CN115466248A - Diterpenoid compound and extraction method and application thereof - Google Patents
Diterpenoid compound and extraction method and application thereof Download PDFInfo
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Abstract
The invention discloses diterpenoid compounds, an extraction method and application thereof. The diterpenoid compound is selected from diterpenoid compound A, diterpenoid compound B or diterpenoid compound C with the following structures. The invention also discloses a separation method of the diterpenoid compound and application of the diterpenoid compound in preparation of a medicament for treating cancer.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to diterpenoid compounds, an extraction method of the diterpenoid compounds from ajuga ciliata and application of the diterpenoid compounds in treating tumors/cancers.
Background
Cancer has become the leading fatal disease worldwide, and cancer can occur in various organs and tissues at any age. Therefore, the development of new anticancer drugs is a problem to be solved urgently.
However, most cancer patients usually find the disease in the middle to late stage, and the overall effect of clinical treatment is poor, especially the multi-drug resistance is continuously appeared, so that the treatment of cancer is difficult. Although some small molecule anticancer chemotherapeutic and antibody drugs have already entered the clinic. However, most of the new anticancer drugs for clinical application are basically imported, and the treatment cost is high. Relatively few new anticancer drugs are independently developed in China. Therefore, the development of novel anticancer drugs with high activity and low side effects to meet clinical needs is urgent. In particular, new anti-tumor candidate compounds found from traditional Chinese medicines are the mainstream of the current medicine research and development.
Ajuga decumbens Thunb belonging to the family Labiatae is an annual herb, also known as Ayuga decumbens. Is mainly distributed in the southern provinces of the Yangtze river of China (such as Jiangsu, anhui, zhejiang, shanghai, sichuan, fujian, hubei, hunan, guangdong, guangxi, guizhou, yunnan, etc.), and dried whole herbs are used as the medicine. Has the functions of clearing away heat and toxic material, cooling blood and calming liver. Can be used for treating upper respiratory infection, tonsillitis, pharyngitis, bronchitis, pneumonia, lung abscess, gastroenteritis, hepatitis, appendicitis, mastitis, acute conjunctivitis, and hypertension; it is used externally to treat traumatic injury, traumatic hemorrhage, carbuncle, furuncle, sore, ulcer, burn, scald, snake bite, etc. The Chinese pharmacopoeia (1977, part of the disclosure) was selected. It is used for treating carbuncle, cellulitis, sore, acute conjunctivitis, acute mastitis, pharyngolaryngitis, gastroenteritis, acute conjunctivitis, scald, dog bite, snake bite, traumatic hemorrhage, etc. in southern China (Fujian medicine material, first volume: fuzhou, fujian people publishing house, 1979.410). People in Tantang island refer to the 'Ajuga ciliata' in the local area as 'domestic antibiotics without side effect'. In view of the deep accumulation of the inventor in the research aspect of natural drugs and good results in the research aspect of small molecular antitumor drugs in recent years, the carpet bugle growing in Tabane island of Fujian province is extracted and separated, so as to discover a new drug candidate compound or lead compound with antitumor activity.
Disclosure of Invention
The invention provides a diterpenoid compound, an extraction method and application thereof.
The invention provides a diterpenoid compound which is selected from a diterpenoid compound A, a diterpenoid compound B or a diterpenoid compound C with the following structures:
according to an embodiment of the invention, the diterpenoid B is a colorless solid having a melting point of 98-99 ℃.
The invention also provides the diterpenoid crystal, for example, the diterpenoid crystal is selected from at least one of the diterpenoid A crystal and the diterpenoid C crystal.
According to an embodiment of the invention, the diterpenoid A crystals belong to the monoclinic system with the space group P2 1 ，
α＝90.00°，β＝94.046(5)°，γ＝90.00°。
According to an embodiment of the invention, the diterpenoid a crystals are colorless needle crystals.
According to an embodiment of the invention, the diterpenoid A crystals have a melting point of 185-186 ℃.
According to an embodiment of the invention, the diterpenoid C crystals belong to the triclinic system with the space group P2 1 ，
α＝90.00°，β＝100.858(2')°，γ＝90.00°。
According to an embodiment of the present invention, the diterpenoid C crystals are colorless columnar crystals.
According to an embodiment of the invention, the diterpenoid C crystals have a melting point of 148-149 ℃.
The invention also provides an extraction method of the diterpenoid compounds or the crystal thereof, which comprises the following steps:
(1) Pulverizing herba Ajugae, extracting with ethanol water solution, and concentrating the obtained extractive solution to obtain total extract;
(2) Dispersing the total extract obtained in the step (1) with water, extracting with an organic solvent, and concentrating the obtained extract to obtain an organic solvent extract;
(3) Performing column separation on the organic solvent extract obtained in the step (2) to obtain a crude component;
(4) And (4) separating and purifying the crude component obtained in the step (3) to obtain the diterpenoid compound or the crystal thereof.
According to an embodiment of the present invention, in step (1), the mass fraction of ethanol in the aqueous ethanol solution may be 50 to 80%, for example, 50%, 60%, 70%, or 80%.
According to an embodiment of the present invention, in the step (1), the aqueous ethanol extraction may be a soaking extraction or a reflux extraction.
Preferably, the soaking extraction conditions include: the soaking temperature is 15-30 ℃, and preferably 20-25 ℃; 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 can be performed by methods known in the art, for example, the reflux extraction conditions include: the reflux temperature is 70-100 ℃, preferably 79-90 ℃; the reflux time is from 10 to 24h, for example from 12 to 15h.
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, e.g. (0.5-2): 1, exemplified by 1:1.
According to an embodiment of the invention, in step (2), the volume ratio of the organic solvent to water is 1 (0.2-3), such as 1 (0.5-2), exemplary 1:2.
According to an embodiment of the present invention, in the step (2), the organic solvent is selected from water-immiscible organic solvents. Preferably, the organic solvent may be selected from at least one including, but not limited to, petroleum ether, ethyl acetate, chloroform and n-butanol.
According to an embodiment of the present invention, in the step (3), the column separation includes, but is not limited to, silica gel column separation, reverse silica gel column separation, macroporous resin column enrichment separation, and/or preparative liquid chromatography separation.
According to an embodiment of the present invention, in the step (4), the separation and purification may be at least one or two or more of column separation, recrystallization, and preparative liquid chromatography separation.
According to an embodiment of the present invention, in step (3) or (4), the silica gel column separation specifically comprises: gradient elution is carried out by adopting a developing solvent to obtain crude components with different polarities.
Preferably, the developing solvent is selected from petroleum ether and/or ethyl acetate, preferably V Petroleum ether /V Ethyl acetate =1:0 to 0:1 gradient elution, i.e. from pure petroleum ether (V) Petroleum ether /V Acetic acid ethyl ester = 1:0) gradually increasing the amount of ethyl acetate while decreasing the amount of petroleum ether, eventually becoming pure ethyl acetate (V) Petroleum ether /V Ethyl acetate = 0:1). Preferably, the gradient elution is performed in a volume ratio of petroleum ether to ethyl acetate of 1:0, 0.9, 0.8, 0.2, 0.7, 0.4, 0.5, 0.6.
Preferably, the column separation is performed at least once, for example twice.
According to an embodiment of the invention, in step (4), the column separation is carried out with the selection of fractions of greater difference in polarity, for example R f The difference is 3-5 components of 0.5-1.
According to an embodiment of the present invention, in step (3) or (4), the conditions for the liquid chromatography separation are preferably as follows: the mobile phase is selected from a mixture of methanol and water containing 0.3wt% phosphoric acid, wherein V Methanol :V Water containing 0.3wt% phosphoric acid =7:3, column temperature 15-30 deg.CPreferably 20 to 30 ℃; the detection wavelength is 200-400nm.
According to an embodiment of the present invention, in the step (4), the solvent for recrystallization is selected from a mixed solvent of tetrahydrofuran, methanol and water. Preferably, the volume ratio of tetrahydrofuran, methanol and water is (1-3) 1:1, e.g., 2.
According to an embodiment of the present invention, diterpenoid compound B is obtained by preparative liquid chromatography separation of steps (3) and/or (4).
According to an embodiment of the present invention, step (4) further comprises separating the different crystals under a microscope to obtain diterpenoid a and diterpenoid C. The different crystals refer to different appearances of the crystals, and the appearances comprise shapes and colors; the shape can be needle, sheet or block.
Preferably, the separation of different crystals under a microscope specifically means that crystals with different crystal forms are selected according to different appearance states of the crystals.
According to an exemplary embodiment of the present invention, the method for extracting diterpenoids or crystals thereof specifically comprises the following steps:
(1) Pulverizing herba Ajugae, 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 respectively, and concentrating the extract liquor with different polarities respectively to obtain petroleum ether extract, ethyl acetate extract and chloroform extract;
(3) Performing silica gel column separation on the ethyl acetate extract obtained in the step (2), and performing gradient elution by using the developing agent to obtain crude components (I) with different polarities; analyzing the crude component (I) by TLC, and combining similar components to obtain crude components (II) with different polarities;
preferably, the ethyl acetate extract obtained in the step (2) is subjected to first silica gel column separation, gradient elution is carried out by adopting the developing solvent, the gradient elution has the meaning as described above, and V is collected Petroleum ether /V Ethyl acetate Washing of =2:1 and 1:1Removing components to obtain crude components (I) with different polarities; analyzing the crude component (I) by TLC, and combining similar components to obtain crude components (II) with different polarities;
(4) Analyzing the crude components (II) with different polarities by HPLC, and combining similar components to obtain crude components (III) with different polarities; separating the crude component (III) by silica gel column, preparing liquid chromatography, and recrystallizing to obtain diterpene compounds A-C;
preferably, the solvent for recrystallization is a mixed solution of tetrahydrofuran, methanol and water, wherein V is Tetrahydrofuran (THF) :V Methanol :V Water (W) ＝2:1:1。
The invention also provides a pharmaceutical composition, which at least comprises the diterpenoid compound, the pharmaceutically acceptable salt thereof or the solvate thereof, or the crystal of the diterpenoid compound.
According to an embodiment of the invention, the pharmaceutical composition contains at least one pharmaceutically acceptable pharmaceutical excipient; for example, the pharmaceutical excipient may be selected from excipients, carriers and/or diluents. Wherein the pharmaceutically acceptable pharmaceutic adjuvant refers to an inert and nontoxic pharmaceutic adjuvant.
According to an embodiment of the invention, the pharmaceutical excipient may also be selected from one or more of the following excipients: fillers, disintegrants, lubricants, glidants, effervescent agents, flavoring agents, preservatives and pharmaceutically acceptable auxiliary materials for coating materials.
The invention also provides a pharmaceutical preparation, which at least comprises the diterpenoid compound, the pharmaceutically acceptable salt thereof or the solvate thereof, or the crystal of the diterpenoid compound.
According to an embodiment of the present invention, the pharmaceutical preparation contains the above pharmaceutical composition.
According to an embodiment of the invention, the pharmaceutical formulation is a solid oral formulation, a liquid oral formulation or an injection.
Preferably, the preparation can be selected from tablets, dispersible tablets, enteric-coated tablets, chewable tablets, orally disintegrating tablets, capsules, granules, oral solutions, water injection for injection, freeze-dried powder injection for injection, large infusion solutions or small infusion solutions.
The invention also provides the application of the diterpenoid compound, the pharmaceutically acceptable salt or the solvate thereof, the crystal of the diterpenoid compound, the pharmaceutical composition or the pharmaceutical preparation in preparing a medicament for treating cancer.
According to an embodiment of the invention, the cancer includes, but is not limited to, lung cancer, gastric cancer, breast cancer or cervical cancer.
Advantageous effects
The 3 diterpenoid compounds A-C obtained by separating ajuga ciliata have strong inhibitory activity on various cancer cells (such as gastric cancer cell strains, large intestine cancer cell strains, breast cancer cell strains and cervical cancer cell strains).
The extraction method of the invention carries out primary purification (such as column separation, plate preparation separation, liquid chromatography preparation separation and the like) on the plant extract, then recrystallizes the plant extract, quickly and accurately obtains the high-purity target compound (the purity can reach 98 percent), and has important significance for quickly separating and identifying specific compounds, especially chiral enantiomers in the plant extract containing complex components.
The inventors have surprisingly found that by the process of the invention different crystalline forms of the compound to be isolated can be obtained, thereby facilitating further isolation, especially by visual observation under a microscope. The method is more practical for separating enantiomers.
According to the invention, a CCK8 method is adopted to carry out in-vitro anti-tumor test on the diterpenoid compounds A-C, and the diterpenoid compounds have inhibitory activity on gastric cancer cell strains, lung cancer cell strains A549, breast cancer cell strains MCF-7 and cervical cancer cell strains.
Drawings
FIG. 1 is an HPLC chromatogram of Compound A;
FIG. 2 is an HPLC chromatogram of Compound B;
FIG. 3 is an HPLC chromatogram of Compound C;
FIG. 4 is a schematic representation of a ball stick model of Compound A;
FIG. 5 is a schematic representation of a ball stick model of Compound C;
in FIGS. 4 and 5, the red sphere represents an oxygen atom O, the white sphere represents a hydrogen atom H, and the black sphere represents a carbon atom C;
FIG. 6 is a HMBC correlation plot for compound A;
FIG. 7 is a graph relating to HMBC for compound B;
FIG. 8 is a graph relating to HMBC for compound C.
Detailed Description
The technical solution of the present invention will be further described in detail with reference to specific embodiments. It is to be understood that the following examples are only illustrative and explanatory of the present invention and should not be construed as limiting the scope of the present invention. All the technologies realized based on the above-mentioned contents of the present invention are covered in the protection scope of the present invention.
Unless otherwise indicated, the raw materials and reagents used in the following examples are all commercially available products or can be prepared by known methods.
The instruments and reagents used in the following examples:
herba ajugae ciliatae (collected from Tansma island in Fujian province in 2018), wherein chemical reagents used in the method are all commercially available chemical pure reagents; CCK8 kit (shanghai beibo biotechnology limited); DMEM high-glucose medium (semer feishale (suzhou) instruments ltd); EDTA (pancreatin) (gibco); fetal Bovine Serum, foetal bone Serum (Biological Industries); phosphate buffered saline; 96-well cell culture plates; a multifunctional microplate reader.
Example 1: extraction, separation and structure identification of diterpenoid A, B, C
The extraction and isolation of the diterpenoid A, B, C are carried out according to the following processes:
(a) Pulverizing 20 kg of dried herba Ajugae (2018, collected from Tansma island in Fujian province), respectively placing into 2 plastic barrels of 20L, adding 10L of ethanol aqueous solution with the mass fraction of 70% to soak at room temperature for 1 month, filtering, and concentrating to obtain extract. Soaking, filtering, concentrating and extracting the two barrels of medicinal materials for 3 times respectively, and combining the obtained extracts to obtain a total extract;
(b) Dispersing 1 kg of the extract obtained in the step (a) in 1000mL of water, and sequentially extracting with petroleum ether, ethyl acetate and chloroform for 5 times respectively, wherein the dosage of the petroleum ether, the ethyl acetate and the chloroform is 500 mL each time. Respectively concentrating the extraction liquid with different polarities to obtain petroleum ether extract, ethyl acetate extract and chloroform extract with different polarities;
(c) Performing first silica gel column separation on the ethyl acetate extract obtained in the step (b), and adopting V Petroleum ether /V Ethyl acetate Gradient elution was performed for 1:0 to 0:1 (1:0, 0.9, 0.8, 0.2, 0.7, 0.6, 0.4, 0.5, 0.4 Petroleum ether /V Ethyl acetate Fractions eluted at 1 1 -Fr 50 )(I)；
(d) The 50 fractions obtained in step (c) were first checked by preliminary TLC and similar fractions were combined to obtain 30 fractions (II). Then, the HPLC qualitative analysis (chromatographic condition: V) was carried out on each of the components (II) Methanol :V Water (containing 0.3% phosphoric acid) 7:3, column temperature room temperature, detection wavelength 200-400 nm), similar components were combined. Then 5 components with large polarity difference (core material retention time difference is about 0.5-5 min) are selected and respectively subjected to first silica gel column separation, the obtained components are subjected to primary TLC analysis, the approximate components are combined, and the components with large polarity difference (R) are selected f The difference value is about 0.2-0.6), then the components after the second column separation are subjected to preparative high performance liquid chromatography, and the components obtained by the preparative chromatography are recrystallized, wherein the recrystallization solvent is a mixed solvent of tetrahydrofuran, methanol and water, and the volume ratio V of the mixed solvent is Tetrahydrofuran (THF) :V Methanol :V Water (W) =2:1:1. the crystals were microscopically sorted out into colorless needles and colorless columns to obtain diterpenoid compound A and compound C.
The inventors have found that only a mixture of compound a and its enantiomer, compound C and its enantiomer can be obtained by column separation only, preparative plate separation or medium pressure preparative chromatographySubstance (one spot by TLC and same retention time by HPLC analysis, but with the exception of 1 HNMR analysis is a mixture), and surprisingly, the mixture is in a mixed solvent of tetrahydrofuran, methanol and water, the compound a and the enantiomer thereof, the compound C and the enantiomer thereof can easily form a large number of crystals, and the crystal appearance states of the compound a and the enantiomer thereof, the compound C and the enantiomer thereof are slightly different under a microscope, so that the separation of the compound a and the enantiomer thereof, and the compound C and the enantiomer thereof can be realized by selecting different crystal forms under the microscope. The compound B and A, C have larger polarity difference and are prepared by separation through preparative liquid chromatography.
The isolated diterpenoid compounds A, B and C were tested for purity by HPLC: purity of a 98.70%, rt =20.187min; purity of B98.809%, rt =14.172min; purity of C99.641%, rt =14.420min; (chromatographic conditions: C 18 A column; mobile phase: v Methanol :V Water (W) =7:3; the detection wavelength is 254nm. The HPLC chromatogram of compound A is shown in 1,B and 2,C and in FIG. 3).
The structure is determined by NMR and high-resolution mass spectrometry, and the crystal form and the absolute configuration are determined by XRD analysis.
The characterization data for diterpenoid a are as follows: colorless needle crystal, melting point: 185-186 ℃ of monoclinic system, and the space group is P2 1 ，
α＝90.00°, β＝94.046(5)°,γ＝90.00°,
(the crystal ball-and-stick model structure determined by XRD is shown in FIG. 4); HR-MS for C 25 H 36 O 6 + Na calculated: 455.2404, experimental values: 455.2404; 1 HNMR(400MHz,DMSO-d 6 ,ppm,J/Hz):δ H 0.66(3H,s,H 3 -19),0.77(3H,d,6.6, H 3 -20),1.03(1H,m,H-3β),1.36(2H,m,H 2 -11),1.40(1H,m,H-10),1.47(1H,m, H-2β),1.49(1H,m,H-7α),1.52(2H,m,H 2 -1),1.56(1H,m,H-8),1.75(3H,d,7.2, H 3 -4'),1.78(3H,s,H 3 -5'),1.85(1H,m,H-2α),1.97(1H,m,H-3α),2.14(1H,m, H-12β),2.26(1H,m,H-12α),2.46(1H,d,J＝3.4Hz,H-17β),3.12(1H,s,H-17α), 3.27(1H,s,OH-6),3.56(1H,m,H-6),4.40(1H,d,J＝11.9Hz,H-18α),4.41(1H,d, J＝11.9Hz,H-18β),4.84(2H,d,J＝1.8Hz,H 2 -16), 5.93 (1h, s, h-14), 6.89 (1h, q, J =7.0hz, h-3'); as shown in fig. 6.
The characterization data of diterpenoid B are as follows: colorless solid, melting point: 98-99 ℃; 1 HNMR(400MHz,CD 3 OD,ppm,J/Hz):δ H 0.79(3H,s,H 3 -19),0.90(3H,d,J＝5.9Hz, H 3 -20),1.16(1H,dd,J＝13.6,1.16Hz,H-1β),1.36(1H,d,J＝15.6Hz,H-11β),1.50(1H, d,J＝3.8Hz,H-7β),1.68(1H,m,H-7α),1.74(3H,d,J＝8.0Hz,H 3 -4'),1.81(3H,s, H 3 -5'),1.94(3H,s,H 3 -2”'),1.98(1H,m,H-8),2.05(2H,m,H 2 -2),2.12(3H,s,H 3 -2”), 2.38(1H,m,H-17β),2.40(1H,m,H-1α),2.48(1H,t,J＝15.6Hz,H-11α),3.03(1H,m, H-17α),3.07(1H,m,H-10),4.48(1H,d,J＝12.5Hz,H-18β),4.78(1H,d,J＝3.2Hz, H-12),4.82(1H,d,J＝3.1Hz,H-6),4.88(2H,m,H 2 -16), 5.03 (1H, d, J =12.5Hz, H-18 α), 5.66 (1H, m, H-3), 5.97 (1H, s, H-14), 6.97 (1H, q, J =8.0Hz, H-3'); as shown in fig. 7.
Characterization data for diterpenoid C are as follows: colorless columnar crystals, melting point: 148-149 deg.C, triclinic system, space group is P2 1 ，
α＝90.00°, β＝100.858(2')°,γ＝90.00°,
(the crystal club model structure determined by XRD is shown in FIG. 5); HR-MS for C 31 H 42 O 11 + Na calculated: 543.2928, experimental values: 543.2928;
1 H NMR(400MHz,CD 3 OD,ppm,J/Hz):δ H 0.80(3H,s,H 3 -19),0.90(3H,d, J＝6.6Hz,H 3 -20),1.17(1H,dd,J＝8.2,2.6Hz,H-3β),1.50(1H,dd,J＝9.5,3.84Hz, H-7β),1.62(1H,d,J＝2.2Hz,H-2β),1.67(1H,d,J＝2.0Hz,H-11β),1.74(1H,d, J＝11.8Hz,H-7α),1.80(3H,dd,J＝7.1,1.2Hz,H 3 -4'),1.86(3H,t,J＝1.3Hz,H 3 -5'),1.93 (3H,s,H 3 -2”),2.11(3H,s,H 3 -2””),2.14(1H,m,H-2α),2.23(3H,s,H 3 -2”'),2.25(1H,d, J＝11.0Hz,H-10),2.37(1H,m,H-17β)2.40(1H,d,J＝4.0Hz,H-3α),2.65(1H,dd, J＝16.1,10.6Hz,H-11α),2.97(1H,dd,J＝4.1,2.1Hz,H-17α),4.49(1H,d,J＝12.5Hz, H-18β),4.71(1H,dd,J＝11.5,4.4Hz,H-6),4.92(2H,m,H 2 -16), 5.01 (1h, d, J =12.5hz, H-18 α), 5.71 (1h, td, J =10.9,4.7hz, H-1), 5.97 (1h, d, J =10.6hz, H-12), 6.03 (1h, q, J =3.3hz, H-14), 7.03 (1h, q, J =7.1hz, H-3'); as shown in fig. 8.
The structure of diterpenoid compounds A-C is confirmed by the above characterization as follows:
application example 1: in vitro antitumor Activity test
The compounds A to C extracted in example 1 were tested for their antitumor activity in vitro, and their inhibitory activities against lung cancer cell line A549 and cervical cancer cell line Hela (cell line source: ningxia medical university) were mainly studied. The specific test process is illustrated by taking the test process of the inhibitory activity of the compound B on the lung cancer cell strain A549 as an example:
1. test sample concentration preparation
Compound B23.7 mg was weighed into a 5mL plastic centrifuge tube and diluted to 1mL with DMSO. Thus obtaining the initial concentration of 23.7 mg/mL. The initial concentration was then diluted in DMSO in multiples to obtain 5 different concentration gradients of 11.85mg/mL,5.93mg/mL,2.96mg/mL,1.48mg/mL,0.74mg/mL in that order, and stored in a refrigerator at 4 ℃ until use.
2. Culture of lung adenocarcinoma cell strain (A549) and test of inhibitory activity
Placing lung adenocarcinoma cell strain (A549) at 37 deg.CAnd humidity, 5% CO 2 After 24 hours in the incubator, when the cells were in logarithmic phase, the supernatant culture was aspirated and digested with 0.25% trypsin-EDTA solution, and then the digestion was terminated using a high-sugar medium. And the cells were seeded in a 96-well plate such that the cell density was 5000 cells/well. The 96-well plate was placed in an incubator for 24 hours. The cell culture medium in the 96-well plate was then aspirated. And 100. Mu.L of the high-sugar medium was supplemented to the 96-well plate, and then 1. Mu.L of the test sample (5 multiple wells per concentration) was added to each well, followed by setting at 37 ℃ for saturation humidity, 5% CO 2 After further incubation in the incubator of (1) for 48h, 10. Mu.L of CCK8 was added to each well and further incubation in the incubator at 37 ℃ for 1-4h was carried out. The absorbance value of each well at a wavelength of 450nm was measured on a multifunctional microplate reader. According to the inhibition ratio% = [ (control cell OD-medicated cell OD)/(control cell OD-blank OD)]X 100. Negative control is V High-sugar culture medium /V DMSO A mixed solution of =10.
3. Culture of cervical cancer cell strain Hela and test of inhibitory activity
The test process of the compound B on the cervical cancer cell strain Hela refers to the culture and inhibition activity test of the lung adenocarcinoma cell strain (A549), and the difference is that the lung adenocarcinoma cell strain (A549) is replaced by the cervical cancer cell strain Hela.
The inhibition rate of compound B on lung adenocarcinoma cell strain (A549) and cervical carcinoma cell strain Hela is shown in Table 1.
TABLE 1 inhibitory Effect of Compound B on two tumor cell lines
| Compound B | Lung adenocarcinoma cell strain (A549) | Cervical cancer cell line Hela |
| IC 50 (μM) | 0.152 | 9.17×10 4 |
The compound A, C and the crystal thereof have the inhibition rate on lung adenocarcinoma cell strains (A549) and cervical cancer cell strains Hela which are basically equivalent to the inhibition rate of the compound B.
The above description is that of the exemplary embodiments of the invention. However, the scope of protection of the present application is not limited to the above-described embodiments. Any modification, equivalent replacement, improvement made by those skilled in the art within the spirit and principle of the present invention shall be included in the protection scope of the present invention.
Claims (10)
1. Diterpenoid compound selected from diterpenoid compound a, diterpenoid compound B or diterpenoid compound C having the following structure:
2. the diterpenoid of claim 1, wherein the diterpenoid B is a colorless solid having a melting point of 98-99 ℃.
3. The crystal of diterpenoid compound according to claim 1 or 2, which is selected from at least one of a crystal of diterpenoid compound a and a crystal of diterpenoid compound C.
Preferably, the diterpenoid A crystal belongs to a monoclinic system, and the space group is P2 1 ，
α＝90.00°，β＝94.046(5)°，γ＝90.00°。
Preferably, the diterpenoid A crystals are colorless needle-like crystals.
Preferably, the melting point of the diterpenoid A crystals is 185-186 ℃.
Preferably, the diterpenoid C crystal belongs to a triclinic system, and the space group is P2 1 ，
α＝90.00°，β＝100.858(2')°，γ＝90.00°。
Preferably, the diterpenoid C crystals are colorless columnar crystals.
Preferably, the melting point of the diterpenoid C crystal is 148-149 ℃.
4. The diterpenoid of claim 1 or 2, or the crystal of claim 3, comprising the steps of:
(1) Pulverizing herba Ajugae, extracting with ethanol water solution, and concentrating the obtained extractive solution to obtain total extract;
(2) Dispersing the total extract obtained in the step (1) by using water, extracting by using an organic solvent, and concentrating the obtained extract to obtain an organic solvent extract;
(3) Performing column separation on the organic solvent extract obtained in the step (2) to obtain a crude component;
(4) And (4) separating and purifying the crude component obtained in the step (3) to obtain the diterpenoid compound or the crystal thereof.
5. The extraction method according to claim 4, wherein in the step (1), the mass fraction of ethanol in the ethanol aqueous solution is 50-80%.
Preferably, in step (1), the aqueous ethanol extraction may be a soaking extraction or a reflux extraction.
Preferably, in the step (2), the mass-to-volume ratio (g/mL) of the total extract to water is (0.2-3): 1.
Preferably, in the step (2), the volume ratio of the organic solvent to the water is 1 (0.2-3).
Preferably, in step (2), the organic solvent is selected from water-immiscible organic solvents. Preferably, the organic solvent may be selected from at least one including, but not limited to, petroleum ether, ethyl acetate, chloroform and n-butanol.
Preferably, in step (3), the column separation includes, but is not limited to, silica gel column separation, reverse silica gel column separation, macroporous resin column enrichment separation, and/or preparative liquid chromatography separation.
Preferably, in the step (4), the separation and purification may be performed by at least one or more of column separation, recrystallization, and preparative liquid chromatography separation.
6. The extraction method according to claim 4 or 5, wherein in step (3) or (4), the silica gel column separation specifically comprises: gradient elution is carried out by adopting a developing solvent to obtain crude components with different polarities. Preferably, the developing solvent is selected from petroleum ether and/or ethyl acetate, preferably V Petroleum ether /V Ethyl acetate =1:0 to 0:1. Preferably, the gradient elution is performed in a volume ratio of petroleum ether to ethyl acetate of 1:0, 0.9, 0.8, 0.2, 0.7, 0.4, 0.5, 0.6.
Preferably, in step (3) or (4), the conditions for the liquid chromatographic separation are as follows: the mobile phase is selected from a mixture of methanol and water containing 0.3wt% phosphoric acid, wherein V Methanol :V Water containing 0.3wt% phosphoric acid =7:3, column temperature 15-30 ℃, preferably 20-30 ℃; the detection wavelength is 200-400nm.
Preferably, in the step (4), the solvent for recrystallization is selected from a mixed solvent of tetrahydrofuran, methanol and water. Preferably, the volume ratio of tetrahydrofuran, methanol and water is (1-3): 1:1.
Preferably, diterpenoid compound B is obtained by liquid chromatography separation of step (3) and/or (4).
Preferably, step (4) further comprises separating the different crystals under a microscope to obtain diterpene compound a and diterpene compound C.
7. The extraction method according to any one of claims 4 to 6, characterized in that it comprises in particular the steps of:
(1) Pulverizing herba Ajugae, 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 respectively, and concentrating the extract liquor with different polarities respectively to obtain petroleum ether extract, ethyl acetate extract and chloroform extract;
(3) Performing silica gel column separation on the ethyl acetate extract obtained in the step (2), and performing gradient elution by using the developing agent to obtain crude components (I) with different polarities; analyzing the crude component (I) by TLC, and combining similar components to obtain crude components (II) with different polarities;
(4) Analyzing the crude components (II) with different polarities by HPLC, and combining similar components to obtain crude components (III) with different polarities; separating the crude component (III) with silica gel column, separating with preparative liquid chromatography, and recrystallizing to obtain diterpene compounds represented by A-C, preferably, the solvent for recrystallization is a mixture of tetrahydrofuran, methanol and water, wherein V is Tetrahydrofuran (THF) :V Methanol :V Water (W) ＝2:1:1。
8. A pharmaceutical composition comprising at least the diterpenoid, the pharmaceutically acceptable salt thereof or the solvate thereof according to claim 1 or 2, or the crystal according to claim 3.
9. A pharmaceutical preparation comprising at least the diterpenoid, the pharmaceutically acceptable salt thereof or the solvate thereof according to claim 1 or 2, or the crystal according to claim 3.
Preferably, the pharmaceutical preparation contains the pharmaceutical composition of claim 8.
10. Use of the diterpenoid, a pharmaceutically acceptable salt thereof or a solvate thereof according to claim 1 or 2, or the crystal according to claim 3, the pharmaceutical composition according to claim 8, or the pharmaceutical formulation according to claim 9 for the preparation of a medicament for the treatment of cancer.
Preferably, the cancer includes, but is not limited to, lung cancer, gastric cancer, breast cancer or cervical cancer.
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| CN116003371B (en) * | 2023-01-04 | 2024-04-16 | 厦门稀土材料研究所 | Terpenoid, and extraction method and application thereof |
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Inventor after: Lu Canzhong Inventor after: Olated olagok Zaks Inventor after: Yong Jianping Inventor after: Lu Zhencan Inventor after: Lu Jian Inventor before: Yong Jianping Inventor before: Lu Canzhong Inventor before: Olated olagok Zaks Inventor before: Lu Zhencan Inventor before: Lu Jian |