CN101851272A - Glaucocalyxin B, derivative, preparation method and application thereof - Google Patents
Glaucocalyxin B, derivative, preparation method and application thereof Download PDFInfo
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- CN101851272A CN101851272A CN200910048553A CN200910048553A CN101851272A CN 101851272 A CN101851272 A CN 101851272A CN 200910048553 A CN200910048553 A CN 200910048553A CN 200910048553 A CN200910048553 A CN 200910048553A CN 101851272 A CN101851272 A CN 101851272A
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- glaucocalyxin
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Abstract
The invention discloses a glaucocalyxin B derivative, a preparation method and application thereof. The glaucocalyxin B (GLB) is used as a main body of the glaucocalyxin B (GLB) derivative which is a diterpenes substance and has an ent-kaurane structure, carbonyl, polypeptide cNGQGEQc and/ or hydroxyl. In the invention, rabdosia amethystoides medicinal material (part on the above-ground) is used as a raw material, and the preparation method of the derivative is as follows: crashing the raw material, extracting, treating solution, passing through a column, recrystallizing and carrying out other steps to obtain the glaucocalyxin B (GLB), and further carrying out acetylation on the glaucocalyxin B (GLB) and the polypeptide cNGQGEQc to obtain the glaucocalyxin B (GLB) derivative. The glaucocalyxin B (GLB) derivative has high anticancer activity, can significantly inhibit a plurality of tumor cells such as cells in lung cancer, liver cancer, nasopharyngeal carcinoma, lymphoma, melanoma and myelomatosis, especially has a targeting effect on the lung caner and can pertinently treat the lung caner.
Description
Technical field
The present invention relates to Glaucocalyxin B (GLB) derivative, particularly contain Glaucocalyxin B (GLB) derivative of polypeptide.
The invention still further relates to described preparation method and the application in the treatment cancer thereof that contains Glaucocalyxin B (GLB) derivative of polypeptide.
Background technology
Herba Rabdosiae glaucocalycis is a Labiatae Rabdosia plant, is distributed widely in provinces such as Henan, Hubei, Sichuan, Guizhou, Shaanxi, Shanxi, Hebei.Effects such as that herb has is antibiotic, anti-inflammatory, acute Jaundice Jaundice, acute cholecystitis, wound, venomous snake bite, the purulence lump rash etc. of being used for the treatment of among the people.Its pharmacological action does not appear in the newspapers.The henry rabdosia leaf crude preparation by using has been carried out Pharmacological action study, and the result shows that it is to having the obvious suppression effect with external ehrlich carcinoma tumour cell in the body.
Labiatae Rabdosia plant.Widely distribute at east Asia and African western part, the whole world has 150 kinds approximately, and China has 90 kinds of 25 mutation approximately.Wherein have approximately 30 kinds among the people as medicinal, as clearing heat and detoxicating, anticancer anti-inflammatory, invigorating the spleen, invigorate blood circulation, Hangzhoupro bacterium medicine uses.People to this platymiscium-the mushroom constituents done research, therefrom extracted over one hundred kind of medicine constituents.Find that through pharmacological screening many diterpene compounds have cell toxicant, antitumor, Hang Yan active function.Also find this platymiscium except that containing two terpene components under study for action, also be rich in triterpene, lipid acid and a spot of flavones, sesquiterpene, chain hydrocarbon etc.
Rabdosia japonica is a Labiatae Rabdosia plant, is distributed in northeast, the North China of China, Korea. Japan, and former Soviet Union the Far East Area, Jilin Province's resource is especially abundant.Rabdosia japonica have be good for the stomach, clearing heat and detoxicating, invigorate blood circulation, antisepsis and anti-inflammation and antitumour activity, be used for gastritis, hepatitis from the beginning of diseases such as, cold, fever, mazoitis, arthrodynia.Modern study finds that herb has certain effect to cardiovascular, and effective constituent wherein has certain influence to platelet aggregation and cancer.From the eighties in last century so far the various countries scholar each side such as its pharmacology and chemistry are studied more with Chinese scholar research.
People such as Yunlong extracts glaucocalyxin A (glaucocalyxinA) and Glaucocalyxin B (glaucocalyxinB) about 1981 from rabdosia japonica; infer that from spectrum glaucocalyxin A has a typical skilful oxygen-16 1 kaurene (ent-15-oxo-I6-kaurene) skeleton of Rabdosia diterpene; and Glaucocalyxin B is 14 1 acetylates of glaucocalyxin A; both are distinguished acetylize; obtain identical acetylate, thereby confirmed both mutual relationships.Become also from the product rabdosia japonica of Jilin, to get this two kinds of diterpene with the later Chao Dynasty is complete.
Liu Chen rivers in 1988 etc. are produced from the Beijing area and are got the rabdosia japonica outside first element and the second element, also get blue calyx third element (glaucocalyxinC), and structure is mapping-7p, 14a, 15a-trihydroxy--16-kauri pine-3-ketone.Dongsa kimls, except therefrom telling glaucocalyxin A, Glaucocalyxin B and blue calyx third element, also told two kinds of new diterpene glaucocalyxinD and glaucocalyxinE and listed the structure infrared and ultraviolet absorption value of these five kinds of compounds, topmost 1H one nucleus magnetic resonance, 13C one chemical shift of NMR value and mass-spectrometric data value.
Kim Yong Ils etc. have been got glaucocalyxin A from the rabdosia japonica root, king elder generation honor etc. is produced the Isodon amethystoides (c) (Isodonamethystoides (Benth) CyWuetHsuan) from Anhui and also got glaucocalyxin A in addition.Human reversed-phased high performace liquid chromatographic external standard methods such as Wang Huifen are measured the content of glaucocalyxin A in different sites and the rabdosia japonica of different acquisition phase, and the result shows that glaucocalyxin A is far above root, stem in the leaf.The different acquisition phase is with 7-8 month content height, 9 the end of month content descend.Zhang Yuantong etc. also adopt reversed-phase HPLC to measure glaucocalyxin A content, and recording glaucocalyxin A content is 1.03%, and average recovery rate is 99.2%.Zhao Quancheng gets p one Sitosterol and ursolic acid from rabdosia japonica.After silica gel column chromatography and HPLC preparative column chromatographic separation obtain 4 compound Oleanolic Acids, arjunolic acid, hair are spent macaque acid B and ursolic acid to rabdosia japonica with extraction using alcohol.People such as Dai Guiming jade also get rutin and and daucosterol from rabdosia japonica.
Day island proper Tianjin AA-670 type atomic absorption spectrophotometer such as Yang Xuejian, the peach leaf that is provided with Chinese Academy of Sciences's cyclisation is a marker, root, stem, the leaf of rabdosia japonica are measured Fe in the stem of finding blue honor Herba Rabdosiae glaucocalycis, the leaf respectively, Cu, Mn, the content of Se are higher, and modern medicine study is verified, the existence of these elements is effective to anti-curing cancers and other some diseases, and the prompting rabdosia japonica has certain effect in this regard.After have human ICP-AES instrument to measure the content of the multiple element of Hebei rabdosia japonica different sites, find 5 kinds of macro elements K in its leaf, Na, P, Ca, the content of Mg is higher than root and stem, 8 kinds of trace elements zns, Fe, S, Ba, Li, Al, V, the content of Ti also are higher in the leaf, find first to contain rare earth element ce in the Herba Rabdosiae glaucocalycis leaf, Y, Yb, the best medicinal part of Notes of Key Data per nnial herb rabdosia japonica may be a leaf.
Liu Landi etc. have studied the influence of cardiac muscle C-FOS genetic expression when rabdosia japonica pours into again and again to the rat global ischemia.Adopt the stripped global ischemia one reperfusion injury model of dortl; the immunohistochemical methods method; with C-fos expression conditions among the corresponding C-fos antibody test myocardial cell; analyze the C-fos expression conditions with the computer image analysis system; the rabdosia japonica crude drug extracts for 3 times with alcohol reflux; extracting solution reclaims ethanol; be added in 0.5% starch paste after concentrating; fling to ethanol; concentration is 1Kg/L; the Herba Rabdosiae glaucocalycis that is divided into high, normal, basic three kinds of different concns is poured into advancing irrigation notes whole-heartedly again and again to ischemic; found that it can obviously reduce apoptosis-related genes C-fos expression of gene; show that myocardial ischemia one reperfusion injury is light, cardiac muscle is subjected to certain protection, finds that simultaneously the high density rabdosia japonica has certain myocardial cell's toxic action.It has the myocardium protecting action relevant with dosage, but is not that concentration is big more effective more.
In sum, rabdosia japonica is abundant in china natural resources.Chemical research shows that it contains diterpene and three terpene components, but is its main activeconstituents with two terpene component glaucocalyxin As, second element, fourth element wherein, has the effect of platelet aggregation-against.Because of its parent nucleus is the ent-kaurene type, similar in appearance to rubescensine A (oridonin), so may have certain antitumous effect.
Summary of the invention
One of purpose of the present invention is to provide can treat cancer, particularly treats Glaucocalyxin B (GLB) derivative of lung cancer, liver cancer, nasopharyngeal carcinoma, lymphoma, melanoma and myelomatosis, this Glaucocalyxin B (GLB) derivative has targeting to lung cancer especially, promptly can treat multiple cancer and just can treat lung cancer drugs targetedly thereby provide a kind of.
Two of purpose of the present invention is to provide the preparation method of a kind of preparation Glaucocalyxin B (GLB) derivative, thereby the bigger defective of toxic side effect of avoiding simple employing organic synthesis to be brought, and the drug effect of avoiding simple employing feedstock purification to bring lower more, can't specific aim treat the defective of a certain particular cancers.
Glaucocalyxin B disclosed in this invention (GLB) derivative is a kind of diterpenes material, has kaurane type structure, carbonyl, polypeptide cNGQGEQc and/or hydroxyl, and the general formula of its molecular structure is as follows:
Wherein, R
1For-cNGQGEQc ,-OH or
R
2For-cNGQGEQc or-OH.
Wherein, four kinds of structures below to the treatment cancer, particularly treat lung cancer, liver cancer, nasopharyngeal carcinoma, lymphoma, melanoma and myelomatosis and have good effect.Wherein introduce polypeptide cNGQGEQc, come the main body diterpene is carried out structural modification by acetylization reaction, thus the Glaucocalyxin B (GLB) after feasible the modification, and promptly Glaucocalyxin B (GLB) derivative has targeting to lung cancer.
Described Glaucocalyxin B (GLB) (C
22H
30O
5) molecular structure as follows:
The present invention adopts Herba Rabdosiae glaucocalycis medicinal material (over-ground part) as raw material; pulverize, extraction, solution-treated, cross post, recrystallization etc. and obtain Glaucocalyxin B (GLB); carry out further acetylization reaction with polypeptide cNGQGEQc again, obtain described Glaucocalyxin B (GLB) derivative.The preparation method of described Glaucocalyxin B (GLB) derivative specifically comprises following steps:
Step 1: pulverize
Get Herba Rabdosiae glaucocalycis medicinal material (over-ground part) and be crushed to 20 orders～50 orders.
Step 2: extract
Step 2.1: the crushed material of step 1 gained was mixed according to volume ratio with 95% ethanol (A.R.) in 1: 6 to 1: 10, and heating refluxed 1～2 hour under 80 ℃～90 ℃ temperature condition, extracted, filtered, and obtained extracting solution and residuum.
Step 2.2: the residuum of step 2.1 gained was mixed according to volume ratio with 95% ethanol (A.R.) in 1: 6 to 1: 10, heating refluxed 1～2 hour under 80 ℃～90 ℃ temperature condition, extracted, filtered, obtain extracting solution and residuum, repeat this step 1～3 time.
Step 2.3: the extracting solution of combining step 2.1 and 2.2 gained.
Step 3: solvent treatment
Step 3.1: with the extracting solution heating of step 2 gained, concentrating under reduced pressure under 55 ℃～65 ℃ temperature condition reclaims ethanol and obtains the elementary enriched material of heavy-gravity.
Step 3.2: the elementary enriched material of step 3.1 gained was mixed according to volume ratio with water in 1: 8～1: 10, stirred 10 minutes with 60～120 rev/mins speed at ambient temperature, left standstill subsequently 6～12 hours, abandoning supernatant obtains lower floor's solid.
Step 3.3: the solid and the ethyl acetate (A.R.) of step 3.2 gained were mixed according to volume ratio in 1: 4～1: 6, under 25 ℃～35 ℃ temperature condition,, left standstill 1～3 hour, filter and obtain filtrate and insolubles with 60～120 rev/mins speed stirring and dissolving.
Step 3.4: the insolubles and the ethyl acetate (A.R.) of step 3.3 gained were mixed according to volume ratio in 1: 4～1: 6, under 25 ℃～35 ℃ temperature condition with 60～120 rev/mins speed stirring and dissolving, left standstill 1～3 hour, and filtered and to obtain filtrate and insolubles, repeat this step 1～3 time.
Step 3.5: the filtrate of combining step 3.3 and 3.4 gained.
Step 3.6: with the filtrate heating of step 3.5 gained, concentrating under reduced pressure under 35 ℃～45 ℃ temperature condition reclaims ethyl acetate (A.R.), obtains solid concentrates.
Step 4: cross post
Step 4.1: the solid concentrates of step 3 gained and 95% ethanol (A.R.) according to 1: 4～1: 6 mixed dissolution of volume ratio, are obtained solution.
Step 4.2: slowly be added to the solution of step 4.1 gained in the resin column, observe the color of resin, when resin column has 2/3 variable color, stop application of sample, and adopt 95% ethanol (A.R.) of 200ml～400ml to wash this resin column, collect all effluent liquid, the weighting material of described resin column is a strongly basic anionic resin and to be saturated to the pH value with sodium hydroxide neutral.
Step 4.3: with step 4.2 gained effluent liquid heating, and under 55 ℃～65 ℃ temperature condition reduced-pressure backflow, do until solvent, obtain solid residue.
Step 5: recrystallization
Step 5.1: with above-mentioned residue with 30 ℃～40 ℃ polar solvent mixed according to volume ratio in 1: 4～1: 6, obtain primary solution, heating, 30 ℃～40 ℃ of temperature condition under concentrating under reduced pressure, under-5 ℃～-8 ℃ temperature condition, leave standstill crystallization, filter and obtain yellow needle crystal.
Described polar solvent is the mixing solutions according to 1: 1～1: 2 blended chloroform (A.R.) of volume ratio and acetone (A.R.).
Step 5.2: the crystallization of step 5.1 gained was mixed according to volume ratio with polar solvent in 1: 4～1: 6, and recrystallization 2～4 times repeatedly under-5 ℃～-8 ℃ temperature condition obtains enriched material.
Described polar solvent is the mixing solutions according to 1: 1～1: 4 blended chloroform (A.R.) of volume ratio and acetone (A.R.).
Step 6: separate and purify
Step 6.1: the enriched material of step 5 gained is added 20ml ethyl acetate (A.R.) dissolving, add 200-300 order silica gel, mix, the room temperature volatile dry is removed solvent ethyl acetate (A.R.).
The add-on of described silica gel is 3 times of described enriched material weight.
Step 6.2: chromatographic silica gel post of wet-filling, 20 times of the enriched material weight that described silica gel consumption is step 5 gained, the pillar blade diameter length ratio is 1: 12, and adopts 2BV chloroform (A.R.) to wash silicagel column repeatedly.
Step 6.3: the enriched material that is mixed with silica gel of step 6.1 gained is joined in the silicagel column of step 6.2 gained, and adopting 2BV chloroform (A.R.) flushing and flow velocity is 1.5BV/ hour.
Step 6.4: adopt the mixing solutions of 3BV chloroform (A.R.) and acetone (A.R.) to wash described silicagel column, and employing TLC tlc analysis method test-target cut Glaucocalyxin B (GLB), when checking corresponding cut, collect the cut that contains Glaucocalyxin B (GLB) principal constituent.
In the mixing solutions of the chloroform of described 3BV (A.R.) and acetone (A.R.), the volume ratio of chloroform (A.R.) and acetone (A.R.) is 20: 1.
Step 6.5: the cut of step 6.4 gained is merged, concentrating under reduced pressure under 30 ℃～40 ℃ temperature condition, reclaim solvent, with the enriched material usefulness chloroform (A.R.) of gained and the mixing solutions recrystallization of acetone (A.R.), etc. white needle-like crystals Glaucocalyxin B (GLB).
The consumption of the mixing solutions of described chloroform (A.R.) and acetone (A.R.) and the volume ratio of described cut are 8: 1～12: 1.
In the mixing solutions of described chloroform (A.R.) and acetone (A.R.), the volume ratio of chloroform (A.R.) and acetone (A.R.) is 1: 1～1: 4.
Step 7: synthetic
Step 7.1: the Glaucocalyxin B (GLB) of step 6 gained and polypeptide (cNGQGEQc) are obtained mixed solution according to the mixed of per 8～17g Glaucocalyxin B (GLB), 0.7～2.3g polypeptide (cNGQGEQc) and 80～150ml anhydrous polar solvent, and described anhydrous polar solvent is a kind of in methyl alcohol (A.R.), ethanol (A.R.), the methylene dichloride (A.R.).
Step 7.2: with the mixing solutions heating of step 7.1 gained, stirred condensing reflux 6～10 hours under 60 ℃～80 ℃ temperature condition, suction filtration leaves standstill under the room temperature while hot, separates out crystal, filters, and isolates crystal, obtains Glaucocalyxin B (GLB) derivative.
Step 8: synthetic
Step 8.1: with the Glaucocalyxin B (GLB) of step 6 gained according to per 8～17g Glaucocalyxin B (GLB), the mixed of per 70～130ml ethanolic soln, (GLB) is dissolved in the ethanolic soln with Glaucocalyxin B, at room temperature to the sodium hydroxide solution that wherein drips 1mol/L, regulate PH=13, continue to stir 5 minutes, again concentrating under reduced pressure, remove ethanolic soln, obtain enriched material.
Step 8.2: the enriched material of step 8.1 gained is added ethyl acetate, at room temperature extract, obtain the ethyl acetate extraction layer, coextraction 2 times obtains extract.
The usage quantity of ethyl acetate is 2 times of amounts (volume ratio) of the usage quantity of sodium hydroxide solution in the step 8.1 described in each extraction.
Step 8.3: the extract of the step 8.2 gained polar solvent with 30 ℃～40 ℃ was mixed according to volume ratio in 1: 4～1: 6, obtain primary solution.
With the primary solution heating that obtains, concentrating under reduced pressure under 30 ℃～40 ℃ temperature condition leaves standstill crystallization under-5 ℃～-8 ℃ temperature condition, filter to obtain crystallization.
Described polar solvent is mixed into the mixing solutions according to 1: 1～1: 2 blended chloroform of volume ratio and acetone.
Step 8.4: the crystallization of step 8.3 gained is mixed with polar solvent, and recrystallization is 2～4 times repeatedly, obtains crystal.
Described polar solvent is mixed into the mixing solutions according to 1: 1～1: 2 blended chloroform of volume ratio and acetone.
Step 8.5:, obtain mixing solutions with the crystal of step 8.4 gained mixed according to per 8～17g gained crystal, 0.7～2.3g polypeptide (cNGQGEQc) and 80～150ml anhydrous polar solvent.
Described anhydrous polar solvent is a kind of in methyl alcohol, ethanol, the methylene dichloride.
Step 8.6: with the heating of step 8.5 gained mixing solutions, stirred condensing reflux 6～10 hours under 60 ℃～80 ℃ temperature condition, suction filtration leaves standstill crystallization under the room temperature while hot, filters, and isolates crystal, obtains Glaucocalyxin B (GLB) derivative.
Through high performance liquid chromatography, LC-MS, NMR analysis confirmation gained crystal is Glaucocalyxin B (GLB) derivative that contains polypeptide cNGQGEQc.
The present invention discloses the described application of Glaucocalyxin B (GLB) derivative in the treatment cancer, the particularly application in treatment lung cancer, liver cancer, nasopharyngeal carcinoma, lymphoma, melanoma and myelomatosis that contains polypeptide cNGQGEQc.Because this Glaucocalyxin B (GLB) derivative is based on Glaucocalyxin B (GLB) and to adopt polypeptide cNGQGEQc by acetylization reaction Glaucocalyxin B (GLB) to be carried out structural modification resultant, it has good targeting for lung cancer.
The Glaucocalyxin B of aforementioned arbitrary structure disclosed in this invention (GLB) derivative can adopt the conventional medicine carrier on the pharmaceutics to make any formulation, includes but not limited to tablet, capsule, flexible glue agent, sprays, gelifying agent, gel inhalation, oral preparation, suspensoid, electuary, patch, ointment, pill, powder, injection, infusion solution, freeze dried injection, lipidosome injection, target administration injection, suppository, sustained release preparation or controlled release preparation.Be preferably freeze dried injection.
Description of drawings
Fig. 1 is the general formula of molecular structure of Glaucocalyxin B of the present invention (GLB) derivative.
Fig. 2 is the molecular structural formula of Glaucocalyxin B of the present invention (GLB).
Fig. 3 is the molecular structural formula of Glaucocalyxin B (GLB) derivative of the embodiment of the invention eight.
Fig. 4 is the molecular structural formula of the intermediate product in Glaucocalyxin B of the present invention (GLB) the derivative preparation process.
Fig. 5 a～5c is the molecular structural formula of three kinds of Glaucocalyxin Bs (GLB) derivative of the embodiment of the invention nine.
Embodiment
According to claim of the present invention and the disclosed content of summary of the invention, technical scheme of the present invention is specific as follows described.
The preparation of embodiment one Glaucocalyxin B (GLB) derivative:
Below each chemical reagent of being adopted in the prepared process as there not being special mark, then be analytical pure.
Adopt Herba Rabdosiae glaucocalycis medicinal material (over-ground part) as raw material, purification Glaucocalyxin B (GLB), and adopt polypeptide cNGQGEQc to modify its structure, the preparation method of preparation Glaucocalyxin B (GLB) derivative specifically comprises following steps:
Step 1: pulverize
Get Herba Rabdosiae glaucocalycis medicinal material (over-ground part) and be crushed to 20 orders～50 orders.
Step 2: extract
Step 2.1: the crushed material of step 1 gained was mixed according to volume ratio with 95% ethanol (A.R.) in 1: 6 to 1: 10, and heating refluxed 1～2 hour under 80 ℃～90 ℃ temperature condition, extracted, filtered, and obtained extracting solution and residuum.
Step 2.2: the residuum of step 2.1 gained was mixed according to volume ratio with 95% ethanol (A.R.) in 1: 6 to 1: 10, heating refluxed 1～2 hour under 80 ℃～90 ℃ temperature condition, extracted, filtered, obtain extracting solution and residuum, repeat this step 1～3 time.
Step 2.3: the extracting solution of combining step 2.1 and 2.2 gained.
Step 3: solvent treatment
Step 3.1: with the extracting solution heating of step 2 gained, concentrating under reduced pressure under 55 ℃～65 ℃ temperature condition reclaims ethanol and obtains the elementary enriched material of heavy-gravity.
Step 3.2: the elementary enriched material of step 3.1 gained was mixed according to volume ratio with water in 1: 8～1: 10, stirred 10 minutes with 60～120 rev/mins speed at ambient temperature, left standstill subsequently 6～12 hours, abandoning supernatant obtains lower floor's solid.
Step 3.3: the solid and the ethyl acetate (A.R.) of step 3.2 gained were mixed according to volume ratio in 1: 4～1: 6, under 25 ℃～35 ℃ temperature condition,, left standstill 1～3 hour, filter and obtain filtrate and insolubles with 60～120 rev/mins speed stirring and dissolving.
Step 3.4: the insolubles and the ethyl acetate (A.R.) of step 3.3 gained were mixed according to volume ratio in 1: 4～1: 6, under 25 ℃～35 ℃ temperature condition with 60～120 rev/mins speed stirring and dissolving, left standstill 1～3 hour, and filtered and to obtain filtrate and insolubles, repeat this step 1～3 time.
Step 3.5: the filtrate of combining step 3.3 and 3.4 gained.
Step 3.6: with the filtrate heating of step 3.5 gained, concentrating under reduced pressure under 35 ℃～45 ℃ temperature condition reclaims ethyl acetate (A.R.), obtains solid concentrates.
Step 4: cross post
Step 4.1: the solid concentrates of step 3 gained and 95% ethanol (A.R.) according to 1: 4～1: 6 mixed dissolution of volume ratio, are obtained solution.
Step 4.2: slowly be added to the solution of step 4.1 gained in the resin column, observe the color of resin, when resin column has 2/3 variable color, stop application of sample, and adopt 95% ethanol (A.R.) of 200ml～400ml to wash this resin column, collect all effluent liquid, the weighting material of described resin column is a strongly basic anionic resin and to be saturated to the pH value with sodium hydroxide neutral.
Described resin column adopts No. 7 resin columns, and post is directly than 1: 12.
Step 4.3: with step 4.2 gained effluent liquid heating, and under 55 ℃～65 ℃ temperature condition reduced-pressure backflow, do until solvent, obtain solid residue.
Step 5: recrystallization
Step 5.1: with above-mentioned residue with 30 ℃～40 ℃ polar solvent mixed according to volume ratio in 1: 4～1: 6, obtain primary solution, heating, 30 ℃～40 ℃ of temperature condition under concentrating under reduced pressure, under-5 ℃～-8 ℃ temperature condition, leave standstill crystallization, filter and obtain yellow needle crystal.
Described polar solvent is the mixing solutions according to 1: 1～1: 2 blended chloroform (A.R.) of volume ratio and acetone (A.R.).
Step 5.2: the crystallization of step 5.1 gained was mixed according to volume ratio with polar solvent in 1: 4～1: 6, and recrystallization 2～4 times repeatedly under-5 ℃～-8 ℃ temperature condition obtains Glaucocalyxin B (GLB).
Described polar solvent is the mixing solutions according to 1: 1～1: 4 blended chloroform (A.R.) of volume ratio and acetone (A.R.).
Step 6: separate and purify
Step 6.1: the enriched material of step 5 gained is added 20ml ethyl acetate (A.R.) dissolving, add 200-300 order silica gel, mix, the room temperature volatile dry is removed solvent ethyl acetate (A.R.).
The add-on of described silica gel is 3 times of described enriched material weight.
Step 6.2: chromatographic silica gel post of wet-filling, 20 times of the enriched material weight that described silica gel consumption is step 5 gained, the pillar blade diameter length ratio is 1: 12, and adopts 2BV chloroform (A.R.) to wash silicagel column repeatedly.
Step 6.3: the enriched material that is mixed with silica gel of step 6.1 gained is joined in the silicagel column of step 6.2 gained, and adopting 2BV chloroform (A.R.) flushing and flow velocity is 1.5BV/ hour.
Step 6.4: adopt the mixing solutions of 3BV chloroform (A.R.) and acetone (A.R.) to wash described silicagel column, and employing TLC tlc analysis method test-target cut Glaucocalyxin B (GLB), when checking corresponding cut, collect the cut that contains Glaucocalyxin B (GLB) principal constituent.
In the mixing solutions of the chloroform of described 3BV (A.R.) and acetone (A.R.), the volume ratio of chloroform (A.R.) and acetone (A.R.) is 20: 1.
Step 6.5: the cut of step 6.4 gained is merged, concentrating under reduced pressure under 30 ℃～40 ℃ temperature condition, reclaim solvent, with the enriched material usefulness chloroform (A.R.) of gained and the mixing solutions recrystallization of acetone (A.R.), etc. white needle-like crystals Glaucocalyxin B (GLB).
The consumption of the mixing solutions of described chloroform (A.R.) and acetone (A.R.) and the volume ratio of described cut are 8: 1～12: 1.
In the mixing solutions of described chloroform (A.R.) and acetone (A.R.), the volume ratio of chloroform (A.R.) and acetone (A.R.) is 1: 1～1: 4.
Step 7: synthetic
Step 7.1: the Glaucocalyxin B (GLB) of step 6 gained and polypeptide (cNGQGEQc) are obtained mixed solution according to the mixed of per 8～17g Glaucocalyxin B (GLB), 0.7～2.3g polypeptide (cNGQGEQc) and 80～150ml anhydrous polar solvent, and described anhydrous polar solvent is a kind of in methyl alcohol (A.R.), ethanol (A.R.), the methylene dichloride (A.R.).
Step 7.2: with the mixing solutions heating of step 7.1 gained, stirred condensing reflux 6～10 hours under 60 ℃～80 ℃ temperature condition, suction filtration leaves standstill under the room temperature while hot, separates out crystal, filters, and isolates crystal, obtains Glaucocalyxin B (GLB) derivative.
Step 8: synthetic
Step 8.1: with the Glaucocalyxin B (GLB) of step 6 gained according to per 8～17g Glaucocalyxin B (GLB), the mixed of per 70～130ml ethanol (A.R.) solution, Glaucocalyxin B (GLB) is dissolved in ethanol (A.R.) solution, at room temperature to the sodium hydroxide solution that wherein drips 1mol/L, regulate PH=13, continue to stir 5 minutes, again concentrating under reduced pressure, remove ethanol (A.R.) solution, obtain enriched material.
Step 8.2: the enriched material of step 8.1 gained is added ethyl acetate (A.R.), at room temperature extract, obtain the ethyl acetate extraction layer, coextraction 2 times obtains extract.
The usage quantity of ethyl acetate (A.R.) is 2 times of amounts (volume ratio) of the usage quantity of sodium hydroxide solution in the step 8.1 described in each extraction.
Step 8.3: the extract of the step 8.2 gained polar solvent with 30 ℃～40 ℃ was mixed according to volume ratio in 1: 4～1: 6, obtain primary solution.
With the primary solution heating that obtains, concentrating under reduced pressure under 30 ℃～40 ℃ temperature condition leaves standstill crystallization under-5 ℃～-8 ℃ temperature condition, filter to obtain crystallization.
Described polar solvent is mixed into the mixing solutions according to 1: 1～1: 2 blended chloroform (A.R.) of volume ratio and acetone (A.R.).
Step 8.4: the crystallization of step 8.3 gained is mixed with polar solvent, and recrystallization is 2～4 times repeatedly, obtains crystal.
Described polar solvent is mixed into the mixing solutions according to 1: 1～1: 2 blended chloroform (A.R.) of volume ratio and acetone (A.R.).
Step 8.5:, obtain mixing solutions with the crystal of step 8.4 gained mixed according to per 8～17g gained crystal, 0.7～2.3g polypeptide (cNGQGEQc) and 80～150ml anhydrous polar solvent.
Described anhydrous polar solvent is a kind of in methyl alcohol, ethanol, the methylene dichloride.
Step 8.6: with the heating of step 8.5 gained mixing solutions, stirred condensing reflux 6～10 hours under 60 ℃～80 ℃ temperature condition, suction filtration leaves standstill crystallization under the room temperature while hot, filters, and isolates crystal, obtains Glaucocalyxin B (GLB) derivative.
Through high performance liquid chromatography, LC-MS, NMR analysis confirmation gained crystal is Glaucocalyxin B (GLB) derivative that contains polypeptide cNGQGEQc.
The preparation of embodiment two Glaucocalyxin Bs (GLB) derivative:
Adopt following technical parameter to improve the preparation method of embodiment one:
In the described step 2.1, be that described crushed material is mixed according to volume ratio with 95% ethanol (A.R.) at 1: 6.5, heating refluxed 1.8 hours under 82 ℃ of temperature condition, extracted and filtered.
In the described step 2.2, be that described residuum is mixed according to volume ratio with 95% ethanol (A.R.) at 1: 9.5, heating refluxed under 88 ℃ of temperature condition 1.2 hours, extracted and filtered, and repeated this step 1 time.
In the described step 3.1, be with described extracting solution heating, concentrating under reduced pressure under 57 ℃ of temperature condition.
In the described step 3.2, be that described elementary enriched material is mixed according to volume ratio with water at 1: 8.5, stir at ambient temperature and leave standstill subsequently and just separated supernatant liquor and solid in 11 hours.
In the described step 3.3, be that described solid is mixed according to volume ratio with ethyl acetate (A.R.) at 1: 5.5, stirring and dissolving under 27 ℃ of temperature condition leaves standstill and refiltered in 1.5 hours.
In the described step 3.4, be that described insolubles is mixed according to volume ratio with ethyl acetate (A.R.) at 1: 4.5, stirring and dissolving under 33 ℃ of temperature condition leaves standstill and refiltered in 2.5 hours, repeats this step 3 time.
In the described step 3.6, be with described filtrate heating, concentrating under reduced pressure under 37 ℃ of temperature condition.
In the described step 4.1, be according to 1: 4.5 mixed dissolution of volume ratio with described solid concentrates and 95% ethanol (A.R.).
In the described step 4.2, the resin column of employing is that weighting material is strongly basic anionic resin and is saturated to pH value neutral resin column with sodium hydroxide.
In the described step 4.2, when described resin column has 2/3 variable color, stop application of sample, and adopt 95% ethanol (A.R.) of 240ml to wash described resin column.
In the described step 4.3, be with the heating of described effluent liquid, and under 57 ℃ of temperature condition reduced-pressure backflow.
In the described step 5.1 and 5.2, described polar solvent is mixed into the mixing solutions according to 1: 1.2 blended chloroform (A.R.) of volume ratio and acetone (A.R.).
In the described step 5.1, be that the polar solvent of described residue with 31 ℃ mixed according to volume ratio at 1: 5.5.
In the described step 5.1, be that concentrating under reduced pressure under 31 ℃ of temperature condition leaves standstill crystallization under-7 ℃ of temperature condition with described primary solution heating.
In the described step 5.2, be that described crystallization is mixed according to volume ratio with polar solvent at 1: 4.5, recrystallization 4 times repeatedly under-6 ℃ of temperature condition.
In the described step 6.5, be concentrating under reduced pressure under 32 ℃ of temperature condition, the consumption of the mixing solutions of chloroform described in the concentrating under reduced pressure process (A.R.) and acetone (A.R.) and the volume ratio of described cut are 11.5: 1.
In the described step 6.5, in the mixing solutions of described chloroform (A.R.) and acetone (A.R.), the volume ratio of chloroform (A.R.) and acetone (A.R.) is 1: 2.
In the described step 7.1, be mixed according to every 10g Glaucocalyxin B (GLB), 0.9g polypeptide (cNGQGEQc) and 90ml anhydrous polar solvent.
In the described step 7.1, described anhydrous polar solvent is methyl alcohol (A.R.).
In the described step 7.2, be with described mixing solutions heating, stirred condensing reflux 9.5 hours under 64 ℃ of temperature condition, suction filtration leaves standstill crystallization under the room temperature while hot.
In the described step 8.1, be Glaucocalyxin B (GLB) with step 6 gained according to every 10g Glaucocalyxin B (GLB), the mixed of every 80ml ethanol (A.R.) solution.
In the described step 8.3, be that the extract of the step 8.2 gained polar solvent with 32 ℃ is mixed according to volume ratio at 1: 4.5, obtain primary solution.
With the primary solution heating that obtains, concentrating under reduced pressure under 32 ℃ of temperature condition leaves standstill crystallization under-5.5 ℃ of temperature condition, filter to obtain crystallization.
Described polar solvent is mixed into the mixing solutions according to 1: 1.2 blended chloroform (A.R.) of volume ratio and acetone (A.R.).
In the described step 8.4, described polar solvent is mixed into the mixing solutions according to 1: 1.2 blended chloroform (A.R.) of volume ratio and acetone (A.R.).
In the described step 8.5, be with the crystal of step 8.4 gained mixed according to every 10g gained crystal, 0.9g polypeptide (cNGQGEQc) and 90ml anhydrous polar solvent.
In the described step 8.6, be, under 64 ℃ of temperature condition, stirred condensing reflux 9.5 hours the heating of step 8.5 gained mixing solutions.
Through high performance liquid chromatography, LC-MS, NMR analysis confirmation gained crystal is Glaucocalyxin B (GLB) derivative that contains polypeptide cNGQGEQc.
The preparation of embodiment three Glaucocalyxin Bs (GLB) derivative:
Adopt following technical parameter to improve the preparation method of embodiment one:
In the described step 2.1, be that described crushed material is mixed according to volume ratio with 95% ethanol (A.R.) at 1: 7.5, heating refluxed 1.6 hours under 84 ℃ of temperature condition, extracted and filtered.
In the described step 2.2, be that described residuum is mixed according to volume ratio with 95% ethanol (A.R.) at 1: 8.5, heating refluxed under 86 ℃ of temperature condition 1.4 hours, extracted and filtered, and repeated this step 1 time.
In the described step 3.1, be with described extracting solution heating, concentrating under reduced pressure under 59 ℃ of temperature condition.
In the described step 3.2, be that described elementary enriched material is mixed according to volume ratio with water at 1: 8.5, stir at ambient temperature and leave standstill subsequently and just separated supernatant liquor and solid in 10 hours.
In the described step 3.3, be that described solid is mixed according to volume ratio with ethyl acetate (A.R.) at 1: 5.5, stirring and dissolving under 29 ℃ of temperature condition leaves standstill and refiltered in 1.5 hours.
In the described step 3.4, be that described insolubles is mixed according to volume ratio with ethyl acetate (A.R.) at 1: 4.5, stirring and dissolving under 31 ℃ of temperature condition leaves standstill and refiltered in 2.5 hours, repeats this step 3 time.
In the described step 3.6, be with described filtrate heating, concentrating under reduced pressure under 39 ℃ of temperature condition.
In the described step 4.1, be according to 1: 4.5 mixed dissolution of volume ratio with described solid concentrates and 95% ethanol (A.R.).
In the described step 4.2, the resin column of employing is that weighting material is strongly basic anionic resin and is saturated to pH value neutral resin column with sodium hydroxide.
In the described step 4.2, when described resin column has 2/3 variable color, stop application of sample, and adopt 95% ethanol (A.R.) of 280ml to wash described resin column.
In the described step 4.3, be with the heating of described effluent liquid, and under 59 ℃ of temperature condition reduced-pressure backflow.
In the described step 5.1 and 5.2, described polar solvent is mixed into the mixing solutions according to 1: 1.4 blended chloroform (A.R.) of volume ratio and acetone (A.R.).
In the described step 5.1, be that the polar solvent of described residue with 33 ℃ mixed according to volume ratio at 1: 5.5.
In the described step 5.1, be that concentrating under reduced pressure under 33 ℃ of temperature condition leaves standstill crystallization under-7 ℃ of temperature condition with described primary solution heating.
In the described step 5.2, be that described crystallization is mixed according to volume ratio with polar solvent at 1: 4.5, recrystallization 4 times repeatedly under-7 ℃ of temperature condition.
In the described step 6.5, be concentrating under reduced pressure under 34 ℃ of temperature condition, the consumption of the mixing solutions of chloroform described in the concentrating under reduced pressure process (A.R.) and acetone (A.R.) and the volume ratio of described cut are 10.5: 1.
In the described step 6.5, in the mixing solutions of described chloroform (A.R.) and acetone (A.R.), the volume ratio of chloroform (A.R.) and acetone (A.R.) is 1: 2.
In the described step 7.1, be mixed according to every 12g Glaucocalyxin B (GLB), 1.3g polypeptide (cNGQGEQc) and 105ml anhydrous polar solvent.
In the described step 7.1, described anhydrous polar solvent is ethanol (A.R.).
In the described step 7.2, be with described mixing solutions heating, stirred condensing reflux 8.5 hours under 68 ℃ of temperature condition, suction filtration leaves standstill crystallization under the room temperature while hot.
In the described step 8.1, be Glaucocalyxin B (GLB) with step 6 gained according to every 12g Glaucocalyxin B (GLB), the mixed of every 90ml ethanol (A.R.) solution.
In the described step 8.3, be that the extract of the step 8.2 gained polar solvent with 34 ℃ is mixed according to volume ratio at 1: 5.5, obtain primary solution.
With the primary solution heating that obtains, concentrating under reduced pressure under 34 ℃ of temperature condition leaves standstill crystallization under-6 ℃ of temperature condition, filter to obtain crystallization.
Described polar solvent is mixed into the mixing solutions according to 1: 1.4 blended chloroform (A.R.) of volume ratio and acetone (A.R.).
In the described step 8.4, described polar solvent is mixed into the mixing solutions according to 1: 1.4 blended chloroform (A.R.) of volume ratio and acetone (A.R.).
In the described step 8.5, be with the crystal of step 8.4 gained mixed according to every 12g gained crystal, 1.3g polypeptide (cNGQGEQc) and 105ml anhydrous polar solvent.
In the described step 8.6, be, under 68 ℃ of temperature condition, stirred condensing reflux 8.5 hours the heating of step 8.5 gained mixing solutions.
Through high performance liquid chromatography, LC-MS, NMR analysis confirmation gained crystal is Glaucocalyxin B (GLB) derivative that contains polypeptide cNGQGEQc.
The preparation of embodiment four Glaucocalyxin Bs (GLB) derivative:
Adopt following technical parameter to improve the preparation method of embodiment one:
In the described step 2.1, be that described crushed material is mixed according to volume ratio with 95% ethanol (A.R.) at 1: 8.5, heating refluxed 1.4 hours under 86 ℃ of temperature condition, extracted and filtered.
In the described step 2.2, be that described residuum is mixed according to volume ratio with 95% ethanol (A.R.) at 1: 7.5, heating refluxed under 84 ℃ of temperature condition 1.6 hours, extracted and filtered, and repeated this step 3 time.
In the described step 3.1, be with described extracting solution heating, concentrating under reduced pressure under 61 ℃ of temperature condition.
In the described step 3.2, be that described elementary enriched material is mixed according to volume ratio with water at 1: 9.5, stir at ambient temperature and leave standstill subsequently and just separated supernatant liquor and solid in 8 hours.
In the described step 3.3, be that described solid is mixed according to volume ratio with ethyl acetate (A.R.) at 1: 4.5, stirring and dissolving under 31 ℃ of temperature condition leaves standstill and refiltered in 2.5 hours.
In the described step 3.4, be that described insolubles is mixed according to volume ratio with ethyl acetate (A.R.) at 1: 5.5, stirring and dissolving under 29 ℃ of temperature condition leaves standstill and refiltered in 1.5 hours, repeats this step 1 time.
In the described step 3.6, be with described filtrate heating, concentrating under reduced pressure under 41 ℃ of temperature condition.
In the described step 4.1, be according to 1: 5.5 mixed dissolution of volume ratio with described solid concentrates and 95% ethanol (A.R.).
In the described step 4.2, the resin column of employing is that weighting material is strongly basic anionic resin and is saturated to pH value neutral resin column with sodium hydroxide.
In the described step 4.2, when described resin column has 2/3 variable color, stop application of sample, and adopt 95% ethanol (A.R.) of 320ml to wash described resin column.
In the described step 4.3, be with the heating of described effluent liquid, and under 61 ℃ of temperature condition reduced-pressure backflow.
In the described step 5.1 and 5.2, described polar solvent is mixed into the mixing solutions according to 1: 1.6 blended chloroform (A.R.) of volume ratio and acetone (A.R.).
In the described step 5.1, be that the polar solvent of described residue with 37 ℃ mixed according to volume ratio at 1: 4.5.
In the described step 5.1, be that concentrating under reduced pressure under 37 ℃ of temperature condition leaves standstill crystallization under-6 ℃ of temperature condition with described primary solution heating.
In the described step 5.2, be that described crystallization is mixed according to volume ratio with polar solvent at 1: 5.5, recrystallization 2 times repeatedly under-6 ℃ of temperature condition.
In the described step 6.5, be concentrating under reduced pressure under 36 ℃ of temperature condition, the consumption of the mixing solutions of chloroform described in the concentrating under reduced pressure process (A.R.) and acetone (A.R.) and the volume ratio of described cut are 9.5: 1.
In the described step 6.5, in the mixing solutions of described chloroform (A.R.) and acetone (A.R.), the volume ratio of chloroform (A.R.) and acetone (A.R.) is 1: 3.
In the described step 7.1, be mixed according to every 14g Glaucocalyxin B (GLB), 1.7g polypeptide (cNGQGEQc) and 125ml anhydrous polar solvent.
In the described step 7.1, described anhydrous polar solvent is ethanol (A.R.).
In the described step 7.2, be with described mixing solutions heating, stirred condensing reflux 7.5 hours under 72 ℃ of temperature condition, suction filtration leaves standstill crystallization under the room temperature while hot.
In the described step 8.1, be Glaucocalyxin B (GLB) with step 6 gained according to every 14g Glaucocalyxin B (GLB), the mixed of every 110ml ethanol (A.R.) solution.
In the described step 8.3, be that the extract of the step 8.2 gained polar solvent with 36 ℃ is mixed according to volume ratio at 1: 4.5, obtain primary solution.
With the primary solution heating that obtains, concentrating under reduced pressure under 36 ℃ of temperature condition leaves standstill crystallization under-7 ℃ of temperature condition, filter to obtain crystallization.
Described polar solvent is mixed into the mixing solutions according to 1: 1.6 blended chloroform (A.R.) of volume ratio and acetone (A.R.).
In the described step 8.4, described polar solvent is mixed into the mixing solutions according to 1: 1.6 blended chloroform (A.R.) of volume ratio and acetone (A.R.).
In the described step 8.5, be with the crystal of step 8.4 gained mixed according to every 14g gained crystal, 1.7g polypeptide (cNGQGEQc) and 125ml anhydrous polar solvent.
In the described step 8.6, be, under 72 ℃ of temperature condition, stirred condensing reflux 7.5 hours the heating of step 8.5 gained mixing solutions.
Through high performance liquid chromatography, LC-MS, NMR analysis confirmation gained crystal is Glaucocalyxin B (GLB) derivative that contains polypeptide cNGQGEQc.
The preparation of embodiment five Glaucocalyxin Bs (GLB) derivative:
Adopt following technical parameter to improve the preparation method of embodiment one:
In the described step 2.1, be that described crushed material is mixed according to volume ratio with 95% ethanol (A.R.) at 1: 9.5, heating refluxed 1.2 hours under 88 ℃ of temperature condition, extracted and filtered.
In the described step 2.2, be that described residuum is mixed according to volume ratio with 95% ethanol (A.R.) at 1: 6.5, heating refluxed under 82 ℃ of temperature condition 1.8 hours, extracted and filtered, and repeated this step 3 time.
In the described step 3.1, be with described extracting solution heating, concentrating under reduced pressure under 63 ℃ of temperature condition.
In the described step 3.2, be that described elementary enriched material is mixed according to volume ratio with water at 1: 9.5, stir at ambient temperature and leave standstill subsequently and just separated supernatant liquor and solid in 7 hours.
In the described step 3.3, be that described solid is mixed according to volume ratio with ethyl acetate (A.R.) at 1: 4.5, stirring and dissolving under 33 ℃ of temperature condition leaves standstill and refiltered in 2.5 hours.
In the described step 3.4, be that described insolubles is mixed according to volume ratio with ethyl acetate (A.R.) at 1: 5.5, stirring and dissolving under 27 ℃ of temperature condition leaves standstill and refiltered in 1.5 hours, repeats this step 1 time.
In the described step 3.6, be with described filtrate heating, concentrating under reduced pressure under 43 ℃ of temperature condition.
In the described step 4.1, be according to 1: 5.5 mixed dissolution of volume ratio with described solid concentrates and 95% ethanol (A.R.).
In the described step 4.2, the resin column of employing is that weighting material is strongly basic anionic resin and is saturated to pH value neutral resin column with sodium hydroxide.
In the described step 4.2, when described resin column has 2/3 variable color, stop application of sample, and adopt 95% ethanol (A.R.) of 360ml to wash described resin column.
In the described step 4.3, be with the heating of described effluent liquid, and under 63 ℃ of temperature condition reduced-pressure backflow.
In the described step 5.1 and 5.2, described polar solvent is mixed into the mixing solutions according to 1: 1.8 blended chloroform (A.R.) of volume ratio and acetone (A.R.).
In the described step 5.1, be that the polar solvent of described residue with 39 ℃ mixed according to volume ratio at 1: 4.5.
In the described step 5.1, be that concentrating under reduced pressure under 39 ℃ of temperature condition leaves standstill crystallization under-6 ℃ of temperature condition with described primary solution heating.
In the described step 5.2, be that described crystallization is mixed according to volume ratio with polar solvent at 1: 5.5, recrystallization 2 times repeatedly under-7 ℃ of temperature condition.
In the described step 6.5, be concentrating under reduced pressure under 38 ℃ of temperature condition, the consumption of the mixing solutions of chloroform described in the concentrating under reduced pressure process (A.R.) and acetone (A.R.) and the volume ratio of described cut are 8.5: 1.
In the described step 6.5, in the mixing solutions of described chloroform (A.R.) and acetone (A.R.), the volume ratio of chloroform (A.R.) and acetone (A.R.) is 1: 3.
In the described step 7.1, be mixed according to every 16g Glaucocalyxin B (GLB), 2.1g polypeptide (cNGQGEQc) and 140ml anhydrous polar solvent.
In the described step 7.1, described anhydrous polar solvent is methyl alcohol (A.R.).
In the described step 7.2, be with described mixing solutions heating, stirred condensing reflux 6.5 hours under 76 ℃ of temperature condition, suction filtration leaves standstill crystallization under the room temperature while hot.
In the described step 8.1, be Glaucocalyxin B (GLB) with step 6 gained according to every 16g Glaucocalyxin B (GLB), the mixed of every 120ml ethanol (A.R.) solution.
In the described step 8.3, be that the extract of the step 8.2 gained polar solvent with 38 ℃ is mixed according to volume ratio at 1: 5.5, obtain primary solution.
With the primary solution heating that obtains, concentrating under reduced pressure under 38 ℃ of temperature condition leaves standstill crystallization under-7.5 ℃ of temperature condition, filter to obtain crystallization.
Described polar solvent is mixed into the mixing solutions according to 1: 1.8 blended chloroform (A.R.) of volume ratio and acetone (A.R.).
In the described step 8.4, described polar solvent is mixed into the mixing solutions according to 1: 1.8 blended chloroform (A.R.) of volume ratio and acetone (A.R.).
In the described step 8.5, be with the crystal of step 8.4 gained mixed according to every 16g gained crystal, 2.1g polypeptide (cNGQGEQc) and 140ml anhydrous polar solvent.
In the described step 8.6, be, under 76 ℃ of temperature condition, stirred condensing reflux 6.5 hours the heating of step 8.5 gained mixing solutions.
Through high performance liquid chromatography, LC-MS, NMR analysis confirmation gained crystal is Glaucocalyxin B (GLB) derivative that contains polypeptide cNGQGEQc.
The preparation (preferred embodiment) of embodiment six Glaucocalyxin Bs (GLB) derivative:
Adopt following technical parameter to improve the preparation method of embodiment one:
In the described step 2.1, be that described crushed material is mixed according to volume ratio with 95% ethanol (A.R.) at 1: 8, heating refluxed 1.5 hours under 85 ℃ of temperature condition, extracted and filtered.
In the described step 2.2, be that described residuum is mixed according to volume ratio with 95% ethanol (A.R.) at 1: 8, heating refluxed under 85 ℃ of temperature condition 1.5 hours, extracted and filtered, and repeated this step 2 time.
In the described step 3.1, be with described extracting solution heating, concentrating under reduced pressure under 60 ℃ of temperature condition.
In the described step 3.2, be that described elementary enriched material is mixed according to volume ratio with water at 1: 9, stir at ambient temperature and leave standstill subsequently and just separated supernatant liquor and solid in 9 hours.
In the described step 3.3, be that described solid is mixed according to volume ratio with ethyl acetate (A.R.) at 1: 5, stirring and dissolving under 30 ℃ of temperature condition leaves standstill and refiltered in 2 hours.
In the described step 3.4, be that described insolubles is mixed according to volume ratio with ethyl acetate (A.R.) at 1: 5, stirring and dissolving under 30 ℃ of temperature condition leaves standstill and refiltered in 2 hours, repeats this step 2 time.
In the described step 3.6, be with described filtrate heating, concentrating under reduced pressure under 40 ℃ of temperature condition.
In the described step 4.1, be according to 1: 5 mixed dissolution of volume ratio with described solid concentrates and 95% ethanol (A.R.).
In the described step 4.2, the resin column of employing is that weighting material is strongly basic anionic resin and is saturated to pH value neutral resin column with sodium hydroxide.
In the described step 4.2, when described resin column has 2/3 variable color, stop application of sample, and adopt 95% ethanol (A.R.) of 300ml to wash described resin column.
In the described step 4.3, be with the heating of described effluent liquid, and under 60 ℃ of temperature condition reduced-pressure backflow.
In the described step 5.1 and 5.2, described polar solvent is mixed into the mixing solutions according to 1: 1.5 blended chloroform (A.R.) of volume ratio and acetone (A.R.).
In the described step 5.1, be that the polar solvent of described residue with 35 ℃ mixed according to volume ratio at 1: 5.
In the described step 5.1, be that concentrating under reduced pressure under 35 ℃ of temperature condition leaves standstill crystallization under-6.5 ℃ of temperature condition with described primary solution heating.
In the described step 5.2, be that described crystallization is mixed according to volume ratio with polar solvent at 1: 5, recrystallization 3 times repeatedly under-6.5 ℃ of temperature condition.
In the described step 6.5, be concentrating under reduced pressure under 35 ℃ of temperature condition, the consumption of the mixing solutions of chloroform described in the concentrating under reduced pressure process (A.R.) and acetone (A.R.) and the volume ratio of described cut are 10: 1.
In the described step 6.5, in the mixing solutions of described chloroform (A.R.) and acetone (A.R.), the volume ratio of chloroform (A.R.) and acetone (A.R.) is 1: 2.5.
In the described step 7.1, be mixed according to every 13g Glaucocalyxin B (GLB), 1.5g polypeptide (cNGQGEQc) and 115ml anhydrous polar solvent.
In the described step 7.1, described anhydrous polar solvent is methylene dichloride (A.R.).
In the described step 7.2, be with described mixing solutions heating, stirred condensing reflux 8 hours under 70 ℃ of temperature condition, suction filtration leaves standstill crystallization under the room temperature while hot.
Through high performance liquid chromatography, LC-MS, NMR analysis confirmation gained crystal is Glaucocalyxin B (GLB) derivative that contains polypeptide cNGQGEQc.
The preparation (preferred embodiment) of embodiment seven Glaucocalyxin Bs (GLB) derivative:
Adopt following technical parameter to improve the preparation method of embodiment one:
In the described step 2.1, be that described crushed material is mixed according to volume ratio with 95% ethanol (A.R.) at 1: 8, heating refluxed 1.5 hours under 85 ℃ of temperature condition, extracted and filtered.
In the described step 2.2, be that described residuum is mixed according to volume ratio with 95% ethanol (A.R.) at 1: 8, heating refluxed under 85 ℃ of temperature condition 1.5 hours, extracted and filtered, and repeated this step 2 time.
In the described step 3.1, be with described extracting solution heating, concentrating under reduced pressure under 60 ℃ of temperature condition.
In the described step 3.2, be that described elementary enriched material is mixed according to volume ratio with water at 1: 9, stir at ambient temperature and leave standstill subsequently and just separated supernatant liquor and solid in 9 hours.
In the described step 3.3, be that described solid is mixed according to volume ratio with ethyl acetate (A.R.) at 1: 5, stirring and dissolving under 30 ℃ of temperature condition leaves standstill and refiltered in 2 hours.
In the described step 3.4, be that described insolubles is mixed according to volume ratio with ethyl acetate (A.R.) at 1: 5, stirring and dissolving under 30 ℃ of temperature condition leaves standstill and refiltered in 2 hours, repeats this step 2 time.
In the described step 3.6, be with described filtrate heating, concentrating under reduced pressure under 40 ℃ of temperature condition.
In the described step 4.1, be according to 1: 5 mixed dissolution of volume ratio with described solid concentrates and 95% ethanol (A.R.).
In the described step 4.2, the resin column of employing is that weighting material is strongly basic anionic resin and is saturated to pH value neutral resin column with sodium hydroxide.
In the described step 4.2, when described resin column has 2/3 variable color, stop application of sample, and adopt 95% ethanol (A.R.) of 300ml to wash described resin column.
In the described step 4.3, be with the heating of described effluent liquid, and under 60 ℃ of temperature condition reduced-pressure backflow.
In the described step 5.1 and 5.2, described polar solvent is mixed into the mixing solutions according to 1: 1.5 blended chloroform (A.R.) of volume ratio and acetone (A.R.).
In the described step 5.1, be that the polar solvent of described residue with 35 ℃ mixed according to volume ratio at 1: 5.
In the described step 5.1, be that concentrating under reduced pressure under 35 ℃ of temperature condition leaves standstill crystallization under-6.5 ℃ of temperature condition with described primary solution heating.
In the described step 5.2, be that described crystallization is mixed according to volume ratio with polar solvent at 1: 5, recrystallization 3 times repeatedly under-6.5 ℃ of temperature condition.
In the described step 6.5, be concentrating under reduced pressure under 35 ℃ of temperature condition, the consumption of the mixing solutions of chloroform described in the concentrating under reduced pressure process (A.R.) and acetone (A.R.) and the volume ratio of described cut are 10: 1.
In the described step 6.5, in the mixing solutions of described chloroform (A.R.) and acetone (A.R.), the volume ratio of chloroform (A.R.) and acetone (A.R.) is 1: 2.5.
In the described step 8.1, be Glaucocalyxin B (GLB) with step 6 gained according to every 13g Glaucocalyxin B (GLB), the mixed of every 100ml ethanol (A.R.) solution.
In the described step 8.3, be that the extract of the step 8.2 gained polar solvent with 35 ℃ is mixed according to volume ratio at 1: 5, obtain primary solution.
With the primary solution heating that obtains, concentrating under reduced pressure under 35 ℃ of temperature condition leaves standstill crystallization under-6.5 ℃ of temperature condition, filter to obtain crystallization.
Described polar solvent is mixed into the mixing solutions according to 1: 1.5 blended chloroform (A.R.) of volume ratio and acetone (A.R.).
In the described step 8.4, described polar solvent is mixed into the mixing solutions according to 1: 1.5 blended chloroform (A.R.) of volume ratio and acetone (A.R.).
In the described step 8.5, be with the crystal of step 8.4 gained mixed according to every 13g gained crystal, 1.5g polypeptide (cNGQGEQc) and 115ml anhydrous polar solvent.
In the described step 8.6, be, under 70 ℃ of temperature condition, stirred condensing reflux 8 hours the heating of step 8.5 gained mixing solutions.
Through high performance liquid chromatography, LC-MS, NMR analysis confirmation gained crystal is Glaucocalyxin B (GLB) derivative that contains polypeptide cNGQGEQc.
Embodiment eight:
Adopt Glaucocalyxin B (GLB) derivative of preparation method's preparation of preferred embodiment six, its molecular structural formula is as follows:
Wherein, described-cNGQGEQc be by former Glaucocalyxin B (GLB) thus on hydroxyl and the acetylization reaction of polypeptide cNGQGEQc introduce.
The Glaucocalyxin B of this structure (GLB) derivative to the treatment cancer, particularly treat lung cancer, liver cancer, nasopharyngeal carcinoma, lymphoma, melanoma and myelomatosis and have good effect.Wherein introduce polypeptide cNGQGEQc, come the main body diterpene is carried out structural modification by acetylization reaction, thus the Glaucocalyxin B (GLB) after feasible the modification, and promptly Glaucocalyxin B (GLB) derivative has targeting to lung cancer.
Embodiment nine:
Adopt Glaucocalyxin B (GLB) derivative of preparation method's preparation of preferred embodiment seven, its general formula of molecular structure is as follows:
C or-OH, R2 is-cNGQGEQc or-OH.
Wherein, described-cNGQGEQc be by former Glaucocalyxin B (GLB) thus on hydroxyl and the acetylization reaction of polypeptide cNGQGEQc introduce.
Wherein, three kinds of structures below to the treatment cancer, particularly treat lung cancer, liver cancer, nasopharyngeal carcinoma, lymphoma, melanoma and myelomatosis and have good effect.Wherein introduce polypeptide cNGQGEQc, come the main body diterpene is carried out structural modification by acetylization reaction, thus the Glaucocalyxin B (GLB) after feasible the modification, and promptly Glaucocalyxin B (GLB) derivative has targeting to lung cancer.
The preparation of embodiment ten Glaucocalyxin Bs (GLB) derivative formulations:
Adopt the conventional medicine carrier on the pharmaceutics, and the conventional preparation method on the employing pharmaceutics, the preparation method who adopts preferred embodiment six and/or preferred embodiment seven is prepared, have in Glaucocalyxin B (GLB) derivative of molecular structure of embodiment eight and/or embodiment nine one or more, be prepared into the regular dosage form on the pharmaceutics, include but not limited to tablet, capsule, the flexible glue agent, sprays, gelifying agent, the gel inhalation, oral preparation, suspensoid, electuary, patch, ointment, pill, powder, injection, infusion solution, freeze dried injection, lipidosome injection, the target administration injection, suppository, sustained release preparation or controlled release preparation.
Wherein the preferred preparation formulation is a freeze dried injection.
The application of embodiment 11 Glaucocalyxin Bs (GLB) derivative:
According among the embodiment one to six arbitrarily the preparation method prepare Glaucocalyxin B (GLB) derivative of gained, be used for treatment for cancer, especially for treatment lung cancer, liver cancer, nasopharyngeal carcinoma, lymphoma, melanoma and myelomatosis.Wherein because Glaucocalyxin B (GLB) derivative of gained; be to introduce polypeptide cNGQGEQc by acetylization reaction; come the main body diterpene is carried out structural modification; thereby the Glaucocalyxin B (GLB) after feasible the modification; be that Glaucocalyxin B (GLB) derivative has targeting to lung cancer, especially being suitable for is the treatment of lung cancer.
The pharmacodynamic experiment of embodiment 12 Glaucocalyxin Bs (GLB) derivatives for treatment lung cancer:
Employing is carried out pharmacodynamic experiment according to the mixture (hereinafter to be referred as GH-02) of preparation method's Glaucocalyxin B (GLB) derivative preparation, that have the molecular structure of embodiment seven of preferred embodiment six, investigates the influence of GH-02 to lung cancer A549 cell.
1. experiment material
1.1 medicine and reagent: tetrazole orchid (MTT)
1.2 instrument: carbonic acid gas incubator, microscope, microplate reader
1.3 animal: do not have
1.4 knurl strain: lung cancer A549 cell is so kind as to give by teacher Luo Jianmin of institute of oncology of tumour hospital of Fudan University
2. experimental technique
2.1A549 cell cultures
The A549 cell is with containing the DMEM substratum of 20% foetal calf serum, places 37 ℃, 5%CO2 incubator to cultivate.
2.2 passage
Take out the 25cm that has covered with cell
2Culturing bottle removes nutrient solution, injects tryptic digestive juice 1ml earlier, discards after jiggling.Add tryptic digestive juice 2ml again, behind its infiltration cell, put into the CO2 incubator and digest 2-3min.Inject the 5ml nutrient solution, blow and beat the culturing bottle basal plane repeatedly with the nutrient solution that dropper is drawn in the culturing bottle.Get 2 aseptic culturing bottles, the nutrient solution that will contain cell evenly part is gone in the culturing bottle, cultivates after adding the 10ml fresh medium respectively.
2.3 cell cryopreservation
Make the cell suspension that contains cell 500,000/ml earlier, after add 10%DMSO, the frozen pipe moderate heat envelope of packing into is positioned in-20 ℃ of refrigerators and takes out behind the 3h, places in-60 ℃ of refrigerators and spends the night, and drops in the liquid nitrogen behind the anchor of packing into after the taking-up and preserves.
2.4 cell recovery
After taking out frozen pipe, the warm water that drops into 37 ℃ rapidly thaws, sucking-off enchylema, centrifugal 1000rpm, 10min abandons supernatant liquor, removes DMSO, change to the nutrient solution that contains 20% foetal calf serum, the diluting cells number is that 20-50 ten thousand/ml branch is planted in culturing bottle, places the CO2 incubator to cultivate.
3. experimental result
Table 1GH-02 to the influence of A549 cell (X ± S, n=4)
Annotate:
*Expression is compared P＜0.05 with the blank group.
*Expression is compared P＜0.01 with the blank group.△ represents to compare P＜0.01 with positive control (Zorubicin).▲ expression is compared P＜0.05 with the PV solubility promoter.▲ ▲ expression is compared P＜0.01 with the PVP solubility promoter.
4. conclusion:
The GH-02 low dose has certain tumor-inhibiting action, and high dose group then has obvious antineoplastic, and comparing difference with model group has significance, compares then difference with positive controls and does not have significance.Though the GH-02 antitumor action is slightly poorer than the positive control Zorubicin, but still demonstrate stronger antitumor action.
The animal model test that embodiment 13 Glaucocalyxin Bs (GLB) derivative influences Mice Bearing Lewis Lung Cancer:
Employing is carried out animal model test according to the mixture (hereinafter to be referred as GH-02) of preparation method's Glaucocalyxin B (GLB) derivative preparation, that have the molecular structure of embodiment seven of preferred embodiment six, investigates the influence of GH-02 to Mice Bearing Lewis Lung Cancer.
1. experiment material,
1.1 medicine and reagent: GH-02 (massfraction 99.8%) dilutes with physiological saline during test.
1.2 instrument: microscope, microplate reader
1.3 animal: 64 of C57 mouse inbred liness, body weight are (20 scholar 2) 9, and the male and female dual-purpose is provided by Shanghai Univ. of Traditional Chinese Medicine's Experimental Animal Center.Laboratory temperature is 22 1 25 ℃, and conventional feed is fed, and amount of drinking water is not limit.
1.4 knurl strain: the Lewis lung cancer solid-type is so kind as to give by teacher Luo Jianmin of institute of oncology of tumour hospital of Fudan University
2. experimental technique
Model group (physiological saline 10mL/kg), positive controls (endoxan 25mg/ml), GH-02 (25mg/ml) is divided into abdominal injection group, intravenous injection group, every group of 8 animals.Experiment is inoculation according to a conventional method, and next day is by drafting dosage ip administration, every day 1 time, 10d continuously after the inoculation.The ascites tumour is curative effect index with the increase in life span, continues after the drug withdrawal to observe 60d, and dead animal calculated with the actual survival time.The solid-type tumour as curative effect index, next day with tumour cuts accurate title quality in drug withdrawal with knurl quality inhibiting rate.Experimental result is represented with tumour inhibiting rate x ± s, is relatively checked with t between group.
3. experimental result:
Experimental result sees the influence of table 2GH-02 to Mice Bearing Lewis Lung Cancer for details.
4. conclusion:
GH-02 has tangible tumor-inhibiting action and antitumor action, and comparing difference with model group has significance, compares then difference with positive controls and does not have significance.The GH-02 antitumor action is better than positive control, demonstrates stronger antitumor action.Abdominal injection and intravenous injection all have tangible tumor-inhibiting action and antitumor action.
Foregoing is exemplifying of specific embodiments of the invention, for the wherein not reagent of detailed description, equipment, working method etc., should be understood to take the existing common and conventional reagent in this area, equipment, working method to wait and implemented.
The above embodiment of the present invention for the usefulness of explanation technical solution of the present invention, is enumerating of technical solution of the present invention only only simultaneously, is not limited to technical scheme of the present invention and protection domain thereof.Adopt the equivalent technologies means, be equal to reagent etc. the improvement of claims of the present invention and the disclosed technical scheme of specification sheets be will be understood that it is not exceed claims of the present invention and the disclosed scope of specification sheets.
Claims (10)
1. a Glaucocalyxin B (GLB) derivative is characterized in that, is a kind of diterpenes material, has kaurane type structure, carbonyl, polypeptide cNGQGEQc and/or hydroxyl, and the general formula of its molecular structure is as follows:
Wherein, R
1For-cNGQGEQc ,-OH or
R
2For-cNGQGEQc or-OH.
2. Glaucocalyxin B as claimed in claim 1 (GLB) derivative is characterized in that molecular structure is as follows:
3. the preparation method as the described Glaucocalyxin B of claim 1～2 (GLB) derivative is characterized in that, comprises following steps:
Step 1: pulverize
Get Herba Rabdosiae glaucocalycis medicinal material (over-ground part) and be crushed to 20 orders～50 orders;
Step 2: extract
Step 2.1: the crushed material of step 1 gained is mixed with 95% ethanol, and reflux is extracted and is filtered, and obtains extracting solution and residuum;
Step 2.2: the residuum of step 2.1 gained is mixed with 95% ethanol, and reflux extract to be filtered, and obtains extracting solution and residuum, repeats this step 1～3 time;
Step 2.3: the extracting solution of combining step 2.1 and 2.2 gained;
Step 3: solvent treatment
Step 3.1: the extracting solution heating concentrating under reduced pressure with step 2 gained, reclaim ethanol and obtain the elementary enriched material of heavy-gravity;
Step 3.2: the elementary enriched material of step 3.1 gained is mixed with water, stir and leave standstill subsequently, abandoning supernatant obtains lower floor's solid;
Step 3.3: the solid of step 3.2 gained is mixed with ethyl acetate, and stirring and dissolving also leaves standstill, and filters to obtain filtrate and insolubles;
Step 3.4: the insolubles of step 3.3 gained is mixed with ethyl acetate, and stirring and dissolving also leaves standstill, and filters to obtain filtrate and insolubles, repeats this step 1～3 time;
Step 3.5: the filtrate of combining step 3.3 and 3.4 gained;
Step 3.6: with the filtrate heating of step 3.5 gained, concentrating under reduced pressure reclaims ethyl acetate, obtains solid concentrates; Step 4: cross post
Step 4.1: solid concentrates and 95% ethanol mixed dissolution with step 3 gained obtain solution;
Step 4.2: the solution of step 4.1 gained slowly is added in the resin column, observes the color of resin, adopt this resin column of 95% alcohol flushing, collect all effluent liquid,
Step 4.3: with step 4.2 gained effluent liquid decompression reflux, do, obtain solid residue until solvent; Step 5: recrystallization
Step 5.1: above-mentioned residue is mixed with polar solvent, obtain primary solution, heating and concentrating under reduced pressure leave standstill crystallization, filter and obtain yellow needle crystal;
Step 5.2: the crystallization of step 5.1 gained is mixed with polar solvent, and recrystallization is 2～4 times repeatedly, obtains enriched material;
Step 6: separate and purify
Step 6.1: the enriched material of step 5 gained is added acetic acid ethyl dissolution, add 200-300 order silica gel, mix, the room temperature volatile dry is removed solvent ethyl acetate;
Step 6.2: chromatographic silica gel post of wet-filling, and adopt chloroform to wash silicagel column repeatedly;
Step 6.3: the enriched material that is mixed with silica gel of step 6.1 gained is joined in the silicagel column of step 6.2 gained, adopt the chloroform flushing;
Step 6.4: adopt the mixing solutions of chloroform and acetone to wash described silicagel column, and adopt TLC tlc analysis method test-target cut Glaucocalyxin B (GLB), when checking corresponding cut, collect the cut that contains Glaucocalyxin B (GLB) principal constituent;
Step 6.5: the cut of step 6.4 gained is merged, and concentrating under reduced pressure reclaims solvent, with the enriched material usefulness chloroform of gained and the mixing solutions recrystallization of acetone, obtains white needle-like crystals Glaucocalyxin B (GLB);
Step 7: synthetic
Step 7.1: the Glaucocalyxin B (GLB) of step 6 gained and polypeptide (cNGQGEQc) and anhydrous polar solvent mixed obtaining mixed solution;
Step 7.2: with the mixing solutions heating of step 7.1 gained, stir condensing reflux, suction filtration leaves standstill while hot, separates out crystal, filters, and isolates crystal, obtains Glaucocalyxin B (GLB) derivative;
Step 8: synthetic
Step 8.1: the Glaucocalyxin B (GLB) of step 6 gained is dissolved in the ethanolic soln, and at room temperature to dropping sodium solution wherein, concentrating under reduced pressure is removed ethanolic soln, obtains enriched material;
Step 8.2: the enriched material of step 8.1 gained is added ethyl acetate, at room temperature extract, obtain the ethyl acetate extraction layer, coextraction 2 times obtains extract;
Step 8.3: the extract of step 8.2 gained is mixed with polar solvent, obtain primary solution, heating and concentrating under reduced pressure leave standstill crystallization, filter and obtain crystallization;
Step 8.4: the crystallization of step 8.3 gained is mixed with polar solvent, and recrystallization is 2～4 times repeatedly, obtains crystal;
Step 8.5: the crystal of step 8.4 gained and polypeptide (cNGQGEQc) and anhydrous polar solvent mixed obtaining mixed solution;
Step 8.6: with the mixing solutions heating of step 8.5 gained, stir condensing reflux, suction filtration leaves standstill while hot, separates out crystal, filters, and isolates crystal, obtains Glaucocalyxin B (GLB) derivative.
4. the preparation method of Glaucocalyxin B as claimed in claim 3 (GLB) derivative is characterized in that, in the described step 2.1, be that described crushed material was mixed according to volume ratio with 95% ethanol in 1: 6 to 1: 10, heating refluxed 1～2 hour under 80 ℃～90 ℃ temperature condition, extracted and filtered;
In the described step 2.2, be that described residuum was mixed according to volume ratio with 95% ethanol in 1: 6 to 1: 10, heating refluxed 1～2 hour under 80 ℃～90 ℃ temperature condition, extracted and filtered.
5. the preparation method of Glaucocalyxin B as claimed in claim 4 (GLB) derivative is characterized in that, in the described step 3.1, is with described extracting solution heating, concentrating under reduced pressure under 55 ℃～65 ℃ temperature condition;
In the described step 3.2, be that described elementary enriched material was mixed according to volume ratio with water in 1: 8～1: 10, stir at ambient temperature and leave standstill subsequently and just separated supernatant liquor and solid in 6～12 hours;
In the described step 3.3, be that described solid was mixed according to volume ratio with ethyl acetate in 1: 4～1: 6, stirring and dissolving under 25 ℃～35 ℃ temperature condition leaves standstill and refiltered in 1～3 hour;
In the described step 3.4, be that described insolubles was mixed according to volume ratio with ethyl acetate in 1: 4～1: 6, stirring and dissolving under 25 ℃～35 ℃ temperature condition leaves standstill and refiltered in 1～3 hour;
In the described step 3.6, be with described filtrate heating, concentrating under reduced pressure under 35 ℃～45 ℃ temperature condition.
6. the preparation method of Glaucocalyxin B as claimed in claim 5 (GLB) derivative is characterized in that, in the described step 4.1, is according to 1: 4～1: 6 mixed dissolution of volume ratio with described solid concentrates and 95% ethanol;
In the described step 4.2, the resin column of employing is that weighting material is strongly basic anionic resin and is saturated to pH value neutral resin column with sodium hydroxide;
In the described step 4.2, when described resin column has 2/3 variable color, stop application of sample, and adopt the described resin column of 95% alcohol flushing of 200ml～400ml;
In the described step 4.3, be with the heating of described effluent liquid, and under 55 ℃～65 ℃ temperature condition reduced-pressure backflow.
7. the preparation method of Glaucocalyxin B as claimed in claim 6 (GLB) derivative is characterized in that, in the described step 5.1 and 5.2, described polar solvent is mixed into the mixing solutions according to 1: 1～1: 2 blended chloroform of volume ratio and acetone; In the described step 5.1, be that the polar solvent of described residue with 30 ℃～40 ℃ mixed according to volume ratio in 1: 4～1: 6;
In the described step 5.1, be that concentrating under reduced pressure under 30 ℃～40 ℃ temperature condition leaves standstill crystallization under-5 ℃～-8 ℃ temperature condition with described primary solution heating;
In the described step 5.2, be that described crystallization was mixed according to volume ratio with polar solvent in 1: 4～1: 6, recrystallization 2～4 times repeatedly under-5 ℃～-8 ℃ temperature condition.
8. the preparation method of Glaucocalyxin B as claimed in claim 7 (GLB) derivative is characterized in that, in the described step 6.1, be that described enriched material is added the 20ml acetic acid ethyl dissolution, and the add-on of described silica gel is 3 times of described enriched material weight;
In the described step 6.2,20 times of the enriched material weight that described silica gel consumption is step 5 gained, the pillar blade diameter length ratio is 1: 12, the consumption of described chloroform is 2BV (2 times of column volume);
In the described step 6.3, the consumption of described chloroform is that 2BV and flow velocity are 1.5BV/ hour;
In the described step 6.4, be to adopt the chloroform of 3BV and the mixing solutions of acetone to wash described silicagel column;
In the chloroform of described 3BV and the mixing solutions of acetone, the volume ratio of chloroform and acetone is 20: 1;
In the described step 6.5, the gained cut is a concentrating under reduced pressure under 30 ℃～40 ℃ temperature condition, and the volume ratio of the consumption of the mixing solutions of chloroform and acetone and described cut is 8: 1～12: 1 in the concentration process;
In the mixing solutions of described chloroform and acetone, the volume ratio of chloroform and acetone is 1: 1～1: 4.
9. the preparation method of Glaucocalyxin B as claimed in claim 8 (GLB) derivative, it is characterized in that, in the described step 7.1, be mixed according to per 8～17g Glaucocalyxin B (GLB), 0.7～2.3g polypeptide (cNGQGEQc) and 80～150ml anhydrous polar solvent;
In the described step 7.1, described anhydrous polar solvent is a kind of in methyl alcohol, ethanol, the methylene dichloride;
In the described step 7.2, be with described mixing solutions heating, stirred condensing reflux 6～10 hours under 60 ℃～80 ℃ temperature condition, suction filtration leaves standstill crystallization under the room temperature while hot;
In the described step 8.1, be according to per 8～17g Glaucocalyxin B (GLB), the mixed of per 70～130ml ethanolic soln, and, regulate PH=13 at room temperature to the sodium hydroxide solution that wherein drips 1mol/L, continue to stir 5 minutes, concentrating under reduced pressure is removed ethanolic soln again, obtains enriched material;
In the described step 8.2, the usage quantity of ethyl acetate is 2 times of amounts (volume ratio) of the usage quantity of sodium hydroxide solution in the step 8.1 described in each extraction;
In the described step 8.3 and 8.4, described polar solvent is mixed into the mixing solutions according to 1: 1～1: 2 blended chloroform of volume ratio and acetone;
In the described step 8.3, be that the polar solvent of described extract with 30 ℃～40 ℃ mixed according to volume ratio in 1: 4～1: 6;
In the described step 8.3, be that concentrating under reduced pressure under 30 ℃～40 ℃ temperature condition leaves standstill crystallization under-5 ℃～-8 ℃ temperature condition with described primary solution heating;
In the described step 8.4, be that described crystallization was mixed according to volume ratio with polar solvent in 1: 4～1: 6, recrystallization 2～4 times repeatedly under-5 ℃～-8 ℃ temperature condition obtains crystal.
In the described step 8.5, be mixed according to per 8～17g step 8.4 gained crystal, 0.7～2.3g polypeptide (cNGQGEQc) and 80～150ml anhydrous polar solvent;
In the described step 8.5, described anhydrous polar solvent is a kind of in methyl alcohol, ethanol, the methylene dichloride;
In the described step 8.6, be with described mixing solutions heating, stirred condensing reflux 6～10 hours under 60 ℃～80 ℃ temperature condition, suction filtration leaves standstill crystallization under the room temperature while hot.
10. as the application of the described Glaucocalyxin B of claim 1～3 (GLB) derivative in the treatment cancer, the particularly application in treatment lung cancer, liver cancer, nasopharyngeal carcinoma, lymphoma, melanoma and myelomatosis.
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| CN102134188A (en) * | 2011-03-22 | 2011-07-27 | 深圳市仙湖植物园管理处 | Tetrahydroanthraquinone compound Prisconnatacin and preparation method and application thereof in preparation of anti-tumor medicaments |
| CN115125190A (en) * | 2022-07-25 | 2022-09-30 | 苏州市中医医院 | Culture medium for promoting vascular endothelial cell proliferation |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102134188A (en) * | 2011-03-22 | 2011-07-27 | 深圳市仙湖植物园管理处 | Tetrahydroanthraquinone compound Prisconnatacin and preparation method and application thereof in preparation of anti-tumor medicaments |
| CN115125190A (en) * | 2022-07-25 | 2022-09-30 | 苏州市中医医院 | Culture medium for promoting vascular endothelial cell proliferation |
| CN115125190B (en) * | 2022-07-25 | 2023-09-05 | 苏州市中医医院 | Culture medium for promoting vascular endothelial cell proliferation |
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