CN101804128B - Medicine composition, and use thereof in preparing medicine for curing inflammatory enteropathy - Google Patents
Medicine composition, and use thereof in preparing medicine for curing inflammatory enteropathy Download PDFInfo
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- CN101804128B CN101804128B CN 200910014317 CN200910014317A CN101804128B CN 101804128 B CN101804128 B CN 101804128B CN 200910014317 CN200910014317 CN 200910014317 CN 200910014317 A CN200910014317 A CN 200910014317A CN 101804128 B CN101804128 B CN 101804128B
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- Medicines Containing Plant Substances (AREA)
Abstract
The invention discloses a medicine composition containing pollen pini and green tea extract and use thereof in preparing medicine for curing inflammatory enteropathy. The invention further discloses a preparation method of green tea extract, specifically comprising the following steps: taking and grinding tea leaves into powder, adding a 10-30 folds amount of extraction solvent, heating for 10-60 minutes for extraction, filtering, recycling filtrate, depositing with calcium chloride, adjusting the pH, carrying out vacuum filtration, dissolving a filter cake with hydrochloric acid solution, and adjusting the pH value of the solution to be about 4. The solution is heated till slight boiling state and is filtered when still hot, and the filtrate is conveyed to a separating funnel after cooled. Acetic ether is added for extraction, and a rotatory evaporator is used for recycling acetic ether, and green tea extract is obtained after evaporation. The content of EGCG is 10%-90%, and preferably the content of EGCG is 30%-70%.
Description
Technical field
The present invention relates to field of medicaments, specifically be meant a kind of pharmaceutical composition and preparation method thereof, and the purposes of this pharmaceutical composition in the medicine of preparation treatment inflammatory bowel.
Background technology
Inflammatory bowel (inflammatory bowel disease is one group of agnogenic chronic enteritis property disease IBD), comprise ulcerative colitis (ulcerative colitis, UC) and Crohn disease (Crohn ' s disease, CD).The sickness rate of IBD is in rising trend both at home and abroad in recent years; Its cause of disease and pathogenesis are that many-sided factor causes; Promptly in physical basis of heredity, because the activation of antigenic stimulus and vivo immuning system, various environmental factorss interactions and the chronic enteritis that causes.The at present definite cause of disease and pathogenesis are still very unclear, and its pathogenetic complexity has caused certain difficulty for clinical radical cure, is classified as one of modern difficult treatment by World Health Organization (WHO).The medicine that is used to treat IBD at present has traditional salicylic acid preparation, 17-hydroxy-11-dehydrocorticosterone, immunosuppressant and emerging biological preparation, and because of the untoward reaction and the price factor of taking for a long time, seeking new Therapeutic Method and the medicine of IBD has become a kind of focus gradually.
The main active of green tea extract is the polyphenols tea polyphenols; Be pale yellow powder; Account for 18～36% of dry weight of tea leaves; The material that content is the highest in the tea polyphenols is that (epigallocatechin-3-gallate, EGCG), EGCG also is the strongest a kind of of biological effect in the catechin compounds to epigallocatechin gallate (EGCG).A large amount of epidemiological studies, experiment in vitro and zoopery confirm that tea polyphenols has very strong antiinflammatory action.Activatory macrophage and lymphocytic emiocytosis cytokine and mediate a series of inflammatory reaction process when being inflamed; These proinflammatory cytokines comprise nuclear transcription factor-kappa B (NF-κ B), interleukin-1 (IL-1), tumor necrosis factor-alpha (TNF-α), chemotactic factor interleukin 8 (IL-8), nitric oxide (NO) and macrophage chemotactic factor etc.; The main mechanism of tea polyphenols antiinflammatory is to reduce the cytokine growing amount, thereby reaches the generation of inflammation-inhibiting or reduce inflammation degree and shortening inflammation persistent period.[Patricia A.Abboud such as Patricia A.Abboud; Paul W.Hake; Timothy J.Burroughs, et al.Therapeutic effect of epigallocatechin-3-gallate in a mousemodel of colitis.European Journal of Pharmacology 2008, (579): 411-417.] confirm through the colitis mouse model; EGCG administration group and blank control group are relatively; Can effectively improve the diarrhoea situation, body weight does not significantly alleviate, and the colon damage only shows edema and congested situation.Evidence [Ran Zhihua; Chen Chixiao; Tree east. epigallocatechin gallate (EGCG) is to the inductive rat colonitis antiinflammatory action of acetic acid Study on Mechanism. [J] gastroenterology, 2008,13 (8): 465～468.]: EGCG can reach antiphlogistic therapeutical effect through regulating immune factor; The colitis reaction is alleviated, and its curative effect is superior to conventional medicament sulfasalazine (SASP).
Pollen Pini medicine name Flos pini, pine Huang are China pinaceae plant Pinus massoniana Lamb Pinus m assoniana Lam b., Pinus tabulaeformis tabulaeform is Carr. or the dry pollen that belongs to several plants together, are foresythia or faint yellow fine powder, and its nature and flavor are sweet flat nontoxic.The history of Pollen Pini dietotherapeutic has exceeded thousand; Be one of two kinds of pollen that record in the ancient mat of the traditional Chinese medical science; Be only food in motherland's medicine and pharmacology treasure-house, medicine dual-purpose pollen kind, the record of " pine is yellow " is just arranged in first pharmacopeia Shennong's Herbal of China, it is classified as top grade; The Tang Dynasty official issues pharmacopeia " Newly Revised Canon of Materia Medica " and also it is included wherein; Ming Dynasty's Li Shizhen (1518-1593 A.D.) is more claimed in Compendium of Material Medica: " Flos pini, sweet, warm, nontoxic.Profit cardiopulmonary, QI invigorating remove the wind hemostasis, also can make wine." as ancient medicine source and nutrient source, health ministry has confirmed that Pollen Pini is a new resource food in recent years, is listed as the bread and cheese management, has taken in 2005 editions " Chinese pharmacopoeia at present.
Pollen Pini contains all nutritional labelings and the life active compound that the pinaster plant is had; Multiple proteins is wherein arranged, seed amino acid surplus in the of 20 (comprise human body necessary 8 kinds), 15 kinds of vitamin, 30 multi mineral prime elements, 11% fat, nearly hundred kinds of enzymes and coenzyme; And nucleic acid, unsaturated fatty acid, lecithin, flavonoid, monosaccharide, polysaccharide, cellulose etc.; Kind surplus total amount reaches 200; And the nutrition that collocation rationally can all replenish, balancing human body is required; Have effects such as the immunity of raising, defying age, resisting fatigue, adjustment organism metabolism, blood fat reducing and skin care, be widely used in fields such as health product, medicine, cosmetics and feed additive at home and abroad.
The occurrence cause and the mechanism of causing a disease of inflammatory bowel are not illustrated so far as yet fully, and research for many years confirms should disease relevant with autoimmune response, the important step that unusual immunoreation or normal immunoregulatory destruction are its morbidities.Contained polysaccharide has immunoloregulation function in the Pollen Pini, can improve intestinal mucosa function reduction or the forfeiture by immunologic dysfunction caused; Wherein contained effective ingredient polysaccharide and multiple nutritional components (aminoacid, trace element and vitamin C etc.) have reparation and Nutrition to impaired intestinal mucosa.Cellulose in the Pollen Pini can reduce the absorbtivity of bile acid, changes the digestion rate and the secretion of digestive juice amount of food.
We find that through test the effect of Pollen Pini and green tea extract being share the treatment inflammatory bowel is better than list with green tea extract or Pollen Pini.
Up to now, we do not see about both and share the report that is used to treat inflammatory bowel.
Summary of the invention
The object of the present invention is to provide a kind of pharmaceutical composition that can be used for treating inflammatory bowel, effective ingredient wherein comprises Pollen Pini and green tea extract.
The present invention further provides this preparation of drug combination method that is used to treat inflammatory bowel.
Another object of the present invention is to provide the purposes of above-mentioned composition in the treatment inflammatory bowel.
In order to solve technical problem of the present invention, the present invention takes following technical scheme:
Green tea is got the preparation of thing:
The preparation of green tea extract:
Get Folium Camelliae sinensis powder and be broken into powder, add the mixture lixiviate 1～3 time of entry, alcohols or water and alcohols.The mixture of water and alcohols, for example concentration is the alcohol of 40～99% (volume ratios).Preferred alcohols comprises methanol, ethanol.During extraction quantity of solvent be 10～30 times of former medicine weight (volume/weight, ml/g).The temperature of extracting is in 30 ℃～90 ℃ scope, preferably at 70 ℃.Extraction time can be 10～60min, and the preferred time is 30min.Collect filtrating and be the lixiviating solution that contains tea polyphenols.Filtrating is reclaimed most of ethanol after the rotary evaporator evaporation and concentration.Add calcium chloride solution, under constantly stirring, adding sodium hydroxide solution adjusting pH is about 7.Solution left standstill after a period of time with cloth formula funnel sucking filtration.Filter cake is used dissolve with hydrochloric acid solution, and the pH value of regulator solution is about 4.Solution is heated to little boiling and filtered while hot, and filtrating cooling back moves into separatory funnel.Add ethyl acetate extraction 1～4 time, decaffeination is further purified tea polyphenols.Combining extraction liquid reclaims ester with rotary evaporator, and evaporate to dryness promptly gets green tea extract.Wherein EGCG content is 10%～90%.Green tea extract is preferably the extract of EGCG content 30%-70%.
The invention still further relates to the pharmaceutical composition that contains Pollen Pini and green tea extract.This pharmaceutical composition can be according to method preparation well known in the art.Can process the peroral dosage form that is suitable for human or animal's use through the mixture by a certain percentage of Pollen Pini and green tea extract and one or more pharmaceutically acceptable solids or liquid excipient and/or adjuvant are combined.The content of the mixture of Pollen Pini and green tea extract in its pharmaceutical composition is generally 0.1～95 weight %.
The weight ratio of Pollen Pini and green tea extract can be 1: 0.1～arbitrary proportion between 1: 1 in the pharmaceutical composition.Wherein Pollen Pini and green tea extract are 3: 1 by weight optimum ratio.
The specific embodiment
Embodiment 1:
1 kilogram of green tea is ground into powder, and with 20 times of amounts, 70% alcoholic solution lixiviate 2 times, each 30min collects filtrating and is the lixiviating solution that contains tea polyphenols.Filtrating is reclaimed most of ethanol after the rotary evaporator evaporation and concentration.Add 500mL 3mol/L calcium chloride solution, under constantly stirring, adding 1mol/L sodium hydroxide solution adjusting pH is about 7.Solution left standstill after a period of time with cloth formula funnel sucking filtration.Filter cake is used the 5mL/L dissolve with hydrochloric acid solution, and the pH value of regulator solution is about 4.Solution is heated to little boiling and filtered while hot, and filtrating cooling back moves into separatory funnel.Add ethyl acetate extraction 3 times, decaffeination is further purified tea polyphenols.Combining extraction liquid reclaims ester with rotary evaporator.Evaporate to dryness gets green tea extract 196g.High effective liquid chromatography for measuring wherein EGCG content is 51.7%.(chromatographic column: Discovery anti-phase C18 chromatographic column; Mobile phase: 0.2% phosphoric acid-acetonitrile (87: 13); Column temperature: 30 ℃; Detect wavelength: 273nm; Flow velocity: 1ml/min)
Embodiment 2:
Take by weighing Pollen Pini 90g, green tea extract (pressing embodiment 1 preparation) 30g, mixing is crossed 80 mesh sieves; Adding microcrystalline Cellulose 200g, starch 90g form pharmaceutical formulation as diluent, mixing, and spraying into 75% ethanol is binding agent system soft material, granulates with 24 mesh sieves; Dry back granulate, mixing, tabletting is processed 1000; Coating promptly gets, every mixture 120mg that contains Pollen Pini and green tea extract.
Embodiment 3:
Take by weighing Pollen Pini 50g, green tea extract (pressing embodiment 1 preparation) 50g, mixing; Cross 80 mesh sieves, be suspended among the soybean oil 400g, grind evenly through colloid mill; As feed liquid,, process 1000 with the compacting of 500mg/ grain; Promptly get every mixture 100mg that contains Pollen Pini and green tea extract through dry, typing, packing.
Embodiment 4:
Take by weighing Pollen Pini 100g, green tea extract (pressing embodiment 1 preparation) 10g, mixing is crossed 80 mesh sieves; Add pregelatinized Starch 280g, microcrystalline Cellulose 80g forms pharmaceutical formulation, mixing as diluent; Spraying into 75% ethanol is binding agent system soft material, granulates with 24 mesh sieves, dry back granulate, mixing; Encapsulated, process 1000, promptly get every mixture 110mg that contains Pollen Pini and green tea extract.
Test Example 1
1. material
Laboratory animal: healthy 3 grades of SD rats, male and female half and half, body weight 200～220g.
Experiment medicine: sulfasalazine; The pharmaceutical composition (hereinafter to be referred as pharmaceutical composition) of Pollen Pini and green tea extract (weight ratio 3: 1); Green tea extract is pressed embodiment 1 preparation.
Reagent and equipment: dehydrated alcohol (analytical pure), TNB (U.S., Sigma company); SOD, MDA detection kit (Beijing Bang Ding Biomedicines, Inc.).TU-1810 ultraviolet-uisible spectrophotometer (Beijing Puxi General Instrument Co., Ltd), Aglient1200 high performance liquid chromatograph (Anjelen Sci. & Tech. Inc), TDL-5-A centrifuge (Anting Scientific Instrument Factory, Shanghai).
The foundation of rat experiment property inflammatory bowel disease model:
Adopt 2,4, the modeling of 6-TNB (TNBS) method:, disposable TNBS (100mg/kg) and 50% alcoholic solution 0.25ml are slowly injected in the enteric cavity at the about 8cm of anus place with the rubber tube for transfusion anaesthetizing behind the rat fasting 24h.The 3rd day treatment group begins administration after the modeling.
Experiment is divided into groups and administration:
Normal group and model control group are given normal saline and are irritated stomach, 1/d, totally 5 weeks; Positive drug matched group (to call the positive drug group in the following text) selects for use sulfasalazine (SASP) by the medium therapeutic dose that is equivalent to the people, irritates stomach and gives 0.3g/kg SASP suspension, and 1/d treated for 5 weeks altogether; Pollen Pini gives 150mg/kg and irritates stomach, 1/d; The green tea extract group gives 50mg/kg and irritates stomach, 1/d; Pharmaceutical composition is irritated stomach by low dose group (100mg/kg), middle dose groups (200mg/kg) and high dose group (300mg/kg) respectively, and 1/d treated for 5 weeks altogether.
Observation index and assay method:
Respectively organize rat after 5 weeks through 10% chloral hydrate (400mg/kg) anesthesia; Open abdomen immediately and separate colon; Cut off enteric cavity along the mesentery edge, get lesions colon 1～2cm quick-freezing in liquid nitrogen, after go to-70 ℃ of preservations; Wait until and survey superoxide dismutase (SOD), malonaldehyde (MDA), with both content as observation index.The homogenate SOD of colon assay adopts xanthine oxidase to measure, and the result representes with U/mg.MDA content adopts the production of thiobarbituric acid reactive colorimetric method for determining, and the result representes with μ mol/g.
The lipid peroxidation of all finding free radical and triggering thereof about the clinical and experimentation of inflammatory bowel plays an important role in its generation, development.And SOD can remove oxygen-derived free radicals effectively, and the ultra oxyradical of catalysis resolves into H
2O
2, and then under catalatic effect, resolve into H
2O and oxygen molecule, thus the lipid peroxidation in the intestinal tissue suppressed.The SOD activity can reflect the lipid peroxidation of body and the ability of anti peroxidation of lipid reaction indirectly.
Body produces oxygen-derived free radicals through enzyme system and non-enzyme system, and the latter can attack the polyunsaturated fatty acid in the biomembrane, causes lipid peroxidation, and so forms MDA and new oxygen-derived free radicals etc.Thereby the test MDA amount usually can reflect the snperoxiaized degree of body inner lipid, reflect the degree of cell injury indirectly.
Statistical procedures:
Experimental data is represented with x ± s, carries out the t check between each group.
2. result
Table 1 is respectively organized SOD and MDA changes of contents in the colon (n=10, x ± s)
Annotate:
△ △ △Compare with normal group P＜0.001;
*P＜0.05,
* *P＜0.01,
* *Compare with model group P＜0.001.
+Compare with the Pollen Pini group P＜0.05;
#Compare with the green tea extract group P＜0.05.
Table 2 is respectively organized the rat colon general form and (n=10, x ± s divide) are compared in Histological injury's scoring
Annotate:
*Compare with model group P＜0.01.
+Compare with the Pollen Pini group P＜0.05;
#Compare with the green tea extract group P＜0.05.
(intestinal tissue damage general form and histology's form methods of marking [Zhu Feng; Qian Jiaming; Pan Guozong. the foundation [J] of cell immune response property inflammatory bowel animal model. Chinese Academy of Medical Sciences's journal; 1998,20 (4): 271.] general form damage scoring index comprises adhesion, contrafluxion, ulcer and inflammation.Adhesion and congested by the weight that has that it's too late 0,1,2 minutes respectively, when inflammation, ulcer number occurring and increasing by 1, ulcer surface＞2cm, every increases of scope 1cm, scoring all adds 1.Histological indices comprises ulcer, inflammation, granuloma, fibrosis and the pathological changes degree of depth, and by the weight that has that it's too late 0,1,2 minutes respectively, pathological changes depth Submucosa flesh layer, placenta percreta were counted respectively 1,2,3 fens.Each item addition gets total points.)
3. conclusion
Experiment shows that the SOD vigor of model group and normal group relatively have significance to reduce (P＜0.001); The SOD vigor of positive drug group and model group relatively have significance rising (P＜0.001); Pollen Pini group and green tea extract group and model group more then have significance rising (P＜0.01); The SOD vigor of the basic, normal, high dose groups of pharmaceutical composition and model group more then have rising (P＜0.05, P＜0.01, P＜0.001), and the SOD vigor of middle and high dose groups and Pollen Pini group and green tea extract group have significant difference (P＜0.05).
The MDA content of model group and normal group relatively have significance rising (P＜0.001); The MAD content of positive drug group with more then have significance to reduce (P＜0.01) with model group; Pollen Pini group and green tea extract group and model group more then have significance to reduce (P＜0.01); The MDA content of the basic, normal, high dose groups of pharmaceutical composition and model group more then have reduction (P＜0.05), and the MDA content of middle and high dose groups and Pollen Pini group and green tea extract group have significant difference (P＜0.05).
Rat colon general form and Histological injury's scoring also show that Pollen Pini, green tea extract and pharmaceutical composition all have therapeutical effect to colitis, and the middle and high dose groups of pharmaceutical composition relatively has significant difference with single with Pollen Pini or green tea extract.
In sum, this experiment proof: 1) pharmaceutical composition has the certain repairing effect to rat pathological changes colonic mucosa; 2) inflammatory bowel rat MAD content behind medicine composite for curing reduces, and the vigor of SOD increases, and is good with the therapeutic effect of middle and high dose groups.3) the middle and high dose groups of pharmaceutical composition is superior to single with Pollen Pini or green tea extract to the therapeutic effect of inflammatory bowel.
Claims (6)
1. a pharmaceutical composition of treating inflammatory bowel is characterized in that, effective ingredient Pollen Pini and green tea extract by weight, proportioning is 3: 1;
Wherein green tea extract prepares according to following method:
Get Folium Camelliae sinensis powder and be broken into powder, add the mixture lixiviate 1～3 time of entry or alcohols or water and alcohols; According to the ml/g ratio, quantity of solvent is 10～30 times of former medicine weight during extraction; The temperature of extracting is in 30 ℃～90 ℃ scope; Extraction time is 10～60min; Filtrating is reclaimed most of ethanol after the rotary evaporator evaporation and concentration; Add calcium chloride solution, under constantly stirring, adding sodium hydroxide solution adjusting pH is 7; Solution left standstill after a period of time with cloth formula funnel sucking filtration; Filter cake is used dissolve with hydrochloric acid solution, and the pH value of regulator solution is 4; Solution is heated to little boiling and filtered while hot, and filtrating cooling back moves into separatory funnel; Add ethyl acetate extraction 1～4 time, decaffeination, combining extraction liquid reclaims ester with rotary evaporator, and evaporate to dryness promptly gets green tea extract, and wherein EGCG content is 10%～90%.
2. pharmaceutical composition according to claim 1 is characterized in that: said alcohols is methanol or ethanol.
3. pharmaceutical composition according to claim 1 is characterized in that: the temperature of extraction is 70 ℃.
4. pharmaceutical composition according to claim 1 is characterized in that: extraction time is 30min.
5. pharmaceutical composition according to claim 1 is characterized in that: EGCG content is 30%-70% in the green tea extract.
6. claim 1 or 2 or 3 or 4 or 5 said pharmaceutical compositions are treated the purposes in the inflammatory bowel medicine in preparation.
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