CN101375937A - Cudrania tricuspidata extract, preparation and application thereof - Google Patents
Cudrania tricuspidata extract, preparation and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of natural medicaments and traditional Chinese medicine extracts, and discloses a tsugeki extract. The weight ratio of kaempferol-7-O-beta-D-glucoside and quercetin-7-O-beta-D-glucoside in the extract is 1: (0.6 to 1.1); on the basis of the weight of the extract, the weight percentage of total flavone is 1.5 to 50 percent, wherein the weight percentage of the kaempferol-7-O-beta-D-glucoside is 0.3 to 20 percent; and the weight percentage of the quercetin-7-O-beta-D-glucoside is 0.2 to 15 percent. The invention further discloses a preparation process and the application for the tsugeki extract. The preparation method for the tsugeki extract has the advantages of perfect process, perfect process index, controllable prepared tsugeki extract and active components of various preparations, as well as remarkable anti-tumor effect, and has broad prospect for market application.
Description
Technical field
The present invention relates to the technical field of natural drug and Chinese medicine extract.
Background technology
Lignum Cudraniae tricuspidatae is Moraceae three-bristle cudrania platymiscium Cudrania tricuspidata (Carr.) Bun. machaka or dungarunga, and its function is a clearing away heat and cooling blood, modern age the digestive tract tumor that is used among the people.Main chemical compositions is flavonoid, xanthone, lignanoid, coumarin and polysaccharide compound, the seventies is to the nineties, Chinese scholars (China, Japan and Korea S.) is studied the Lignum Cudraniae tricuspidatae antitumaous effect, the basic confirmation flavone compound be Lignum Cudraniae tricuspidatae anticancer, press down the cancer effective ingredient, polysaccharide has participated in immunomodulating, has a synergism to anticancer.In addition about kaempferol and Quercetin and their glycoside compound, generally believe in the world have antioxidation, antiinflammatory, anti-cardiovascular disease and physiologically active (Elliott Middleton et al Pharmacological Reviews52 (4): 673-751,2002) such as anticancer.
At home, the drug development of relevant Lignum Cudraniae tricuspidatae starts from the sixties, and researching and developing successful preparation is syrup, and records in drug standard promulgated by the ministries or commissions of the Central Government.Through clinical use, confirm that the Lignum Cudraniae tricuspidatae syrup has the auxiliary treatment effect to Infusion in Patients with Digestive chemotherapy or radiotherapy, and can improve patient's quality of life.Yet as a kind of Chinese medicine preparation of age restriction at that time that is subjected to, people are to its effective ingredient and insufficient understanding, let alone the quantitative analysis of setting up effective ingredient in the preparation; Also there are quality unstable factors such as aerogenesis, precipitation in said preparation, needs to add a certain amount of antiseptic in the preparation; This medicine carries and transports inconvenience in addition, has influenced medicine and has used widely.In view of the foregoing, continue exploratory development Lignum Cudraniae tricuspidatae effective ingredient, develop all kinds of more scientific and reasonable Lignum Cudraniae tricuspidatae preparations and have very big realistic meaning, it not only can improve the up-to-dateness of tcm product, the demand that can adapt to modern medication again, being the inexorable trend of tcm product secondary development, also is the road that must have that Chinese medicine moves towards the international world.
Deep modern pharmacological research finds that increasing Chinese medicine or crude drug are arranged, and its effective biological activity mainly is to come from its inner composite parts, but not single component.Effectively extract in order to reach various compositions, need to set up the production technology that adapts separately; Secondly the multicomponent characteristics of Chinese patent medicine make the quality control of medicine and the pharmacology of Chinese medicine set forth more than the next complexity of chemical medicine, yet the complexity of this series have constituted the peculiar property that Chinese patent medicine self is had just.
Chinese patent 03114766.6 discloses a kind of solid preparation that contains cudrania tricuspidata extract, in the middle of mentioned cudrania tricuspidata extract, it is perfect inadequately that but this patent prepares the method purifying process of cudrania tricuspidata extract, technic index is also still unsound, the cudrania tricuspidata extract quality that obtains is influenced greatly by initial medical material, a little less than the controllability relative thin.
Summary of the invention
It is perfect to the purpose of this invention is to provide a kind of purifying process, quality controllable cudrania tricuspidata extract, its preparation method and application.
The inventor attempts by the exploration to various extracting method, and to the mainly analysis and research of flavone component in the extract that meets medicinal requirements of various extracting method acquisitions, having summed up with kaempferol-7-O-β-D-glycoside and Quercetin-7-O-β-D-glycoside is the perfect production technology of a cover of quality control index.It is evident in efficacy to utilize production technology of the present invention to obtain cudrania tricuspidata extract, and quality controllability is good, very suitable suitability for industrialized production.
First aspect present invention, a kind of cudrania tricuspidata extract is disclosed, the weight ratio of the kaempferol in the extract-7-O-β-D-glycoside and Quercetin-7-O-β-D-glycoside is 1:0.6-1.1, preferred 1:0.6-0.9, based on extract weight, the total flavones percentage by weight is 1.5-50%, preferred 14-50%, wherein kaempferol-7-O-β-D-glycoside percentage by weight is 0.3-20%, preferred 2.5-20%, Quercetin-7-O-β-D-glycoside percentage by weight is 0.2-15%, and is preferred 1.7-15%, and satisfactory extract obtains through preparation technology of the present invention.
7-glucose-kaempferol glycoside (populnin) is the present known unique flavonoid glycoside composition that is used for the Quality Control of Lignum Cudraniae tricuspidatae preparation, and this all has report in patent application document CN1515294 and CN1726962.Quercetin-7-O-glycoside is another flavonoid glycoside composition of Lignum Cudraniae tricuspidatae, and the inventor also obtains this material in effective ingredient separates.Retrieval finds still do not have the report that other plant contains nimbecetin-7-O-glycoside and Quercetin-7-O-glycoside binary simultaneously so far according to system documentation.This inventor thought to significantly improve Quality Control project specificity if can in Lignum Cudraniae tricuspidatae and preparation Quality Control thereof, detect above-mentioned binary simultaneously, and enlarged effective ingredient and control the size, help further improving the quality control system of Lignum Cudraniae tricuspidatae and preparation thereof.
The determination of total flavonoids employing is the UV method of contrast with kaempferol-7-O-β-D-glycoside in the cudrania tricuspidata extract of the present invention, and kaempferol-7-O-β-D-glycoside and Quercetin-7-O-β-D-glycoside content adopts the HPLC method to measure respectively.
Second aspect present invention discloses the preparation method of above-mentioned cudrania tricuspidata extract, comprises the following steps:
A) Lignum Cudraniae tricuspidatae extraction process: get the Lignum Cudraniae tricuspidatae medical material, with 5-10 times of 0%-70% alcohol-water solution to initial medical material weight, reflux, extract, 2-4 time, each 1-5 hours, merge extractive liquid, was evaporated to relative density 1.06-1.07 (70 ℃ ± 10);
B) Lignum Cudraniae tricuspidatae purifying process: the extraction concentrated solution that step a is made, cross a chromatographic column that macroporous resin is housed, through 3-6 times of water washings to the resin bed volume, with 2-4 times of 20-80% alcoholic solution gradient elutions (what choose among the embodiment is 20%, 40%, 60% and 80% gradient) to the resin bed volume, collect 40-60% ethanol stream part, concentrating under reduced pressure, vacuum drying obtains extract powder.
Alcohol in the above-mentioned alcohol-water solution is selected from lower aliphatic alcohols, as methanol, ethanol, propanol etc.
Preferable, macroporous resin hangs down polarity in being, and the weight of the macroporous resin of adorning is 1/2-1/4 of initial medical material weight in the chromatographic column.
Improved, after above-mentioned steps b obtains macroporous resin chromatographic column 40-60% ethanol stream part concentrated solution, after a chromatographic column that polyamide (initial medical material amount 1/3-1/8) is housed, through 3-6 times of water washings to the column volume amount, with 1-4 times of 20-50% ethanol gradient elutions (what choose among the embodiment is 20%, 30%, 40% and 50% gradient), collect 30-50% ethanol stream part, concentrating under reduced pressure to the column volume amount, vacuum drying obtains extract powder.
Preferable, preferred 40-80 orders of polyamide.The weight of the polyamide of adorning is 1/3-1/8 of initial medical material weight in the chromatographic column.
Further improved, after above-mentioned steps is crossed the polyamide chromatographic column and is obtained 30-50% ethanol stream part concentrated solution, after a chromatographic column that gel is housed, 50% washing with alcohol through 4-8 times column volume amounts, 95% ethanol elution of the column volume amount that reuse is 4-8 times is collected 95% ethanol stream part, concentrating under reduced pressure, vacuum drying obtains extract powder.
Improved equally, also can be after above-mentioned steps b obtains macroporous resin 40-60% chromatographic column ethanol stream part concentrated solution, directly mistake one is equipped with the chromatographic column of gel, 50% washing with alcohol through 4-8 times of column volume amounts, 95% ethanol elution of 4-8 times of column volume amounts of reuse is collected 95% ethanol stream part, concentrating under reduced pressure, vacuum drying obtains extract powder.
Preferable, above-mentioned gel is a glucose gel, preferred hydroxypropyl glucose gel.The weight of the gel of adorning is that the upper prop concentrated solution is always measured (being dry weight) 10-50 times admittedly in the chromatographic column.
Among the present invention, various pure percentage ratios are percent by volume.
Studies show that the extract that above-mentioned three kinds of technologies obtain all can reach the technical specification of aforementioned kaempferol-7-O-β-D-glycoside and Quercetin-7-O-β-D-glycoside.
A third aspect of the present invention openly is used to prepare the antitumor pharmaceutical formulation with above-mentioned cudrania tricuspidata extract.Described pharmaceutical formulation comprises dosage forms such as granule, tablet, capsule, dry syrup or freeze-dried powder.
Fourth aspect present invention discloses a kind of pharmaceutical composition, is made up of the above-mentioned cudrania tricuspidata extract and the pharmaceutic adjuvant of treatment effective dose, and the weight ratio of cudrania tricuspidata extract and pharmaceutic adjuvant is 3-8:7-2.
Pharmaceutic adjuvant of the present invention is preparation solid preparation adjuvant commonly used, as: starch, dextrin, lactose, microcrystalline Cellulose, low-substituted hydroxypropyl cellulose, sucrose, sorbitol, mannitol, glycine, carboxymethyl starch sodium, magnesium stearate, Pulvis Talci, soluble starch, maltodextrin, micropowder silica gel and correctives etc.
Adopt Lignum Cudraniae tricuspidatae extraction process purifying process of the present invention perfect, technic index is sound, cudrania tricuspidata extract of the present invention and all kinds of preparation thereof, and active component is controlled, and antitumous effect is remarkable, has wide market application prospect.
The specific embodiment
Further set forth the present invention below in conjunction with embodiment.Should be understood that these embodiment only are used to illustrate the present invention, but not limit the scope of the invention.
Each assay method of the present invention is as follows:
Determination of total flavonoids:
With kaempferol-7-O-β-D-glycoside is contrast, adopts the UV standard measure to measure, and step is as follows:
(1) configuration kaempferol-7-O-β-D-glycoside reference substance solution;
(2) preparation standard curve;
(3) for the test agent formulations prepared from solutions: precision takes by weighing or measures an amount of Lignum Cudraniae tricuspidatae powder or extract or preparation, makes sample through routine extraction or dissolution process;
(4) algoscopy: add AlCl at sample solution
3And before and after the liquor kalii acetici, use the trap at spectrophotometric determination 408nm wavelength place respectively, with the weight that the difference of both traps is read kaempferol the need testing solution-7-O-β-D-glycoside from standard curve, calculate, promptly.
Quercetin-7-O-β-D-glycoside assay:
Adopt the HPLC method to measure, operating procedure is carried out according to high performance liquid chromatography (appendix VID of Chinese Pharmacopoeia version in 2005):
(1) chromatographic condition and system suitability test: filler closes silica gel for the octadecylsilane base, mobile phase is that volume ratio is methanol-water solution or the aqueous acetic acid of methanol-percent by volume 0.3% or the trifluoroacetic acid aqueous solution of methanol-percent by volume 0.1% of 38:62, the detection wavelength is 257nm, number of theoretical plate is pressed Quercetin-7-O-β-D-glycoside and is calculated, and should be not less than 1000;
(2) configuration Quercetin-7-O-β-D-glycoside reference substance solution;
(3) for the test agent formulations prepared from solutions: precision takes by weighing or measures an amount of Lignum Cudraniae tricuspidatae powder or extract or preparation, extracts or dissolution process through routine, uses the macroporous resin column purification process earlier, obtains through filtering with microporous membrane again;
(4) algoscopy: difference is accurate to be drawn reference substance solution and supplies test agent solution 5-15 μ l, injects chromatograph of liquid, measures, promptly.
Kaempferol-7-O-β-D-glycoside assay:
Adopt the HPLC method to measure, operating procedure is carried out according to high performance liquid chromatography (appendix VID of Chinese Pharmacopoeia version in 2005):
(1) chromatographic condition and system suitability test: filler closes silica gel for the octadecylsilane base, and mobile phase is methanol-water solution or the aqueous acetic acid of methanol-0.3% or the trifluoroacetic acid aqueous solution of methanol-0.1% of volume ratio 44:56; The detection wavelength is 265nm, and number of theoretical plate is pressed kaempferol-7-O-β-D-glycoside and calculated, and should be not less than 1000;
(2) configuration kaempferol-7-O-β-D-glycoside reference substance solution;
(3) for the test agent preparation: precision takes by weighing or measures an amount of Lignum Cudraniae tricuspidatae powder or extract or preparation, extracts or dissolution process through routine, uses the macroporous resin column purification process earlier, obtains through filtering with microporous membrane again;
(4) algoscopy: precision is drawn reference substance solution and is supplied test agent solution 5-15 μ l respectively, injects chromatograph of liquid, mensuration, promptly.
The separating and differentiate of Quercetin in embodiment 1 Lignum Cudraniae tricuspidatae-7-O-β-D-glycoside and kaempferol-7-O-β-D-glycoside
The Lignum Cudraniae tricuspidatae of water or rare alcohol extraction of learning from else's experience extracts concentrated solution, directly cross the macroporous resin column of a water processing, after the abundant eluting of water is clean, with 30%～95% alcoholic solution gradient elution, to 30%～50% ethanol stream part part of collecting, after concentrating, a last silicagel column, with ethyl acetate-methanol (0～30% methanol) gradient elution, eluent is a detection means with the TLC method, the unfolding condition that silica gel tlc detects: ethyl acetate-acetone-formic acid-water (5:3:0.5:0.5), color condition: spray AlCl3 ethanol liquid, thin plate volatilizes the back and desires separated component whether on the position identical with Quercetin-7-O-β-D-glycoside or kaempferol-7-O-β-D-glycoside contrast chromatograph inspecting under the 365nm fluorescent, showing has identical yellow-green fluorescence speckle, has and then confirms as the stream part that contains Quercetin-7-O-β-D-glycoside or kaempferol-7-O-β-D-glycoside.
Merge the stream part that contains kaempferol-7-O-β-D-glycoside and Quercetin-7-O-β-D-glycoside respectively, after the stream part of being rich in kaempferol-7-O-β-D-glycoside and Quercetin-7-O-β-D-glycoside concentrates respectively, make eluent with methanol, repeatedly cross gel column and separate, collect the isolating kaempferol of desire-7-O-β-D-glycoside and Quercetin-7-O-β-D-glycoside.The kaempferol that obtains-7-O-β-D-glycoside is further purified through anti-phase preparation liquid phase respectively with Quercetin-7-O-β-D-glycoside crude product and separates, and refining back two reference substance purity reaches 98% (HPLC normalization method).
Kaempferol-7-O-β-D-glycoside and Quercetin-7-O-β-D-glycoside chemical constitution is confirmed through UV, EI-MS, ESI-MS, H-NMR and C-NMR spectrum analysis research.
Identify: separated kaempferol-7-O-β-D-glycoside: pale yellow powder (MeOH), mp.259～261 ℃ (proofreading and correct), uv absorption λ max (MeOH): 252,265 and 369nm.Mass spectrum: EI M/Z286 (strong peak, glycoside unit quasi-molecular ions), 448 (faint, molecular ion peaks), ESI (M-1)/Z 447.2 (highest peak), (M+1)/and Z 449.0 (highest peak), theoretical molecular 448.37.Nuclear magnetic resonance, NMR: 1H-NMR (DMSO-d6) δ (ppm): 5.11 (1H, protons on the glycoside key), 6.38 (1H, d, C6-H), 6.77 (1H, d, C8-H), 6.90 (2H, d, C3 '-H, C5 '-H), 8.04 (2H, d, C2 '-H, and C6 '-H), 9.53 (1H, C3-OH), 10.13 (1H, C4 '-OH), 12.45 (1H, C5-OH); 13C-NMR (DMSO-d6) δ (ppm) data see Table 1.Uv absorption, mass spectrum and hydrogen, nuclear magnetic resonance of carbon data and kaempferol-7-O-β-D-glycoside literature value (Zhang Yuehua, Ren Wanwei, Wan Shuwen etc. Lignum Cudraniae tricuspidatae chemical constitution study [J]. medical industry (3): 15,1980) all corresponding coincideing, the wherein data and the consistent (K.R.Markham of literature value height of carbon spectrum, B.Ternal, R.Stanley, H.Geiger and T.J.Mabry CARBON-13 NMRSTUDIES OF FLAVONOIDS-III[J] .Tetrahedron.1978,34:1389-1397).
Separated Quercetin-7-O-β-D-glycoside: pale yellow powder (MeOH), mp.219 ~ 223 ℃ (proofreading and correct), uv absorption λ max (MeOH): 208,257 and 375nm.Mass spectrum: ESI (M-1)/Z463.24 (highest peak), theoretical molecular 464.37.Nuclear magnetic resonance, NMR: 1H-NMR (DMSO-d6) δ (ppm): 5.15 (1H, d, protons on the glycoside key), 6.41 (1H, d, C6-H), 6.76 (1H, d, C8-H), 6.91 (1H, d, C5 '-H), 7.55 (1H, dd, C2 '-H), 7.71 (1H, d, C6 '-H), 9.10 (3H, m, C3, C3 ', C4 '-OH), 12.47 (1H, s, C5-OH); 13C-NMR (DMSO-d6) δ (ppm) data see Table 1.Mass spectrum and hydrogen, nuclear magnetic resonance of carbon data and Quercetin-7-O-β-D-glycoside literature value (Zhang Xiaofeng, Hu Bailin, Zhou Bingnan. the active substance research [J] of Tibetan medicine Heterostemonous Biebersteinia. Acta Pharmaceutica Sinica.1995,30 (3): 211-214) all corresponding coincideing.The determination data of carbon spectrum and document (K.R.Markham, B.Ternal, R.Stanley, H.Geiger and T.J.MabryCARBON-13NMR STUDIES OF FLAVONOIDS-III[J] .Tetrahedron.1978,34:1389-1397) highly consistent.
Table 1. Compound I (kaempferol-7-O-β-D-glycoside)
And II (Quercetin-7-O-β-D-glycoside)
13The chemical shift data (ppm) that C-NMR measures
Confirm that through above-mentioned test separating the chemical compound that obtains from Lignum Cudraniae tricuspidatae is respectively kaempferol-7-O-β-D-glycoside and Quercetin-7-O-β-D-glycoside, purity reaches 98%.This experiment has also illustrated and contained kaempferol-7-O-β-D-glycoside and Quercetin-7-O-β-D-glycoside in the Lignum Cudraniae tricuspidatae simultaneously.
The preparation of embodiment 2-5 cudrania tricuspidata extracts (cross the macropore resin bed and analyse post)
Extraction process: 100kg Lignum Cudraniae tricuspidatae fritter, with 5-10 times of 0%-70% alcohol-water solution, reflux, extract, 2 times to initial medical material weight, each 1-5 hours, merge extractive liquid,, being evaporated to medicinal liquid does not have the alcohol flavor.
Purifying process: concentrated solution is crossed a neutral macroporous resin column (HPD-100, precious grace chemical industry), use the pure water eluting of 3-6 times of column volume amounts then, use 20%, 40%, 60%, 80% ethanol elution of 2-4 times of column volume amounts respectively, collect 40-60% ethanol elution and concentrate drying.
The result that the extraction process parameter is selected and obtained:
Composition one: kaempferol-7-O-β-D-glycoside;
Composition two: Quercetin-7-O-β-D-glycoside
The preparation of embodiment 6 cudrania tricuspidata extracts (crossing macroporous resin and gel chromatography column)
Get 40-60% ethanol stream part partial concentration liquid that embodiment 3 obtains, after one gel (Sephadex LH-20 is housed, Pharmacia) chromatographic column, 50% washing with alcohol through 4-8 times of column volume amounts, 5% ethanol elution of 4-8 times of column volume amounts of reuse, collect 95% ethanol stream part, concentrating under reduced pressure, vacuum drying obtains extract powder 100g, wherein, general flavone content 35%, kaempferol-7-O-β-D-glycoside 9%, Quercetin-7-O-β-D-glycoside 8%, the weight ratio of kaempferol-7-O-β-D-glycoside and Quercetin-7-O-β-D-glycoside is 1:0.88.
The preparation of embodiment 7 cudrania tricuspidata extracts (crossing macroporous resin and polyamide chromatographic column)
Get 40-60% ethanol stream part partial concentration liquid that embodiment 3 obtains, after a chromatographic column that polyamide is housed, through 3-6 times of water washings to the column volume amount, with 1-4 times to 20% of column volume amount, 30%, 40% and 50% ethanol gradient elution, collect 30-50% ethanol stream part, concentrating under reduced pressure, vacuum drying, obtain extract powder 500g, wherein, general flavone content 30%, kaempferol-7-O-β-D-glycoside 7%, Quercetin-7-O-β-D-glycoside 6%, the weight ratio of kaempferol-7-O-β-D-glycoside and Quercetin-7-O-β-D-glycoside is 1:0.86.
The preparation of embodiment 8 cudrania tricuspidata extracts (crossing macroporous resin, polyamide and gel chromatography column)
Get 30-50% ethanol stream part partial concentration liquid that embodiment 7 obtains, after a chromatographic column that gel is housed, 50% washing with alcohol through 4-8 times of column volume amounts, 95% ethanol elution of 4-8 times of column volume amounts of reuse, collect 95% ethanol stream part, concentrating under reduced pressure, vacuum drying, obtain extract powder 100g, wherein, general flavone content 50%, kaempferol-7-O-β-D-glycoside 20%, Quercetin-7-O-β-D-glycoside 15%, the weight ratio of kaempferol-7-O-β-D-glycoside and Quercetin-7-O-β-D-glycoside is 1:0.75.
The preparation of embodiment 9 tablets
Get the dry extract (flavones content 18%) 45% that embodiment 3 makes, add microcrystalline Cellulose 35%, low-substituted hydroxypropyl cellulose 8%, starch 8%, mixing granulation, in add micropowder silica gel, magnesium stearate each 0.5%, tabletting, film coating 3%, above-mentioned percentage ratio is weight percentage.The heavy 0.5g of sheet, every contains flavone 40mg, kaempferol-7-O-β-D-glycoside 6.5mg wherein, Quercetin-7-O-β-D-glycoside 4.5mg.
Embodiment 10 capsular preparations
Get the extract powder (general flavone content 18%) 60% that embodiment 3 makes, dextrin 20%, starch 10% mix, and other gets 10% amount starch slurrying, is used for wet granulation, dry back filling capsule, and above-mentioned percentage ratio is weight percentage.The heavy 0.4g of grain, every contains total flavones 40mg, kaempferol-7-O-β-D-glycoside 7.0mg wherein, Quercetin-7-O-β-D-glycoside 4.5mg.
The preparation of embodiment 11 injectable powder
Get the extract powder 50g that embodiment 8 makes, add glucose 50g,, divide to be filled in the bottle with the dissolving of 1000ml aquesterilisa, sterilising filtration, every bottle of 2ml, lyophilization then, sealing is promptly.Every bottle of powder pin contains total flavones 50mg, kaempferol-7-O-β-D-glycoside 20mg, Quercetin-7-O-β-D-glycoside 15mg.
Embodiment 12 anti-tumor activity tests (intestinal cancer)
1. be subjected to the reagent thing: cudrania tricuspidata extract
The cudrania tricuspidata extract of sample 1: embodiment 3
The cudrania tricuspidata extract of sample 2: embodiment 6
The cudrania tricuspidata extract of sample 3: embodiment 7
The cudrania tricuspidata extract of sample 4: embodiment 8
2. tumor cell line: HCT-116 (humanized's colorectal cancer, national drug screening center).
3. experimental technique: leech acyl rhodamine B (Sulforhodamine B, SRB) protein staining method.Be 72 hours action time.
4. experimental result: see Table 1.
Table 1. Lignum Cudraniae tricuspidatae extracts the suppression ratio (%) of 1～4 couple of people HCT-116 of sample growth of tumour cell
5. experiment conclusion
The growth activity that Lignum Cudraniae tricuspidatae extracts 1～4 couple of people HCT-116 of sample intestinal cancer has the obvious suppression effect.
Embodiment 13 anti-tumor activity tests (gastric cancer)
1. be subjected to the reagent thing: cudrania tricuspidata extract sample 1～4 (with embodiment 12)
2. tumor cell line: HGC-7901 (humanized's gastric cancer, national drug screening center).
3. experimental technique: leech acyl rhodamine B (Sulforhodamine B, SRB) protein staining method.Be 72 hours action time.
4. experimental result: see Table 2.
Table 2. Lignum Cudraniae tricuspidatae extracts the suppression ratio (%) of 1～5 couple of people HGC-7901 of sample growth of tumour cell
5. experiment conclusion: the growth activity that Lignum Cudraniae tricuspidatae extracts 1～5 couple of people HGC-7901 of sample gastric cancer has the obvious suppression effect.
Above-mentioned test shows, cudrania tricuspidata extract of the present invention and all kinds of preparation thereof, and its active component is controlled, and antitumous effect is remarkable, has wide market application prospect.
Embodiment 14 toxicologic studies
4 sample samples for embodiment 12, carried out maximum dosage-feeding mensuration: extract is irritated stomach respectively and is given mice, dosage doubles to enhance, wherein the dosage of sample 2 be respectively 0.4,0.8,1.6g/kg, the suitable crude drug 510g/kg of its maximum dose level, be 101 times of clinical using dosage, tangible toxicity is not also found in none death of animal as a result.
Claims (15)
1. cudrania tricuspidata extract, it is characterized in that, the weight ratio of the kaempferol in the extract-7-O-β-D-glycoside and Quercetin-7-O-β-D-glycoside is 1:0.6-1.1, based on extract weight, the total flavones percentage by weight is 1.5-50%, kaempferol-7-O-β-D-glycoside percentage by weight is 0.3-20%, and Quercetin-7-O-β-D-glycoside percentage by weight is 0.2-15%, and extract is made by the method that comprises the following steps:
A) Lignum Cudraniae tricuspidatae extraction process: get the Lignum Cudraniae tricuspidatae medical material, with 5-10 times of 0%-70% alcohol-water solution, reflux, extract, 2-4 times to initial medical material weight, each 1-5 hours, merge extractive liquid, was evaporated to relative density 1.06-1.07, wherein, described alcohol is selected from lower aliphatic alcohols;
B) Lignum Cudraniae tricuspidatae purifying process: the extraction concentrated solution that step a is made, cross a chromatographic column that macroporous resin is housed, through 3-6 times of water washings to the resin bed volume, with 2-4 times of 20-80% alcoholic solution gradient elutions to the resin bed volume, collect 40-60% ethanol stream part, concentrating under reduced pressure, vacuum drying obtains extract powder.
2. as claim cudrania tricuspidata extract according to claim 1, it is characterized in that, the weight ratio of the kaempferol in the described extract-7-O-β-D-glycoside and Quercetin-7-O-β-D-glycoside is 1:0.6-0.9, based on extract weight, the total flavones percentage by weight is 14-50%, kaempferol-7-O-β-D-glycoside percentage by weight is 2.5-20%, and Quercetin-7-O-β-D-glycoside percentage by weight is 1.7-15%.
3. cudrania tricuspidata extract according to claim 1, it is characterized in that, described step b is after obtaining macroporous resin chromatographic column 40-60% ethanol stream part concentrated solution, after a chromatographic column that polyamide is housed, through 3-6 times of water washings, with 1-4 times of 20-50% ethanol gradient elutions to the column volume amount to the column volume amount, collect 30-50% ethanol stream part, concentrating under reduced pressure, vacuum drying obtains extract powder.
4. as cudrania tricuspidata extract as described in the claim 3, it is characterized in that, at described polyamide chromatographic column excessively and after obtaining 30-50% ethanol stream part concentrated solution step, after a chromatographic column that gel is housed, through 50% washing with alcohol of 4-8 column volume amounts, 95% ethanol elution of reuse 4-8 column volume amounts, collect 95% ethanol stream part, concentrating under reduced pressure, vacuum drying obtains extract powder.
5. cudrania tricuspidata extract according to claim 1, it is characterized in that, after described step b obtains macroporous resin chromatographic column 40-60% ethanol stream part concentrated solution, cross a chromatographic column that gel is housed, through 50% washing with alcohol of 4-8 times of column volume amounts, 95% ethanol elution of 4-8 times of column volume amounts of reuse, collect 95% ethanol stream part, concentrating under reduced pressure, vacuum drying obtains extract powder.
6. as the preparation method of cudrania tricuspidata extract as described in arbitrary claim in the claim 1～5, comprise the following steps:
A) Lignum Cudraniae tricuspidatae extraction process: get the Lignum Cudraniae tricuspidatae medical material, with 5-10 times of 0%-70% alcohol-water solution, reflux, extract, 2-4 times to initial medical material weight, each 1-5 hours, merge extractive liquid, was evaporated to relative density 1.06-1.07, wherein, described alcohol is selected from lower aliphatic alcohols;
B) Lignum Cudraniae tricuspidatae purifying process: the extraction concentrated solution that step a is made, cross a chromatographic column that macroporous resin is housed, through 3-6 times of water washings to the resin bed volume, with 2-4 times of 20-80% alcoholic solution gradient elutions to the resin bed volume, collect 40-60% ethanol stream part, concentrating under reduced pressure, vacuum drying obtains extract powder.
7. as the preparation method of cudrania tricuspidata extract as described in the claim 6, it is characterized in that, described macroporous resin be in low polar macroporous resin, the volume of the macroporous resin of adorning is 1/2-1/4 of an initial medical material volume in the chromatographic column.
8. as the preparation method of cudrania tricuspidata extract as described in claim 6 or 7, it is characterized in that, described step b is after obtaining macroporous resin chromatographic column 40-60% ethanol stream part concentrated solution, after a chromatographic column that polyamide is housed, through 3-6 times of water washings, with 1-4 times of 20-50% ethanol gradient elutions to the column volume amount to the column volume amount, collect 30-50% ethanol stream part, concentrating under reduced pressure, vacuum drying obtains extract powder.
9. as the preparation method of cudrania tricuspidata extract as described in the claim 8, it is characterized in that, preferred 40-80 order polyamide of described polyamide, the weight of the polyamide of adorning is 1/3-1/8 of initial medical material weight in the chromatographic column.
10. as the preparation method of cudrania tricuspidata extract as described in the claim 8, it is characterized in that, at described polyamide chromatographic column excessively and after obtaining 30-50% ethanol stream part concentrated solution step, after a chromatographic column that gel is housed, through 50% washing with alcohol of 4-8 column volume amounts, 95% ethanol elution of reuse 4-8 column volume amounts, collect 95% ethanol stream part, concentrating under reduced pressure, vacuum drying obtains extract powder.
11. preparation method as cudrania tricuspidata extract as described in the claim 6, it is characterized in that, after described step b obtains macroporous resin chromatographic column 40-60% ethanol stream part concentrated solution, cross a chromatographic column that gel is housed, through 50% washing with alcohol of 4-8 times of column volume amounts, 95% ethanol elution of 4-8 times of column volume amounts of reuse, collect 95% ethanol stream part, concentrating under reduced pressure, vacuum drying obtains extract powder.
12. the preparation method as cudrania tricuspidata extract as described in claim 10 or 11 is characterized in that described gel is a glucose gel, the weight of the gel of adorning is 10-50 times of upper prop concentrated solution dry weight in the chromatographic column.
13. the described cudrania tricuspidata extract of arbitrary claim is used to prepare the antitumor pharmaceutical formulation in the claim 1～5.
14. the purposes as cudrania tricuspidata extract as described in the claim 13 is characterized in that described pharmaceutical formulation is selected from granule, tablet, capsule, dry syrup or freeze-dried powder.
15. a pharmaceutical composition is made up of described cudrania tricuspidata extract of arbitrary claim and pharmaceutic adjuvant in the claim 1～5 of treatment effective dose, the weight ratio of cudrania tricuspidata extract and pharmaceutic adjuvant is 3-8:7-2.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102659853A (en) * | 2012-05-08 | 2012-09-12 | 济南圣泉唐和唐生物科技有限公司 | Method for purifying substances by utilizing chromatographic column |
| CN104758197A (en) * | 2014-01-03 | 2015-07-08 | 伽蓝（集团）股份有限公司 | Cudrania amboinesis extract product and applications thereof |
| CN105796647A (en) * | 2014-12-30 | 2016-07-27 | 暨南大学 | Cudrania tricuspidata extract and application thereof to preparation of RXR receptor transcription inhibitor |
| CN106404955A (en) * | 2016-09-10 | 2017-02-15 | 长治市食品药品检验所 | Method for measuring content of quercetin-7-glucoside in sorghum red pigment |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1515294A (en) * | 2003-01-07 | 2004-07-28 | 上海市中药研究所 | Solid preparation containing extract of three-bristle cudrania and its preparation method |
| CN1726962A (en) * | 2005-07-27 | 2006-02-01 | 濮桂宝 | Method for controlling quality of cudrania wood and preparation |
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2007
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102659853A (en) * | 2012-05-08 | 2012-09-12 | 济南圣泉唐和唐生物科技有限公司 | Method for purifying substances by utilizing chromatographic column |
| CN102659853B (en) * | 2012-05-08 | 2014-10-08 | 济南圣泉唐和唐生物科技有限公司 | Method for purifying substances by utilizing chromatographic column |
| CN104758197A (en) * | 2014-01-03 | 2015-07-08 | 伽蓝（集团）股份有限公司 | Cudrania amboinesis extract product and applications thereof |
| CN104758197B (en) * | 2014-01-03 | 2018-02-09 | 伽蓝（集团）股份有限公司 | A kind of cudrania tricuspidata extract and its application |
| CN105796647A (en) * | 2014-12-30 | 2016-07-27 | 暨南大学 | Cudrania tricuspidata extract and application thereof to preparation of RXR receptor transcription inhibitor |
| CN106404955A (en) * | 2016-09-10 | 2017-02-15 | 长治市食品药品检验所 | Method for measuring content of quercetin-7-glucoside in sorghum red pigment |
| CN106404955B (en) * | 2016-09-10 | 2019-12-17 | 长治医学院 | Method for determining content of quercetin-7-glucoside in sorghum red pigment |
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| CN101375937B (en) | 2012-03-21 |
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