2 were changed from d 1.62, d 1.23 to d 1.65, d 1.24, indicating that C-1 wasSConfiguration; f₂Is/are as follows¹In the H-NMR spectrum, d 3.76 (s, 2H 3' -CH)₂OH) becomes a doublet of peaks at d 3.50 and d 3.48, indicating that C-3' is alsoSConfiguration.

NOESY spectra show d 0.77 and 1.45(1H, m 8 a-CH)₃) 1.24 (1H, m H-2), 1.94 (1H, mH-4), d 3.54 (1H, m H-1) and d 1.93 (1H, m 3a), 0.91 (3H, s 3-CH)₃) 1.62 (1H, dd, J =4, 12Hz H-2), 6.00 (1H, d, J =1.5Hz H-8) and suggest that the azulene skeleton is trans and the stereoconfiguration of each chiral carbon is 1: (d), J =1.5Hz H-8), respectivelyS), 3(S), 3a(R), 8a(S), C-1’(S), C-3’(S)。

Therefore, the structure of the drynarin C is determined to be 1, 2, 3, 3a, 4, 8a-haxahydro-3- [2 ' -ene-3 ' -methyl- (1 ')b-D-glucopyranoside)-cyclopentyl]3, 7, 8a-trimetyl-1-azulenol, which is a diterpene alcohol glycoside compound with a novel framework.

According to the extraction method, the purity of the obtained compound of the drynaria fortunei C is as follows: 96.7 percent. The obtained compound, drynarin C, is characterized in FIGS. 1-7.

EXAMPLE 2 Activity test of Compounds

Experimental Material

Calf serum; thiazole blue (MTT) (CAS 298-93-1), dimethyl sulfoxide (national pharmaceutical group chemical Co., Ltd.); streptomycin, penicillin; the breast cancer cells MCF-7, ovarian cancer cells SKOV3 and promyelocytic leukemia cells HL-60 were provided by Shanghai cells of the Chinese academy of sciences.

Experimental methods

Taking breast cancer cells MCF-7, ovarian cancer cells SKOV3 and promyelocytic leukemia cells HL-60, and mixing the cells with a suitable culture solution (MCF-7 cells: fetal bovine serum: MEM: double antibody =5:45: 0.5; SKOV3 cells: fetal bovine serum: M5A:diabody =5:45: 0.5; HL-60 cells: fetal bovine serum: RPMI-1640: diabolo =5:45: 0.5), 5% CO at 37 ℃₂Culturing in an incubator. Cells grown in log phase were randomly selected and adjusted to 3X 10 cell concentration⁴And/ml, inoculating on a 96-well plate, placing 100 mu l of the plate in an incubator for 24 h. Adding test sample 20 μ g/ml and positive control drug cisplatin 5 μ g/ml, adding cell suspension equal to the experimental group into negative control group, adding culture solution equal to the experimental group into blank control group, and culturing for 48 hr respectively. Adding 20 μ l of 5mg/ml MTT solution, culturing for 4 hr, removing MTT solution, adding 150 μ l of dimethyl sulfoxide into each well, shaking on a shaking table for 10 min, mixing well for dissolving, and measuring OD value at 490 nm with enzyme labeling instrument. Inhibition =1- (experimental OD value/control OD value) × 100% results are shown in table 1.

TABLE 1 inhibition ratio of Dryopterin C to tumor cells (%)

	MCF-7	SKOV3	HL-60
				Dryopteris C (20 ug/ml)	67.6	40.2	81.5
Cisplatin (5. mu.g/ml)	32.9	46.5	63.7

The activity experiment result shows that the new compound of the drynaria fortunei C can obviously inhibit the proliferation and survival of various tumor cells, has certain tumor inhibition activity and has potential value as an anticancer lead compound.

Claims

1. A diterpene alcohol glycoside compound is characterized in that the molecular formula is C₂₆H₄₀O₇The structure is shown as formula 1;

formula 1.

2. A method for producing the diterpene glycoside compound according to claim 1, which comprises: extracting from Aleuritopteris argentea fee;

comprises the following steps of (a) carrying out,

(1) weighing the entire plant of the Tongdong creeper, performing reflux extraction twice by using ethanol with the volume fraction of 80% to obtain an extracting solution, performing reduced pressure concentration on the extracting solution until the volume of the extracting solution is 2-5 times that of the Tongdong creeper, centrifuging for 5-10 minutes, extracting supernatant obtained by centrifuging by using ethyl acetate with the same volume as that of the supernatant, and recovering a solvent to obtain an ethyl acetate extract;

(2) dissolving an ethyl acetate extract by adding methanol, loading the ethyl acetate extract on a silica gel chromatographic column, eluting by using a petroleum ether-ethyl acetate mixed solvent firstly, and then eluting by using an ethyl acetate-methanol mixed solvent, wherein the volume ratio of the petroleum ether to the ethyl acetate is 5:1 → 4:1 → 3:1 → 2:1 → 1:1 → 0:1, the volume ratio of the ethyl acetate to the methanol is 9:1 → 8:1 → 7:1 → 6:1 → 5:1, and collecting components of each segment by thin-layer chromatography tracking identification;

(3) taking a mixed solvent elution part of ethyl acetate and methanol with a volume ratio of 9:1, applying a silica gel column, eluting with a mixed solvent of cyclohexane-chloroform-methanol with a volume ratio of 10:1:0.1 → 10:2:1 → 5:1:1 → 5:2:1 → 5:3:2, collecting an elution part of cyclohexane-chloroform-methanol with a volume ratio of 5:2:1, collecting the elution part, applying a reverse phase silica gel column, performing gradient elution with 30% -65% of methanol with a volume fraction of 30% -40% -50% -60% -65%, collecting a 50% methanol elution part, applying a Sephadex LH-20 gel column, performing gradient elution with 35% -55% of methanol with a volume fraction of 35% -40% -45% -50% -55%, collecting the methanol elution part with the volume fraction of 50%, recovering the solvent, and concentrating under reduced pressure to obtain an extract;

(4) drying the extract obtained in the step (3) to obtain a white amorphous powdery monomer compound shown in the formula 1.

3. The method of claim 2, wherein: performing reflux extraction in the step (1), adding ethanol with the volume 15 times that of the tongjincao for the first time, heating and refluxing for 2 hours, adding ethanol with the volume 10 times that of the tongjincao for the second time, heating and refluxing for 1 hour, wherein the ethanol used for refluxing is 80% ethanol in volume fraction; vacuum degree of decompression concentration is 0.08-0.1 MPa; the rotation speed during centrifugation was 4000 rpm.

4. The method of claim 2, wherein: and (3) adding methanol in the step (2) to dissolve the ethyl acetate extract, wherein the adding amount of the methanol is 2-3 times of the volume of the ethyl acetate extract.

5. The method of claim 2, wherein: in the step (4), the drying temperature is 45-50 ℃, and the drying time is 3-4 hours.

6. The use of diterpene glycosides of claim 1 in the preparation of a medicament for treating tumors selected from the group consisting of MCF-7, SKOV3, and HL-60.