CN115477678A - Preparation method of triterpene diglycoside compound and application of triterpene diglycoside compound in preparation of antitumor drugs - Google Patents
Preparation method of triterpene diglycoside compound and application of triterpene diglycoside compound in preparation of antitumor drugs Download PDFInfo
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- CN115477678A CN115477678A CN202211065395.4A CN202211065395A CN115477678A CN 115477678 A CN115477678 A CN 115477678A CN 202211065395 A CN202211065395 A CN 202211065395A CN 115477678 A CN115477678 A CN 115477678A
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- arabinopyranoside
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- oleanolic acid
- xylopyranosyl
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- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
- C07H15/20—Carbocyclic rings
- C07H15/24—Condensed ring systems having three or more rings
- C07H15/256—Polyterpene radicals
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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Abstract
The invention discloses a preparation method of a triterpene diglycoside compound and application of the triterpene diglycoside compound in preparation of antitumor drugs. The potential potent and low-toxicity anti-tumor active substance oleanolic acid-3-O-beta-D-xylopyranoside- (1 → 2) -alpha-L-arabinopyranoside is extracted and separated from the plant such as akebia trifoliata, the source of the plant material is rich, the extraction and preparation are easy to operate, and pharmacological experiments show that the compound oleanolic acid-3-O-beta-D-xylopyranoside- (1 → 2) -alpha-L-arabinopyranoside has obvious activity of inhibiting the growth of tumor cells in vitro, and the monomer compound is stable and easy to store, so the compound has the potential of developing and preparing anti-tumor drugs or is a lead compound for developing effective low-toxicity anti-tumor drugs, and has potential good application and development prospects.
Description
The technical field is as follows:
the invention belongs to the field of biological medicine, and particularly relates to a preparation method of oleanolic acid-3-O-beta-D-xylopyranosyl- (1 → 2) -alpha-L-arabinopyranoside and application of the oleanolic acid-3-O-beta-D-xylopyranosyl- (1 → 2) -alpha-L-arabinopyranoside in preparation of antitumor drugs.
Background art:
tumors are the second-ranked health killers of cardiovascular and cerebrovascular diseases that seriously compromise human health. With the development of economic society, the incidence of tumors is increasing continuously in the global scope, the number of the tumor incidence in China also shows a trend of increasing sharply in recent years, and the tumors cause heavy burden and huge loss to the health of the people in China and the national economy.
Chemical synthesis of cytotoxic antineoplastic drugs has been in use for decades. However, synthetic antitumor drugs often have significant toxic and side effects, and are always difficult in tumor treatment. At present, the tumor treatment by the existing clinical drugs approaches or reaches the plateau stage, and research and development of new tumor treatment drugs with effectiveness and low toxicity are urgently needed.
Natural active compounds in medicinal and edible plants have relatively low toxicity to human beings, and have important potential in the aspect of researching and developing high-efficiency low-toxicity antitumor drugs. The akebiaceae akebia plant akebia trifoliate akebia stem and the like are Chinese traditional important Chinese medicinal materials, and the rattan and mature fruits of the akebia trifoliate akebia stem have the various effects of clearing heat, promoting urination, promoting blood circulation, dredging collaterals, resisting bacteria, diminishing inflammation and the like. In recent years, the publication reports that Akebia plant also has anticancer and antitumor effects (Song Liren, hong Xun, ding Zhuliang, etc.. Modern Chinese medicine dictionary (II), beijing: people health Press, 2001, 335-337), shows that it has important potential in the discovery of tumor therapeutic drugs with both effectiveness and low toxicity.
The compound oleanolic acid-3-O-beta-D-xylopyranosyl- (1 → 2) -alpha-L-arabinopyranoside is a disaccharide saponin compound using oleanane type pentacyclic triterpene as aglycone, which has been isolated from the fruit of Akebia trifoliata Koidz (Akebia trifolia, thumb.) by [ Dan Jiang, she-Po Shi, ji-Juan Cao, qi-Pin Gao, peng-Fei Tu. However, no report on the antitumor activity has been made so far, and no literature is available on the separation of Akebia trifoliata and related varieties Akebia alba and Akebia longicalycis.
The invention content is as follows:
the first purpose of the invention is to provide the application of the compound oleanolic acid-3-O-beta-D-xylopyranosyl- (1 → 2) -alpha-L-arabinopyranoside in preparing anti-tumor drugs.
The oleanolic acid-3-O-beta-D-xylopyranosyl- (1 → 2) -alpha-L-arabinopyranoside is proved to have obvious inhibition activity (IC) on human central nervous system tumor cells SF-268, breast cancer cells MCF-7, liver cancer cells HepG2 and lung cancer cells A549 by in vitro pharmacological experiments 50 The values are respectively 9.43 +/-0.09, 11.75 +/-0.31, 9.38 +/-0.47 and 12.66 +/-0.02 mu M), and has no obvious toxic or side effect on normal cells. Therefore, the derivative has the potential of being developed to be used for preparing antitumor drugs with effective low toxicity or being used as a lead compound of the antitumor drugs.
Therefore, the compound oleanolic acid-3-O-beta-D-xylopyranosyl- (1 → 2) -alpha-L-arabinopyranoside can be applied to the preparation of antitumor drugs.
The anti-tumor drug is preferably a drug for resisting nervous system tumor, breast cancer, liver cancer or liver cancer.
The second purpose of the invention is to provide an anti-tumor drug which is characterized by comprising an effective amount of oleanolic acid-3-O-beta-D-xylopyranosyl- (1 → 2) -alpha-L-arabinopyranoside as an active ingredient and pharmaceutically common auxiliary materials or carriers.
The third object of the present invention is to provide a method for preparing oleanolic acid-3-O-beta-D-xylopyranosyl- (1 → 2) -alpha-L-arabinopyranoside, characterized in that the compound oleanolic acid-3-O-beta-D-xylopyranosyl- (1 → 2) -alpha-L-arabinopyranoside is prepared and isolated from the stems, leaves or fruits of Akebia trifoliata (thumb.) Koidz) or its variant Arthropoda nivea (Akebia trifoliata (thumb.) Koidz.Var.australis (Diels.) Rehd) and Akebia trifoliata (thumb.) Koidz.
The fruit can be dried or fresh stem leaf or fruit material, preferably dried fruit.
It should be noted that the pericarp and the seed are the components of the fruit, so the preparation of the compound oleanolic acid-3-O-beta-D-xylopyranosyl- (1 → 2) -alpha-L-arabinopyranoside by using the pericarp and the seed as raw materials also belongs to the protection scope of the invention.
The specific steps are preferably as follows:
a. preparing a total extract: crushing stems, leaves or fruits of akebia trifoliata or akebia quinata long-sequence, extracting with an ethanol water solution, concentrating an extracting solution to remove ethanol to obtain a total extract crude extract, suspending the total extract crude extract in water, extracting with petroleum ether and then ethyl acetate, and concentrating the ethyl acetate extract to obtain an ethyl acetate total extract;
b. separation and purification: performing normal-phase silica gel column chromatography on the ethyl acetate total extract, performing gradient elution by using chloroform/methanol as an eluent from a solvent which is 100 percent of a solvent with the volume ratio of 8.
Preferably, the ethanol aqueous solution is 95% ethanol aqueous solution.
The fourth purpose of the invention is to provide the application of the stems, leaves or fruits of akebia trifoliata, akebia trifoliata and akebia longicalyx to the preparation of oleanolic acid-3-O-beta-D-xylopyranosyl- (1 → 2) -alpha-L-arabinopyranoside.
The compound of oleanolic acid-3-O-beta-D-xylopyranoside- (1 → 2) -alpha-L-arabinopyranoside can be combined with pharmaceutically common auxiliary materials or drug carriers to prepare the drug or drug composition which has the anti-tumor activity of oleanolic acid-3-O-beta-D-xylopyranoside- (1 → 2) -alpha-L-arabinopyranoside and can be used for preventing and treating tumors. The medicine or the pharmaceutical composition can adopt wettable powder, tablets, granules, capsules, oral liquid, dripping pills, injection, aerosol and other formulations; controlled or sustained release formulations or nano-formulations well known in the modern pharmaceutical industry may also be used.
The invention adopts the plant tissues such as akebia trifoliata which are widely distributed in China to extract and separate the oleanolic acid-3-O-beta-D-xylopyranoside- (1 → 2) -alpha-L-arabinopyranoside with obvious anti-tumor activity, the material sources are rich, the preparation process is easy to operate, the monomer compound is stable and easy to store, and because akebia trifoliata and other plants are medicinal and edible plants, the obtained compound oleanolic acid-3-O-beta-D-xylopyranoside- (1 → 2) -alpha-L-arabinopyranoside is probably an anti-tumor active compound with effectiveness and low toxicity to human bodies, so the invention has better application and development potential.
Description of the drawings:
FIG. 1 is a schematic representation of the compound oleanolic acid-3-O-beta-D-xylopyranosyl- (1 → 2) -alpha-L-arabinopyranoside 1 An H NMR spectrum;
FIG. 2 is a drawing showing the synthesis of oleanolic acid-3-O-beta-D-xylopyranosyl- (1 → 2) -alpha-L-arabinopyranoside 13 C NMR spectrum.
The specific implementation mode is as follows:
the following examples are further illustrative of the present invention and are not intended to be limiting thereof, and all simple modifications made to the invention in light of the spirit thereof are intended to be included within the scope of the present invention as claimed.
Example 1: preparation of oleanolic acid-3-O-beta-D-xylopyranosyl- (1 → 2) -alpha-L-arabinopyranoside in akebia trifoliata fruits
1.1 plant origin and identification
Rattan samples of plant material Akebia trifoliata (thumb.) Koidz for extraction were collected from Hunan province in 9 months of 2020, and the retained samples were stored in the applicant's laboratory.
1.2 extraction and separation
Pulverizing a sample (2.0 kg of akebia trifoliata stem dry product), extracting with 95% ethanol by volume fraction at room temperature for three times, filtering, combining filtrates, concentrating under reduced pressure to remove organic solvent ethanol, and obtaining a total extract crude extract. Suspending the total extract in 500ml water, extracting with petroleum ether of the same volume for three times, extracting with ethyl acetate, and concentrating the ethyl acetate extractive solution under reduced pressure to obtain ethyl acetate total extract (27 g). Dissolving the ethyl acetate total extract with methanol (250 mL), adding normal phase silica gel (80-100 meshes) to mix the mixture in a weight ratio of 1.5, volatilizing, carrying out dry loading on a dry loading column (200-300 meshes, 550 g), eluting with chloroform/methanol as an eluent from a solvent with a volume ratio of 2,7.
1.3 Structure identification of Compound Oleanolic acid-3-O-beta-D-xylopyranosyl- (1 → 2) -alpha-L-arabinopyranoside
The obtained compound 1 is a white powdery solid with a molecular formula of C 40 H 64 O 11 Which is 1 H NMR spectrum and 13 the C NMR spectra are shown in FIG. 1 and FIG. 2, respectively; ESI-MS (+) m/z 2, 743M + Na] + ；ESI-MS(-)m/z 719[M-H] – ,755[M+Cl] - ； 1 H NMR(400MHz,CD 3 OD):δ5.24(1H,br s.,H-12),1.16(3H,s),1.03(3H,s),0.95(3H,s),0.94(3H,s),0.91(3H,s),0.84(3H,s),0.81(3H,s)； 13 C NMR(100MHz,CD 3 OD):δ39.8(CH 2 ,C-1),26.4(CH 2 ,C-2),90.8(CH,C-3),39.8(C,C-4),57.0(CH,C-5),19.3(CH 2 ,C-6),31.7(CH 2 ,C-7),40.3(C,C-8),48.3(CH,C-9),37.9(C,C-10),24.5(CH 2 ,C-11),123.7(CH,C-12),145.2(C,C-13),42.7(C,C-14),28.8(CH 2 ,C-15),24.0(CH 2 ,C-16),47.6(C,C-17),42.8(CH,C-18),47.2(CH 2 ,C-19),31.6(C,C-20),34.0(CH 2 ,C-21),33.8(CH 2 ,C-22),28.3(CH 3 ,C-23),16.6(CH 3 ,C-24),15.9(CH 3 ,C-25),17.7(CH 3 ,C-26),26.4(CH 3 ,C-27),181.9(C,C-28),23.9(CH 3 ,C-29),21.2(CH 3 ,C-30),105.4(CH,Ara-C-1),80.6(CH,Ara-C-2),73.6(CH,Ara-C-3),68.9(CH,Ara-C-4),65.3(CH 2 ,Ara-C-5),106.2(CH,Xyl-C-1),75.9(CH 2 ,Xyl-C-2),77.8(CH,Xyl-C-3),71.2(CH,Xyl-C-4),67.1(CH 2 ,Xyl-C-5)。
According to the comprehensive analysis of the mass spectrum data and the nuclear magnetic spectrum data and the comparison with the literature, the chemical structure of the compound 1 can be analytically deduced to be oleanolic acid-3-O-beta-D-xylopyranosyl- (1 → 2) -alpha-L-arabinopyranoside.
Example 2: preparation of oleanolic acid-3-O-beta-D-xylopyranosyl- (1 → 2) -alpha-L-arabinopyranoside in akebia trifoliata fruits
2.1 plant origin and identification:
fruit samples of plant material Akebia trifoliata (thumb.) Koidz for extraction were collected from Hunan province in 9 months of 2020, and the retained samples were stored in the applicant's laboratory.
2.2 extraction and separation:
pulverizing a sample (1.5 kg of dried fruits of akebia trifoliata Thunb), extracting with 95% ethanol by volume fraction at room temperature for three times, filtering, combining filtrates, concentrating under reduced pressure to remove organic solvent ethanol to obtain a total extract crude extract. Suspending the total extract in 500ml water, extracting with petroleum ether of the same volume for three times, extracting with ethyl acetate, and concentrating the ethyl acetate extractive solution under reduced pressure to obtain ethyl acetate total extract (26.4 g). Dissolving the ethyl acetate total extract with methanol (250 mL), adding normal phase silica gel (80-100 meshes) to mix the mixture in a weight ratio of 1.5, volatilizing, carrying out dry loading on a dry loading column (200-300 meshes, 550 g), eluting with chloroform/methanol as an eluent from a solvent with a volume ratio of 2,7.
Example 3: preparation of oleanolic acid-3-O-beta-D-xylopyranosyl- (1 → 2) -alpha-L-arabinopyranoside in akebia trifoliata leaves
2.3 plant origin and identification:
leaf samples of plant material Akebia trifoliata (thumb.) Koidz for extraction were collected from Hunan province in 9 months of 2020, and the retained samples were stored in the applicant's laboratory.
2.4, extraction and separation:
pulverizing a sample (akebia trifoliata She Ganpin 1.2.2 kg), extracting with 95% ethanol by volume fraction at room temperature for three times, filtering, combining filtrates, and concentrating under reduced pressure to remove organic solvent ethanol to obtain a concentrated total extract crude extract. Suspending the total extract in 500ml water, extracting with petroleum ether of the same volume for three times, extracting with ethyl acetate, and concentrating the ethyl acetate extractive solution under reduced pressure to obtain ethyl acetate total extract (29.4 g). Dissolving the ethyl acetate total extract with methanol (250 mL), adding normal phase silica gel (80-100 meshes) to mix the mixture in a weight ratio of 1.5, volatilizing, carrying out dry loading on a dry loading column (200-300 meshes, 550 g), eluting with chloroform/methanol as an eluent from a volume ratio of 2,7.
Example 4:
the oleanolic acid-3-O-beta-D-xylopyranosyl- (1 → 2) -alpha-L-arabinopyranoside can be obtained by separating stem and leaf dry products of akebia quinata and akebia trifoliate as materials according to the extraction and separation method of example 1.
Example 5:
the oleanolic acid-3-O-beta-D-xylopyranosyl- (1 → 2) -alpha-L-arabinopyranoside can be obtained by separating the dried fruits of akebia quinata and akebia trifoliate as materials according to the extraction and separation method of the embodiment 2.
Example 6: oleanolic acid-3-O-beta-D-xylopyranosyl- (1 → 2) -alpha-L-arabinopyranoside antitumor activity detection 6.1 instrument and material
Cell line and cell culture solution: the four human tumor cell lines, namely central nervous system tumor cell (SF-268), breast cancer cell (MCF-7), liver cancer cell (HepG 2) and lung cancer cell (NCI-H460), are from Guangdong province microbial institute; RPMI1640 medium (Gibaco), fetal bovine serum (FBS, gibaco), trypsin (Trypsin 1, amersco), PBS (laboratory preparations).
An experimental instrument: the microplate reader Genois microplate reader (Tecan GENios, swizerland).
Reagent and sample: dimethyl sulfoxide (DMSO, analytical grade), 3- (4,5-dimethylthiazole-2) -2,5-diphenyltetrazolium bromide salt (MTT, sigma), doxorubicin (Pfizer Italiai SRL); oleanolic acid-3-O- β -D-xylopyranosyl- (1 → 2) - α -L-arabinopyranoside was prepared in the above experimental examples.
6.2 test method:
a) Tumor cell culture: 4 human tumor cell lines were adherent cells, and the culture medium was RPMI164 medium, 10% FBS and 1% double antibody, and the cells were cultured at 37 deg.C and 5% CO 2 Saturated humidity CO 2 An incubator. When the cells are 90% of the bottom of the culture dish, the cells can be passaged. Cells in logarithmic growth phase were taken for experiments.
b) Preparation of a test sample: the oleanolic acid-3-O-beta-D-xylopyranosyl- (1 → 2) -alpha-L-arabinopyranoside and doxorubicin are prepared into 10mg/ml solutions respectively from dimethyl sulfoxide (DMSO), and then diluted with a culture medium to a final treatment concentration.
c) The half inhibition concentration determination of the compound oleanolic acid-3-O-beta-D-xylopyranosyl- (1 → 2) -alpha-L-arabinopyranoside on tumor cells is completed by adopting a 96-well plate by adopting an MTT method.
First, the cells in logarithmic growth phase are digested and collected, and the cell suspension concentration is adjusted to 5X 10 4 Cells/ml were plated in 96-well plates with 100. Mu.l per well. The assay was carried out in 96-well plates at 37 ℃ and 5% CO 2 Incubate 24h. After the supernatant was aspirated, 100. Mu.l of fresh medium and 100. Mu.l of medium containing the sample solution at the concentration required for the experiment were added to each well and mixed thoroughly to give a final concentration of 200. Mu.l per well. The experiment was divided into a test sample group, a blank control group (no sample added), and a positive control group (doxorubicin) at different concentrations. The tested sample group is provided with 6 concentration gradients per sample and 4 multiple wells per concentration. At 37 ℃ C, 5% CO 2 Culturing for 72h in an incubator, adding 20 μ l (5 mg/ml) of MTT into each well, further incubating for 4h, centrifuging to remove the upper layer culture solution, adding 150 μ l of DMSO into each well, sufficiently shaking on a shaker to dissolve the generated crystals, and after 15min, placing in a microplate reader to measure the absorbance (OD) at a wavelength of 570 nm. The inhibition rate of the compound is calculated by the following formula: inhibition (%) = (OD) control –OD treated )/OD control X 100%. Wherein the half Inhibitory Concentration (IC) of the test compound on tumor cells 50 ) Using IC 50 The calculation software (LOGIT method) calculates (Xu, X.Y.; xie, H.H.; hao, J.; jiang, Y.M.; wei, X.Y.Eudesmane setipertepen polysaccharides from biochee seed and the same cytoxic activity. Food chem.,2010, 123.
6.3 Experimental data see Table 1:
TABLE 1 in vitro antitumor cell Activity IC of Oleanolic acid-3-O-beta-D-xylopyranosyl- (1 → 2) -alpha-L-arabinopyranoside 50 (μM)
6.4 conclusion of the experiment:
the oleanolic acid-3-O-beta-D-xylopyranosyl provided by the inventionThe (1 → 2) -alpha-L-arabinopyranoside has obvious inhibition activity (IC) on human central nervous system tumor cell SF-268, breast cancer cell MCF-7, liver cancer cell HepG2 and lung cancer cell NCI-H460 as proved by in vitro pharmacological experiments 50 The values are respectively 9.43 +/-0.09, 11.75 +/-0.31, 9.38 +/-0.47 and 12.66 +/-0.02 mu M, and the product can be used as a glucoside component separated from edible plant resources such as akebia trifoliata and the like, has multiple excellent characteristics of low toxicity, good water solubility and the like, and therefore, has the potential of developing and preparing related antitumor drugs, or can be used as a lead compound for developing the related antitumor drugs.
Claims (9)
1. The application of oleanolic acid-3-O-beta-D-xylopyranosyl- (1 → 2) -alpha-L-arabinopyranoside in preparing antitumor drugs, wherein the structural formula of the oleanolic acid-3-O-beta-D-xylopyranosyl- (1 → 2) -alpha-L-arabinopyranoside is shown as the formula (I):
2. the use of claim 1, wherein the anti-neoplastic agent is an anti-tumor agent against nervous system, breast, liver or lung cancer.
3. An antitumor drug is characterized by comprising an effective amount of oleanolic acid-3-O-beta-D-xylopyranosyl- (1 → 2) -alpha-L-arabinopyranoside as an active ingredient and pharmaceutically common auxiliary materials or carriers;
the oleanolic acid-3-O-beta-D-xylopyranosyl- (1 → 2) -alpha-L-arabinopyranoside has a structural formula shown in a formula (I):
4. the antitumor drug according to claim 3, wherein the antitumor drug is a drug against central nervous system tumor, breast cancer, liver cancer or lung cancer.
5. A method for preparing oleanolic acid-3-O-beta-D-xylopyranosyl- (1 → 2) -alpha-L-arabinopyranoside, characterized in that the compound oleanolic acid-3-O-beta-D-xylopyranosyl- (1 → 2) -alpha-L-arabinopyranoside is prepared and separated from stems, leaves or fruits of Akebia trifoliata (Akebia trifolia (thumb.) Koidz) or its variant Akebia trifolia (thumb.) Koidz.var.australis (Diels) Rehd) and Akebia trifolia (thumb.) Koidz.Longia H.N.Qin);
the oleanolic acid-3-O-beta-D-xylopyranosyl- (1 → 2) -alpha-L-arabinopyranoside has a structural formula shown in a formula (I):
6. the preparation method according to claim 5, comprising the following steps:
a. preparing a total extract: crushing stems, leaves or fruits of akebia trifoliata or akebia quinata long-sequence, extracting with an ethanol water solution, concentrating an extracting solution to remove ethanol to obtain a total extract crude extract, suspending the total extract crude extract in water, extracting with petroleum ether and then ethyl acetate, and concentrating the ethyl acetate extract to obtain an ethyl acetate total extract;
b. separation and purification: subjecting the ethyl acetate total extract to normal-phase silica gel column chromatography, eluting with chloroform/methanol as an eluent in a gradient manner from a solvent with the volume ratio of 8.
7. The process according to claim 6, wherein the aqueous ethanol solution is a 95% aqueous ethanol solution.
8. The method according to claim 6, wherein the stem leaves or the fruits are dried or fresh.
9. Application of stems, leaves or fruits of caulis Akebiae, caulis Akebiae and caulis Akebiae in preparing oleanolic acid-3-O-beta-D-xylopyranosyl- (1 → 2) -alpha-L-arabinopyranoside is provided.
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| CN1927878A (en) * | 2005-09-09 | 2007-03-14 | 徐先祥 | Clematis chinensis total saponin and treatment action thereof for rheumatoild disease |
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