CN116003371A - Terpenoid, and extraction method and application thereof - Google Patents
Terpenoid, and extraction method and application thereof Download PDFInfo
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Medicines Containing Plant Substances (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses a terpenoid shown in a formula (I), and an extraction method and application thereof. The invention also discloses a separation method of the terpenoid and application of the terpenoid in preparing a medicament for treating cancer.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to a terpenoid, an extraction method and application thereof.
Background
Cancer has become the leading fatal disease worldwide, and can occur in various organs and tissues at any age.
Therefore, development of new anticancer drugs is a challenge to be solved.
However, most cancer patients usually find the disease to be middle to late stage, and the overall clinical treatment effect is poor, especially the continuous occurrence of multi-drug resistance, so that the treatment difficulty of the cancer is serious. Although some small molecule anticancer chemotherapeutics and antibody drugs have entered the clinic. However, most of the new anticancer drugs used in clinic are basically imported and have high treatment cost. The number of new anticancer drugs independently developed in China is relatively small. Therefore, development of a novel anticancer drug with high activity and low side effect is urgent to meet clinical demands. Especially, the discovery of new antitumor candidate compounds from traditional Chinese medicines is the mainstream of current drug development.
The jasmine root is a dry root of a dicotyledon Jasminum sambac (L.) Ait. Warm nature, bitter taste, toxic and original in India. The Chinese is mainly produced in Jiangsu, zhejiang, fujian, taiwan, guangdong, sichuan and other places. According to the ancient book records in China, jasmine roots are used for fracture, dislocation and bone fracture, and have analgesic effect and certain anesthetic effect. The jasmine root has the effects of improving immunity, clearing heat and detoxicating, and relieving swelling and pain. Has good therapeutic effect on patients with dysentery, abdominal pain, enteropathy, and conjunctivitis. It is used for treating fracture, injury of tendons, dental caries, and parietal headache.
The invention extracts and separates jasmine root growing in Ningde of Fujian province, so as to find new drug candidate compound or lead compound with anti-tumor activity.
Disclosure of Invention
The terpenoid with novel structure is separated from jasmine roots, and has strong inhibitory activity on tumors.
The invention provides a terpenoid shown in a formula (I):
the invention also provides an extraction and separation method of the terpenoid shown in the formula (I), which comprises the following steps:
(1) Crushing jasmine roots, extracting with ethanol water solution, and concentrating the extract to obtain a total extract;
(2) Dispersing the total extract obtained in the step (1) with water, extracting with organic solvents with different polarities, and concentrating the extract to obtain extracts with different polarities;
(3) Carrying out column separation on the extractum with different polarities obtained in the step (2) to obtain a crude component; and
(4) And (3) separating and purifying the crude component obtained in the step (3) to obtain the terpenoid shown in the formula (I).
In an embodiment according to the present invention, the aqueous ethanol extraction in step (1) may be a soaking or reflux extraction.
According to an embodiment of the invention, the soaking extraction: the soaking temperature is 15-30deg.C, preferably room temperature; the soaking time is 20-40 days, preferably 25-35 days, such as 28 days, 29 days, 30 days, 31 days, and 32 days.
According to an embodiment of the invention, the reflux extraction: the reflux time may be 10-24 hours, for example 12-15 hours.
According to an embodiment of the present invention, the organic solvents of different polarity used in step (2) are selected from petroleum ether, ethyl acetate, chloroform or n-butanol.
According to an embodiment of the present invention, the chromatographic separation in step (3) includes, but is not limited to, silica gel column separation, preparative liquid chromatography separation, and any combination thereof.
According to an embodiment of the present invention, the separation and purification in step (4) is selected from the group consisting of silica gel column separation, preparative plate separation, or preparative liquid chromatography separation, and any combination thereof.
According to an embodiment of the present invention, the method for extracting and separating the compound represented by formula (I) comprises the steps of:
(1) Pulverizing radix Jasmini sambac, soaking in ethanol water solution, filtering, and concentrating to obtain total extract;
(2) Dispersing the total extract obtained in the step (1) with water, sequentially extracting with petroleum ether, ethyl acetate and chloroform for 3-5 times, and concentrating the extractive solutions with different polarities to obtain petroleum ether extract, ethyl acetate extract and chloroform extract;
(3) Separating the ethyl acetate extract obtained in the step (2) by a silica gel column, and performing gradient elution by using a developing agent to obtain coarse components with different polarities;
(4) Analyzing and combining the crude components with different polarities in the step (3) by HPLC, separating by silica gel column, preparative plate or preparative liquid chromatography, and combining R f The terpene compound shown in the formula (I) is obtained after repeated recrystallization of the components with the same or same retention time.
According to an embodiment of the present invention, in the step (1), the mass fraction of ethanol in the aqueous ethanol solution may be 50-80%, for example 50%, 60%, 70% or 80%.
According to an embodiment of the invention, in step (2), the mass to volume ratio (g/mL) of the total extract to water is (0.2-3): 1, for example (0.5-2): 1, exemplary 1:1.
According to an embodiment of the invention, in said step (2), the volume ratio of organic solvent to water used for extraction is 1 (0.2-3), for example 1 (0.5-2), and is exemplified by 1:2.
According to an embodiment of the present invention, the developing agent in step (3) is petroleum ether and/or ethyl acetate, and starting from pure petroleum ether, the amount of ethyl acetate is gradually increased while the amount of petroleum ether is reduced, and finally pure ethyl acetate is obtained. Preferably, the volume ratio of petroleum ether to ethyl acetate is 1:0, 0.9:0.1, 0.8:0.2, 0.7:0.3, 0.6:0.4, 0.5:0.5, 0.4:0.6, 0.3:0.7, 0.2:0.8, 0.1:0.9, 0:1.
According to an embodiment of the invention, in step (4), the HPLC analysis conditions are as follows: mobile phase: v (V) Methanol :V Water (0.3% phosphoric acid) The column temperature was room temperature and the detection wavelength was 200-400nm integration wavelength =7:3.
According to an embodiment of the present invention, in the step (4), the separation and purification are performed by selecting a component having a larger polarity difference (e.g., selecting R f 3-5 components with a difference of 0.5-1).
According to an embodiment of the present invention, in step (4), the separation and purification may be repeated.
The invention also provides application of the terpenoid shown in the formula (I) in preparing a medicament for treating and/or preventing cancers.
According to an embodiment of the invention, the cancer is selected from lung cancer, stomach cancer, breast cancer or cervical cancer.
Advantageous effects
The terpenoid compound with novel structure is separated from jasmine roots, and has strong inhibitory activity on various cancer cells (such as gastric cancer cell lines, colorectal cancer cell lines, breast cancer cell lines and cervical cancer cell lines).
The extraction method provided by the invention has the advantages that the plant extract is subjected to primary purification (such as column separation, preparation plate separation, preparation liquid chromatography separation and the like), then recrystallization is carried out, and the target compound with high purity (the purity can be up to 98%) is rapidly and accurately obtained, so that the extraction method has important significance in rapid separation and identification of specific compounds, particularly chiral enantiomers, in the plant extract containing complex components.
Drawings
FIG. 1 is an HPLC chromatogram of a compound of formula (I);
FIG. 2 is a HMBC correlation diagram of a compound of formula (I);
FIG. 3 is a ROESY correlation diagram of a compound of formula (I);
FIG. 4 is a diagram showing the structure of spherical crystals of the compound represented by formula (I).
Detailed Description
The technical scheme of the invention will be further described in detail below with reference to specific embodiments. It is to be understood that the following examples are illustrative only and are not to be construed as limiting the scope of the invention. All techniques implemented based on the above description of the invention are intended to be included within the scope of the invention.
Unless otherwise indicated, the starting materials and reagents used in the following examples were either commercially available or may be prepared by known methods.
Instrument and reagent:
jasmine root (10 months of 2021 is collected from Fujian province), and other chemical reagents are domestic chemical pure reagents; CCK8 (shanghai Bei Bo biotechnology limited); DMEM high sugar medium (sameifer's instruments limited); EDTA (pancreatin) (gibco); foetal Bovine Serum (Biological Industries); phosphate buffer salt solution; 96-well cell culture plates; multifunctional enzyme labeling instrument.
Example 1: terpenoid extraction separation and structural identification shown in formula (I)
The extraction and separation of the compounds are carried out according to the following procedures:
(a) 20 kg of dried jasmine roots (2021, 10 months from Fujian province) are crushed, respectively filled into 3 plastic barrels of 20L, respectively added with 15L of 70% ethanol/water solution for soaking for 2 months at room temperature, and filtered and concentrated to obtain extract.
(b) Dispersing 1 kg of the extract obtained in the step a in 2L of water, and extracting with petroleum ether, ethyl acetate and chloroform respectively for 5 times in sequence, wherein the dosage of petroleum ether, ethyl acetate and chloroform is 1000 ml each time. Concentrating the extractive solutions of different polarities to obtain extract of different polarities.
(c) C, performing primary silica gel column separation on the ethyl acetate extract obtained in the step b, and adopting V Petroleum ether /V Acetic acid ethyl ester Gradient elution with =1:0 to 0:1, collection V Petroleum ether /V Acetic acid ethyl ester The elution fractions =2:1 and 1:1 gave 50 sites of different polarity (Fr 1 -Fr 50 )(I)。
(d) The 50 fractions obtained in step (c) were first examined by preliminary TLC and similar fractions were combined to obtain 30 fractions (II). Then subjecting the component (II) to HPLC qualitative analysis (chromatographic conditions: V) Methanol :V Water (0.3% phosphoric acid) 7:3, column temperature is room temperature, detection wavelength is 200-400 nm), and similar components are combined. Then selecting 5 components with larger polarity difference (core material retention time difference about 0.5-5 min), separating with first silica gel column, performing primary TLC analysis, combining the similar components, and selecting polarThe difference in the properties is larger (R f Separating the components with difference of about 0.2-0.6) with a second silica gel column, separating the components with the second column by preparative plate separation or preparative high performance liquid chromatography, separating the same R with that obtained by preparative chromatography f And combining the components with the same value or the same retention time, and then recrystallizing to obtain the terpenoid shown in the formula (I).
For isolated = terpenoid, its purity was determined by HPLC: purity 94.34%, rt= 18.880min; (chromatographic conditions: C) 18 A column; mobile phase: v (V) Methanol :V Water and its preparation method 7:3; detection wavelength: 254 nm), and HPLC chromatogram thereof is shown in figure 1.
The structure was determined by 1D NMR and 2D NMR, high resolution mass spectrometry, and the absolute configuration was determined by ROESY and XRD analysis methods. The HMBC of the compound shown in formula (I) is shown in figure 2, the ROESY is shown in figure 3, and the XRD spherical pattern is shown in figure 4.
The characterization data of the terpenoids shown in formula (I) are as follows: yellow crystals, melting point: 147-148 ℃; HR-MS for C 30 H 32 O 6 +na ": 511.2091, experimental values: 511.2091; 1 H NMR(400MHz,CD 3 OD,ppm,J/Hz):0.95(3H,d,J＝7.1Hz,H-13),1.29(2H,s,H-8),1.35(2H,m,H-7),1.40(1H,m,H-9),1.61(1H,m,H-5'β),1.70(3H,s,H-14'),2.21(1H,m,H-5'α),2.27(3H,s,H-17'),2.41(2H,m,H-6),2.88(1H,m,H-6'α),3.05(1H,m,H-6'β),3.96(1H,d,J＝10.6Hz,H-15'β),4.47(1H,d,J＝10.6Hz,H-15'α),4.77(1H,d,J＝2.3Hz,H-12β),4.97(1H,d,J＝2.3Hz,H-12α),5.15(1H,s,H-4),5.25(2H,d,J＝19.7Hz,H-18'),6.11(1H,s,H-2'),6.80(1H,s,H-7'). 13 C NMR(100MHz):17.7(C-7),18.6(C-13),23.6(C-6),24.0(C-6'),24.87(C-5'),24.89(C-14'),24.94(C-17'),29.5(C-8),34.7(C-9),47.8(C-13'),55.4(C-4'),64.6(C-15'),102.62(C-12),108.1(C-4),113.7(C-10'),117.3(C-18'),121.7(C-7'),126.7(C-2'),127.0(C-12'),136.7(C-11'),140.1(C-11),142.5(C-10),143.66(C-8'),143.68(C-16'),147.69(C-9'),147.71(C-3'),156.6(C-1),171.3(C-1'),172.4(C-3),190.3(C-5)。
example 2: in vitro antitumor Activity test
The in vitro antitumor activity test is carried out on the terpenoid shown in the formula (I), and the inhibition activity of the terpenoid on lung cancer cell strains A549 and cervical cancer cell strains Hela is mainly studied.
1. Concentration preparation of test sample
13.0mg of the compound represented by formula (I) was weighed into a 5mL plastic centrifuge tube, and diluted to 1mL with DMSO. The initial concentration of 13.0mg/mL was obtained. Then the initial concentration is diluted by DMSO in a multiple ratio to obtain 6.5mg/mL,3.25mg/mL,1.625mg/mL,0.8125mg/mL and 0.40625mg/mL of 5 different concentration gradients in sequence, and the concentration gradients are stored in a refrigerator at 4 ℃ for standby.
2. Culture of cancer cell lines and test of inhibitory Activity
The lung adenocarcinoma cell line (A549) was subjected to saturation humidity at 37℃with 5% CO 2 After culturing in the incubator for 24 hours, the supernatant medium was aspirated when the cells were in the logarithmic growth phase, and digested with 0.25% trypsin-EDTA solution, the digestion was stopped with high sugar medium. And cells were seeded in 96-well plates such that the cell density was 5000 cells/well. The 96-well plate was placed in an incubator for 24 hours. With consequent pipetting of the cell culture broth in the 96-well plate. And 100. Mu.L of high sugar culture medium is added into a 96-well plate, then 1. Mu.L of test samples with different concentrations (5 compound wells are arranged for each concentration) are added into each well, and then the mixture is placed at 37 ℃ and saturated humidity, and 5% CO 2 After 48h of continued incubation in the incubator of (C), 10. Mu.L of CCK8 was added to each well and incubation was continued in the incubator at 37℃for 1-4 h. The absorbance value per well at a wavelength of 450nm was measured on a multifunctional microplate reader. According to the inhibition ratio = [ (control cell OD-dosing cell OD)/(control cell OD-blank OD)]X 100. Negative control is V High sugar culture medium /V DMSO 10:1 mixed solution.
The test procedure of the compound shown in the formula (I) on the culture of cervical cancer cell strain Hela and breast cancer cell strain MCF-7 is the same as that of the test procedure of the inhibition activity.
IC of Compound of formula (I) against Breast cancer cell line MCF-7 50 The value was 75.44. Mu.g/mL.
The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiment. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (7)
1. A terpenoid represented by formula (I):
2. the method for extracting and separating a terpenoid represented by the formula (I) according to claim 1, comprising the steps of:
(1) Crushing jasmine roots, extracting with ethanol water solution, and concentrating the extract to obtain a total extract;
(2) Dispersing the total extract obtained in the step (1) with water, extracting with organic solvents with different polarities, and concentrating the extract to obtain extracts with different polarities;
(3) Carrying out column separation on the extractum with different polarities obtained in the step (2) to obtain a crude component; and
(4) And (3) separating and purifying the crude component obtained in the step (3) to obtain the terpenoid shown in the formula (I).
3. The extraction and separation method according to claim 2, wherein,
the ethanol water solution in the step (1) is extracted by soaking or reflux extraction;
preferably, the soaking extraction temperature is 15-30deg.C, such as room temperature; the soaking time is 20-40 days, preferably 25-35 days, such as 28 days, 29 days, 30 days, 31 days, and 32 days;
preferably, the reflux extraction time is 10-24 hours, for example 12-15 hours;
and/or the organic solvents of different polarity used in step (2) are selected from petroleum ether, ethyl acetate, chloroform or n-butanol;
and/or, chromatographic separations in step (3) include, but are not limited to, silica gel column separations, preparative liquid chromatographic separations, and any combination thereof;
and/or the separation and purification in step (4) is selected from the group consisting of silica gel column separation, preparative plate separation, and preparative liquid chromatography separation, and any combination thereof.
4. The extraction and separation method according to claim 2 or 3, wherein,
the extraction and separation method of the compound shown in the formula (I) comprises the following steps:
(1) Pulverizing radix Jasmini sambac, soaking in ethanol water solution, filtering, and concentrating to obtain total extract;
(2) Dispersing the total extract obtained in the step (1) with water, sequentially extracting with petroleum ether, ethyl acetate and chloroform for 3-5 times, and concentrating the extractive solutions with different polarities to obtain petroleum ether extract, ethyl acetate extract and chloroform extract;
(3) Separating the ethyl acetate extract obtained in the step (2) by a silica gel column, and performing gradient elution by using a developing agent to obtain coarse components with different polarities;
(4) Analyzing and combining the crude components with different polarities in the step (3) by HPLC, separating by silica gel column, preparative plate or preparative liquid chromatography, and combining R f The terpene compound shown in the formula (I) is obtained after repeated recrystallization of the components with the same or same retention time.
5. The extraction and separation method as claimed in any one of claims 2 to 4, wherein,
in step (1), the mass fraction of ethanol in the aqueous ethanol solution may be 50-80%, such as 50%, 60%, 70% or 80%;
and/or in step (2), the mass to volume ratio (g/mL) of the total extract to water is (0.2-3): 1, for example (0.5-2): 1, exemplary 1:1;
and/or, in the step (2), the volume ratio of the organic solvent used for extraction to water is 1 (0.2-3), for example 1 (0.5-2), and is exemplified by 1:2;
and/or, the developing agent in the step (3) is petroleum ether and/or ethyl acetate, and starting from pure petroleum ether, gradually increasing the amount of ethyl acetate and simultaneously reducing the amount of petroleum ether, and finally, the pure ethyl acetate is obtained; preferably, the volume ratio of petroleum ether to ethyl acetate is 1:0, 0.9:0.1, 0.8:0.2, 0.7:0.3, 0.6:0.4, 0.5:0.5, 0.4:0.6, 0.3:0.7, 0.2:0.8, 0.1:0.9, 0:1;
and/or, in step (4), the HPLC analysis conditions are as follows: mobile phase: v (V) Methanol :V Water (0.3% phosphoric acid) =7:3, column temperature is room temperature, detection wavelength is 200-400nm integration wavelength;
and/or, in the step (4), the separation and purification are performed by selecting a component having a larger polarity difference (e.g., selecting R f 3-5 components with a difference of 0.5-1);
and/or, in the step (4), the separation and purification may be repeated.
6. Use of a terpenoid represented by formula (I) according to claim 1 for the preparation of a medicament for the treatment and/or prophylaxis of cancer.
7. The use according to claim 6, wherein the cancer is selected from lung cancer, stomach cancer, breast cancer or cervical cancer.
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