A method for preparing a triterpene diglycoside compound and its application in preparing anti-tumor drugs

Abstract

The invention discloses a preparation method of a triterpene diglycoside compound and its application in the preparation of anti-tumor drugs. The invention extracts and separates a potential strong and low-toxic anti-tumor active substance oleanolic acid-3-O-β-D-xylpyranyl-(1→2)-α-L-arabinopyranoside from plants such as Akebia trifoliata. The plant material source is rich, the extraction and preparation are easy to operate, and pharmacological experiments show that the compound oleanolic acid-3-O-β-D-xylpyranyl-(1→2)-α-L-arabinopyranoside has significant in vitro activity of inhibiting tumor cell growth, and the monomer compound is stable and easy to store. Therefore, the compound has the potential to be developed for the preparation of anti-tumor drugs, or as a lead compound for the development of effective low-toxic anti-tumor drugs, and has potential good application development prospects.

Description

Technical field:

[0001] The invention belongs to the field of biomedicine, and specifically relates to a preparation method of oleanolic acid-3-O-β-D-xylose pyranyl-(1→2)-α-L-arabinopyranoside and an application thereof in the preparation of anti-tumor drugs. Background technology:

[0002] Tumors are the second leading health killer after cardiovascular and cerebrovascular diseases that seriously endanger human health. With the development of economy and society, the incidence of tumors is increasing worldwide. The number of tumor cases in my country has also shown a sharp increase in recent years. Tumors are causing heavy burdens and huge losses to the health of the Chinese people and the national economy.

[0003] Chemically synthesized cytotoxic anti-tumor drugs have been used for decades. However, synthetic anti-tumor drugs often have significant toxic side effects, which has always been a problem in tumor treatment. At present, the existing clinical drug treatment of tumors has approached or reached a plateau, and there is an urgent need to research and develop new tumor treatment drugs that are both effective and low in toxicity.

[0004] The natural active compounds in medicinal and edible plants have relatively low toxicity to humans, and they have great potential in the development of highly effective and low-toxic anti-tumor drugs. Akebia trifoliata, a plant of the genus Akebia in the family Akebiae, is an important traditional Chinese medicinal material in my country. Its vines and mature fruits have various effects such as clearing away heat and promoting diuresis, promoting blood circulation and promoting blood circulation, and antibacterial and anti-inflammatory. In recent years, literature reports have pointed out that Akebia plants also have anti-cancer and anti-tumor effects (Song Liren, Hong Xun, Ding Zhuliang, et al. Modern Chinese Medicine Dictionary (II), Beijing: People's Medical Publishing House, 2001, 335-337), indicating that they have important potential in the development of tumor treatment drugs that are both effective and low-toxic.

[0005] The compound oleanolic acid-3-O-β-D-xylpyranyl-(1→2)-α-L-arabinopyranoside is a disaccharide saponin compound with oleanane-type pentacyclic triterpenoid as aglycone. The compound has been isolated from the fruit of Akebia trifolia (Thumb.) Koidz [Dan Jiang, She-Po Shi, Ji-Juan Cao, Qi-Pin Gao, Peng-Fei Tu. Triterpene saponins from the fruits of Akebia quinata. Biochemical Systematics and Ecology, 2008, 36: 138-141], and its chemical structure is shown in the following formula (I). However, there has been no report on its anti-tumor activity so far, and there is no literature on its isolation from Akebia trifolia (Thumb.) Koidz and its related variants, Akebia trifolia (Thumb.) Koidz.

[0006] Summary of the invention:

[0007] The first object of the present invention is to provide the use of the compound oleanolic acid-3-O-β-D-xylose pyranyl-(1→2)-α-L-arabinopyranoside in the preparation of anti-tumor drugs.

[0008] Oleanolic acid-3-O-β-D-xylose pyranose-(1→2)-α-L-arabinopyranoside of the present invention has been confirmed by in vitro pharmacological experiments to have significant inhibitory activity (IC 50 The values ​​were 9.43±0.09, 11.75±0.31, 9.38±0.47 and 12.66±0.02 μM, respectively), and had no significant toxic side effects on normal cells. Therefore, it has the potential to be developed for the preparation of anti-tumor drugs with both efficacy and low toxicity or as a lead compound for anti-tumor drugs.

[0009] Therefore, the compound oleanolic acid-3-O-β-D-xylose pyranyl-(1→2)-α-L-arabinopyranoside of the present invention can be used in the preparation of anti-tumor drugs.

[0010] The anti-tumor drug is preferably a drug for nervous system tumors, breast cancer, liver cancer or hepatocellular carcinoma.

[0011] The second object of the present invention is to provide an anti-tumor drug, characterized in that it contains an effective amount of oleanolic acid-3-O-β-D-xylopyranyl-(1→2)-α-L-arabinopyranoside as an active ingredient and a commonly used pharmaceutical excipient or carrier.

[0012] The third object of the present invention is to provide a method for preparing oleanolic acid-3-O-β-D-xylopyranyl-(1→2)-α-L-arabinopyranoside, characterized in that the compound oleanolic acid-3-O-β-D-xylopyranyl-(1→2)-α-L-arabinopyranoside is prepared and separated from the stems, leaves or fruits of Akebia trifolia (Thumb.) Koidz or its variant Akebia trifolia (Thumb.) Koidz. Var. australis (Diels) Rehd and Akebia trifolia (Thumb.) Koidz..subsp. Longisepala HNQin.

[0013] Specifically, it can be a dried or fresh product of stems, leaves or fruits, preferably a dried fruit product.

[0014] It should be noted that the peel and seeds are components of the fruit, and therefore the preparation of the compound oleanolic acid-3-O-β-D-xylopyranyl-(1→2)-α-L-arabinopyranoside using the peel and seeds as raw materials also falls within the protection scope of the present invention.

[0015] The specific steps are preferably:

[0016] a. Preparing a total extract: crushing the stems, leaves or fruits of Akebia trifoliata, Akebia dasyphylla or Akebia longifolia and extracting them with ethanol aqueous solution, concentrating the extract to remove ethanol to obtain a total extract crude extract, suspending the total extract crude extract in water, first extracting with petroleum ether and then with ethyl acetate, and concentrating the ethyl acetate extract to obtain an ethyl acetate total extract;

[0017] b. Separation and purification: The total ethyl acetate extract was subjected to normal phase silica gel column chromatography, with chloroform / methanol as the eluent, and gradient eluted from volume ratios of 8:2, 7:3, 6:4, and 0:100 in sequence, and the fractions eluted with chloroform / methanol 7:3 were collected, and then separated by reverse phase medium pressure column chromatography, first eluted with 40% methanol-water, then eluted with 55% methanol-water and the eluate was collected, and then separated and purified by Sphadex LH-20 gel column with methanol as the eluent to obtain the target compound oleanolic acid-3-O-β-D-xylopyranyl-(1→2)-α-L-arabinopyranoside.

[0018] Preferably, the ethanol aqueous solution is 95% ethanol aqueous solution.

[0019] The fourth object of the present invention is to provide an application of the stems, leaves or fruits of Akebia trifoliata, Akebia scabra and Akebia longifolia in the preparation of oleanolic acid-3-O-β-D-xylopyranyl-(1→2)-α-L-arabinopyranoside.

[0020] The compound oleanolic acid-3-O-β-D-xylose pyranyl-(1→2)-α-L-arabinopyranoside described in the present invention can be combined with common pharmaceutical excipients or drug carriers to prepare a drug or drug composition having the anti-tumor activity of oleanolic acid-3-O-β-D-xylose pyranyl-(1→2)-α-L-arabinopyranoside, which can be used to prevent and treat tumors. The drug or drug composition can be in the form of wettable powder, tablet, granule, capsule, oral liquid, dripping pill, injection, aerosol and the like; it can also be in the form of controlled release or sustained release or nano-preparation known in the modern pharmaceutical industry.

[0021] The invention uses plant tissues such as Akebia trifoliata widely distributed in my country to extract and separate oleanolic acid-3-O-β-D-xylopyranyl-(1→2)-α-L-arabinopyranoside with significant anti-tumor activity. The material source is abundant, the preparation process is easy to operate, and the monomer compound is stable and easy to store. In addition, because plants such as Akebia trifoliata are medicinal and edible plants, the obtained compound oleanolic acid-3-O-β-D-xylopyranyl-(1→2)-α-L-arabinopyranoside is very likely to be an anti-tumor active compound with both effectiveness and low toxicity to the human body, and thus has good application development potential. Description of the drawings:

[0022] Figure 1 It is the compound oleanolic acid-3-O-β-D-xylose pyranyl-(1→2)-α-L-arabinopyranoside. 1 HNMR spectrum;

[0023] Figure 2 It is the compound oleanolic acid-3-O-β-D-xylose pyranyl-(1→2)-α-L-arabinopyranoside. 13 CNMR spectrum. Specific implementation method:

[0024] The following examples are provided to further illustrate the present invention, rather than to limit the present invention. All simple improvements made to the present invention based on the essence of the present invention fall within the scope of protection claimed by the present invention.

[0025] Example 1: Preparation of oleanolic acid-3-O-β-D-xylose pyranyl-(1→2)-α-L-arabinopyranoside from Akebia trifoliata fruit

[0026] 1.1 Plant origin and identification

[0027] The vine stem samples of Akebia trifolia (Thumb.) Koidz., the plant material for extraction, were collected in Hunan Province in September 2020, and the retained samples were stored in the applicant's laboratory.

[0028] 1.2 Extraction and separation

[0029] The sample (dry stem of Akebia trifoliata, weight 2.0 kg) was crushed and extracted three times with 95% ethanol at room temperature, and the filtrate was filtered and combined, and the organic solvent ethanol was concentrated under reduced pressure to obtain a crude total extract. The total extract was suspended in 500 ml of water, extracted three times with an equal volume of petroleum ether, and then extracted with ethyl acetate. The ethyl acetate extract was concentrated under reduced pressure to obtain an ethyl acetate total extract (27 g). The total ethyl acetate extract was dissolved in methanol (250 mL), normal phase silica gel (80-100 mesh) was added and mixed with the sample at a weight ratio of 1:1.5, and evaporated to dryness. The sample was dry loaded on a column (200-300 mesh, 550 g), and chloroform / methanol was used as the eluent. The gradient elution was sequentially from a volume ratio of 8:2, 7:3, 6:4, and 0:100. The fraction eluted with chloroform / methanol 7:3 was collected, and then separated by reverse phase medium pressure column chromatography, first eluted with 40% methanol-water, then eluted with 55% methanol-water and the eluate was collected. Then, the pure compound oleanolic acid-3-O-β-D-xylopyranyl-(1→2)-α-L-arabinopyranoside (8.2 mg) represented by formula (I) was obtained.

[0030] 1.3 Structural identification of the compound oleanolic acid-3-O-β-D-xylopyranyl-(1→2)-α-L-arabinopyranoside

[0031] The obtained compound 1 is a white powdery solid with the molecular formula of C 40 H 64 O 11 ,That 1 H NMR spectra and 13 C NMR spectra are shown in Figure 1 and Figure 2 As shown; ESI-MS (+) m / z 743 [M+Na] + ; ESI-MS(-)m / z 719[MH] – ,755[M+Cl] - ; 1H NMR (400MHz, CD3OD): 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, CD3OD): δ39.8(CH2,C-1),26.4(CH2,C-2),90.8(CH,C-3),39.8(C,C-4),57.0( CH,C-5),19.3(CH2,C-6),31.7(CH2,C-7),40.3(C,C-8),48.3(CH,C-9),37.9(C,C-10),24. 5(CH2,C-11),123.7(CH,C-12),145.2(C,C-13),42.7(C,C-14),28.8(CH2,C-15),24.0(CH2 ,C-16),47.6(C,C-17),42.8(CH,C-18),47.2(CH2,C-19),31.6(C,C-20),34.0(CH2,C-21), 33.8(CH2,C-22),28.3(CH3,C-23),16.6(CH3,C-24),15.9(CH3,C-25),17.7(CH3,C-26),26 .4(CH3,C-27),181.9(C,C-28),23.9(CH3,C-29),21.2(CH3,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(CH2,Ara-C-5),106.2(CH,X yl-C-1), 75.9 (CH2, Xyl-C-2), 77.8 (CH, Xyl-C-3), 71.2 (CH, Xyl-C-4), 67.1 (CH2, Xyl-C-5).

[0032] Based on the comprehensive analysis of the above mass spectrometry and NMR spectral data and comparison with the literature, the chemical structure of compound 1 can be deduced to be oleanolic acid-3-O-β-D-xylopyranyl-(1→2)-α-L-arabinopyranoside.

[0033] Example 2: Preparation of oleanolic acid-3-O-β-D-xylose pyranyl-(1→2)-α-L-arabinopyranoside from Akebia trifoliata fruit

[0034] 2.1 Plant origin and identification:

[0035] The fruit samples of Akebia trifolia (Thumb.) Koidz., the plant material for extraction, were collected in Hunan Province in September 2020, and the retained samples were stored in the applicant's laboratory.

[0036] 2.2 Extraction and separation:

[0037] The sample (1.5 kg of dried fruit of Akebia trifoliata) was crushed and extracted three times with 95% ethanol at room temperature, and the filtrate was filtered and combined and concentrated under reduced pressure to remove the organic solvent ethanol to obtain a crude total extract. The total extract was suspended in 500 ml of water, extracted three times with an equal volume of petroleum ether, and then extracted with ethyl acetate. The ethyl acetate extract was concentrated under reduced pressure to obtain ethyl acetate total extract (26.4 g). The total ethyl acetate extract was dissolved in methanol (250 mL), normal phase silica gel (80-100 mesh) was added and mixed with the sample at a weight ratio of 1:1.5, and evaporated to dryness. The sample was dry loaded on a column (200-300 mesh, 550 g), and chloroform / methanol was used as the eluent. The gradient elution was sequentially from a volume ratio of 8:2, 7:3, 6:4, and 0:100. The fraction eluted with chloroform / methanol 7:3 was collected, and then separated by reverse phase medium pressure column chromatography, first eluted with 40% methanol-water, then eluted with 55% methanol-water and the eluate was collected. Then, the pure compound oleanolic acid-3-O-β-D-xylopyranyl-(1→2)-α-L-arabinopyranoside (7.1 mg) represented by formula (I) was obtained.

[0038] Example 3: Preparation of oleanolic acid-3-O-β-D-xylopyranyl-(1→2)-α-L-arabinopyranoside from leaves of Akebia trifoliata

[0039] 2.3 Plant origin and identification:

[0040] The leaf samples of Akebia trifolia (Thumb.) Koidz., the plant material for extraction, were collected in Hunan Province in September 2020, and the retained samples were stored in the applicant's laboratory.

[0041] 2.4 Extraction and separation:

[0042] The sample (1.2 kg of dried leaves of Akebia trifoliata) was crushed and extracted three times with 95% ethanol at room temperature, and the filtrate was filtered and combined and concentrated under reduced pressure to remove the organic solvent ethanol to obtain a concentrated total extract. The total extract was suspended in 500 ml of water, extracted three times with an equal volume of petroleum ether, and then extracted with ethyl acetate. The ethyl acetate extract was concentrated under reduced pressure to obtain ethyl acetate total extract (29.4 g). The total ethyl acetate extract was dissolved in methanol (250 mL), normal phase silica gel (80-100 mesh) was added and mixed with the sample at a weight ratio of 1:1.5, and evaporated to dryness. The sample was dry loaded on a column (200-300 mesh, 550 g), and chloroform / methanol was used as the eluent. The gradient elution was sequentially from a volume ratio of 8:2, 7:3, 6:4, and 0:100. The fraction eluted with chloroform / methanol 7:3 was collected, and then separated by reverse phase medium pressure column chromatography, first eluted with 40% methanol-water, then eluted with 55% methanol-water and the eluate was collected. Then, the pure compound oleanolic acid-3-O-β-D-xylopyranyl-(1→2)-α-L-arabinopyranoside (3.8 mg) represented by formula (I) was obtained.

[0043] Embodiment 4:

[0044] Using the dried stems and leaves of Akebia trifoliata and Akebia serrata as materials, oleanolic acid-3-O-β-D-xylopyranyl-(1→2)-α-L-arabinopyranoside can be isolated according to the extraction and separation method of Example 1.

[0045] Embodiment 5:

[0046] Using the dried fruits of Akebia trifoliata and Akebia serrata as materials, oleanolic acid-3-O-β-D-xylopyranyl-(1→2)-α-L-arabinopyranoside can be separated according to the extraction and separation method of Example 2.

[0047] Example 6: Antitumor Activity Detection of Oleanolic Acid-3-O-β-D-Xylopyranyl-(1→2)-α-L-Arabinopyranoside 6.1 Instruments and Materials

[0048] Cell lines and cell culture medium: Four human tumor cell lines, namely central nervous system tumor cells (SF-268), breast cancer cells (MCF-7), liver cancer cells (HepG2), and lung cancer cells (NCI-H460), were derived from the Guangdong Institute of Microbiology; RPMI1640 medium (Gibaco), fetal bovine serum (FBS, Gibaco), trypsin (Trypsin 1:250, Amersco), and PBS (prepared in the laboratory).

[0049] Experimental instrument: Genois microplate reader (Tecan GENios, Switzerland).

[0050] Reagents and samples: dimethyl sulfoxide (DMSO, analytical grade), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma), doxorubicin (Pfizer Italia SRL); oleanolic acid-3-O-β-D-xylopyranyl-(1→2)-α-L-arabinopyranoside was prepared as in the above experimental example.

[0051] 6.2 Test method:

[0052] a) Tumor cell culture: All four human tumor cell lines were adherent cells, cultured with RPMI164 medium plus 10% FBS and 1% double antibody, and cultured in a CO2 incubator at 37°C and 5% CO2 saturated humidity. Cells can be passaged when they cover 90% of the bottom of the culture dish. Cells in the logarithmic growth phase were used for the experiment.

[0053] b) Preparation of test samples: The compounds oleanolic acid-3-O-β-D-xylopyranyl-(1→2)-α-L-arabinopyranoside and doxorubicin were prepared into 10 mg / ml solutions with dimethyl sulfoxide (DMSO) and then diluted with culture medium to the final treatment concentration.

[0054] c) The MTT method was used to determine the half-maximal inhibitory concentration of the compound oleanolic acid-3-O-β-D-xylopyranyl-(1→2)-α-L-arabinopyranoside on tumor cells using a 96-well plate.

[0055] First, digest and collect cells in the logarithmic growth phase and adjust the cell suspension concentration to reach 5×10 4Cells / ml were inoculated into 96-well plates, and 100μl was added to each well. The 96-well plate was incubated in a 37℃, 5% CO2 incubator for 24h. The supernatant was aspirated, and then 100μl of fresh culture medium and 100μl of culture medium containing the sample solution of the required concentration for the experiment were added to each well, and mixed completely to make the final concentration of each well 200μl. The experiment was divided into different concentration test sample groups, blank control group (no sample added), and positive control group (doxorubicin). Six concentration gradients were set for each sample in the test sample group, and 4 replicates were set for each concentration. Incubate in a 37℃, 5% CO2 incubator for 72h, add MTT 20μl (5mg / ml) to each well, continue incubation for 4h, then centrifuge to remove the upper culture solution, add 150μl of DMSO to each well, and fully shake on the oscillator to dissolve the generated crystals. After 15min, put it into the microplate reader to measure the absorbance value (OD) at a wavelength of 570nm. The formula for calculating the compound inhibition rate is as follows: Inhibition rate (%) = (OD control –OD treated ) / OD control ×100%. Among them, the half inhibitory concentration (IC 50 ) Using IC 50 Calculation was performed using computer software (LOGIT method) (Xu, XY; Xie, HH; Hao, J.; Jiang, YM; Wei, XY. Eudesmanesesquiterpene glucosides from lychee seed and their cytotoxic activity. Food Chem., 2010, 123: 1123–1126).

[0056] 6.3 Experimental data see Table 1:

[0057] Table 1. In vitro antitumor cell activity IC of oleanolic acid-3-O-β-D-xylopyranyl-(1→2)-α-L-arabinopyranoside 50 (μM)

[0058]

[0059]

[0060] 6.4 Experimental conclusion:

[0061] Oleanolic acid-3-O-β-D-xylose pyranose-(1→2)-α-L-arabinopyranoside provided by the present invention has been confirmed by in vitro pharmacological experiments to have significant inhibitory activity (IC 50The values ​​were 9.43±0.09, 11.75±0.31, 9.38±0.47 and 12.66±0.02 μM, respectively. As a glycoside component isolated from edible plant resources such as Akebia trifoliata, it has multiple advantages such as low toxicity and good water solubility. Therefore, it has the potential to be developed for the preparation of related anti-tumor drugs, or can be used as a lead compound for the development of related anti-tumor drugs.

Claims

1. Oleanolic acid-3-O- β -D-Xylopyranyl-(1→2)- α -L-arabinopyranoside in the preparation of anti-tumor drugs, wherein the anti-tumor drugs are drugs for treating nervous system tumors, breast cancer or liver cancer, and the oleanolic acid-3-O- β -D-Xylopyranyl-(1→2)- α The structural formula of -L-arabinopyranoside is shown in formula (I): Formula (I).

2. An anti-tumor drug, characterized in that: Contains an effective amount of oleanolic acid-3-O- β -D-Xylopyranyl-(1→2)- α -L-arabinopyranoside as an active ingredient and a commonly used pharmaceutical excipient or carrier; the anti-tumor drug is a drug for central nervous system tumors, breast cancer or liver cancer The oleanolic acid-3-O- β -D-Xylopyranyl-(1→2)- α The structural formula of -L-arabinopyranoside is shown in formula (I): Formula (I).

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