CN111875662B - Extraction method and application of clematis midriflora stem chemical extract - Google Patents
Extraction method and application of clematis midriflora stem chemical extract Download PDFInfo
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Abstract
The invention relates to the technical field of medicines, in particular to an extraction method and application of a clematis meyeriana stem chemical extract with a molecular formula of C 47 H 76 O 16 The compound is determined to be 3-O-beta-D-glucopyranose- (1 → 4) - [ alpha-L-rhamnopyranose- (1 → 2) by adopting spectral analysis and chemical means]- β -D-xylopyranosyl-oleanolic acid glycoside. In vitro antitumor experiment shows that the compound has antitumor effect on HL-60 leukemia cells and HepG 2 The liver cancer cell has stronger cytotoxicity, and the half inhibition concentrations of the liver cancer cell to HL-60 leukemia cells and HepG2 liver cancer cells are 5.04 mu M and 3.12 mu M respectively.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to an extraction method and application of a clematis midriflora stem chemical extract.
Background
The Clematis aethiopica (Clematis potaninii) is a plant of the genus Clematis (Clematis L.) in the family Ranunculaceae (Ranunculaceae), and is grown in a shady and humid place such as a hillside with an altitude of 1700-3000 m or under a valley. Distributed in Tibet, Yunnan, Sichuan, Gansu, Shaanxi, etc. of China. The product can grow on hillside, valley, forest or stream side, and has abundant resources.
Plants of the genus Nelumbo (Clematis L.) of the family Ranunculaceae (Ranunculaceae) have more than 350 varieties and are distributed in all continents. More than 150 species exist in China, and are distributed all over the country, particularly in the southwest region. The plant has high ornamental value and is called as the queen of vine flower. This plant has high medicinal value and is frequently used in korea to treat asthma, beriberi and dysuria; as a traditional herbal medicine in india for the treatment of burns; in China, the roots or stems of the traditional Chinese medicine are commonly used as medicines for treating stranguria, edema, rheumatism, meridian paralysis and other diseases; in recent years, researches show that the clematis plants have important effects in the aspects of tumor resistance, bacteria resistance, virus resistance, oxidation resistance and the like.
To date, scholars at home and abroad have conducted chemical composition research on more than 20 plants in the genus, and have isolated various compounds from the plants, but no research report on chemical compositions of clematis maidenhair is available.
Disclosure of Invention
Aiming at the technical defects, the invention aims to provide an extraction method and application of a clematis meyeriana stem chemical extract, and aims to provide a compound with a novel structure and good activity, so as to provide a theoretical basis and an experimental basis for application and research of clematis meyeriana in antitumor drugs.
In order to solve the technical problems, the invention adopts the following technical scheme:
an application of a chemical extract of clematis beauty-Linn stem in preparing an anti-tumor drug is disclosed, wherein the chemical structure of the chemical extract of clematis beauty-Linn stem is shown as the formula (I):
the chemical name of the structure of the formula (I) is 3-O-beta-D-glucopyranose- (1 → 4) - [ alpha-L-rhamnopyranose- (1 → 2) ] -beta-D-xylopyranose-oleanolic acid glycoside, which is hereinafter referred to as ARXO.
Preferably, the chemical extract ARXO of clematis madaio stem is used for inhibiting HL-60 leukemia cells and HepG2 liver cancer cells.
Preferably, the chemical extract of clematis madaiotaeni stem can be used alone or matched with other medicines in preparation of antitumor medicines to prepare various clinically usable medicines in different dosage forms.
Preferably, the dosage form is injection, powder, pill, capsule, tablet, microcapsule, soft capsule, membrane, paste, tincture, granule or aerosol.
The invention also provides an extraction method of the chemical extract of clematis armandii stem, which comprises the following steps:
(1) extracting medicinal materials:
pulverizing dried cauliflower stalk, adding 3-5 times of 70% ethanol, heating and refluxing for 2-3 times at a weight-volume ratio for 2-3 hr each time, mixing extractive solutions, and concentrating under reduced pressure to obtain extractive solution; dispersing the extractive solution in water 2-3 times the weight volume ratio, extracting with petroleum ether 2-3 with equal volume to water, separating to obtain water phase, extracting with saturated n-butanol 3-5 with equal volume to water, mixing n-butanol extractive solutions, and concentrating under reduced pressure to obtain n-butanol layer extract;
(3) isolation of the compound:
separating the n-butanol layer extract obtained in the step (1) by adopting a 100-sand 200-mesh silica gel column chromatography, performing gradient elution by using a chloroform-methanol-water mixed solvent with the volume ratio of 50:1:0-6:3:0.5 to obtain a mixture A containing the compound ARXO shown in the formula I, and performing gradient elution by adopting a 100-sand 200-mesh silica gel column chromatography, using an ether-water mixed solvent with the volume ratio of 1:2-3 to obtain a mixture B containing the compound ARXO shown in the formula I;
(4) extraction of ARXO:
and (3) purifying the mixture B obtained in the step (2) by using an ODS (ODS) reverse phase silica gel column chromatography and a high performance liquid chromatography in sequence, wherein the ODS reverse phase silica gel column chromatography uses methanol as an eluent, the methanol solution with the concentration of 30% -68% is sequentially subjected to gradient elution according to the increasing sequence of the concentration, the methanol eluent with the concentration of 50% -68% is collected, the high performance liquid chromatography is used for purifying ethanol as the eluent, the ethanol solution with the concentration of 55% -75% is sequentially subjected to gradient elution according to the increasing sequence of the concentration, and the ethanol eluent with the concentration of 65% -75% is collected to obtain the ARXO.
Compared with the prior art, the invention has the beneficial effects that:
1. the compound ARXO of the invention is firstly separated from the plant of the genus, the compound ARXO is determined to be a new compound through the search of a SciFinder database, and the 3-position sugar chain connecting sequence (sugar chains are connected at the 2 and 4 positions of xylose) of the compound ARXO is less common in the plant saponin of the genus, the invention also carries out the research of the in vitro anti-tumor activity of the new compound, and the result shows that: the compound ARXO has remarkable activity on HL-60 leukemia cells and HepG2 liver cancer cells, and half Inhibitory Concentration (IC) 50 Value) is divided intoThe obvious inhibition effect of the compound ARXO shown in the formula I on various HL-60 leukemia cells and HepG2 liver cancer cells is also shown to further serve as a new anti-tumor drug to be researched and developed, wherein the inhibition effect is respectively 5.04 mu M and 3.12 mu M.
2. The invention preliminarily discusses the anti-tumor structure-activity relationship of the oleanane type triterpenoid saponin through in vitro anti-tumor activity screening.
3. The applicant of the present invention also published a paper entitled "study of saponin components of aerial parts of clematis meihuashanensis" in the mid-South pharmaceutical field, april 2017, vol.15 No.4, in 2017, 4.4, in zhongnan, 4.4, the inventors are heck, grand rain, plum blossom, continental clouds, royal jade plum, and thonghai peak, specifically disclosing 5 new compounds separated from clematis meihuashanensis for the first time, compared with the present application, the compounds separated in the present application are separated for the first time, and are different from the 5 new compounds in the paper; secondly, the applicant also carries out in-vitro anti-tumor activity research on the novel compound ARXO separated by the application, and the result shows that the compound ARXO has obvious activity and half Inhibition Concentration (IC) on HL-60 leukemia cells and HepG2 liver cancer cells 50 Value) was 5.04. mu.M and 3.12. mu.M, respectively; the invention provides scientific basis for reasonably developing the medicinal resources of clematis merriflora, accumulates experimental materials for further researching active substances of the clematis merriflora, provides new data for expanding the medicinal plant resources of clematis, and provides a valuable lead compound for developing new antitumor medicines.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.
FIG. 1 is an ESI-MS mass spectrum of a compound ARXO extracted from Clematis maydis stems according to the present invention;
FIG. 2 is a high resolution mass spectrogram of HR-ESI-MS of the compound ARXO extracted from clematis menhadiana stems according to the present invention;
FIG. 3 is a hydrogen spectrum of a compound ARXO extracted from the cauliflower of clematis maidenhair of the present invention;
FIG. 4 is a carbon spectrum of the compound ARXO extracted from the cauliflower of clematis maidenhair of the present invention;
FIG. 5 is the DEPT spectrum of the compound ARXO extracted from the cauliflower stalk of the clematis maidenhair of the present invention;
FIG. 6 is a HSQC spectrum of compound ARXO extracted from Clematis maydis stems according to the present invention;
FIG. 7 is an HMBC spectrum of a compound ARXO extracted from the cauliflower of the clematis maidenhair of the present invention;
FIG. 8 shows the compound ARXO extracted from Clematis maydis stems 1 H- 1 HCOSY spectrum;
FIG. 9 is a nomos spectrum of the compound ARXO extracted from the cauliflower of the invention;
FIG. 10 is a Turkes spectrum of the compound ARXO extracted from the cauliflower of Clematis maydis of the present invention.
Detailed Description
In order to make the technical solutions of the present invention better understood and enable those skilled in the art to practice the present invention, the following embodiments are further described, but the present invention is not limited to the following embodiments.
The medicinal materials used in the research of the invention are collected from Taibai mountain of Shaanxi province in 2015 8 months, and are identified as stems of Clematis beauty (Clematis potaninii) by Shaanxi Chinese medicine university. Sample specimens (No.20150831) were stored in the Natural pharmacy research laboratory of the fourth university of military medical science.
Example 1
A method for extracting a chemical extract of clematis meyeriana stem comprises the following steps:
(1) extracting medicinal materials:
pulverizing 7.5kg of dried cauliflower, adding 3-5 times of 70% ethanol, heating and reflux-extracting for 3 times (2 hr each time), mixing extractive solutions, and vacuum distilling and concentrating to obtain about 481g of extractive solution. Dispersing the extractive solution in (10L) water, extracting with petroleum ether with the same volume as water for 4 times, separating to obtain water phase, extracting with saturated n-butanol with the same volume as water for 5 times, mixing n-butanol extractive solutions, and concentrating under reduced pressure to obtain n-butanol layer extract 171.83 g.
(2) Isolation of the compounds:
weighing 170g of the butanol layer extract obtained in the step (1), separating by silica gel column chromatography (100-200 meshes), performing gradient elution by using a chloroform-methanol-water (100:1:0-6:3:0.5) mixed solvent, combining similar components by TLC to obtain a mixture A containing the compound ARXO of the formula I, selecting a plurality of components with better point property in TLC, performing separation by using 100-200 meshes silica gel column chromatography, and performing gradient elution by using an ether-water mixed solvent with a volume ratio of 1:2.5 to obtain a mixture B containing the compound ARXO of the formula I;
(3) extraction of ARXO:
and (3) purifying the mixture B obtained in the step (2) by using an ODS (ozone depleting substance) reverse phase silica gel column chromatography and a high performance liquid chromatography in sequence, wherein the ODS reverse phase silica gel column chromatography uses methanol as an eluent, the methanol solutions with the concentration of 30%, 50% and 68% are gradually and gradually eluted in sequence according to the increasing sequence, the methanol eluates with the concentration of 50% and 68% are collected, the mixture is purified by using the high performance liquid chromatography, the ethanol as the eluent, the ethanol solutions with the concentration of 55%, 65% and 75% are gradually and gradually eluted in sequence according to the increasing sequence, the fractions with the concentration of 65% and 75% are collected, and the ARXO is obtained after mixing.
The following technical characterization is carried out on 3-O-beta-D-glucopyranose- (1 → 4) - [ alpha-L-rhamnopyranose- (1 → 2) ] -beta-D-xylopyranose-oleanolic acid glycoside (compound ARXO for short) by the following specific method:
compound ARXO:
white amorphous powder, readily soluble in pyridine and methanol. The Liebermann-Burchard and Molish reactions both show positive results, and the compound is supposed to be a triterpene or a steroid saponin compound. ESI-MS (negative) gives M/z 895[ M-H ]] - ESI-MS (positive) gave M/z 919[ M + Na ]] + Peak of excimer ion [ M + Na ] in HR-ESI-MS (positive ion mode)] + Found m/z is 919.5032 (calculated value C) 47 H 76 O 16 Na,919.5031) was combined with the spectroscopic data to determine the formula of the compoundC 47 H 76 O 16 。
Analysis of the Compound ARXO 1 H-NMR Spectrum (500MHz, pyridine-d) 5 ) The middle, high field region gives 7 methyl unimodal hydrogen signals: δ 1.19, δ 1.10, δ 0.86, δ 1.01, δ 1.32, δ 0.98, δ 1.04(all in s, each 3H), and 1 alkene hydrogen signal δ 5.49(br.s,1H) appears in the low field region. Analysis in combination with HSQC Spectroscopy 13 C-NMR Spectrum (125MHz, pyridine-d) 5 ) The high field region correspondingly exhibits 7 methyl carbon signals: δ 28.5, δ 17.5, δ 016.0, δ 18.0, δ 26.7, δ 33.9, δ 24.3, the low field region shows 1 methine carbon signal δ 89.2 with oxygen, 2 characteristic alkene carbon signals δ 122.7 and δ 145.7, and 1 carboxycarbonyl carbon signal δ 181.5. Through the analysis, the compound ARXO can be preliminarily deduced to be oleanane type triterpene saponin, and the aglycone is oleanolic acid. Comprehensively analyze HSQC, HMBC, and, 1 H- 1 Two-dimensional spectrograms of HCOSY, TOCSY, NOESY and the like are used for fully attributing carbon signals and hydrogen signals of aglycone parts (shown in table 1), and then according to C-3 signals delta 89.2 and C-28 signals delta 181.5, the compound ARXO can be judged to be oleanane type triterpene saponin, the aglycone is oleanolic acid and is 3-position glycosidic monosaccharide saponin. In the NOESY spectrum, it can be seen that H-3 has NOE correlation with H-5 and H-23, thereby determining that the relative configuration of C-O bond at position 3 is beta.
The compound ARXO is subjected to acid hydrolysis by trifluoroacetic acid to prepare a sugar nitrile acetate derivative, and the sugar nitrile acetate derivative is detected to contain D-xylose (Xyl), L-rhamnose (Rha) and D-glucose (Glc) in a ratio of 1:1:1 by GC analysis and comparison with a derivative prepared by a standard sugar method. From the point of origin, the rhamnose contained in the saponin isolated from the Nelumbo plant is L-type, xylose and glucose are D-type, and the glycosyl type of the compound ARXO can also be considered as the above configuration. Observation of Compound ARXO 13 C-NMR Spectrum (125MHz, pyridine-d) 5 ) The low field region presents a terminal carbon signal of 3 sugars: : δ 105.4(XylC-1), δ 102.3(Rha C-1), δ 106.8(Glc C-1). Combined with HSQC spectra in 1 H-NMR Spectrum (500MHz, pyridine-d) 5 ) Shows a terminal hydrogen signal of 3 sugars: δ 4.79(1H, d, J ═ 6.4Hz, H-1 of Xyl), δ 6.19(1H, s, H-1 of Rha), δ 5.15(1H, d, J ═ 8.0Hz, H-1 of Glc). The relative configuration of Xyl and Glc is beta and that of Rha is alpha, as judged by the coupling constant of the sugar terminal hydrogens and the chemical shift value of the relevant carbons. The two-dimensional spectra of HSQC, HMBC, 1H-1HCOSY, TOCSY and NOESY, etc. were analyzed in a comprehensive manner to perform a total assignment of carbon and hydrogen signals for each sugar, as shown in Table 1.
Determination of the order of linkage of sugar chains, δ, by HMBC Spectroscopy H 4.79(H-1 of Xyl) and delta C 89.2(C-3) there is a remote correlation, δ H 6.19(H-1 of Rha) and δ C 76.8(C-2 of Xyl) there is a remote correlation, δ H 5.15(H-1 of Glc) and δ C 80.1(C-4 of Ara) there is a long-range correlation, which indicates that Xyl is linked to the 3-position of aglycon, Rha is linked to the 2-position of Xyl, and Glc is linked to the 4-position of Xyl. The above-described attachment was further verified by NOESY spectral analysis.
Process for preparation of ARXO as compound in Table 1 1 H-NMR Spectrum (500MHz, pyridine-d) 5 ) And 13 C-NMR Spectroscopy (125MHz, pyridine-d) 5 ) Data of
| C | δ C | δ H | δ C | C | δ H |
| 1 | 39.4 | 0.93m,1.49m | 3-O-sugar | / | / |
| 2 | 27.1 | 1.83m,2.05m | Xyl-1 | 105.4 | 4.79(d,J＝6.4Hz) |
| 3 | 89.2 | 3.22(dd,J＝12.0,4.8) | 2 | 76.8 | 4.54m |
| 4 | 40.0 | / | 3 | 74.4 | 4.33m |
| 5 | 56.5 | 0.77(d,J＝12.0Hz) | 4 | 80.1 | 4.32m |
| 6 | 19.0 | 1.31m,1.48m | 5 | 65.0 | 3.84(d,J＝11.4Hz),4.44m |
| 7 | 31.5 | 1.21m,1.46m | Rha-1 | 102.3 | 6.19s |
| 8 | 40.2 | / | 2 | 72.8 | 4.76m |
| 9 | 49.1 | / | 3 | 73.0 | 4.64m |
| 10 | 37.5 | / | 4 | 74.5 | 4.30m, |
| 11 | 24.4 | 1.92m,2.03m | 5 | 70.3 | 4.64m |
| 12 | 122.7 | 5.49br.s | 6 | 19.1 | 1.66(d,J＝6.4Hz) |
| 13 | 145.7 | / | |||
| 14 | 42.7 | / | Glc-1 | 106.8 | 5.15(d,J＝8.0Hz) |
| 15 | 28.9 | 2.22m | 2 | 76.0 | 4.06(t,J＝8.0Hz) |
| 16 | 24.4 | 2.92m,2.03m | 3 | 79.3 | 3.92m |
| 17 | 47.3 | / | 4 | 71.7 | 4.27m |
| 18 | 42.6 | 4.34(dd,J＝13.6,4.0) | 5 | 79.0 | 4.23(t,J＝8.8Hz) |
| 19 | 47.2 | 1.30m,1.83m | 6 | 63.0 | 4.38m,4.52m |
| 20 | 30.5 | / | / | / | / |
| 21 | 34.9 | 1.21m,1.50m | / | / | / |
| 22 | 33.7 | 1.31m,1.46m | / | / | / |
| 23 | 28.5 | 1.19(3H,s) | / | / | / |
| 24 | 17.5 | 1.11(3H,s) | / | / | / |
| 25 | 16.0 | 0.86(3H,s) | / | / | / |
| 26 | 18.0 | 1.01(3H,s) | / | / | / |
| 27 | 26.7 | 1.32(3H,s) | / | / | / |
| 28 | 181.5 | / | / | / | |
| 29 | 33.9 | 0.98(3H,s) | / | / | / |
| 30 | 24.3 | 1.04(3H,s) | / | / | / |
Note: "/" indicates that there is no such item of data.
In conclusion, the compound ARXO is 3-O-beta-D-glucopyranosyl- (1 → 4) - [ alpha-L-rhamnopyranosyl- (1 → 2) ] -beta-D-glucopyranosyl oleanic acid. The SciFinder database searches and determines that the compound is a new compound.
The first compound ARXO obtained is studied for its antitumor activity and tested for its antitumor activity in vitro, as follows:
(1) and (3) testing a sample: the compound ARXO;
(2) the experimental method comprises the following steps:
2.1 preparation of the solution
MTT working solution: weighing 50mg MTT powder, dissolving with PBS and fixing the volume to 10mL to prepare 5mg/mL stock solution, filtering and sterilizing with a 0.22 mu m filter membrane, and storing at 4 ℃ for later use.
Phosphate Buffered Saline (PBS): dissolving 8.0g of sodium chloride, 0.2g of potassium dihydrogen phosphate and 3.48g of disodium hydrogen phosphate in 800mL of deionized water, stirring for dissolving, adjusting the pH value to 7.2-7.4, adding deionized water to a constant volume of 1000mL, autoclaving, and storing at 4 ℃ for later use.
2.2 culture of cell lines
At 37 ℃ 5% CO 2 In an incubator with saturated humidity, the human hepatoma HepG2 cells, which are adherent cells, were cultured in DMEM medium containing 10% fetal bovine serum, 100u/mL penicillin and 100u/mL streptomycin. The culture method of HL-60 cells is the same as that of human liver cancer HepG2 cells, but the HL-60 cells adopt RPMI1640 culture medium, and the cells are suspension cells.
2.3 MTT assay to determine the Effect of Compounds on the proliferation of HL-60 cells
HL-60 cells in the logarithmic growth phase were collected, counted and made into 1X 105/mL suspensions, and 200. mu.l per well of 96-well culture plates were added. After 24h, the drug was administered into a positive control group and a test drug group (80, 40,20,10,5 and 2.5. mu. mol/L, respectively), each group having 3 multiple wells, in a cell culture chamber (37 ℃, 5% CO) 2 ) After the medium incubation for 72h, 20 mul MTT test solution is added for incubation for 4h, after the cell culture solution is discarded, 150 mul DMSO solution is added and the shaking is carried out for 10min, the absorbance value is measured by an enzyme linked immunosorbent assay detector at the wavelength of 490nm, and the cell inhibition rate is calculated. The positive control is adriamycin.
2.4 MTT assay for the Effect of Compounds on the proliferation of HepG2 cells
HepG2 cells in the exponential growth phase were digested with 0.25% trypsin, adherent cells were detached and a cell suspension of 5X 104 cells/mL was prepared. The cells were inoculated in a 96-well plate at 200. mu.l/well in a cell incubator (37 ℃ C., 5% CO) 2 ) And (4) performing medium incubation for 24 h. Every other day, the solution was changed, divided into positive control group and test drug group (80, 40,20,10,5 and 2.5. mu. mol/L, respectively), each group was plated with 3 duplicate wells and cultured for 72 hours. MTT was added to a 96-well plate at 20. mu.l/well and reacted in an incubator for 4 hours. The supernatant was aspirated off, and 150. mu.l of DMSO solution was added to each well and shaken for 10 min. The ELISA measures the absorbance of each well at 490nm and counts the cellsThe inhibition rate. The positive control is adriamycin.
2.5 formula for calculating cell inhibition ratio
Cell inhibition rate (negative control OD value-treatment OD value)/negative control OD value × 100%). Calculating half Inhibitory Concentration (IC) by SPSS software 50 )。
3 results and discussion of the experiments
According to the types of aglycone and sugar chain of the compound, the compound ARXO is selected for primary screening of antitumor activity, and the specific table is shown in table 2.
TABLE 2 in vitro killing Activity of the Compound ARXO against human hepatoma HepG2 cells and human leukemia HL-60 cells (IC) 50 ，μM)
Research shows that the mixture B has an inhibiting effect on HL-60 leukemia cells and HepG2 liver cancer cells, based on the results, the mixture B is separated again to obtain a compound ARXO, the compound ARXO has strong cytotoxicity on the HL-60 leukemia cells and the HepG2 liver cancer cells, the compound ARXO has obvious activity on the HL-60 leukemia cells and the HepG2 liver cancer cells, and IC is IC 50 5.04. mu.M and 3.12. mu.M, respectively.
Compared with the adriamycin in the prior art, although the cytotoxicity of the adriamycin is better than that of the ARXO obtained by separation, the adriamycin has certain side effects on normal cells and organisms, and the specific expression is as follows: affecting bone marrow hematopoiesis, and reducing platelets and leucocytes; has cardiotoxicity, and can cause heart failure when severe; can be used for treating nausea, emesis, stomatitis, alopecia, hyperpyrexia, phlebitis and skin pigmentation; with few fever, hemorrhagic erythema and impaired liver function; the ARXO obtained by separation in the application is a natural substance, and the laboratory animal experiment result shows that: the ARXO has no side effect on rat body basically, and is a safer antitumor compound.
According to the structure of the compound, C-28 position of the compound ARXO is biglycan saponin forming ester-glycosidic bond, the activity of the compound ARXO is strong, the reason is probably related to the number of glycosyl, and the activity of increasing the number of glycosyl is weakened. The strong activity of the compound ARXO we speculate that it may be the presence of a branched sugar group at the C-3 position of the compound ARXO.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations. The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of protection is not limited thereto. The equivalents and modifications of the present invention which may occur to those skilled in the art are within the scope of the present invention as defined by the appended claims.
Claims (3)
1. The application of the chemical extract of clematis beauty-Linn stem in preparing the antitumor drugs is characterized in that the chemical structure of the chemical extract of clematis beauty-Linn stem is shown as the formula (I):
the chemical name of the structure of the formula (I) is 3-O-beta-D-glucopyranose- (1 → 4) - [ alpha-L-rhamnopyranose- (1 → 2) ] -beta-D-xylopyranose-oleanolic acid glycoside, which is hereinafter referred to as ARXO;
the chemical extract of the clematis meihuashanensis stem is extracted according to the following steps:
(1) extracting medicinal materials:
pulverizing dried cauliflower stalk, adding 3-5 times of 70% ethanol, heating and refluxing for 2-3 times at a weight-volume ratio for 2-3 hr each time, mixing extractive solutions, and concentrating under reduced pressure to obtain extractive solution; dispersing the extractive solution in water 2-3 times the weight volume ratio, extracting with petroleum ether 2-3 with equal volume to water, separating to obtain water phase, extracting with saturated n-butanol 3-5 with equal volume to water, mixing n-butanol extractive solutions, and concentrating under reduced pressure to obtain n-butanol layer extract;
(2) isolation of the compound:
separating the n-butanol layer extract obtained in the step (1) by adopting a 100-200-mesh silica gel column for chromatography, performing gradient elution by using a chloroform-methanol-water mixed solvent with the volume ratio of 50:1:0-6:3:0.5 to obtain a mixture A containing the compound ARXO of the formula I, and performing gradient elution by adopting a 100-200-mesh silica gel column with the volume ratio of 1:2-3 to obtain a mixture B containing the compound ARXO of the formula I;
(3) extraction of ARXO:
and (3) purifying the mixture B obtained in the step (2) by using an ODS (ozone depleting substance) reverse phase silica gel column chromatography and a high performance liquid chromatography in sequence, wherein the ODS reverse phase silica gel column chromatography uses methanol as an eluent, the methanol solution with the concentration of 30% -68% is sequentially subjected to gradient elution according to the increasing sequence of the concentration, the methanol eluent with the concentration of 50% -68% is collected, the high performance liquid chromatography is used for purifying the mixture B by using ethanol as an eluent, the ethanol solution with the concentration of 55% -75% is sequentially subjected to gradient elution according to the increasing sequence of the concentration, and the ethanol eluent with the concentration of 65% -75% is collected, so that the ARXO is obtained.
2. The application of the chemical extract of clematis armandii stem ARXO in preparing the antitumor drugs according to claim 1, wherein the chemical extract of clematis armandii stem ARXO is used for inhibiting HL-60 leukemia cells and HepG2 liver cancer cells.
3. The application of the chemical extract of clematis filamentosa Dunn stem in preparing the antitumor drug as claimed in claim 1, wherein the dosage form is injection, powder, pill, capsule, tablet, microcapsule, membrane, paste, tincture, granule or aerosol.
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| US6444233B1 (en) * | 1998-05-19 | 2002-09-03 | Research Development Foundation | Triterpene compositions and methods for use thereof |
| CN102247393A (en) * | 2011-05-30 | 2011-11-23 | 江西本草天工科技有限责任公司 | Preparation method of oleanolic acid saponin component and application thereof |
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| CN103169723A (en) * | 2011-12-22 | 2013-06-26 | 江西本草天工科技有限责任公司 | Preparation method and application of oleanolic acid disaccharide saponin component |
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| US6444233B1 (en) * | 1998-05-19 | 2002-09-03 | Research Development Foundation | Triterpene compositions and methods for use thereof |
| CN102247393A (en) * | 2011-05-30 | 2011-11-23 | 江西本草天工科技有限责任公司 | Preparation method of oleanolic acid saponin component and application thereof |
| CN103169723A (en) * | 2011-12-22 | 2013-06-26 | 江西本草天工科技有限责任公司 | Preparation method and application of oleanolic acid disaccharide saponin component |
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