CN105749154B - A kind of probiotics fermention Traditional Chinese medicine compound composition that treating liver cancer and its preparation and detection method - Google Patents

A kind of probiotics fermention Traditional Chinese medicine compound composition that treating liver cancer and its preparation and detection method Download PDF

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CN105749154B
CN105749154B CN201610138496.8A CN201610138496A CN105749154B CN 105749154 B CN105749154 B CN 105749154B CN 201610138496 A CN201610138496 A CN 201610138496A CN 105749154 B CN105749154 B CN 105749154B
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chinese medicine
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medicine compound
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CN105749154A (en
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吴炳新
甄宗武
褚新红
牛纪江
孙筱林
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SANZHU fur PHARMACEUTICAL Co Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/23—Apiaceae or Umbelliferae (Carrot family), e.g. dill, chervil, coriander or cumin
    • A61K36/233—Bupleurum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/56—Materials from animals other than mammals
    • A61K35/58—Reptiles
    • A61K35/586—Turtles; Tortoises, e.g. terrapins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/56—Materials from animals other than mammals
    • A61K35/618—Molluscs, e.g. fresh-water molluscs, oysters, clams, squids, octopus, cuttlefish, snails or slugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/745—Bifidobacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/06—Fungi, e.g. yeasts
    • A61K36/07—Basidiomycota, e.g. Cryptococcus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/25—Araliaceae (Ginseng family), e.g. ivy, aralia, schefflera or tetrapanax
    • A61K36/258—Panax (ginseng)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/41—Crassulaceae (Stonecrop family)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
    • A61K36/484—Glycyrrhiza (licorice)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/53—Lamiaceae or Labiatae (Mint family), e.g. thyme, rosemary or lavender
    • A61K36/536—Prunella or Brunella (selfheal)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/53—Lamiaceae or Labiatae (Mint family), e.g. thyme, rosemary or lavender
    • A61K36/537—Salvia (sage)
    • A—HUMAN NECESSITIES
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    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/53—Lamiaceae or Labiatae (Mint family), e.g. thyme, rosemary or lavender
    • A61K36/539—Scutellaria (skullcap)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/66—Papaveraceae (Poppy family), e.g. bloodroot
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/88—Liliopsida (monocotyledons)
    • A61K36/906—Zingiberaceae (Ginger family)
    • A61K36/9066—Curcuma, e.g. common turmeric, East Indian arrowroot or mango ginger
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/90—Plate chromatography, e.g. thin layer or paper chromatography
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/10—Preparation or pretreatment of starting material
    • A61K2236/19—Preparation or pretreatment of starting material involving fermentation using yeast, bacteria or both; enzymatic treatment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/30—Extraction of the material
    • A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
    • A61K2236/331—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using water, e.g. cold water, infusion, tea, steam distillation, decoction
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/30—Extraction of the material
    • A61K2236/39—Complex extraction schemes, e.g. fractionation or repeated extraction steps

Abstract

The invention discloses a kind of probiotics fermention Traditional Chinese medicine compound composition for treating liver cancer and its preparation and detection methods, nutrients and the inorganic salts etc. of different ratio are added in Chinese medicine compound prescription medicinal material after water mentions, high-temperature sterilization accesses compound enteron aisle probiotics fermention rear stabilization and handles up to probiotics fermention Traditional Chinese medicine compound composition；The Chinese medicine compound prescription are as follows: radix bupleuri, radix scutellariae, red ginseng, hypericum japonicum, stringy stonecrop, stir-baked CARAPAX TRIONGCIS with sand then treated with vinegar, Radix Salviae Miltiorrhizae, Prunella vulgaris, raw oyster, rhizoma corydalis, rhizoma curcumae longae concisa, umbellate pore furgus, radix glycyrrhizae preparata.The invention also discloses the identification of the probiotics fermention Traditional Chinese medicine compound composition and detection methods.It is demonstrated experimentally that probiotics fermention Traditional Chinese medicine compound composition of the invention can significantly improve the curative effect of Chinese medical extract, tumour inhibiting rate is improved, has conspicuousness effect in terms of inhibiting liver neoplasm growth.The present invention is remarkably improved the mouthfeel of Chinese medicinal compound extract, mitigates Chinese medical extract to the irritation of stomach, can improve the appetite of patient, improve the constitution of patient.

Description

A kind of probiotics fermention Traditional Chinese medicine compound composition that treating liver cancer and its preparation and detection Method
Technical field
The present invention relates to a kind of Chinese medical extracts through fermentation composition made from enteron aisle probiotics fermention and the fermentation group It closes object and is preparing the application in the drug for treating liver cancer, belong to technical field of biological fermentation.
Background technique
Hepatocellular carcinoma (HCC) is one of China's common cancer, and the death rate occupies in alimentary system malignant tumour Three, it is only second to gastric cancer and the cancer of the esophagus, disease incidence is on the rise, seriously endangers human health and existence.As the modern times are raw The development of object medical technology, Clinical Surgery's technology, minimally-invasive treatment technology etc., the clinical diagnosis and treatment working level of liver cancer present steadily and Lasting raising.In the latest 20 years, with medico physics, iconography, anesthesiology, immunology, molecular biology continuous hair Exhibition and surgical technic it is ripe day by day, the treatment of China's primary carcinoma of liver achieves significant progress, Resection Rate, postoperative Survival rate and postoperative life quality improve a lot.But compared with other oncotherapies, the curative effect of liver cancer such as people's will, is studied carefully far away Its reason is in addition to the biological characteristics of liver cancer itself determine its prognosis, selection, the patient even treatment of doctor for the treatment of method Idea is also an important factor for affecting the treatment.
Traditional Chinese medical herbal treatment liver cancer takes diagnosis and treatment based on an overall analysis of the illness and the patient's condition, the method that tonification and purgation in combination, often with other therapy fit applications, with Body's resistance to disease is improved, overall health of patients and symptom are improved, mitigates chemotherapy, radiotherapy adverse reaction.Should say, many liver cancer patients and Its family members shows special preference to Chinese medicine, because they know Western medicine " palliative ", and toxic side effect is very big, so they are not Want the disease with western medicine oneself, wants to be improved and treated with the Chinese medicine of " strengthening vital QI to eliminate pathogenic factors, treating both manifestation and root cause of disease ".But traditional Chinese medicine Decoction also has deficiency, that is, smell is unpleasant, and many patients are difficult to be resistant to if long-term use, or even smell flavour of a drug and just think It spits.Though " Good medicine for health tastes bitter to the mouth ", if things go on like this affects patient's appetite, it is unfavorable for improvement, the disease of patient's constitution instead The recovery of feelings.
Probiotics is a kind of active microorganism beneficial to host, is to be colonized in human body intestinal canal, in reproductive system, can generate Definite health efficacy so as to improve host's microecological balance, play the active beneficial microorganism general name of beneficial effect.Human body moves Beneficial bacterium or fungi mainly have in object: clostridium butyricum, lactic acid bacteria, Bifidobacterium, lactobacillus acidophilus, actinomyces, yeast Bacterium etc..
Chemotherapeutics and ray can kill probiotics, and intestinal flora is caused to be lacked of proper care.Show as abdominal distension, constipation, nutriment It loses and toxin is absorbed, not only influence Rehabilitation, it is also possible to it forces chemotherapy, radiotherapy to be interrupted, influences the appetite of patient, into One step reduces the immunity of patient, forms vicious circle.
Summary of the invention
In order to overcome the shortcomings of that the prior art treats liver cancer, it is an object of the present invention to provide a kind of benefits for treating liver cancer Raw bacterium fermented tcm compound, the composition are made after enteron aisle probiotics fermention by Chinese medicinal compound extract.It uses Technical solution are as follows: nutrients and the inorganic salts etc. of different ratio are added in Chinese medicine compound prescription medicinal material after water mentions, and high-temperature sterilization, access is again Beneficial bacteria of intestinal tract fermentation rear stabilization is closed to handle up to probiotics fermention Traditional Chinese medicine compound composition.
Chinese medicine compound prescription of the present invention is made of (by weight) the following raw material medicine:
Radix bupleuri 12-40 parts by weight, red ginseng 4-24 parts by weight, hypericum japonicum 48-96 parts by weight, are hung down at radix scutellariae 12-40 parts by weight Basin grass 48-96 parts by weight, stir-baked CARAPAX TRIONGCIS with sand then treated with vinegar 36-80 parts by weight, Radix Salviae Miltiorrhizae 36-48 parts by weight, Prunella vulgaris 24-60 parts by weight, raw oyster 36- 80 parts by weight, rhizoma corydalis 12-40 parts by weight, rhizoma curcumae longae concisa 12-36 parts by weight, umbellate pore furgus 24-40 parts by weight, radix glycyrrhizae preparata 8-36 weight Part.
According to a preferred embodiment of the invention, Chinese medicine compound prescription of the present invention is made of the following raw material medicine (by weight Measure part meter):
20 parts by weight of radix bupleuri, 20 parts by weight of radix scutellariae, 8 parts by weight of red ginseng, 48 parts by weight of hypericum japonicum, 48 parts by weight of stringy stonecrop, 36 parts by weight of stir-baked CARAPAX TRIONGCIS with sand then treated with vinegar, 36 parts by weight of Radix Salviae Miltiorrhizae, 24 parts by weight of Prunella vulgaris, 40 parts by weight of raw oyster, 24 parts by weight of rhizoma corydalis, piece ginger Yellow 24 parts by weight, 24 parts by weight of umbellate pore furgus, 8 parts by weight of radix glycyrrhizae preparata
It is a further object to provide a kind of preparation sides for preparing above-mentioned probiotics fermention Traditional Chinese medicine compound composition Method, its step are as follows:
(a) above-mentioned 13 taste Chinese medicine, takes stir-baked CARAPAX TRIONGCIS with sand then treated with vinegar to crush, and fineness is in 20 mesh~40 mesh；Add water with other 12 taste Chinese medicines It decocts twice, for the first time plus 12 times of amount water impregnate 1.0 hours, and it decocts, is kept after boiling slightly boiled 1.0 hours, second plus 10 times It measures water to decoct, be kept after boiling slightly boiled 1.0 hours, collecting decoction, filtered, it is 1.01~1.05 that filtrate, which is concentrated into relative density, (60 DEG C), obtain traditional Chinese medicine compound extract；Supernatant can be obtained after being subcooled and sinking in gained traditional Chinese medicine compound extract；
(b) to the traditional Chinese medicine compound extract that step (a) obtains or its it is cold it is heavy after be added in obtained supernatant it is a certain amount of Glucose, peptone, yeast extract, yeast powder and inorganic salts, addition or do not add sweetener, constant volume, adjust pH value to 6.5~ 7.0, in 115 DEG C of autoclave sterilization 50min, obtain sterile Chinese medicinal compound extract.
(c) when above-mentioned sterile Chinese medicinal compound extract temperature is down to 39 DEG C, each percent by volume for being inoculated with preculture is 0.5% one or two kinds of and above Bifidobacterium bacterium solutions after routine culture about 3~6 hours, are inoculated with each body of preculture again Product percentage be 0.5% one or two kinds of and above lactobacillus bacterium solutions, continue fermentation 22~27 hours, when pH value be down to 4.1 with When lower, terminate fermentation, stabilization processes are to get the probiotics fermention Traditional Chinese medicine compound composition.
According to the present invention, probiotics used in Chinese medicinal compound extract fermentation is selected from Bifidobacterium and lactobacillus.
According to a preferred embodiment of the invention, the Bifidobacterium for fermented tcm compound extract selects From bifidobacterium bifidum, bifidobacterium breve, bifidobacterium adolescentis and bifidobacterium infantis；Wherein the lactobacillus is selected from acidophilus (strain source is in Chinese Typical Representative culture for lactobacillus, Lactobacillus delbrueckii, lactobacillus plantarum, Lactobacillus casei and Lactobacillus rhamnosus Object collection (CCTCC) (China, Wuhan) or China General Microbiological culture presevation administrative center (CGMCC) (China, north Capital) or these isolated bacterium bacterial strains from the excrement or intestinal submucosa tissue of normal person).
The additional amount of the glucose is 0.7g/100ml, and the additional amount of yeast extract is 0.13g/100ml, and yeast powder adds Entering amount is 0.07g/100ml, and the additional amount of peptone is 0.4g/100ml.
To improve mouthfeel, in above-mentioned preparation method, sweetener type described in step (b) and dosage are sucrose 3.0g/ 100ml, Sucralose 0.008g/100ml.
It is also another object of the present invention to provide the identification of the fermentation composition and detection methods:
[identification]
(1) probiotics fermention Traditional Chinese medicine compound composition 5ml of the invention is taken to add ethyl acetate to shake with hydrochloric acid tune pH to 1-2 Shake extraction 2 times, each 5ml, combined ethyl acetate liquid, water bath method, residue methanol constant volume to 2ml, as test solution. Scutelloside reference substance separately is taken, adds methanol 1ml the is made solution containing 1mg, as reference substance solution.According to thin-layered chromatography (middle traditional Chinese medicines 0502) allusion quotation version four in 2015 is tested, draw test solution 2ul, reference substance solution 1ul is put respectively in same polyamide film On, with ethyl acetate-butanone-formic acid-water (5:3:1:1) for solvent, it is unfolded, takes out, dry, set under ultraviolet lamp (365nm) It inspects.In sample chromatogram, at the position corresponding to the chromatogram of the reference substance, identical dark spots are shown.
(2) test solution under [identification] (1) item is taken, as test solution.Protocatechualdehyde reference substance separately is taken, adds methanol Solution of every 1ml containing 1mg is made, as reference substance solution.According to thin-layered chromatography (Chinese Pharmacopoeia 2015 version four 0502) examination It tests, draws test solution 10ul, reference substance solution 2ul, put respectively on same silica gel g thin-layer plate, with chloroform-acetic acid Ethyl ester-methyl alcohol-formic acid (12:2:1:0.8) is solvent, is unfolded, and takes out, dries, and is sprayed with 3% ferric trichloride ethanol solution.For examination In product chromatography, at the position corresponding to the chromatogram of the reference substance, the spot of identical bluish grey color is shown.
(3) probiotics fermention Traditional Chinese medicine compound composition 20ml of the invention is taken, water saturated n-butanol shaking is added to extract 2 Secondary, each 15ml merges n-butanol liquid, is washed 2 times, each 20ml with ammonia solution, then water washing 2 times be saturated with n-butanol, Each 20ml, water bath method n-butanol liquid, residue methanol constant volume to 2ml, as test solution.Separately take radix bupleuri control medicinal material Fine powder 0.5g adds methanol 30ml, ultrasonic extraction 30min, and filtration, filtrate water bath method, residue adds water 20ml to make to dissolve, same to legal system At control medicinal material solution.According to thin-layered chromatography (Chinese Pharmacopoeia 2015 version four 0502) test, draw test solution 10ul, Radix bupleuri control medicinal material solution 5ul is put respectively on same silica gel g thin-layer plate, with lower layer's solution of chloroform-methanol-water (13:7:2) For solvent, it is unfolded, takes out, dry, sprays with 40% sulfuric acid solution of 2% paradime thylaminobenzaldehyde, be heated to spot at 60 DEG C Colour developing is clear.In sample chromatogram, on position corresponding with reference medicine chromatography, identical punctation is shown.
(4) probiotics fermention Traditional Chinese medicine compound composition 20ml of the invention is taken to be added diethyl ether with strong ammonia solution tune pH to 9-10 Shaking is extracted 2 times, each 15ml, and ether solution, water bath method, residue methanol constant volume to 1ml, as test solution are merged. Tetrahydropalmatine reference substance separately is taken, adds methanol that solution of every 1ml containing 0.4mg is made, as reference substance solution.According to thin-layered chromatography (Chinese Pharmacopoeia 2015 version four 0502) test, draws test solution 5ul, reference substance solution 2ul, is put respectively in same use On the silica gel g thin-layer plate of 0.5% sodium hydroxide solution preparation, with n-hexane-chloroform-methanol (10:6:1) for solvent, exhibition It opens, takes out, dry, set to fumigate in iodine cylinder and about take out after five minutes, after waving the iodine adsorbed on most plate, set and examined under ultraviolet lamp (365nm) Depending on.In sample chromatogram, at the position corresponding to the chromatogram of the reference substance, the fluorescence spot of identical yellowish green color is shown.
[detection method]
(1) it is appropriate to weigh control substance of Rutin for the preparation of reference substance solution, accurately weighed, adds methanol to dissolve, every 1ml is made Solution containing about 0.2mg to get.
(2) the preparation precision of standard curve measures reference substance solution 1ml, 2ml, 3ml, 4ml, 5ml, 6ml, sets respectively In 25ml measuring bottle, 6ml is added water to, adds 5% sodium nitrite solution 1ml, is mixed, places 6 minutes, adds 10% aluminum nitrate solution 1ml, It shakes up, places 6 minutes, adding sodium hydroxide test solution 10ml is added water to scale, shaken up, and is placed 15 minutes, with corresponding reagent work Blank measures absorbance according to UV-VIS spectrophotometry (Chinese Pharmacopoeia 2015 version four 0401) at 500nm wavelength, Using absorbance as ordinate, concentration is abscissa, draws standard curve.
(3) measuring method precision measures probiotics fermention Traditional Chinese medicine compound composition 5ml of the invention, is slowly added into anhydrous second Alcohol 7.5ml, the shaking of side edged, then plus 60% ethyl alcohol 12ml, shake up, seal, centrifugation, supernatant is transferred in 25ml volumetric flask, adds A small amount of 60% ethyl alcohol cleaning precipitates and is settled to scale.Precision measures 10ml and water is added to be settled to 100ml, shakes up molten to get test sample Liquid.Precision draws test solution 5ml, sets in 25ml volumetric flask, the method under the preparation of sighting target directrix curve, " adds water to certainly 6ml " rises, and operates according to methods, and another accurate absorption test solution 5ml is diluted with water to 25ml, shakes up, make blank, measurement is inhaled in accordance with the law Luminosity, from standard curve read test sample in rutin amount, calculate to get.
The every 1ml of this product presses rutin (C containing general flavone27H30O16) meter, 2.8mg must not be less than.
Beneficial effects of the present invention:
The present invention simulates enteron aisle biotic environment, accesses enteron aisle composite reactive probiotics, fermented tcm compound extract can be with The mouthfeel of Chinese medicinal compound extract is significantly improved, mitigates Chinese medical extract to the irritation of stomach, the appetite of patient can be improved, Improve the constitution of patient.
In the present invention, Chinese medicinal compound extract can significantly improve the curative effect of Chinese medical extract after probiotics fermention, mention High tumour inhibiting rate.For the antitumous effect of the probiotics fermention Traditional Chinese medicine compound composition of detection treatment liver cancer of the invention, the present invention The research of anti-tumor activity is unfolded using transplanted hepatoma in rats for experimental example, and interpretation of result shows treatment liver cancer of the invention Probiotics fermention Traditional Chinese medicine compound composition can improve the vital sign of rat, mitigate ascites degree.Fermentation composition group tumor stove Interior necrosis situation overweights group of not fermenting, and so as to cause tumor quality mitigation, fermentation group tumor control rate is high, and especially middle and high dose Amount group is more apparent, there is significant difference (P < 0.05) compared with not fermenting group with same concentration, and illustrating can be significant after probiotics fermention The antihepatocarcinoma effect of this compound preparation is improved, especially has conspicuousness effect in terms of inhibiting liver neoplasm growth.
Detailed description of the invention
Fig. 1 is the thin-layer chromatogram of radix scutellariae in probiotics fermention Traditional Chinese medicine compound composition of the invention；In figure: 1,2,3 points Not Wei radix scutellariae test solution a1, b1, c1,4,5 be scutelloside reference substance, and 6,7 is lack radix scutellariae negative sample.
Fig. 2 is the thin-layer chromatogram of Radix Salviae Miltiorrhizae in probiotics fermention Traditional Chinese medicine compound composition of the invention；In figure: 1,2,3 points Not Wei Radix Salviae Miltiorrhizae test solution a2, b2, c2,4 be protocatechualdehyde reference substance, and 5 is lack Radix Salviae Miltiorrhizae negative sample.
Fig. 3 is the thin-layer chromatogram of radix bupleuri in probiotics fermention Traditional Chinese medicine compound composition of the invention；In figure: 1 is scarce bavin Hu negative sample, 2,3,4 be respectively radix bupleuri test solution a3, b3, c3, and 5 be radix bupleuri control medicinal material.
Fig. 4 is the thin-layer chromatogram of rhizoma corydalis in probiotics fermention Traditional Chinese medicine compound composition of the invention；In figure: 1,2,3 Respectively rhizoma corydalis test solution a4, b4, c4,4,5 be tetrahydropalmatine reference substance, and 6,7 be scarce rhizoma corydalis negative sample.
Specific embodiment
Embodiment 1: the preparation of probiotics fermention Traditional Chinese medicine compound composition A
Each traditional Chinese medicine ingredients: radix bupleuri 20g are weighed by following weight, radix scutellariae 20g, red ginseng 8g, hypericum japonicum 48g, stringy stonecrop 48g, Stir-baked CARAPAX TRIONGCIS with sand then treated with vinegar 36g, Radix Salviae Miltiorrhizae 36g, Prunella vulgaris 24g, raw oyster 40g, rhizoma corydalis 24g, rhizoma curcumae longae concisa 24g, umbellate pore furgus 24g, radix glycyrrhizae preparata 8g.It takes Stir-baked CARAPAX TRIONGCIS with sand then treated with vinegar crushes, and crosses 20 meshes；It is added water to cook twice with other 12 taste Chinese medicines, for the first time plus 12 times of amount water impregnate 1.0 hours, It decocts, is kept after boiling slightly boiled 1.0 hours, second plus 10 times of amount water decoctions keep slightly boiled 1.0 hours after boiling, merge and decoct Liquid, filtration, it is 1.01~1.05(60 DEG C that filtrate, which is concentrated into relative density), obtain traditional Chinese medicine compound extract；To above-mentioned Chinese medicine compound prescription Glucose 0.7g/100ml, peptone 0.4g/100ml, yeast extract 0.13g/100ml, yeast powder 0.07g/ are added in extracting solution 100ml, inorganic salts (K2HPO4•3H2O 0.1g/100ml、KH2PO40.1g/100ml, ferrous sulfate 7H2O 5.0mg/ 100ml, zinc sulfate 7H2O 4.4mg/100ml, magnesium sulfate 7H2O 50mg/100ml), sucrose 3.0g/100ml and Sucralose 0.008g/100ml is settled to 1000ml, adjusts pH value to 6.5, in 115 DEG C of autoclave sterilization 50min, it is multiple to obtain sterile Chinese medicine Formula extraction.When above-mentioned sterile Chinese medicinal compound extract temperature is down to 39 DEG C, the percent by volume for being inoculated with preculture is 0.5% Bifidobacterium breve (AS 1.2213) bacterium solution, after routine culture 5 hours, again be inoculated with preculture each percent by volume be 0.5% Lactobacillus delbrueckii (AS 1.1480) and lactobacillus acidophilus (AS 1.1854) bacterium solution continues 37 DEG C and ferments 24 hours, when When pH value is down to 4.1 or less, terminate fermentation, filling rear stabilization processing is multiple to get probiotics fermention Chinese medicine under aseptic condition Square composition A.General flavone content in this fermentation composition are as follows: 4.34mg/ml.
Embodiment 2: the preparation of probiotics fermention Traditional Chinese medicine compound composition B
Each traditional Chinese medicine ingredients: radix bupleuri 20g are weighed by following weight, radix scutellariae 20g, red ginseng 8g, hypericum japonicum 48g, stringy stonecrop 48g, Stir-baked CARAPAX TRIONGCIS with sand then treated with vinegar 36g, Radix Salviae Miltiorrhizae 36g, Prunella vulgaris 24g, raw oyster 40g, rhizoma corydalis 24g, rhizoma curcumae longae concisa 24g, umbellate pore furgus 24g, radix glycyrrhizae preparata 8g.It takes Stir-baked CARAPAX TRIONGCIS with sand then treated with vinegar crushes, and crosses 30 meshes；It is added water to cook twice with other 12 taste Chinese medicines, for the first time plus 12 times of amount water impregnate 1.0 hours, It decocts, is kept after boiling slightly boiled 1.0 hours, second plus 10 times of amount water decoctions keep slightly boiled 1.0 hours after boiling, merge and decoct Liquid, filtration, it is 1.01~1.05(60 DEG C that filtrate, which is concentrated into relative density), obtain traditional Chinese medicine compound extract.To above-mentioned Chinese medicine compound prescription Glucose 0.7g/100ml, peptone 0.4g/100ml, yeast extract 0.13g/100ml, yeast powder 0.07g/ are added in extracting solution 100ml, inorganic salts (K2HPO4•3H2O 0.1g/100ml、KH2PO40.1g/100ml, ferrous sulfate 7H2O 5.0mg/ 100ml, zinc sulfate 7H2O 4.4mg/100ml, magnesium sulfate 7H2O 50mg/100ml), be settled to 1000ml, adjust pH value to 7.0, in 115 DEG C of autoclave sterilization 50min, obtain sterile Chinese medicinal compound extract.When above-mentioned sterile Chinese medicinal compound extract temperature When degree is down to 39 DEG C, the bifidobacterium adolescentis (AS 1.2190) and baby that each percent by volume for being inoculated with preculture is 0.5% are double Discrimination bacillus (AS 1.853) bacterium solution after routine culture 3 hours, is inoculated with the sandlwood that each percent by volume of preculture is 0.5% again Sugared lactobacillus (AS 1.22) bacterium solution continues 37 DEG C and ferments 26 hours, when pH value is down to 4.1 or less, terminates fermentation, in sterile Under the conditions of the processing of filling rear stabilization to get probiotics fermention Traditional Chinese medicine compound composition B.General flavone content in this fermentation composition Are as follows: 4.63mg/ml.
Embodiment 3: the preparation of probiotics fermention Traditional Chinese medicine compound composition C
Each traditional Chinese medicine ingredients: radix bupleuri 20g are weighed by following weight, radix scutellariae 20g, red ginseng 8g, hypericum japonicum 48g, stringy stonecrop 48g, Stir-baked CARAPAX TRIONGCIS with sand then treated with vinegar 36g, Radix Salviae Miltiorrhizae 36g, Prunella vulgaris 24g, raw oyster 40g, rhizoma corydalis 24g, rhizoma curcumae longae concisa 24g, umbellate pore furgus 24g, radix glycyrrhizae preparata 8g.It takes Stir-baked CARAPAX TRIONGCIS with sand then treated with vinegar crushes, and crosses 40 meshes；It is added water to cook twice with other 12 taste Chinese medicines, for the first time plus 12 times of amount water impregnate 1.0 hours, It decocts, is kept after boiling slightly boiled 1.0 hours, second plus 10 times of amount water decoctions keep slightly boiled 1.0 hours after boiling, merge and decoct Liquid, filtration, it is 1.01~1.05(60 DEG C that filtrate, which is concentrated into relative density), traditional Chinese medicine compound extract is obtained, by above-mentioned Chinese medicine compound prescription It extracts after liquid cooling is sunk 5 hours and takes supernatant, glucose 0.7g/100ml, peptone 0.4g/100ml, yeast extract 0.13g/ is added 100ml, yeast powder 0.07g/100ml, inorganic salts (K2HPO4•3H2O 0.1g/100ml、KH2PO40.1g/100ml, sulfuric acid are sub- Iron 7H2O 5.0mg/100ml, zinc sulfate 7H2O 4.4mg/100ml, magnesium sulfate 7H2O 50mg/100ml), it is settled to 1000ml adjusts pH value to 6.5, in 115 DEG C of autoclave sterilization 50min, obtains sterile Chinese medicinal compound extract.When above-mentioned sterile When Chinese medicinal compound extract temperature is down to 39 DEG C, the bifidobacterium bifidum that the percent by volume for being inoculated with preculture is 0.5% is (normal Human body separation, is identified through Institute of Microorganism, Academia Sinica) bacterium solution, after routine culture 6 hours, it is inoculated with each of preculture again The Lactobacillus delbrueckii (AS 1.1480) and lactobacillus plantarum (AS 1.19) bacterium solution that percent by volume is 0.5% continue 37 DEG C of fermentations 23 hours, when pH value is down to 4.1 or less, terminate fermentation, filling rear stabilization processing is under aseptic condition to get probiotics hair Ferment Traditional Chinese medicine compound composition C.General flavone content in this fermentation composition are as follows: 5.18mg/ml.
Experimental example 1: probiotics fermention Traditional Chinese medicine compound composition of the present invention is compared with the anti-tumor activity of non-fermentation composition
For this experimental example using the research of transplanted hepatoma in rats expansion anti-tumor activity, the model experiment period is short, Modeling is stablized, it is most important that the biological behaviour that liver cancer orthotopic liver transplantation is proliferated in liver is similar to human primary's liver cancer.
One, materials and methods:
1, animal: Walker256 lotus knurl young rat (3-4 week old), weight (70 ± 10) g, 4.Male SD young rat (70 ± 10) g, is purchased from Shandong Traditional Chinese Medicine University's Experimental Animal Center by 5, SPF grades of health male SD rat (190 ± 10) g.
2, given the test agent: given the test agent is probiotics fermention Traditional Chinese medicine compound composition A, B, the C prepared in above-described embodiment And its corresponding non-fermented tcm compound.Given the test agent human body recommended amounts are the 250ml/(day for human beings), by 60kg human body Batheroom scale, i.e. 4.17ml/(kg day), the equivalent dose of rat is equivalent to 10 times of human body recommended dose, recommends by this human body 5 times, 10 times, 20 times of dosage are set to low dose group, middle dose group and high dose group.Low dose group: 2.08ml/ (100g days), middle dose group: 4.17ml/(100g days), high dose group: 8.34ml/(100g days).
3, grouping and modeling:
3.1 groupings: healthy male SD rat is divided into 3 groups.Wherein normal group, sham-operation group each 20, remaining is big Mouse is cooked transplanting operation of liver cancer, postoperative to be grouped survival rats at random again: being respectively model group, the combination of probiotics fermention Chinese medicine compound prescription The low, middle and high dose groups of object A, B, C and its corresponding non-fermented tcm compound, each 20.
3.2 modelings: tumor mouse abdomen is sterilized, extract the peritoneal fluid containing tumour cell, adjustment cell concentration be (4-6) × 109Cells/L is spare, and it is subcutaneous that aforementioned peritoneal fluid containing tumour cell is injected into armpit, subcutaneous after each injection point 0.3ml, 7d Tumor diameter about 1cm or so；It after subcutaneous transplantation knurl model is successfully prepared, is anaesthetized, is soaked into whole body in 75% ethyl alcohol and sterilizes, Subcutaneous tumors block is cut in superclean bench, is selected the eugonic flesh of fish sample tissue in periphery, is cut into the tumor mass of 1mm × 1mm size, It is placed in RPMI-1640 culture solution and saves backup for 4 DEG C, all tumor mass must transplant in 2h.Adult SD rats are anaesthetized, on Abdomen hits exactly conventional preserved skin, opens abdomen exposure left lobe of liver and right lobe of liver, is cut in its diaphragmatic surface with ophthalmic tweezers and puncture Glisson's capsule, made under coating The tumor tissue block got ready is implanted by the tunnel of 0.5cm long, and confirmation tumor tissue does not exit, without layer-by-layer suture stomach wall after bleeding.Artificial hand Art group opens tunnel, but is not implanted into tumor mass.
4. medication: 3d starts stomach-filling after completing modeling operation, normal group, sham-operation group, model group give 1ml/ (100g Day) pure water stomach-filling；Administration group is given the 1ml/ dosage stomach-filling of (100g days) by test dose respectively.
5. observation index and detection method
1 week (1w) after medication, 2 weeks (2w), 3 weeks (3w) each groups extracted 6 rat materials immediately respectively.Observation is big Mouse feed, drinking-water, activity, dorsal body setae, weight, the state of mind.The quality of tumor tissues in liver is recorded, and calculates tumor quality Inhibiting rate (%)=(1- administration group tumor quality/model group tumor quality) × 100%.
Two, result:
1, general status is observed
The rat state of mind quick slip in a short time after modeling, hair color is withered, and activity significantly reduces, food-intake and drinking-water It reduces.The feeding tested material group rat state of mind generally takes a turn for the better, and laughs and frolick between accidental rat, food-intake is more.
2, given the test agent influences transplanted hepatoma rat tumor quality inhibiting rate
The tumor quality inhibiting rate of 1 each group of table compares
Note: the number 1,2,3 in table after group A, B, C respectively corresponds the basic, normal, high dosage of the non-fermentation composition of each group Group, 4,5,6 respectively correspond the low, middle and high dose groups of each group fermentation composition
* there are significant difference, P < 0.05 compared with model group
▲ fermentation group has significant difference, P < 0.05 compared with the group of not fermenting of same concentration
Three, interpretation of result:
Tumor quality inhibiting rate: the middle and high dosage group of fermentation composition A at 1 week, the middle dose group of fermentation composition B with Comparison there were significant differences property of the corresponding non-fermentation composition with concentration；The high dose group of fermentation composition B, fementative composition at 2 weeks The high dose group of object C there were significant differences compared with corresponding non-fermentation composition is with concentration property；At 3 weeks in fermentation composition C, High dose group there were significant differences compared with corresponding non-fermentation composition is with concentration property.
The interpretation of result of this experimental example shows that the probiotics fermention Traditional Chinese medicine compound composition for the treatment of liver cancer of the invention can improve The vital sign of rat mitigates ascites degree.Downright bad situation overweights group of not fermenting in fermentation composition group tumor stove, so as to cause swollen Tumor quality mitigates, and fermentation group tumor control rate is high, and especially middle and high dosage group is more apparent, has compared with not fermenting group with same concentration Significant difference (P < 0.05) illustrates the antihepatocarcinoma effect that can significantly improve this compound preparation after probiotics fermention, especially exists Inhibiting liver neoplasm to increase aspect has conspicuousness effect.
Experimental example 2: measuring is carried out to probiotics fermention Traditional Chinese medicine compound composition A, B, C
1, identify
1.1 instruments and test solution
Instrument: the ultraviolet device of ZF-2 type three, electric heating constant-temperature blowing drying box, METTLER AB204 type electronic analytical balance, Iodine cylinder, sample applicator etc..
Test solution: ethyl acetate, n-butanol, ether, hydrochloric acid, ferric trichloride, paradime thylaminobenzaldehyde, strong ammonia solution, iodine, Scutelloside reference substance (715-9405, for identifying), protocatechualdehyde (0810-9402, for identifying), yanhusu B prime (726- 8903, for identifying), radix bupleuri control medicinal material (120992-201108) etc..
1.2 radix scutellariaes identify
1.2.1 prepared by sample liquid
(1) preparation of test solution: probiotics fermention composition each 5ml of A, B, C in Example, with hydrochloric acid tune pH To 1-2, ethyl acetate shaking is added to extract 2 times, each 5ml, combined ethyl acetate liquid, water bath method, residue with methanol constant volume extremely 2ml, as test solution a1, b1, c1.
(2) it lacks the preparation of radix scutellariae negative control sample solution: taking the corresponding scarce radix scutellariae feminine gender sample of probiotics fermention composition A Product 5ml, ibid prepared by method to lack radix scutellariae negative sample liquid.
(3) preparation of scutelloside reference substance solution: scutelloside (715-9405, for identifying) 3.0mg is taken, methanol 2ml is added Dissolution, as reference substance solution.
1.2.2 thin layer identifies
According to thin-layered chromatography (Chinese Pharmacopoeia 2015 version four 0502) test, draws each test solution, lacks radix scutellariae yin Each 2ul of property control sample solution, reference substance solution 1ul are put respectively on same polyamide film, with ethyl acetate-butanone-first Acid-water (5:3:1:1) is solvent, and (temperature: 16 DEG C of humidity: 50%) exhibition, takes out away from are as follows: 7cm, dries, set ultraviolet lamp for expansion It is inspected under (365nm).
Interpretation of result: attached drawing 1 is seen in scutelloside Rf=3.6/7, and as can be seen from the figure the solvent can separate respectively well Ingredient, preferably, Rf value is moderate, and specificity is strong for spot separation, negative noiseless, and this method can be used as yellow in the present composition The discrimination method of a kind of reed mentioned in ancient books.
It is also solvent with 36% acetic acid, separating effect is slightly poor, therefore does not use in experimentation.
1.3, Radix Salviae Miltiorrhizae identifies
1.3.1 prepared by sample liquid
(1) preparation of test solution: take under radix scutellariae item test sample solution as this test solution a2, b2, c2.
(2) it lacks the preparation of Radix Salviae Miltiorrhizae negative control sample solution: taking the corresponding scarce Radix Salviae Miltiorrhizae feminine gender sample of probiotics fermention composition A Product 5ml, ibid prepared by method to lack Radix Salviae Miltiorrhizae negative sample liquid.
(3) protocatechualdehyde reference substance solution: taking protocatechualdehyde reference substance (0810-9402) 2mg, and methanol is added to make to dissolve constant volume To 2ml.
1.3.2 thin layer identifies
According to thin-layered chromatography (Chinese Pharmacopoeia 2015 version four 0502) test, draws each test solution, lacks Radix Salviae Miltiorrhizae yin Each 10ul of property control sample solution, reference substance solution 2ul are put respectively on same silica gel g thin-layer plate, with chloroform-acetic acid Ethyl ester-methyl alcohol-formic acid (12:2:1:0.8) is solvent, and (temperature: 21 DEG C of humidity: 53%) exhibition, takes out, dries in the air away from 9 cm for expansion Dry, spray is developed the color with 3% ferric trichloride ethanol solution to clear spot.
Interpretation of result: separating effect is preferable, negative noiseless.See attached drawing 2, as can be seen from the figure characteristic spots clearly divide Bright, Rf value is moderate, and specificity is strong, and negative noiseless, this method can be used as the discrimination method of Radix Salviae Miltiorrhizae in the present composition.
Once it tested with chloroform-acetone-formic acid (10:1:1) as solvent, expansion effect is poor, therefore does not use.
In addition, also having tested using polyamide film as carrier, toluene-ethyl acetate-methyl alcohol-formic acid (10:3:1:2) is exhibition Agent is opened, as a result: feminine gender is slightly interfered, therefore is not used.
1.4, radix bupleuri identifies
1.4.1 prepared by sample liquid
(1) preparation of test solution: probiotics fermention composition each 20ml of A, B, C in Example adds water saturation N-butanol shaking extract 2 times, each 15ml, merge n-butanol liquid, washed 2 times, each 10ml with ammonia solution, then use n-butanol The water washing of saturation 2 times, each 10ml, water bath method n-butanol liquid, residue methanol constant volume to 2ml, as test solution a3、b3、c3。
(2) it lacks the preparation of radix bupleuri negative control sample solution: taking the corresponding scarce radix bupleuri feminine gender sample of probiotics fermention composition A Product 20ml, ibid prepared by method to lack radix bupleuri negative sample liquid.
(3) preparation of radix bupleuri control medicinal material solution: radix bupleuri control medicinal material (120992-201108) 0.5g is weighed, methanol is added 30ml, ultrasonic extraction 30min, filtration, filtrate water bath method, residue add water 20ml to make to dissolve, and water saturated n-butanol is added to shake It extracts 2 times, each 15ml, merges n-butanol extracting liquid, washed 2 times, each 10ml with ammonia solution, then the water being saturated with n-butanol Washing 2 times, each 10ml, water bath method n-butanol liquid, residue methanol constant volume to 2ml, as control medicinal material solution.
1.4.2 thin layer identifies
According to thin-layered chromatography (Chinese Pharmacopoeia 2015 version four 0502) test, draw that test solution, to lack radix bupleuri negative Each 10ul of control sample solution, radix bupleuri control medicinal material solution 5ul are put respectively on same silica gel g thin-layer plate, with chloroform-methanol- Lower layer's solution of water (13:7:2) is solvent, and (temperature: 15 DEG C of humidity: 50%) exhibition, is taken out, dried, spray away from are as follows: 9cm for expansion With 40% sulfuric acid solution of 2% paradime thylaminobenzaldehyde, it is clear that spot development is heated at 60 DEG C.
Interpretation of result: separating effect is preferable, negative noiseless, sees attached drawing 3, as can be seen from the figure the solvent can be very well Each spot of separation, Rf value is moderate, and specificity is strong, and negative noiseless, this method can be used as radix bupleuri in the present composition Discrimination method.
Once solvent under 2015 editions one middle Radix Bupleuri item of Chinese Pharmacopoeia: ethyl acetate, alcohol and water (8:2:1) is tested, It is unfolded ineffective, feminine gender has interference, therefore does not use.
1.5, rhizoma corydalis identifies
1.5.1 prepared by sample liquid
(1) preparation of test solution: probiotics fermention composition each 20ml of A, B, C in Example is tried with dense ammonia Liquid tune pH to 9-10, add diethyl ether shaking extract 2 times, each 15ml, merge ether solution, water bath method, residue with methanol constant volume extremely 1ml, as test solution a4, b4, c4.
(2) it lacks the preparation of rhizoma corydalis negative control sample solution: taking the corresponding scarce rhizoma corydalis yin of probiotics fermention composition A Property sample 20ml, ibid method prepare lack rhizoma corydalis negative sample liquid.
(3) reference substance: tetrahydropalmatine (726-8903, for identifying) 2.0mg is taken, adds methanol 5ml to dissolve, as control Product solution.
1.5.2 thin layer identifies
According to thin-layered chromatography (Chinese Pharmacopoeia 2015 version four 0502) test, draws each test solution, lacks rhizoma corydalis Each 5ul of negative control sample solution, reference substance solution 2ul are put respectively in the same silica G prepared with 0.5% sodium hydroxide solution On lamellae, with n-hexane-chloroform-methanol (10:6:1) be solvent, expansion (exhibition away from: 8.5cm, temperature: 14 DEG C are wet Degree: 62%), taking out, dry, and sets to fumigate in iodine cylinder and about take out after five minutes, after waving the iodine adsorbed on most plate, sets ultraviolet lamp It is inspected under (365nm).
Interpretation of result: separating effect is preferable, negative noiseless, sees attached drawing 4, as can be seen from the figure uses the solvent exhibition After opening and being smoked with iodine, each spot separating effect is preferable, and spot development is clear, and negative noiseless, specificity is strong, and this method can Using the discrimination method as rhizoma corydalis in the present composition.
Once test is unfolded using the silica gel g thin-layer plate without sodium hydroxide, and expansion is ineffective, and spot development is unclear It is clear, therefore do not use.
2, content detection is tested
2.1 instruments and reagent
Instrument: assay balance (METTLER TOLEDO AB204), L5 ultraviolet-uisible spectrophotometer (Shanghai instrument electroanalysis Instrument Ltd.).
Reagent: control substance of Rutin (080-9303, Nat'l Pharmaceutical & Biological Products Control Institute), ethyl alcohol, aluminum nitrate, nitrous acid Sodium, sodium hydroxide are that analysis is pure.
The investigation of 2.2 linear relationships
It accurately weighs 102mg control substance of Rutin to set in 50ml volumetric flask, 60% ethyl alcohol 35ml is added, ultrasonic treatment makes completely Dissolution, lets cool, adds 60% ethyl alcohol to scale, shake up.The reference substance solution for being 2.04mg/ml up to mass concentration.Precision measures 5ml sets in 50ml volumetric flask, adds water to scale, shakes up the reference substance dilute solution for being 0.204mg/ml to get mass concentration.
Precision measures reference substance dilute solution 1ml, 2ml, 3ml, 4ml, 5ml, 6ml, is set in 25ml volumetric flask respectively, adds water To 6ml, add 5% sodium nitrite solution 1ml, mix, place 6 minutes, add 10% aluminum nitrate solution 1ml, shake up, places 6 minutes, Add 4% sodium hydroxide test solution 10ml, add water to scale, shake up, place 15 minutes, blank is made with corresponding reagent, according to it is ultraviolet- Visible spectrophotometry measures absorbance at 500nm wavelength.It the results are shown in Table 2
2 linear relationship of table investigates result
Regression equation y=0.0113x+0.0092, correlation coefficient r=0.9998.
Test result shows that control substance of Rutin is in good linear pass with trap between 8.16~48.96 μ g/ml System.
The investigation of 2.3 sample solution preparation methods
The sample that methodological study uses is the probiotics fermention Traditional Chinese medicine compound composition A prepared in embodiment 1
Probiotics fermention Traditional Chinese medicine compound composition A5ml is taken, dehydrated alcohol 7.5ml is added, edged shaking in side adds 60% ethyl alcohol 12ml shakes up, and seals, and centrifugation, supernatant is transferred in 25ml volumetric flask, and a small amount of 60% ethyl alcohol cleaning is added to precipitate and be settled to quarter Degree.Precision measures 10ml and water is added to be settled to 100ml, shakes up to get test solution.Precision draws test solution 2ml, 5ml, It is set in 25ml volumetric flask respectively, adds water to 6ml, add 5% sodium nitrite solution 1ml, mixed, placed 6 minutes, add 10% aluminum nitrate Solution 1ml, shakes up, and places 6 minutes, adds 4% sodium hydroxide test solution 10ml, add water to scale, shake up, placement 15 minutes, separately Precision draws test sample dilute solution 2ml, 5ml, is set in 25ml volumetric flask respectively, water is added to be settled to scale, shaken up, make blank, Absorbance is measured in accordance with the law.It is shown in Table 3
3 sampling amount of table investigates result
The result shows that: both in the linear range, but in view of sampling accurately has differences between each batch, therefore, Take test sample solution 5ml measurement trap reasonable.
The experiment of 2.4 instrument precisions
Control substance of Rutin dilute solution 3ml sample METHOD FOR CONTINUOUS DETERMINATION 6 times under 2.2 are taken, its trap is surveyed.It the results are shown in Table 4.
4 instrument precision test result of table
The result shows that: instrument precision is good.(RSD < 2.0%)
The experiment of 2.5 sample stabilities
The test solution 5ml sample prepared under 2.3 is taken, every the trap of measurement in 3 minutes, investigates 18 points altogether Clock calculates RSD, to observe the stability of test solution ingredient to be measured in the detection process.It the results are shown in Table 5.
5 stability test result of table
The result shows that: test solution is good in 18 minutes internal stabilities, can satisfy measurement needs.(RSD<2.0%)
2.6 repeatability investigate experiment
Sample probiotics fermention Traditional Chinese medicine compound composition B is taken, according to the preparation method of test solution, parallel 6 parts of preparation Sample is measured with condition according to the above method, calculates the content of every part of test sample.It the results are shown in Table 6.
6 repeatability of table investigates result
Find out from measurement result, the repeatability of this product content assaying method is good.(RSD<2.0%)
The experiment of 2.7 sample recovery rates
It is 6.68mg/ that precision, which is drawn and predicted the sample probiotics fermention Traditional Chinese medicine compound composition B(general flavone content of content, Ml) 3ml, is separately added into dehydrated alcohol 4.5ml by totally 6 parts, and side edged shakes, accurate in every portion that control substance of Rutin solution is added 10ml(mass concentration is 2.04mg/ml), it shakes up, is centrifuged, supernatant is set respectively in 25ml volumetric flask, adds 60% ethanol washing heavy It forms sediment and is settled to scale, shake up, the accurate 5ml that measures is set in 100ml volumetric flask respectively, is diluted with water to scale, is shaken up to get confession Test sample solution.It is measured by said determination method, calculates the rate of recovery.It the results are shown in Table 7.
7 rutin sample recovery rate test result of table
It is good with the measuring method rate of recovery that test result shows that general flavone with rutin compares product.(RSD < 2.0%)
The measurement of 2.8 samples
(1) preparation of reference substance solution: it is appropriate to weigh control substance of Rutin, accurately weighed, adds methanol to dissolve, every 1ml is made Solution containing about 0.2mg to get.
(2) preparation of standard curve: precision measures reference substance solution 1ml, 2ml, 3ml, 4ml, 5ml, 6ml, sets respectively In 25ml measuring bottle, 6ml is added water to, adds 5% sodium nitrite solution 1ml, is mixed, places 6 minutes, adds 10% aluminum nitrate solution 1ml, It shakes up, places 6 minutes, adding sodium hydroxide test solution 10ml is added water to scale, shaken up, and is placed 15 minutes, with corresponding reagent work Blank, according to UV-VIS spectrophotometry (annexA), at 500nm wavelength measure absorbance, using absorbance as ordinate, Concentration is abscissa, draws standard curve.
(3) measuring method precision measure embodiment in probiotics fermention Traditional Chinese medicine compound composition each 5ml of A, B, C, slowly plus Enter dehydrated alcohol 7.5ml, the shaking of side edged, then plus 60% ethyl alcohol 12ml, shake up, seal, centrifugation, supernatant is transferred to 25ml appearance In measuring bottle, a small amount of 60% ethyl alcohol cleaning is added to precipitate and be settled to scale.Precision measures 10ml and water is added to be settled to 100ml, shakes up, i.e., Obtain each test solution.Precision draws each test solution 5ml, sets in 25ml volumetric flask, under the preparation of sighting target directrix curve Method is operated according to methods from " adding water to 6ml ", and another accurate each test solution 5ml of absorption is diluted with water to 25ml, shakes up, Make blank, measure absorbance in accordance with the law, from standard curve read test sample in rutin amount, calculate to get.
General flavone content through detecting, in probiotics fermention Traditional Chinese medicine compound composition A are as follows: 4.34mg/ml, fermentation composition General flavone content in B are as follows: general flavone content in 4.63mg/ml, fermentation composition C are as follows: 5.18mg/ml.

Claims (7)

1. a kind of identification and detection method of the probiotics fermention Traditional Chinese medicine compound composition for treating liver cancer, which is characterized in that step It is as follows:
Discrimination method:
(1) probiotics fermention Traditional Chinese medicine compound composition 5ml is taken, with hydrochloric acid tune pH to 1-2, adds ethyl acetate to shake extraction 2 times, often Secondary 5ml, combined ethyl acetate liquid, water bath method, residue methanol constant volume to 2ml, as test solution；Scutelloside is taken to compare Product add methanol 1ml the is made solution containing 1mg, as reference substance solution；It is tested according to thin-layered chromatography, draws test solution 2ul, reference substance solution 1ul are put respectively on same polyamide film, are with ethyl acetate-butanone-formic acid-water of 5:3:1:1 Solvent is unfolded, and takes out, dries, set and inspect under ultraviolet lamp 365nm；In sample chromatogram, in position corresponding with reference substance chromatography It sets, shows identical dark spots；
(2) taking test solution under discrimination method (1) item is test solution；Protocatechualdehyde reference substance is taken, methanol is added to be made often Solution of the 1ml containing 1mg, as reference substance solution；
It is tested according to thin-layered chromatography, draws test solution 10ul, reference substance solution 2ul, put respectively in same silica gel g thin-layer plate On, using chloroform-acetate-methanol-formic acid of 12:2:1:0.8 as solvent, it is unfolded, takes out, dry, sprays with 3% 3 Iron chloride ethanol solution；In sample chromatogram, at the position corresponding to the chromatogram of the reference substance, the spot of identical bluish grey color is shown；
(3) probiotics fermention Traditional Chinese medicine compound composition 20ml is taken, adds water saturated n-butanol shaking to extract 2 times, each 15ml, closes And n-butanol liquid, it is washed 2 times, each 20ml with ammonia solution, then water washing 2 times, each 20ml be saturated with n-butanol, water-bath is steamed Dry n-butanol liquid, residue methanol constant volume to 2ml, as test solution；Radix bupleuri control medicinal material fine powder 0.5g is taken, methanol is added 30ml, ultrasonic extraction 30min, filtration, filtrate water bath method, residue add water 20ml to make to dissolve, are made in the same way of control medicinal material solution； It is tested according to thin-layered chromatography, draws test solution 10ul, radix bupleuri control medicinal material solution 5ul is put respectively in same silica G thin layer On plate, using chloroform-methanol-water lower layer's solution of 13:7:2 as solvent, it is unfolded, takes out, dry, spray with 2% pair of dimethylamino It is clear to be heated to spot development at 60 DEG C for 40% sulfuric acid solution of benzaldehyde；In sample chromatogram, with reference medicine chromatography phase On the position answered, identical punctation is shown；
(4) probiotics fermention Traditional Chinese medicine compound composition 20ml is taken, with strong ammonia solution tune pH to 9-10, the shaking that adds diethyl ether extracts 2 Secondary, each 15ml merges ether solution, water bath method, residue methanol constant volume to 1ml, as test solution；Take rhizoma corydalis second Plain reference substance adds methanol that solution of every 1ml containing 0.4mg is made, as reference substance solution；It is tested according to thin-layered chromatography, draws and supply Test sample solution 5ul, reference substance solution 2ul, put respectively in it is same with 0.5% sodium hydroxide solution prepare silica gel g thin-layer plate on, Using n-hexane-chloroform-methanol of 10:6:1 as solvent, be unfolded, take out, dry, set in iodine cylinder fumigate about take after five minutes Out, it after waving the iodine adsorbed on most plate, sets and is inspected under ultraviolet lamp 365nm；In sample chromatogram, in position corresponding with reference substance chromatography It sets, shows the fluorescence spot of identical yellowish green color；
Detection method:
A, the preparation of reference substance solution: it is appropriate to weigh control substance of Rutin, accurately weighed, and methanol is added to dissolve, be made every 1ml containing about The solution of 0.2mg to get；
B, the preparation of standard curve: precision measures reference substance solution 1ml, 2ml, 3ml, 4ml, 5ml, 6ml, sets 25ml measuring bottle respectively In, 6ml is added water to, 5% sodium nitrite solution 1ml is added, is mixed, is placed 6 minutes, is added 10% aluminum nitrate solution 1ml, shake up, put It sets 6 minutes, adding sodium hydroxide test solution 10ml is added water to scale, shaken up, and is placed 15 minutes, is made blank with corresponding reagent, is shone UV-VIS spectrophotometry measures absorbance at 500nm wavelength, and using absorbance as ordinate, concentration is abscissa, is drawn Standard curve processed；
C, measuring method: precision measures probiotics fermention Traditional Chinese medicine compound composition 5ml of the invention, is slowly added into dehydrated alcohol 7.5ml, the shaking of side edged, then plus 60% ethyl alcohol 12ml, shake up, seal, centrifugation, supernatant is transferred in 25ml volumetric flask, adds few The cleaning of 60% ethyl alcohol is measured to precipitate and be settled to scale；Precision measures 10ml and water is added to be settled to 100ml, shakes up molten to get test sample Liquid；Precision draws test solution 5ml, sets in 25ml volumetric flask, the method under the preparation of sighting target directrix curve, " adds water to certainly 6ml " rises, and operates according to methods, and another accurate absorption test solution 5ml is diluted with water to 25ml, shakes up, make blank, measurement is inhaled in accordance with the law Luminosity, from standard curve read test sample in rutin amount, calculate to get；
Every 1ml presses rutin C containing general flavone in probiotics fermention Traditional Chinese medicine compound composition27H30O16Meter, must not be less than 2.8mg；
The probiotics fermention Traditional Chinese medicine compound composition of the treatment liver cancer is after mentioning Chinese medicine compound prescription medicinal material progress water, to be added Glucose, peptone, yeast extract, yeast powder and inorganic salts add or do not add sweetener, high-temperature sterilization, through probiotics fermention Rear stabilization processing is made；
The Chinese medicine compound prescription is made of the bulk pharmaceutical chemicals of following parts by weight:
Radix bupleuri 12-40 parts by weight, radix scutellariae 12-40 parts by weight, red ginseng 4-24 parts by weight, hypericum japonicum 48-96 parts by weight, stringy stonecrop 48-96 parts by weight, stir-baked CARAPAX TRIONGCIS with sand then treated with vinegar 36-80 parts by weight, Radix Salviae Miltiorrhizae 36-48 parts by weight, Prunella vulgaris 24-60 parts by weight, raw oyster 36-80 weight Measure part, rhizoma corydalis 12-40 parts by weight, rhizoma curcumae longae concisa 12-36 parts by weight, umbellate pore furgus 24-40 parts by weight, radix glycyrrhizae preparata 8-36 parts by weight；
The probiotics is selected from Bifidobacterium and lactobacillus.
2. the identification and detection method of the probiotics fermention Traditional Chinese medicine compound composition for the treatment of liver cancer according to claim 1, It is characterized in that, the Chinese medicine compound prescription is made of the bulk pharmaceutical chemicals of following parts by weight:
20 parts by weight of radix bupleuri, 20 parts by weight of radix scutellariae, 8 parts by weight of red ginseng, 48 parts by weight of hypericum japonicum, 48 parts by weight of stringy stonecrop, toast soft-shelled turtle 36 parts by weight of first, 36 parts by weight of Radix Salviae Miltiorrhizae, 24 parts by weight of Prunella vulgaris, 40 parts by weight of raw oyster, 24 parts by weight of rhizoma corydalis, rhizoma curcumae longae concisa 24 Parts by weight, 24 parts by weight of umbellate pore furgus, 8 parts by weight of radix glycyrrhizae preparata.
3. the identification and detection method of the probiotics fermention Traditional Chinese medicine compound composition for the treatment of liver cancer according to claim 1, It is characterized in that, the additional amount of the glucose is 0.7g/100ml, the additional amount of yeast extract is 0.13g/100ml, yeast The additional amount of powder is 0.07g/100ml, and the additional amount of peptone is 0.4g/100ml.
4. the identification and detection method of the probiotics fermention Traditional Chinese medicine compound composition for the treatment of liver cancer according to claim 1, It is characterized in that, the inorganic salts are K2HPO4•3H2O 0.1g/100ml、KH2PO40.1g/100ml, ferrous sulfate 7H2O 5.0mg/100ml, zinc sulfate 7H2O 4.4mg/100ml, magnesium sulfate 7H2O 50mg/100ml。
5. the identification and detection method of the probiotics fermention Traditional Chinese medicine compound composition for the treatment of liver cancer according to claim 1, It is characterized in that, being also added with sweetener to improve mouthfeel, the sweetener is sucrose 3.0g/100ml, Sucralose 0.008g/100ml。
6. the identification and detection method of the probiotics fermention Traditional Chinese medicine compound composition for the treatment of liver cancer according to claim 1, It is characterized in that, the Bifidobacterium is selected from bifidobacterium bifidum, bifidobacterium breve, bifidobacterium adolescentis and baby's bifid bar Bacterium；The lactobacillus is selected from lactobacillus acidophilus, Lactobacillus delbrueckii, lactobacillus plantarum, Lactobacillus casei and Lactobacillus rhamnosus.
7. the identification and inspection of the probiotics fermention Traditional Chinese medicine compound composition for the treatment of liver cancer according to claim 1-6 Survey method, which is characterized in that the probiotics fermention Traditional Chinese medicine compound composition of the treatment liver cancer the preparation method is as follows:
Stir-baked CARAPAX TRIONGCIS with sand then treated with vinegar is taken to crush, fineness adds water to cook twice in 20 mesh~40 mesh with other 12 taste Chinese medicines, and for the first time plus 12 times are measured Water impregnates 1 hour, decocts, keeps after boiling slightly boiled 1 hour, and second plus 10 times of amount water decoctions, keep slightly boiled 1 small after boiling When, collecting decoction, filtration, it is 1.01~1.05 that filtrate, which is concentrated into 60 DEG C of relative densities, obtains traditional Chinese medicine compound extract；Gained Chinese medicine Supernatant can be obtained after being subcooled and sinking in compound extract；
B, to the step A traditional Chinese medicine compound extract prepared or its it is cold it is heavy after supernatant in glucose, peptone, yeast is added Cream, yeast powder and inorganic salts, add or do not add sweetener, and constant volume adjusts pH value to 6.5~7.0, in 115 DEG C of high temperature and pressure Sterilize 50min, obtains sterile Chinese medicinal compound extract；
C, when the obtained sterile Chinese medicinal compound extract temperature of step B is down to 39 DEG C, it is inoculated with each percent by volume of preculture For 0.5% one or two kinds of and above Bifidobacterium bacterium solutions, after routine culture about 3~6 hours, it is inoculated with each of preculture again One or two kinds of and above lactobacillus bacterium solutions that percent by volume is 0.5%, continue fermentation 22~27 hours, when pH value is down to 4.1 When following, terminate fermentation, stabilization processes to get.
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