CN105859805B - A kind of preparation method and purposes of new phenolic glycoside compound in green peel of walnut - Google Patents
A kind of preparation method and purposes of new phenolic glycoside compound in green peel of walnut Download PDFInfo
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- CN105859805B CN105859805B CN201610296216.6A CN201610296216A CN105859805B CN 105859805 B CN105859805 B CN 105859805B CN 201610296216 A CN201610296216 A CN 201610296216A CN 105859805 B CN105859805 B CN 105859805B
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- C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
- C07H15/20—Carbocyclic rings
- C07H15/203—Monocyclic carbocyclic rings other than cyclohexane rings; Bicyclic carbocyclic ring systems
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- C—CHEMISTRY; METALLURGY
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- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
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Abstract
The invention belongs to pharmaceutical technology field, is related to from Juglans mandshurica（Juglans mandshuricaMaxim）Chinese olive skin in extract isolated a kind of new phenolic glycoside compound with anti tumor activity in vitro and preparation method thereof.The method of the present invention, by alcohol extracting, macroporous resin enrichment purifying, silica gel column chromatography and gel filtration chromatography, purifies to have obtained a kind of new 4 hydroxypropiophenonepreparation of phenolic glycoside compound, 4 O using green peel of walnut as raw material in conjunction with HPLC is preparedβD glucopyranosyls (1 → 6)βD glucopyranosides [4 hydroxypropiophenone, 4 Oβ‑D‑glucopyranosyl(1→6)‑β‑D‑glucopyranoside].Raw material according to the present invention easily largely obtains, and the noval chemical compound is verified by experiments and is respectively provided with good inhibiting effect to cells such as stomach cancer, liver cancer and breast cancer.
Description
Technical field
The invention belongs to pharmaceutical technology field, and in particular to a kind of preparation of the noval chemical compound with inhibition of cancer cell effect
Method and the application in tumor is prepared.
Background technology
Malignant tumour is a kind of disease of serious threat human life, the World Health Organization (WHO) scholarly forecast, extremely
Population in the world will be up to 20,000,000 up to 8,000,000,000 cancer new cases within 2020, will have 12,000,000 people to die of cancer.Cancer into
To jeopardize the primary killers of human health.So far, the treatment method of the Common Chemotherapy of tumour is exactly to use antitumor change
Learn medicine to treat tumor patient, which has therapeutic effect, but shortcoming to primary tumor, transfer stove and subclinical transfer stove
And it is fairly obvious, such as to cells, there is powerful toxic side effect, develop immunity to drugs.So theoretically,
Chemotherapy cannot kill all tumour cell, patient's body in vivo and always have residual tumor cell, be its recurrence, the root of transfer
Source.Therefore develop new excellent effect medicine meet the needs of clinical treatment be pendulum in face of medical personal one it is very urgent
Task.
Confirmed through substantial amounts of clinical report, pericarpium juglandis there are the Several Kinds of Malignancy such as esophagus cancer and stomach cancer, liver cancer and breast cancer
Obviously inhibitory action, can alleviate the clinical symptoms of tumour patient, mitigate its pain.But antitumor work is produced to it at present
It is still inconsistent with the definite opinion of effective substance, it would be highly desirable to build pericarpium juglandis active ingredient molecule storehouse.
The content of the invention
The purpose of the present invention is to solve the above problem, expand resource, the source of tumor, there is provided Yi Zhongxin
Have inhibiting tumour cells effect compound.
In order to achieve the above object, the present invention provides 4- hydroxypropiophenonepreparation -4-O- β-D- glucopyranosyls (1 → 6)
β-D- glucopyranosides, its structural formula are as follows：
Present invention also offers 4- hydroxypropiophenonepreparations -4-O- β-D- glucopyranosyls (1 → 6)-β-D- glucopyranoses
The preparation method of glycosides：Using green peel of walnut as raw material, alcohol extracting is passed sequentially through, column chromatography is prepared.
Above-mentioned column chromatography includes macroporous resin column, normal phase silicagel column, sephadex column and prepares HPLC successively.
4- hydroxypropiophenonepreparations -4-O- β-D- glucopyranosyls (1 → 6)-β-D- glucopyranosides of the present invention are specifically made
Standby step is as follows：
（1）Alcohol extracting：By green peel of walnut（Source is the Chinese olive skin of Juglans mandshurica Juglans mandshurica Maxim）Fresh goods
The drying of 40 DEG C of low temperature, obtained 5kg dry products be raw material, are extracted 3 times using alcohol reflux, and respectively, the 1st time with 30L 60%
Ethanol extracts 2.5h, and the 2nd 60% ethanol of 25L extracts 2h, extracts 2h with 60% ethanol of 20L the 3rd time, and filtering, merges 3 filters
Liquid, is recovered under reduced pressure solvent, is dried under reduced pressure, and get dry extract shape extract；
（2）Enriching and purifying：By above-mentioned steps（1）Middle alcohol extracting gets dry extract, and shape extract is water-dispersible to be to relative density
1.30 ± 0.05 solution, through AB-8 type macroporous resin column chromatography enriching and purifyings, respectively with water, 20% ethanol, 30% ethanol successively
Elution, collects 30% ethanol eluate, solvent is recovered under reduced pressure and obtains 30% ethanol elution part；
（3）Purification on normal-phase silica gel post separation：By above-mentioned steps（2）Middle 30% ethanol elution part of gained carries out normal phase silicagel column color
Spectrum separation, respectively using volume proportion as 8:1、3:1、1:1 and 0:1 methylene chloride-methanol mixed solvent gradient elution, it is each
A ratio elutes 3.5 column volumes, will methylene chloride-methanol volume proportion be wherein 3:1 methylene chloride-methanol mixed solvent
Elution fraction is recovered under reduced pressure solvent and obtains separation product；
（4）Sephadex post separation：Learn from else's experience above-mentioned steps（3）Product after separation, by sephadex column, with first
Alcohol:Water=2:3（V/V）3 column volumes are eluted, discard first column volume eluent.Two column volume eluents below are collected,
Recycling design, obtains crude product；
（5）Prepare HPLC separation：By above-mentioned steps（4）Middle gained crude product enters preparation HPLC using methanol dissolving, flows
Dynamic is mutually that volume ratio is 32:68 methanol and water mixed solution, elution flow rate 3mL/min, retention time tR=22.6min~
After collecting cut in the 22.8min stages, recycling is drying to obtain.
Present invention also offers 4- hydroxypropiophenonepreparations -4-O- β-D- glucopyranosyls (1 → 6)-β-D- glucopyranoses
Application of the glycosides in terms of prevention and treatment tumour medicine is prepared.Preferably preparing prevention and treatment human gastric cancer, liver cancer and mammary gland
Application in terms of cancer drug.
There has been no patent or document report for above achievement in research so far.
Beneficial effects of the present invention and meaning are：The raw material used is usually taken as waste for the black cloth of walnut shell
Abandon, carry out compound extraction as raw material, the plant resources can be made full use of；In addition, this research carries out green peel of walnut
Go deep into component research and development, search out with unique chemical moieties and the stronger compound of activity, provided newly for clinical research
Type antitumor drug.
Brief description of the drawings
Fig. 1 is the chemical structural formula of the compounds of this invention；
Fig. 2 is the positivity HR-ESI-MS spectrograms of the compounds of this invention；
Fig. 3 is the compounds of this invention1H-NMR spectrum；
Fig. 4 is the compounds of this invention13C-NMR spectrograms；
Fig. 5 is the hsqc spectrum figure of the compounds of this invention；
Fig. 6 is the HMBC spectrograms of the compounds of this invention.
Embodiment
Disclosed technology contents according to the present invention, those skilled in the art will be apparent that other embodiments of the present invention,
Following embodiments only make example.In the case where not violating present subject matter and scope, various adjustment can be carried out to the present invention
And improvement.These changes should be within the scope of the present invention.With reference to specific embodiment, the present invention is described in detail.
The preparation method of one the compounds of this invention of case study on implementation：
（1）Alcohol extracting：By green peel of walnut（Source is the Chinese olive skin of Juglans mandshurica Juglans mandshurica Maxim）Fresh goods
The drying of 40 DEG C of low temperature, obtained 5kg dry products be raw material, are extracted 3 times using alcohol reflux, and respectively, the 1st time with 30L 60%
Ethanol extracts 2.5h, and the 2nd 60% ethanol of 25L extracts 2h, extracts 2h with 60% ethanol of 20L the 3rd time, and filtering, merges 3 filters
Liquid, is recovered under reduced pressure solvent, is dried under reduced pressure, and get dry extract shape extract 311g；
（2）Enriching and purifying：By above-mentioned steps（1）Middle alcohol extracting gets dry extract, and shape extract is water-dispersible to be to relative density
1.30 ± 0.05 solution, through AB-8 type macroporous resin column chromatography enriching and purifyings（Chromatography column internal diameter 6cm long 1.30m, wherein setting
Fat effective height 0.95m）, eluted successively with water, 20% ethanol, 30% ethanol respectively, collect 30% ethanol eluate, be recovered under reduced pressure
Solvent obtains 30% ethanol elution part 22.5g；
（3）Purification on normal-phase silica gel post separation：By above-mentioned steps（2）Middle 30% ethanol elution part of gained uses normal phase silicagel column（Color
Compose column internal diameter 3cm long 1.5m, wherein silica gel effective height 1.0m）, successively using methylene chloride-methanol（8:1, V/V, 3.5 cylinders
Product）→ methylene chloride-methanol（3:1, V/V, 3.5 column volumes）→ methylene chloride-methanol（1:1, V/V, 3.5 column volumes）→ methanol
Solution（3.5 column volumes）Carry out system gradient elution, will methylene chloride-methanol volume proportion be wherein 3:1 dichloromethane-
Solvent is recovered under reduced pressure to doing in methanol mixed solvent elution fraction, and weigh 0.98 g；
（4）Sephadex post separation：Learn from else's experience above-mentioned steps（3）Product after purification on normal-phase silica gel separation, is coagulated by glucan
Rubber column gel column（Internal diameter 1.5cm, column length 1.5m）, with methanol:Water=2:3（V/V）3 column volumes are eluted, discard first column volume elution
Liquid.Two column volume eluents, recycling design below are collected, obtains crude product 0.22g；
（5）Prepare HPLC separation：By above-mentioned steps（4）Middle gained crude product is further prepared with preparation HPLC（Waters
515-2414, SunFireTMThe mm of Prep C18,250 mm × 10 i.d., 5 μm）, it is 32 by volume ratio of mobile phase:68
Methanol and water mixed solution, elution flow rate 3mL/min, retention time tRCut is collected in=22.6min~22.8min stages
Afterwards, recycling is drying to obtain sterling 11.5mg.
The characterization of two the compounds of this invention of case study on implementation：
The compounds of this invention is white amorphous powder, is soluble in methanol.In UV spectrum（MeOH）Presented most at middle 256nm
It is big to absorb.In positivity HR-ESI-MS spectrums, visible at m/z 475.1865 [M+H]+Quasi-molecular ions, shows the compounds of this invention
Molecular weight is 474.With reference to1H-NMR、13The spectral datas such as C-NMR speculate that the compounds of this invention molecular formula is C21H30O12, calculate it
Degree of unsaturation is 7.Its specific spectral data is shown in Table 1.
Effect example
MTT（Tetrazolium salts）Method measures 4- hydroxypropiophenonepreparations -4-O- β-D- glucopyranosyls (1 → 6)-β-D- glucopyras
Lethal effect of the glucosides to human tumor cells.
（1）Tumour cell：Human gastric cancer, human liver cancer HepG-2 cells and MCF-7 Human Breast Cancer Cells.
（2）Test concrete operation method：Tumour cell is in DMEM matrix is incubated at（Contain 10% L-Glutamine
Hyclone, 100 μ gmL−1Penicillin, 100 μ gmL−1Streptomysin）.The tumour cell in exponential phase is taken,
0.25% Trypsin Induced is added, using concentration as 10 × 104A mL-1, take the cell suspension of 180 μ L to be placed in 96 orifice plates, often
Group is all provided with 3 parallel holes, separately sets blank well and control wells, the non-inoculating cell of blank well, control wells is not
The nutrient solution of drug containing.In 37 DEG C, 5%CO2Under the conditions of cultivate 24h after, sample to be tested is added in nutrient solution（Sample
Product are dissolved in DMSO, are progressively diluted with culture medium, add DMSO final concentration≤1% of cell herb liquid）, make medicine final concentration
For 0,10,30,50,70,90 μM.Solution continues in 37 DEG C of 5% CO2Co-incubation 48h in incubator.20 μ L are added per hole
MTT solution（5 mg/mL, are dissolved in PBS, continue after cultivating 4 h, terminate culture.Careful inhale abandons supernatant, is added per hole
150 μ L DMSO, shake 10min on shaking table, crystal is cmpletely dissolved.Surveyed with microplate reader at 570nm per hole
Light absorption value（A）, calculate cell survival inhibiting rate：Cell survival inhibiting rate %=[1- (experimental group A- blank group A)/（Control group A-
Blank group A）]×100%.Data are handled using SPSS software analysis systems.
（3）Experimental result：Survival inhibiting rate experimental data of the various concentrations noval chemical compound to tumour cell is shown in Table 2.Knot
Fruit shows that noval chemical compound has preferable growth inhibition effect to above-mentioned tumour cell, to human gastric cancer, people liver
The IC50 of cancer HepG-2 cells and human breast carcinoma MCF-7 are respectively 28.15 μM and 22.64 μM and 35.62 μM.
In conclusion isolated phenolic glycoside compound 4-hydroxy base propiophenone -4-O- from pericarpium juglandis of the present invention
β-D- glucopyranosyls (1 → 6)-β-D- glucopyranosides have the prospect for preparing clinical cancer therapy medicine.
Claims (2)
- The preparation side of compound 4-hydroxy 1. base propiophenone -4-O- β-D- glucopyranosyls (1 → 6)-β-D- glucopyranosides Method, it is characterised in that using green peel of walnut as raw material, pass sequentially through alcohol extracting, column chromatography is prepared.
- 2. the preparation method of compound described in claim 1, it is characterised in that using green peel of walnut as raw material, pass sequentially through alcohol extracting, Macroporous resin column, normal phase silicagel column, sephadex column and HPLC are prepared, and specifically comprise the following steps：（1）Alcohol extracting：By green peel of walnut, i.e. source is the Chinese olive skin of Juglans mandshurica Juglansmandshurica Maxim, and fresh goods is low 40 DEG C of drying of temperature, obtained 5kg dry products be raw material, are extracted 3 times using alcohol reflux, and respectively, the 1st time with 60% second of 30L Alcohol extracting 2.5h, the 2nd 60% ethanol of 25L extract 2h, extract 2h with 60% ethanol of 20L the 3rd time, and filtering, merges 3 filtrates, Solvent is recovered under reduced pressure, is dried under reduced pressure, get dry extract shape extract；（2）Enriching and purifying：By above-mentioned steps（1）Middle alcohol extracting get dry extract shape extract it is water-dispersible to relative density for 1.30 ± 0.05 solution, through AB-8 type macroporous resin column chromatography enriching and purifyings, is eluted successively with water, 20% ethanol, 30% ethanol respectively, is received Collect 30% ethanol eluate, solvent is recovered under reduced pressure and obtains 30% ethanol elution part；（3）Purification on normal-phase silica gel post separation：By above-mentioned steps（2）Middle 30% ethanol elution part of gained carries out normal phase silica gel column chromatography point From respectively using volume proportion as 8:1、3:1、1:1 and 0:1 methylene chloride-methanol mixed solvent gradient elution, each ratio Example 3.5 column volumes of elution, will methylene chloride-methanol volume proportion be wherein 3:1 methylene chloride-methanol mixed solvent elution Part reduced pressure recycling design obtains separation product；（4）Sephadex post separation：Learn from else's experience above-mentioned steps（3）Product after separation, by sephadex column, with methanol: Water=2:3（V/V）3 column volumes are eluted, discard first column volume eluent；Collect two column volume eluents below, recycling Solvent, obtains crude product；（5）Prepare HPLC separation：By above-mentioned steps（4）Middle gained crude product enters preparation HPLC, mobile phase using methanol dissolving It is 32 for volume ratio:68 methanol and water mixed solution, elution flow rate 3mL/min, retention time tR=22.6min~ After collecting cut in the 22.8min stages, recycling is drying to obtain.
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| CN106841433B (en) * | 2017-01-17 | 2019-06-11 | 广州浩意万医药科技有限公司 | A kind of green peel reference extract and its application |
| CN109705188B (en) * | 2018-12-27 | 2020-04-07 | 黑龙江中医药大学 | Triterpenoid compound in exocarpium Juglandis Immaturum, and preparation method and application thereof |
| CN111349134B (en) * | 2020-04-23 | 2021-08-20 | 黑龙江中医药大学 | Preparation method of dammarane type triterpene compound in walnut green husk |
| CN111704639B (en) * | 2020-06-03 | 2021-09-03 | 江南大学 | Separation method and application of phenolic acid glucoside compounds in diaphragma juglandis fructus |
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| WO2009105936A1 (en) * | 2008-02-28 | 2009-09-03 | Wang Tao | The use of phenolic glycosides derivatives in the manufacture of compositions for treating cell proliferation diseases |
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