CN113583080B - Compound and preparation method and application thereof - Google Patents
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Abstract
The invention discloses a compound and a preparation method and application thereof. The compound provided by the invention is separated from the ethyl acetate extraction part of the kiwi fruit root, and belongs to one of triterpenoid compounds. The compound can effectively inhibit the proliferation activity of pancreatic cancer cells, and has a wide application prospect in preparation of medicaments for treating pancreatic cancer.
Description
Technical Field
The invention belongs to the technical field of organic compounds, and particularly relates to a compound and a preparation method and application thereof.
Background
Pancreatic cancer is one of the malignant digestive tract tumors with high mortality, the five-year survival rate is less than 10%, and the incidence and mortality of pancreatic cancer have been on the rise in the last decade. Pancreatic cancer has no specific symptoms at the early stage, and clinically shows symptoms such as macula lutea and weight loss and the like along with the development of pancreatic cancer, so the diagnosis is generally in the late stage, which is one of the main reasons for high pancreatic cancer death rate; in addition, the pancreas is a cryptic digestive organ located behind the anterior peritoneum of the lumbar vertebra, so that diagnosis and treatment of pancreatic cancer are difficult.
At present, the clinical treatment mode of pancreatic cancer is mainly chemotherapy, and the chemotherapeutics are mainly single-drug chemotherapy mainly comprising fluorouracil drugs and gemcitabine or combined chemotherapy with other drugs; therefore, the number of the medicines which can be clinically used for treating the pancreatic cancer is very small, and the treatment effect of the existing medicines for treating the pancreatic cancer is quite limited; in the medication process, patients also have side effects such as bone marrow suppression, vomiting, dizziness, liver function damage, adverse reaction of digestive tract and the like.
Natural products have been an important component of anticancer drugs, and more than half of approved anticancer drugs are natural products. In recent years, traditional Chinese medicines have achieved good clinical effects in cancer treatment. Therefore, researchers focus on traditional Chinese medicines, and natural products with anti-pancreatic cancer activity are expected to be searched from the traditional Chinese medicines. Compared with the traditional chemotherapy drugs, the traditional Chinese medicine has rich chemical components and relatively more combined targets, so that the traditional Chinese medicine is not easy to generate drug resistance, and the traditional Chinese medicine has unique advantages in treating pancreatic cancer. And the traditional Chinese medicine has abundant clinical cases, and the safety of the medicine is fully guaranteed.
In recent years, the traditional Chinese medicine and the chemotherapeutic medicine are combined to achieve good clinical curative effect on pancreatic cancer. The traditional Chinese medicine is used for adjuvant therapy, so that the adverse reaction of immunity reduction after chemotherapy of a patient can be obviously relieved, the physical state of the patient is adjusted, the pain of the patient is relieved, and the life cycle of the patient is obviously prolonged. For example, the study of combining the minor radix bupleuri modified prescription with the chemotherapeutic drug to treat the pancreatic cancer patient finds that the survival time of the patient is prolonged, and the adverse reaction degree is reduced; for another example, Actinidia chinensis Planch is the root of evergreen trees and plants in actinidiaceae, has the effects of promoting urination, promoting digestion, preventing vomiting, stopping bleeding, promoting lactation and the like, and a plurality of prescriptions record that Actinidia chinensis root has a good effect of treating digestive tract tumors.
The traditional Chinese medicine for treating pancreatic cancer has the advantages of more side effects, various mechanisms for treating pancreatic cancer by using traditional Chinese medicines, rich varieties of active ingredients, obvious advantages in the aspect of treating pancreatic cancer, and extremely high clinical value and practical significance in the deep research of the action mode of treating pancreatic cancer by using traditional Chinese medicines. Specifically, it is urgent to find a lead compound having an anti-pancreatic cancer effect from a Chinese medicine and develop a patent drug. However, basic research on the substances which have the effect of treating pancreatic cancer by using traditional Chinese medicines is few, and the effect of the traditional Chinese medicines for treating pancreatic cancer which are researched is not obvious enough.
Disclosure of Invention
The present invention has been made to solve at least one of the above-mentioned problems occurring in the prior art. Therefore, the invention provides a compound which can effectively inhibit the proliferation activity of pancreatic cancer cells.
The invention also provides a preparation method of the compound.
The invention also provides a kiwi fruit root extract containing the compound.
The invention also provides a medicament for treating pancreatic cancer, which comprises the compound.
The invention also provides a medicine for treating pancreatic cancer, which contains the kiwi fruit root extract.
According to one aspect of the present invention, there is provided a compound having the formula:
according to a preferred embodiment of the invention, at least the following advantages are achieved:
the half Inhibitory Concentration (IC) of the compound provided by the invention on MP and Patu8988t pancreatic cancer cells 50 ) About 25mg/L (about 50. mu.M); IC on Panco2 pancreatic cancer cells 50 About 160mg/L (83. mu.M); the data show that the compound provided by the invention has obvious activity of inhibiting the proliferation of pancreatic cancer cells.
In some embodiments of the invention, the compound, in the dry state, is a white powder.
In some embodiments of the invention, the compound, in the crystalline state, is amorphous.
In some embodiments of the invention, the compound has methanol solubility.
According to a further aspect of the present invention, there is provided a process for the preparation of said compound, characterized in that it comprises the steps of:
s1, taking ethyl acetate as a solvent to obtain a crude extract of Chinese actinidia root, and drying the crude extract to obtain a crude extract;
s2, using a mixed solution of dichloromethane and methanol as an eluent, carrying out silica gel column chromatography on the crude extract, detecting a product through thin-layer chromatography, and combining similar components to obtain a kiwi root extract;
s3, mixing dichloromethane, ethyl acetate, methanol and water in a volume ratio of (7-9): (1-3): 1 to form a four-phase system, mixing, separating a lower layer solution, carrying out silica gel column chromatography on the kiwi fruit root extract, detecting a product through thin layer chromatography, and combining similar components to obtain an intermediate component;
and S4, carrying out medium-pressure preparation liquid phase separation on the intermediate component to obtain the compound.
The preparation method according to a preferred embodiment of the present invention has at least the following advantageous effects:
the preparation method provided by the invention has the advantages of simplicity and easiness in operation. The preparation method provided by the invention obtains 13 known compounds and one unknown compound from the kiwi fruit root extract.
In some embodiments of the present invention, the preparation method further comprises, before step S1, sequentially pre-treating the actinidia chinensis planch with petroleum ether and dichloromethane.
In some embodiments of the invention, the pre-treatment is aimed at removing the dichloromethane and petroleum ether soluble fraction of the actinidia chinensis root to obtain a purer fraction of ethyl acetate extract.
In some embodiments of the present invention, in step S1, the actinidia chinensis planch root is dried powder.
In some embodiments of the present invention, the dried powder of actinidia chinensis planch root may pass through an 80 mesh screen.
In some preferred embodiments of the present invention, the dried powder of the actinidia chinensis planch may pass through a 60 mesh screen.
In some embodiments of the present invention, in step S1, the crude extract is obtained by percolation.
In some embodiments of the present invention, the percolation method is specifically operated by adding the actinidia chinensis roots into a percolation barrel to form a column; and percolating and extracting the Chinese actinidia root by using ethyl acetate as eluent.
In some embodiments of the present invention, in the percolation method, the elution speed of the ethyl acetate is 3.5 to 4.5 h/column volume (i.e. the volume of the column formed by the actinidia chinensis planch roots).
In some embodiments of the invention, the percolation method is one in which the elution rate of ethyl acetate is 4 h/column volume.
In some embodiments of the invention, the percolation method further comprises eluting the ethyl acetate with a total of 7 to 9 column volumes.
In some preferred embodiments of the invention, the ethyl acetate co-elutes 8 column volumes in the percolation method.
In some embodiments of the invention, in step S1, the drying is performed by sequentially performing solvent recovery and vacuum drying on a rotary evaporator.
In some embodiments of the invention, in step S2, the volume ratio of dichloromethane to methanol is 30-20:1 when obtaining the kiwi root extract.
In some preferred embodiments of the present invention, in step S2, the volume ratio of dichloromethane to methanol is about 30:1 when obtaining the kiwi root extract.
In some embodiments of the present invention, in step S2, the flow rate of the eluent in the silica gel column chromatography is 1-2 column volumes/h.
In some embodiments of the present invention, step S2 further includes drying the obtained material after combining the similar components, so as to obtain the kiwi fruit root extract.
In some embodiments of the present invention, in step S2, the state of the obtained kiwi root extract is white powder (hereinafter, referred to as F2).
The mass ratio of the kiwi fruit root extract to the crude extract is 60-62%.
The mass proportion of the kiwi fruit root extract in the crude extract is about 61%.
In some embodiments of the present invention, before obtaining the kiwi root extract, in step S2, a mixed eluent of dichloromethane and methanol is formed at a volume ratio of about 50:1, the crude extract is eluted, the product is detected by thin layer chromatography, similar components are combined, and after removing the solvent, a yellow oily extract is obtained (hereinafter referred to as F1).
The mass proportion of the F1 component in the crude extract was about 6%.
In some embodiments of the present invention, in step S2, after obtaining the actinidia chinensis root extract, the method further comprises eluting the crude extract with mixed eluents of dichloromethane and methanol at a volume ratio of about 3:1 and 1:10, detecting the product by thin layer chromatography, combining similar components, and removing the solvent to obtain brown powdered extract (hereinafter referred to as F3) and dark brown powdered extract (hereinafter referred to as F4).
The mass proportion of the F3 component in the crude extract is about 21%; the mass proportion of the F4 component in the crude extract is about 7%; because of loss in the separation process, the sum of the mass of the F1-F4 components is less than or equal to the mass of the crude extract.
In some embodiments of the invention, in step S3, the volume ratio of dichloromethane, ethyl acetate, methanol and water in the four-phase eluent is about 8:2:2: 1.
In some embodiments of the present invention, in step S3, the flow rate of the eluent in the silica gel column chromatography is 1-2 column volumes/h.
In some embodiments of the invention, in step S4, the liquid phase in the medium-pressure preparative liquid-phase separation is a mixed system of water and methanol.
In some embodiments of the invention, in the medium pressure preparative liquid phase separation, the liquid phase is water and methanol mixed in a (35-45): 55-65) volume ratio.
In some preferred embodiments of the invention, the liquid phase in the medium pressure preparative liquid phase separation is water and methanol mixed in a volume ratio of about 40: 60.
In some preferred embodiments of the invention, the flow rate of the eluent in the medium pressure preparative liquid phase separation is from 2 to 5 ml/min.
Unless otherwise specified, the column used for the medium-pressure preparation according to the invention is a C18 column (Eclipse XDB-C18 Semi-Prep) produced by Agilent, and the specific dimensions are as follows: 9.4mm (diameter). times.250 mm (length), 5 μm (filler particle diameter).
According to a further aspect of the invention, a kiwifruit root extract comprising said compound is proposed.
The preparation method according to a preferred embodiment of the present invention has at least the following advantageous effects:
the kiwi fruit root extract provided by the invention has IC (integrated circuit) on MP, Patu8988t and HPAC pancreatic cancer cells 50 About 10 g/L; the data show that the kiwi fruit root extract provided by the invention has obvious effect of inhibiting pancreatic cancer cell proliferation activity.
In some embodiments of the present invention, the actinidia root extract further comprises 13 other triterpenoids.
In some embodiments of the present invention, the actinidia chinensis root extract is an ethyl acetate extract of actinidia chinensis root.
In some embodiments of the present invention, the triterpene compounds in the actinidia root extract have a molecular formula of C 30 H 48 O 6 。
According to a further aspect of the invention, there is provided a medicament for treating pancreatic cancer, which comprises the compound as a starting material.
According to another aspect of the invention, a medicament for treating pancreatic cancer is provided, and the preparation raw material of the medicament comprises the kiwi fruit root extract.
Drawings
The invention is further described with reference to the following figures and examples, in which:
FIG. 1 is a structural formula of a compound obtained in example 2 of the present invention;
FIG. 2 is a mass spectrum total ion flow graph of a kiwi fruit root extract obtained in the test example of the present invention;
FIG. 3 is a LC-MS analysis spectrum of the components of the kiwi fruit root extract obtained in the test example of the present invention;
FIG. 4 is a graph relating to HMBC of the compound obtained in example 2 of the present invention;
FIG. 5 is a NOESY correlation chart of the compound obtained in example 2 of the present invention;
fig. 6 is a structural formula of 14 main components contained in the actinidia chinensis planch root extract obtained in example 1 of the present invention.
Detailed Description
The concept and technical effects of the present invention will be clearly and completely described below in conjunction with the embodiments to fully understand the objects, features and effects of the present invention. It is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments, and those skilled in the art can obtain other embodiments without inventive effort based on the embodiments of the present invention, and all embodiments are within the protection scope of the present invention.
In the description of the present invention, reference to the description of the terms "one embodiment," "some embodiments," "an illustrative embodiment," "an example," "a specific example," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
Example 1
The embodiment prepares the kiwi fruit root extract, and the specific process comprises the following steps:
D1. pulverizing Chinese actinidia root, and sieving with 80 mesh sieve to obtain Chinese actinidia root dry powder;
D2. sequentially eluting with petroleum ether and dichloromethane by percolation, percolating the dried powder of radix Actinidiae chinensis with ethyl acetate, and collecting ethyl acetate percolate, wherein the flow rates of petroleum ether, dichloromethane and ethyl acetate are 4 h/column volume and the amount of ethyl acetate is 8 times of column volume during extraction;
D3. d, recovering the extracting solution obtained in the step D2 by using a rotary evaporator, and drying by using a vacuum drying oven to obtain a crude extract;
D4. and D3, performing silica gel column chromatography on the crude extract obtained in the step D3, eluting with a dichloromethane and methanol system, firstly eluting at a ratio of 50:1 (eluent flow rate of 1.5 column volumes/h), discarding the eluent, and then performing column chromatography on the crude extract obtained in the step D3 at a ratio of 30: eluting with eluent of 1 (eluent flow rate of 1.5 column volume/h), detecting the obtained eluates by thin layer chromatography, mixing, and removing solvent to obtain white powdered radix Actinidiae chinensis extract.
Example 2
This example prepares a compound by the specific process of:
t1, performing silica gel column chromatography on the kiwi fruit root extract obtained in the example 1, detecting a product through thin-layer chromatography, and combining similar components to obtain an intermediate component; wherein the eluent proportion of the silica gel column chromatography is dichloromethane, ethyl acetate, methanol and water which are mixed according to the volume ratio of 8:2:2:1, the lower layer solution is separated after the uniform mixing, and the flow rate of the eluent is 1.5 column volume/h;
t2, performing medium-pressure mass spectrum liquid phase separation on the intermediate product obtained in the step T1 to obtain the intermediate product; wherein the liquid phase is water and methanol mixed in a volume ratio of 40: 60; the flow rate of the eluent is 2-5ml/min, and similar technical effects can be achieved in the range.
The structural formula of the compound obtained in the example is shown in figure 1.
Test example 1
This experimental example tested the performance of the kiwifruit root extract prepared in example 1.
The testing method is mass spectrometry, and the specific detection method comprises the following steps: the column was selected from a C18 column (4.6mm (diameter) 250mm (length), 5 μm (packing particle size)) made by Agilent. The experiment was carried out with 0.12% phosphoric acid-acetonitrile as mobile phase, acetonitrile ratio of 5% to 100% for 40min, followed by pure acetonitrile elution for 20 min. The flow rate of the mobile phase is 1.0mL/min, the column temperature is controlled to be room temperature, the experiment is carried out at 25 ℃, the sample injection amount is 5 mu L, and the wavelength is 210 nm. The total ion flow diagram of the obtained mass spectrum is shown in FIG. 2; the LC-MS analysis spectrum of the extract of radix Actinidiae chinensis is shown in FIG. 3.
From the results obtained in fig. 3, the mass spectrometry data of kiwi root extraction is shown in table 1.
TABLE 1 Peak position analysis results of LC-MS analysis spectra of Actinidia chinensis planch extract component
| Mass to charge ratio (m/z) | Number of charges (z) | Ion abundance (Abund) | Chemical Formula (Formula) | Ion (Ion) |
| 489.3567 | 1 | 48241.54 | C 30 H 48 O 5 | (M+H) + |
| 490.3603 | 1 | 15185.65 | C 30 H 48 O 5 | (M+H) + |
| 506.3835 | 1 | 86168.52 | C 30 H 48 O 5 | (M+NH 4 ) + |
| 507.3866 | 1 | 28167.44 | C 30 H 48 O 5 | (M+NH 4 ) + |
| 511.3392 | 1 | 183167.41 | C 30 H 48 O 5 | (M+Na) + |
| 512.3424 | 1 | 57907.83 | C 30 H 48 O 5 | (M+Na) + |
| 513.3458 | 1 | 11813.46 | C 30 H 48 O 5 | (M+Na) + |
| 977.7073 | 1 | 106904.27 | C 30 H 48 O 5 | (2M+H) + |
| 978.7104 | 1 | 69735.08 | C 30 H 48 O 5 | (2M+H) + |
| 979.7131 | 1 | 23955.35 | C 30 H 48 O 5 | (2M+H) + |
By analyzing the results shown in FIGS. 2-3 and Table 1, it was found that the molecular formula of the compound of the actinidia chinensis root extract is mostly C 30 H 48 O 5 The relative molecular mass is mostly 488, and further, the research of combined literature finds that the compounds of the kiwi fruit root extract component obtained by the invention are mostly triterpenoids.
In addition, the triterpenes of the Chinese actinidia root have the activities of resisting tumor, oxidation, inflammation and bacteria, reducing blood sugar, protecting liver and the like, and the anti-tumor effect of the triterpenes is always a hotspot of research; therefore, the ethyl acetate extract of the kiwi fruit root provided by the invention has the effect of resisting pancreatic cancer.
Test example 2
This experimental example tested the inhibitory ability of the actinidia root extract prepared in example 1 on the proliferative activity of three pancreatic cancer cells, MP, Patu8988t, HPAC.
The specific test method comprises the following steps: pancreatic cancer cell activity evaluation was performed on the kiwi fruit root extract obtained in the present invention by cell proliferation assay (CCK8 method). Three independent cell experiments were performed in triplicate using three pancreatic cancer cells, MP, Patu8988t, and HPAC, as cell evaluation models. The cellular data are shown in table 2 below:
table 2 pancreatic cancer cell activity evaluation results of the kiwi fruit root extract obtained in example 1
Wherein, IC 50 Represents the half inhibitory concentration, and SD represents the standard deviation.
As can be seen from the data in Table 2, the kiwi fruit root extract provided by the invention has proliferation inhibiting activity on MP, Patu8988t and HPAC pancreatic cancer cells, has strong activity, and is a compound with anti-pancreatic cancer activity.
In addition, from the determination of the main components in the ethyl acetate extract of the Chinese actinidia root in the test example 1 and the results of the test example 2, it can be known that the material basis of the Chinese actinidia root extract, which has the effect of resisting pancreatic cancer, provided by the invention can be a triterpenoid group.
As can be seen from example 1, in example 1, step D1 also obtained petroleum ether extract and dichloromethane extract of actinidia chinensis planch root, and further obtained 80% ethanol extract from actinidia chinensis planch root used in step D1 after obtaining ethyl acetate extract; the test example also tests the inhibition effect of the petroleum ether, dichloromethane and 80% ethanol extracts on pancreatic cancer cells, and the result shows that the three extracts have no inhibition effect on HPAC pancreatic cancer cells.
Test example 3
This test example determined the defined structural formula of the compound obtained in example 2, and the specific test method included the following steps:
in the first step, a nuclear magnetic hydrogen spectrum of the compound obtained in example 2 was obtained ( 1 HNMR spectrum), the results show: the compound obtained in example 2 has four methyl groups; having three [ delta ] s directly attached to quaternary carbon H 1.25(3H,s,H-27),1.09(3H,s,H-25),0.88(3H,s,H-26)；δ C 22.6(C-27),15.9(C-25),16.4(C-26)](ii) a Having one [ delta ] methyl group directly attached to a tertiary carbon H 1.04(3H,d,J＝6.0Hz,H-29)；δ C 15.38(C-29)]；
Having a methylene [ delta ] group bound to oxygen H 3.90(1H,d,J＝10.8Hz,H-23a),3.86(1H,d,J＝10.8Hz,H-23β)；δ C 60.75(C-23)]And a pair of terminal olefinic hydrogen proton signals [ delta ] H 4.66(1H,brs,H-30a),4.70(1H,brs,H-30b)；δ C 103.8(C-30)]；
Simultaneously has an endoolefin hydrogen proton signal [ delta ] H 5.30(1H,t,J＝3.6Hz,H-12)]；
The above results suggest that the basic structure of the compound obtained in example 2 may be a triterpenoid;
in addition to this, the present invention is, 1 the HNMR spectrum also shows an aldehyde group proton signal [ delta ] H 9.70(1H,brs,H-24)；δ C 206.97(C-24)](ii) a Two methine groups [ delta ] bound to a hydroxy group H 3.84(1H,ddd,J＝11.2,4.4,2.4Hz,H-2),δ C 65.5(C-2)；δ H 4.11(1H,d,J＝2.4Hz,H-3),δ C 70.11(C-3)]And a carboxyl signal delta C 179.92(C-28)；
In summary, 1 the results of HNMR spectroscopy suggest that the compound obtained in example 2 may be a novel ursane triterpene compound.
Second, a nuclear magnetic carbon spectrum of the compound obtained in example 2 was obtained ( 13 CNMR spectra) and DEPT (distortion-free polarization transfer enhancement), which are 13 One detection technique in C-nmr spectroscopy) spectra, the results show:
example 2 the resulting compound contains a carboxyl carbon signal delta C 179.92(C-28)；
4 olefin carbon signals [ delta ] C 103.8(C-30),125.41(C-12),138.21(C-13),153.2C-20]；
4 methyl carbon signals [ delta ] C 22.6(C-27),15.9(C-25),16.4(C-26),15.38(C-29)]；
Total of 9 quaternary carbon signals [ delta ] C 58.3(C-4),39.5(C-8),37.4(C-10),138.2(C-13),42.0(C-14),47.6(C-17),153.2(C-20),179.9(C-28),206.97(C-24)]；
7 tertiary carbon signals [ delta ] C 65.5(C-2),70.1(C-3),42.0(C-5),47.8(C-9),125.4(C-12),55.2(C-18),37.1(C-19)]；
10 secondary carbon signals [ delta ] C 40.5(C-1),19.9(C-6),32.8(C-7),23.0(C-11),27.7(C-15),23.9(C-16),31.9(C-21),39.0(C-22),60.7(C-23),103.8(C-30)]；
13 The CNMR spectrum and the DEPT spectrum further prove that the compound isUrsane triterpenoid with aldehyde group.
Thirdly, an HSQC (heteronuclear single quantum relationship) spectrum of the compound obtained in example 2 is obtained; HSQC spectrum data attribute proton signals (hydrogen spectra) and carbon spectra signals to each other to determine the hydrocarbon signal type of the compound obtained in example 2; firstly, the aldehyde carbon and aldehyde hydrogen [ delta ] are assigned by HSQC spectrum H 9.70(1H,brs,H-24)；δ C 206.97(C-24)](ii) a Second to double bond hydrocarbon [ delta ] H 4.66(1H,brs,H-30a),4.70(1H,brs,H-30b)；δ C 103.8(C-30)；δ H 5.30(1H,t,J＝3.6Hz,H-12)；δ C 125.41(C-12)](ii) a Then carrying out signal attribution on oxygen connecting carbon with delta c of 50.0-80.0 and carbon on 18 position [ delta ] H 3.84(1H,ddd,J＝11.2,4.4,2.4Hz,H-2),δ C 65.5(C-2)；δH 4.11(1H,d,J＝2.4Hz,H-3),δ C 70.11(C-3)；δ H 3.90(1H,d,J＝10.8Hz,H-23a),3.86(1H,d,J＝10.8Hz,H-23b)；δ C 60.75(C-23)]；δ H 1.00-2.80, because a plurality of methylene and methine proton signal overlapping peaks exist between the two, the recognition degree is not high.
Therefore, when HMBC (heteronuclear multi-carbon correlation spectroscopy of 1H is carried out, the 1H nucleus and the remotely coupled 13C nucleus are related, and the coupling information of protons and carbons of 2-3 bonds is more generally) is carried out, the proton signal in the region is not taken as a starting point; a plurality of methyl signals exist in a high field area, and the spectrogram signals are obvious and clear and are easy to observe and analyze. Such as [ delta ] H 1.25(3H,s,H-27),1.09(3H,s,H-25),0.88(3H,s,H-26),1.04(3H,d,J＝6.0Hz,H-29)；δ C 22.6(C-27),15.9(C-25),16.4(C-26),15.38(C-29)]。
The compound obtained in example 2 was resolved by HMBC remote coupling. Firstly, analyzing the position of aldehyde group, and displaying a spectrogram: delta H 9.70(H-24) and δ C 58.3(C-4),70.1(C-3),42.0(C-5) relative, indicating that the aldehyde carbon may be attached to the carbon at the 4-position; delta H 4.11(H-3) and δ C 206.97(C-24) correlation allows for the determination that the aldehyde carbon is attached to the carbon at the 4 position. Delta H 5.30(H-12) and δ C 42.0(C-14),47.8(C-9),55.2 (C-18); delta C 15.9(C-25) and δ C 40.5(C-1),42.0(C-5),47.8(C-9),37.4 (C-10);δ H 3.90(H-23a),3.86(H-23b) and δ C 58.3(C-4),70.1(C-3),42.0(C-5),206.97 (C-24); delta C 16.4(C-26) and δ C 32.8(C-7),39.5(C-8),47.8(C-9),42.0(C-14), the basic structures of the A and B rings can be determined by H-25 and H-26 correlations. Delta H 1.25(H-27) and δ C 39.5(C-8),138.2(C-13),42.0(C-14),27.7(C-15), the proton signal of the methyl group at position 27 can be determined by correlating H-27 with C-13. The basic structure of the C/D loop is determined by the HMBC signal of H-27. Passing through delta H 2.30(H-18) and δ C 138.2(C-13),37.1(C-19),47.6(C-17),39.0(C-22),15.38(C-29), 153.2(C-20) correlate and determine the basic structure of the E-ring.
Through J H-2β/H-3β H-2 and H-3 are homoconfigurated as can be seen at 2.40Hz, via J H-18/H-19 The configuration of C-18 and C-19 can be obtained at 12.4 Hz. C-25 is beta configuration, and delta is found by observing the signal of NOESY (revealing the mutual close relation between protons and spaces) spectrum H 1.09(H-25) and δ H 3.84(H-2) related; delta H 1.09(H-25) and δ H 3.90(H-23) related; delta. for the preparation of a coating H 3.84(H-2) and δ H 3.90(H-23) related; delta H 4.11(H-3) and δ H 3.90(H-23) correlation, the absolute configuration of C-2 and C-3 as well as C-23 can be determined. The absolute configurations of C-28 and C-29 were also determined by comparing the carbon spectrum chemical shifts of similar compounds. The compound obtained in example 2 was identified as (2 α,3 α,4 β) -2,3, 23-Trihydroxy-24-oxidans-12, 20(30) -dien-28-oic acid.
The HMBC correlation chart of the compound obtained in example 2 is shown in FIG. 4; the NOESY correlation diagram is shown in fig. 5.
The 1H NMR and 13C NMR data of the compound obtained in example 2 are shown in Table 3.
TABLE 3 1H NMR and 13C NMR data of the compound obtained in example 2
The kiwi fruit root extract obtained by the separation method similar to that in example 2 and the test method similar to that in the test example also contains other 13 triterpenoids, and the structural formula of the extract is shown in figure 6, wherein the structural formula 1 in figure 6 is the compound obtained by the invention.
Test example 4
This test example tested the activity inhibition test of the compound obtained in example 2 on MP pancreatic cancer cells, Patu8988t pancreatic cancer cells, Panco2 pancreatic cancer cells. The specific test method comprises the following steps: the obtained novel compound was subjected to cell evaluation by the CCK8 method, and MP pancreatic cancer cells, Patu8988t pancreatic cancer cells, and Panco2 pancreatic cancer cells were selected as cell evaluation models. The test results are shown in table 4.
TABLE 4 test results of inhibition of pancreatic cancer cell Activity by the Compound obtained in example 2
IC in this table 50 Is in units of μ M, and IC in Table 2 50 The units of (A) are different, and the IC of the compound obtained in example 2 in terms of g/L is converted into the IC of MP pancreatic cancer cells and Patu8988t pancreatic cancer cells 50 The value of about 0.025g/L shows that the compound prepared from the Chinese actinidia root has better inhibition capability on the proliferation activity of pancreatic cancer cells than the Chinese actinidia root extract obtained in example 1.
The test results of the test example 2 and the test example 4 show that the kiwi fruit root extract and the compound prepared from the kiwi fruit root have excellent effect of inhibiting pancreatic cancer cell proliferation, and have good application prospect in the preparation of medicaments for treating pancreas.
The embodiments of the present invention have been described in detail with reference to the drawings, but the present invention is not limited to the embodiments, and various changes can be made within the knowledge of those skilled in the art without departing from the gist of the present invention. Furthermore, the embodiments of the present invention and the features of the embodiments may be combined with each other without conflict.
Claims (13)
1. A method for preparing a compound, comprising the steps of:
s1, taking ethyl acetate as a solvent to obtain a crude extract of Chinese gooseberry roots, and drying the crude extract to obtain a crude extract;
s2, using a mixed solution of dichloromethane and methanol as an eluent, carrying out silica gel column chromatography on the crude extract, detecting a product through thin-layer chromatography, and combining similar components to obtain a kiwi root extract; in the eluent, the volume ratio of the dichloromethane to the methanol is 30-20: 1;
s3, (7-9) and (1-3) are mixed with water in a volume ratio of 1 to form a four-phase system, the four-phase system is mixed, then a lower layer solution is separated, the kiwi fruit root extract is subjected to silica gel column chromatography, products are detected by thin layer chromatography, and similar components are combined to obtain an intermediate component;
s4, carrying out medium-pressure preparation liquid phase separation on the intermediate component to obtain the compound;
in the medium-pressure preparation liquid phase separation, the liquid phase is water and methanol which are mixed according to the volume ratio of (35-45) to (55-65);
the chromatographic column adopted for medium-pressure preparation is a C18 chromatographic column produced by Agilent company, and the model is Eclipse XDB-C18 Semi-Prep;
the compound has a formula as shown in the following formula:
2. the method according to claim 1, wherein the compound is a white powder in a dry state.
3. The method according to claim 1, wherein the compound is in an amorphous state in a crystalline state.
4. The method of claim 1, wherein the compound has methanol solubility.
5. The method of claim 1, wherein the actinidia chinensis planch root is dried powder in step S1.
6. The method of claim 5, wherein the dry powder is passed through an 80 mesh screen.
7. The method of claim 1, wherein the drying step S1 comprises a rotary evaporation step and a vacuum drying step.
8. The method according to claim 1, wherein the crude extract is obtained by percolation in step S1.
9. The method according to claim 1, wherein in step S2, the volume ratio of dichloromethane to methanol is 30: 1.
10. The method according to claim 1, wherein in step S3, the volume ratio of dichloromethane, ethyl acetate, methanol and water in the four-phase eluent is 8:2:2: 1.
11. A kiwi root extract, which comprises a compound obtained by the preparation method according to any one of claims 1 to 10.
12. A medicament for treating pancreatic cancer, characterized in that the raw materials for preparation comprise the compound prepared by the preparation method of any one of claims 1 to 10.
13. A medicament for treating pancreatic cancer, wherein the raw materials for preparation comprise the kiwi root extract of claim 11.
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