CN1243103C - Preparation process of burduck protoglucoside of anti virus and antiptmour natural medicine - Google Patents
Preparation process of burduck protoglucoside of anti virus and antiptmour natural medicine Download PDFInfo
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- CN1243103C CN1243103C CN 200410015465 CN200410015465A CN1243103C CN 1243103 C CN1243103 C CN 1243103C CN 200410015465 CN200410015465 CN 200410015465 CN 200410015465 A CN200410015465 A CN 200410015465A CN 1243103 C CN1243103 C CN 1243103C
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Abstract
The present invention relates to a preparation process of burduck protoglucoside of natural anti-virus and anti-tumor medicines, which is characterized in that a Chinese medicinal material called burdock fruit is pulverized and degreased by organic solvent to obtain a crude arctiin product; the crude arctiin product make fermentation and enzymolysis by glusulase in an enzyme reaction system; finally, an enzymolysis reaction is terminated by alcohol-containing water-soluble organic solvent and enzymolysis products are extracted; the enzymolysis products are separated by a silica gel column chromatography and solvent recrystallization and purified to obtain a pure burduck protoglucoside product. The manufacturing method of natural anti-virus and anti-tumor medicines has the progress and the practicability that the yield of the burduck protoglucoside is increased by at last over 20 times as compared with that of separating natural-existing burduck protoglucoside in plants such as burdock fruit, etc. The present invention can achieve the purpose of producing the burduck protoglucoside with low costs industrially and is economical and practical.
Description
Technical field
The present invention relates to process for preparing medicine, specifically, relate to a kind of novel preparation method of antiviral and active compound for anti tumor l-arctigenin (l-arctigenin, English name Arctigenin).Method of the present invention is meant by the method for enzymatic conversion in conjunction with chromatographic separation, makes l-arctigenin from arctinin.
The advance and the practical value of manufacture method of medicine of the present invention be, its yield is than l-arctigenin that separating natural from plants such as Great Burdock Achene exists, improve 20 at least surplus times.Can reach the purpose of low-cost suitability for industrialized production l-arctigenin, economical and practical.
Background technology
L-arctigenin is a kind of natural organic-compound with antiviral, antitumor and immunoregulation effect.Can be used for treating influenza, common cold and acquired immune deficiency syndrome (AIDS), or neoplastic diseases such as treatment lung cancer, leukemia.
Great Burdock Achene is traditional Chinese medical science dispelling wind-heat commonly used, detumescence relieve sore throat Chinese medicine.Be used for the treatment of illnesss such as common cold due to wind-heat, swelling and pain in the throat, macula are not saturating, carbuncle sore tumefacting virus as the prescription composition.For example, Chinese medicine preparations such as Lonicerae and Forsythiae Powder often use Great Burdock Achene, and have followed traditional medication (decoction) or extracting method (water boiling and extraction).The ordinary skill of this area can both be understood, and l-arctigenin (Arctigenin) belongs to Lignanoids compounds, coexists as in the medicinal material Great Burdock Achene with its glycoside arctinin (Arctiin), and the latter is the main chemical compositions of medicinal material.Regulation arctinin content should reach more than 5% of quality of medicinal material under Chinese Pharmacopoeia (version in 2000) the burdock subitem; Lack a part glucosyl residue than arctinin on the arctinin meta structure, the content in the Great Burdock Achene medicinal material is about 0.2% or lower.Because the relation of structure, l-arctigenin and arctinin belong to fat-soluble cpds, be soluble in ethanol, methyl alcohol, acetone, vinyl acetic monomer and organic solvents such as methylene dichloride, chloroform, be insoluble in water and hot water (l-arctigenin) or only be partially soluble in hot water (arctinin).Therefore, with regard to extracting l-arctigenin and arctinin, it is unfavorable using water as solvent." Chemistry for Chinese Traditional Medicine " (Science and Technology of Shanghai press,, 109 pages in 1987) have been put down in writing the extracting method of arctinin.Processing condition comprise that the Great Burdock Achene meal adds diethyl ether, and extract (sloughing grease) with soxhlet extraction, and the medicinal material slag adds alcohol reflux, and extract is dissolved in hot water, adds the removal of impurity of plumbic acetate solution, logical hydrogen sulfide deleading, and step such as crystallization obtains arctinin in the aqueous solution; Acta Pharmaceutica Sinica (1992 27 the 7th phases of volume) had once been reported by arctinin synaptase enzymolysis, and can have been produced l-arctigenin with the method for silica gel column chromatography.Processing condition comprise with synaptase and mixing, and are dissolved in water, placed 48 hours at 37 ℃, and enzymolysis mixture dichloromethane extraction, silica gel column chromatography on the extract with methylene dichloride-vinyl acetic monomer (5: 4) wash-out, separates obtaining the l-arctigenin crystallization.
Summary of the invention
Purpose of the present invention is intended to overcome the deficiencies in the prior art and a kind of inflammable and explosive narcotic ether, deleterious heavy metal lead salt got rid of is provided, and utilizes the commodity helicase that is easy to obtain that the arctinin crude product is carried out enzymolysis and prepare the method for antiviral and antitumor natural drug l-arctigenin.
The object of the present invention is achieved like this:
A kind of method for preparing antiviral and antitumor natural drug l-arctigenin, be that the Chinese medicinal materials Great Burdock Achene is pulverized the back with hanging down the polar organic solvent degreasing, get the arctinin crude product, make its enzymolysis that in enzymatic reaction system, ferments with helicase again, at last to contain alcohol-soluble and water-soluble organic solvent enzymolysis reaction and to extract enzymolysis product, recycle silicon glue column chromatography is separated enzymolysis product with solvent recrystallization, purifying and obtain the pure product of l-arctigenin.
The organic solvent of described Great Burdock Achene degreasing is advisable with industrial naptha or sherwood oil (boiling spread is preferably 60 ℃-90 ℃), and consumption is 6～15 times of Great Burdock Achene powder.
The enzymatic reaction system of described fermentation enzymolysis is aqua sterilisa or stroke-physiological saline solution or aseptic saliferous phosphoric acid buffer, 25～45 ℃ of temperature, and PH3～8, in 12～48 hours reaction times, the suitable consumption of helicase is 1/2.5～1/10 of a substrate arctinin consumption.
Described enzymolysis reaction and extract enzymolysis product contain the alcohol-soluble and water-soluble organic solvent with methyl alcohol, ethanol contain methyl alcohol or the alcoholic acid acetone mixed solvent for well, consumption is 6～10 times of enzymatic hydrolysis system volume.
After described separation to enzymolysis product, purifying are meant that enzymolysis stops, get and contain alcohol-soluble and water-soluble organic solution evaporated under reduced pressure, residue adds the chloroform dissolving, get chloroform solution, add anhydrous sodium sulfate dehydration, filtration, evaporated under reduced pressure gets the mixture that mainly contains l-arctigenin again, separate this aglycon with silica gel column chromatography again, and with organic solvent recrystallizations such as methyl alcohol.
Described silica gel column chromatography is meant the one or many column chromatography, and packing material is a silica gel, and eluent is chloroform/methanol (100: 0～95: 5).
At first, the invention discloses a kind of method of extracting arctinin, it is characterized in that from raw medicinal material Great Burdock Achene (Arctium lappa L.), extracting arctinin crude product (containing arctinin and a small amount of l-arctigenin).Detailed process comprises pulverizing, degreasing, alcohol extracting.Feature is with pulverizing medicinal materials, with industrial naptha or sherwood oil (30 ℃-90 ℃ of boiling spreads, be preferably 60 ℃-90 ℃) extraction (fully degreasing), with containing three carbon atoms following lower alcohol extraction l-arctigenin and arctinin, extracting mode comprises cold soaking (diacolation), reflux and supersound extraction again; Optimal extraction solvent is the medicinal alcohol that contains ethanol 60%-95% (volume ratio).Different with document is at degreasing process, substitutes ether with industrial naptha or sherwood oil.
Secondly, the invention discloses a kind of method of making l-arctigenin, it is characterized in that carrying out enzymolysis with helicase under given conditions from arctinin crude product or pure product, binding silica gel column chromatography and recrystallization obtain high conversion, the pure product of highly purified l-arctigenin.Feature is with any one processing in the following dual mode of arctinin crude product employing, to make and to obtain the antiviral natural medicine l-arctigenin of capacity.
First method: adopt 1 time or the method for silica gel column chromatography (column chromatography) repeatedly, obtain the pure product of a small amount of l-arctigenin and a large amount of pure product of arctinin respectively.Method with enzymically hydrolyse makes arctinin be converted into l-arctigenin again.Used column chromatography (column chromatography) comprises with silica gel to be the normal-phase chromatography of packing material and to be the reversed phase chromatography of packing material with alkanisation silica gel; Used enzymically hydrolyse method is characterised in that uses commercially available commodity helicase, be the enzyme reaction system of medium with aqua sterilisa, sterile saline or sterilization saliferous phosphoric acid buffer, in optimal temperature, appropriate pH value, the reflection suitable time, add then and contain alcohol-soluble and water-soluble organic solvent (refer in particular to: methyl alcohol or ethanol or contain methyl alcohol or alcoholic acid acetone) precipitation enzymolysis product, get the filtrate evaporate to dryness, residue obtains pure product l-arctigenin through recrystallization and/or column chromatography.
Second method: directly will contain a large amount of arctinins and a small amount of l-arctigenin and the arctinin crude product, make it to be converted into l-arctigenin with the method for enzymically hydrolyse.Used enzymically hydrolyse method is characterised in that uses commercially available commodity helicase, be the enzyme reaction system of medium with aqua sterilisa, sterile saline or sterilization saliferous phosphoric acid buffer, in optimal temperature, appropriate pH value, the reflection suitable time, add then and contain alcohol-soluble and water-soluble organic solvent (refer in particular to: methyl alcohol or ethanol or contain methyl alcohol or alcoholic acid acetone) precipitation enzymolysis product, get the filtrate evaporate to dryness, residue obtains pure product l-arctigenin through recrystallization and/or column chromatography.
The yield of enzymolysis process can reach more than 75% usually.
The method of the invention, different with existing patent or document is (1) at the silica gel column chromatography separation circuit, what the present invention used is that sherwood oil-acetone is as eluting solvent; (2) adopted new enzymolysis operation, the present invention discloses the utilization of helicase in producing the l-arctigenin process first; Optimize the condition of enzyme digestion reaction simultaneously by careful research, comprised ratio, temperature, time, enzymolysis medium of substrate and enzyme etc.
Different with existing patent or document and improve to some extent local as follows: the one, find that arctinin (arctinin) only is partially dissolved in hot water, it is very big that alcohol extract will change the hot water amount who is dissolved in the hot water needs, further handle used plumbic acetate consumption and also must strengthen, technological operation has tangible irrationality; The 2nd, avoid having used the heavy metallic salt plumbic acetate, make product remove the residual of heavy metal and harmful element from, also removed the loaded down with trivial details technology of removing sulfide naturally from; The 3rd, adopt when stopping enzymolysis to contain alcohol-soluble and water-soluble organic solvent (refer in particular to: methyl alcohol or ethanol or contain methyl alcohol or alcoholic acid acetone) precipitation helicase and stripping l-arctigenin, avoided a large amount of use noxious solvent methylene dichloride or chloroform; The 4th, the inventive method is particularly suitable for directly carrying out with high performance liquid chromatography the mensuration of content and enzymolysis yield; The 5th, be with chloroform/methanol (100: 0-95: 5) wash-out during with the silica gel column chromatography separating purification aglycon.
Practicality of the present invention is, because enzyme digestion reaction is converted into l-arctigenin expeditiously with high-load arctinin, increased from the unit mass medicinal material amount that obtains l-arctigenin greatly, more than the pure product l-arctigenin yield that obtains of two kinds of methods all than surplus original content improves 20 at least in the medicinal material times.
Description of drawings: table 1, table 2 are respectively the level of factor table and the test-results tables of arctinin enzymolysis orthogonal test.
Embodiment
Describe the present invention for example with embodiment below, but these embodiment must not be interpreted as the restriction to claim of the present invention of going up in all senses.
The extraction and the enzymolysis of embodiment 1 arctinin crude product
With the Great Burdock Achene pulverizing medicinal materials, add 6-15 and doubly measure industrial naptha or sherwood oil (60 ℃-90 ℃ of boiling spreads), cold soaking or refluxing extraction 3 times filter.The powder of getting it filled falls solvent evaporates.Add 6-15 and doubly measure 75% medicinal alcohol, cold soaking under the room temperature (diacolation) or supersound extraction, or heating and refluxing extraction.Filter, merging filtrate, decompression and solvent recovery, residue promptly get the arctinin crude product through vacuum-drying.
Adopt snail enzymolysis arctinin to produce l-arctigenin.Adopt orthogonal design to investigate the top condition of enzymolysis.With substrate/enzyme mass ratio, reaction times, reaction medium, solvent pH is four investigation factors, and the yield that generates l-arctigenin with reaction is an index, investigates the influence of each factor to reacting under three different levelss.
Precision takes by weighing each 3 parts of arctinin crude product 200mg, adds water, physiological saline, each 60ml of saliferous phosphoric acid buffer respectively, boils dissolving, is cooled to room temperature.Add reaction medium under the aseptic condition and replenish steam output.Every kind of solution is divided into 3 parts again, accurate each 15ml (wherein containing arctinin 50mg) that draws is adjusted to design pH value with 0.1% hydrochloric acid or 0.1% sodium hydroxide solution respectively, adds the helicase of set amount, put in the constant temperature oscillator, cultivate according to setting-up time in 37 ℃.
Add 8 times of amount methyl alcohol termination reactions, placement is spent the night, and filters.Get subsequent filtrate, through 0.45 μ m filtering with microporous membrane, automatic sampling 5 μ L.The results are shown in Table 1,2.Learn that by table 2 suitable enzymatic hydrolysis condition is A
1B
2-3C
1-2D
1-3, promptly at 37 ℃, when substrate is 5 times of amounts of the helicase commodity bought, in the sterilized water of pH5 or physiological saline enzymolysis 36-48 hour the most suitable.Further carrying out single factor of enzyme dosage on this basis investigates.3 levels of substrate/enzyme (1: 1,2.5: 1,5: 1) are compared, and the yield of l-arctigenin is followed successively by 54.83%, 71.64% and 78.06%.Illustrate that substrate/enzyme (5: 1) ratio still is best enzyme dosage.
According to A
1B
2C
1D
1Condition is amplified the enzymolysis test.Get the aqueous methanol solution after enzymolysis stops, evaporated under reduced pressure.Residue adds the chloroform dissolving, gets chloroform solution, adds anhydrous sodium sulfate dehydration, filters, and evaporated under reduced pressure promptly gets all aglucone.Get all aglucone silica gel (200-300 order) column chromatography (1-2 time), eluent is a chloroform/methanol (100: 0-95: 5), indicate with TLC (chloroform/methanol (97: 3) expansion), collection is the cut 00 of principal constituent with the l-arctigenin, recrystallizing methanol gets colourless platelet, and HPLC checks that purity is 99%, mp 99-101 ℃, [α]
D-32 ° (EtOH).
Present embodiment has determined to use the method for commercially available helicase from arctinin production aglycon, and determined condition is satisfied for the enzymolysis yield of arctinin, reaches 72-76%, and technology is convenient to amplify.
The enzymolysis yield determination of embodiment 2 l-arctigenins
1, material and instrument
The pure product of arctinin are off-white powder for self-control, [α]
D-47 ° (c1.8, EtOH).Thin-layer chromatography (TLC) is shown as single spot, and high performance liquid chromatography (HPLC) records purity 99%.Its TLC behavior and spectrum such as IR, MS are consistent with the arctinin reference substance.Helicase is purchased in source, Shanghai consor thing Science and Technology Ltd. (lot number 011129).Silica gel G F
254The TLC plate is Haiyang Chemical Plant, Qingdao's product.The instrument that uses also comprises: HZQ-C constant temperature oscillator (Harbin Donglian Electronic ﹠ Technology Development Co., Ltd.), the Dionex high performance liquid chromatograph, the P680 infusion pump, the ASI-100 automatic sampler, the PDA-100 photodiode array detector, the Chromeleon chromatographic working station, PHS-25 digital display PH counts (Shanghai Precision Scientific Apparatus Co., Ltd), HN1006 ultrasonic cleaning machine (Chinese south China ultrasonic equipment factory).Water is tri-distilled water, and physiological saline is commercially available injection physiological saline, and the saliferous phosphate buffer solution is pressed two appendix method preparations of Chinese Pharmacopoeia.
2, content assaying method
2.1 the content of arctinin is measured according to a burdock subitem below of Chinese Pharmacopoeia version in 2000 method.
2.2 the assay condition of l-arctigenin
Chromatographic column: Kromasil KR100-5C
18Post (250 * 4.6mm, 5 μ m); 30 ℃ of column temperatures.Moving phase: methanol-water=60: 40 (v/v).Flow velocity: 1.0mL/min; Detect wavelength: 280nm.
Typical curve
It is an amount of that precision takes by weighing the l-arctigenin reference substance, adds dissolve with methanol, is mixed with the solution of 0.5mg/mL, and redilution becomes 0.25,0.125,0.0625,0.0315, the reference substance solution of 0.016mg/mL.Sample size 5 μ L measure peak area, are X-coordinate with reference substance concentration, and peak area is an ordinate zou, and calculate regression equation and be: Y=83.2861C+0.3309, r=0.99960 shows that l-arctigenin content has favorable linearity between 0.08～2.5 μ g scope.
Recovery test
According to A
1B
2C
1D
1The condition enzymolysis, add l-arctigenin reference liquid (5mg/ml) 1,2,3mL, carry out recovery test according to basic, normal, high three levels, the l-arctigenin rate of recovery (%) that records is respectively 101.3 ± 1.84,94.1 ± 0.57 and 95.1 ± 2.12 (the l-arctigenin amount is 13.66mg in the sample liquid).
The enzyme digestion reaction theory must be measured=50 * (C
21H
24O
6/ C
27H
34O
11)=34.83 (mg);
L-arctigenin yield Y (%)=(W/34.83) * 100%, W is the amount of recording.
Present embodiment determined l-arctigenin content and rate get measuring method, possess accurate, reliable, easy characteristics.
Described enzymolysis reaction and extract enzymolysis product contain the alcohol-soluble and water-soluble organic solvent with methyl alcohol, ethanol contain methyl alcohol or the alcoholic acid acetone mixed solvent for well, consumption is 6～10 times of enzymatic hydrolysis system volume.
After described separation to enzymolysis product, purifying are meant that enzymolysis stops, get and contain alcohol-soluble and water-soluble organic solution evaporated under reduced pressure, residue adds the chloroform dissolving, get chloroform solution, add anhydrous sodium sulfate dehydration, filtration, evaporated under reduced pressure gets the mixture that mainly contains l-arctigenin again, separate this aglycon with silica gel column chromatography again, and with organic solvent recrystallizations such as methyl alcohol.
Described silica gel column chromatography is meant the one or many column chromatography, and packing material is a silica gel, and eluent is chloroform/methanol (100: 0～95: 5).
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| CN100443493C (en) * | 2006-06-02 | 2008-12-17 | 辽宁中医药大学 | A kind of method for preparing arctiin and its aglycone |
| CN101392279B (en) * | 2008-10-21 | 2011-06-29 | 浙江工业大学 | A kind of preparation method of arctigenin |
| EP2412377B1 (en) * | 2009-03-27 | 2018-12-26 | Kracie Holdings Ltd. | Burdock fruit extract containing arctigenin at high content and process for producing same |
| CN101736050B (en) * | 2009-12-08 | 2012-07-18 | 辽宁中医药大学 | Preparation method of arctigenin |
| CN102210653B (en) * | 2010-04-03 | 2014-06-04 | 鲁南制药集团股份有限公司 | Burdock aglycone microemulsion |
| WO2012043549A1 (en) | 2010-09-27 | 2012-04-05 | クラシエ製薬株式会社 | Arctigenin-containing bardanae fructus extract and method for producing same |
| CN102440986B (en) * | 2010-10-08 | 2014-12-03 | 鲁南制药集团股份有限公司 | Application of arctigenin in preparation of medicines for preventing and treating bone marrow suppression caused by radiation or chemicals |
| CN102464635B (en) * | 2010-10-30 | 2015-07-22 | 山东新时代药业有限公司 | Separation and purification method of arctigenin |
| CN102154417A (en) * | 2010-12-13 | 2011-08-17 | 天津中医药大学 | Method for preparing periplocymarin |
| CN102352401A (en) * | 2011-07-26 | 2012-02-15 | 苏州宝泽堂医药科技有限公司 | Method for preparing periplogenin through enzymatic hydrolysis |
| CN103356477A (en) * | 2012-04-05 | 2013-10-23 | 山东新时代药业有限公司 | Arctigenin-containing subcutaneous injection and application thereof |
| ITMI20131495A1 (en) | 2013-09-10 | 2015-03-11 | Ce R C Ar Di Paolo Maestri | USEFUL COMPOSITIONS FOR CHEMOTHERAPY-RESISTANT CANCER TREATMENT |
| EP3169364A1 (en) | 2014-07-17 | 2017-05-24 | Probiotical S.p.a. | Compositions comprising melatonin and flavonoids for use in the treatment of tumours resistant to chemotherapy |
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