CN104689129A

CN104689129A - Anti-tuberculous traditional Chinese medicine fritillary and burdock fruit tuberculosis-eliminating extractive and application

Info

Publication number: CN104689129A
Application number: CN201510142873.0A
Authority: CN
Inventors: 吴雪琼; 刘光陵; 梁艳; 阳幼荣; 张俊仙; 白雪娟; 陈实兵; 刘军; 杨春玮; 罗华锋
Original assignee: QIFANG PHARMACEUTICAL CO Ltd GUANGDONG PROVINCE; 309th Hospital of PLA
Current assignee: QIFANG PHARMACEUTICAL CO Ltd GUANGDONG PROVINCE; 309th Hospital of PLA
Priority date: 2015-03-27
Filing date: 2015-03-27
Publication date: 2015-06-10

Abstract

The invention relates to an anti-tuberculous traditional Chinese medicine fritillary and burdock fruit tuberculosis-eliminating extractive and application and belongs to the field of traditional Chinese medicine treatment preparations. Alcohol water, water and/or alcohol are adopted to extract three extractives, adopted raw medicinal materials include, by weight, 3-5 parts of burdock fruits, 5-10 parts of common bletilla pseudobulb, 3-5 parts of fritillary fruits, 3-5 parts of platycodon grandiflorum, 3-10 parts of sticky rice and 3-5 parts of houttuynia cordata and are weighed in proportion, ethyl alcohol and/or water are/is added into the raw medicinal materials, heating is performed for reflux extraction after soaking, filtration is performed to obtain an extracting solution, pressure reduction is performed to recover a solvent, the solvent is concentrated to form thick paste, pressure-reduction drying is performed to form dry paste, and the dry paste is smashed and screened to obtain the extractive. The fritillary and burdock fruit tuberculosis-eliminating extractive serves as a traditional Chinese medicine treatment preparation and enables the weight of a mouse to be inordinately increased than the weight before the mouse is treated, and the weight is more than the weight increased by means of existing medicine powder combinations. Bacterial quantity of the spleen tissues can be remarkably decreased, the lung lesions are inordinately relieved, and a normal lung structure can be seen. The fritillary and burdock fruit tuberculosis-eliminating extractive is better than existing medicine powder in overall treatment effect and can be used for preparation of medicines for treating tuberculosis of patients.

Description

Antituberculous Chinese Traditional Medicine cattle shellfish disappears nuclear extract and Synthesis and applications thereof

Technical field

The present invention relates to Antituberculous Chinese Traditional Medicine cattle shellfish to disappear nuclear extract and Synthesis and applications thereof, particularly three kinds to disappear nuclear extract and Synthesis and applications thereof for antibacterial especially antiphthisic Chinese medicine cattle shellfish, belong to treatment by Chinese herbs formulation art.

Background technology

Tuberculosis (Tuberculosis, TB) is the respiratory infectious disease that the class that caused by mycobacterium tuberculosis (Mycobacterium tuberculosis, MTB) is serious.Although the case load of global tuberculosis patient reduces, but multi-drug resistance tuberculosis (Multidrug-resistant tuberculosis, MDR-TB) and extensively resistant tuberculosis (Extensively drug-resistant tuberculosis, XDR-TB) patient is still continuing to increase.WHO in 2013 whole world tuberculosis is reported the result display: 2012, the whole world increased tuberculosis patient 8,700,000 people newly, dies from tuberculosis patient 1,300,000 people.The national multi-drug resistance tuberculosis number of cases of 27 multi-drug resistance tuberculosis high burden rise, and MDR-TB number be 450,000 people, its death toll is 170,000 people.China arranges the 2nd in global tuberculosis high burden country, in 27 multi-drug resistance tuberculosis high burden countries, also arrange the 2nd.Therefore, tuberculosis is global serious public health problem.

Pulmonary infection is the modal complication of pulmonary tuberculosis, is also that serious pulmonary tuberculosis brings out dead key factor.This complication is apt to occur in the lunger that immunologic hypofunction and lung mechanics sexually revise, especially gerontal patient.The Clinic X-ray findings of most of patients is not true to type, and along with the application of a large amount of extensive pedigree antibiotic and immunosuppressant, the sickness rate that pulmonary tuberculosis merges pulmonary infection also rises thereupon.The pulmonary infection how effectively preventing and treating lunger's secondary is also problem in the urgent need to address.

In treatment, current western medical treatment mainly adopts combined chemotherapy, but the toxic and side effects of western medical treatment is large, and long-term prescription all has infringement in various degree to liver function, renal function, gastrointestinal tract, joint etc.; The appearance of the especially extensive resistant tuberculosis of multi-drug resistance tuberculosis, make tuberculotherapy face without medicine can hard case.Chinese medicine is the medical science rarity of the Chinese nation, Chinese medicine tuberculosis has the history of more than 1000 year, have suppress to kill mycobacterium tuberculosis, alleviate chemotherapeutics side effect, effectively alleviate tuberculosis poisoning symptom, enhancing human body immunity power, reduce the advantage of the incidence rate of resistant tuberculosis.China tuberculosis prevention and treatment worker excavated always diligently, explored, and has made some progress so far the 1950's.The effect and effective ingredient thereof that were once used for the treatment of nearly thousand kinds of Chinese medicines lungy are studied, found much Chinese medicine and the growth of composition to mycobacterium tuberculosis inhibited, as Herba Houttuyniae, Folium Artemisiae Argyi, the Radix Stemonae, Radix Scutellariae, Herba Lysionoti Pauciflori, Rhizoma Polygonati, Herba humuli scandentis, berberine, baicalin, matrine, Shi Sheng Herba Polygoni Avicularis, Fructus Cnidii, dragon's tongue, Flos Lonicerae, Fructus Psoraleae, Radix Ginseng, Radix Bupleuri, Radix Glycyrrhizae, Fructus Forsythiae, the Radix Pulsatillae, garlicin, Semen Ginkgo, Golden seal, the Radix Euphorbiae Fischerianae (Radix Euphorbiae Ebracteolatae) etc.; Much Chinese medicine and composition thereof is also had to play tuberculosis effect by improving immunity of organisms, as astragalus polysaccharides, Ganoderma, Bulbus Allii, Spica Prunellae, Herba Andrographis and andrographolide, the Radix Pulsatillae, Radix Ranunculi Ternati element etc.Developing many Chinese patent medicine preparations in order to auxiliary tuberculosis chemotherapy, as treated the tuberculosis cream of the tuberculosis of cervical lymph nodes, treating phthisical tuberculous bolus, anti-consumptive disease ball, Feitai capsule, stilbene shellfish capsule etc.The traditional Chinese medical science, doctor trained in Western medicine two broad theory system organically combine, jointly develop, and will become tuberculosis especially multi-drug resistance tuberculosis, the refractory tuberculosis active path of getting rid of poverty.

The cattle shellfish core that disappears is pure Chinese medicinal preparation, that the present inventor is under instruction of Chinese Medicine theory, in conjunction with the characteristics of incidence of long-term clinical practice and tuberculosis self, sum up a practical experience side by Bulbus Fritillariae Cirrhosae, Radix Platycodonis, Pseudobulbus Bletillae (Rhizoma Bletillae), Fructus Arctii, Herba Houttuyniae, Oryza glutinosa Six-element medicine compatibility.Wherein Bulbus Fritillariae Cirrhosae, Pseudobulbus Bletillae (Rhizoma Bletillae) two medicine are mutually auxiliary as monarch drug, and Herba Houttuyniae, Radix Platycodonis, Fructus Arctii three taste are all ministerial drug, with the Oryza glutinosa of tonifying the spleen and stomach, lung benefiting gas for adjuvant.Bulbus Fritillariae Uninbracteatae is carried in Qing Dynasty Wu Yi Lip river " Bencao Congxin rolls up a careless portion ": " a surname, eliminating stagnation relieves inflammation or internal heat, lung moistening, dryness-transformation expectorant, to be sweetly slightly cold ... river is produced best ".Effect that " book on Chinese herbal medicine compilation " Yun Qiyou " controls cough due to consumptive disease, haematemesis hemoptysis "; Liu Wansu's cloud: " this is Herba Houttuyniae, is commonly called as Herba Houttuyniae, function heat-clearing and toxic substances removing, expelling phlegm for arresting cough, eliminating carbuncle evacuation of pus.This is fresh goods, and its gas is smelt as of rotten fish, and after drying in the shade, stink smell disappears "; The Tang Dynasty Zhen Quan " property of medicine opinion " carries Radix Platycodonis: " can control the inferior dysentery, removing blood stasis, remove pneumatosis, removing food stagnancy gathers sputum, main lung qi, and tachypnea is coughed inverse "; Bright Li Shizhen (1518-1593 A.D.) Compendium of Material Medica cloud: " Pseudobulbus Bletillae (Rhizoma Bletillae) is puckery and receive, the order of get Qiu Jin, therefore enters lung hemostasis, and granulation promoting controls skin ulcer also "; Effect that " Bencao Congxin " cloud Fructus Arctii has " eliminating stagnation that expels the heat-evil removes wind, lung qi dispersing gas, and disappear throat, reason phlegmatic cough "; " herbal classic is met former " carries Oryza glutinosa: " Oryza glutinosa, QI invigorating spleen reinforcing lung, but pulverizing does rare gruel, multitudinous not viscous, and diuresis, to grow lung, gas is descending ".Therefore, our drug interaction makes it reach heat-clearing and toxic substances removing, softening the hard massization knot, nourishing YIN and moistening the lung, relieving cough and resolving phlegm, holds in the palm effect of old tissue regeneration promoting, detumescence hemostasis, anti-inflammation; Mycobacterium tuberculosis is killed, inhibitory action, low grade fever can be eliminated, become thin, chest pain, cough, the tuberculosis symptoms such as spitting of blood.The clinical various pulmonary tuberculosis caused for mycobacterium tuberculosis, especially multi-drug resistance tuberculosis and extensive resistant tuberculosis.

For thousands of years Chinese medicine mainly direct crude drug or decoction pieces be made into compound recipe, prepare decoction by patient oneself and take, current the method is still in extensive use.This traditional administrated method exists takes the shortcomings such as inconvenience, curative effect is unstable, quality is uncontrollable.Cattle shellfish disappears core Empirical formula in clinical practice also with oral administration after medicinal powder water tune, and taking dose is large, patient's poor compliance, also limits its clinical practice and promotes.Along with the development of medicine science and technology, refining Chinese medicine extract comes into one's own.For overcoming the above-mentioned weak point of this proved recipe, the present inventor is through physical chemistry extraction and isolation process, the effective ingredient that directed acquisition and Antituberculous Chinese Traditional Medicine cattle shellfish of concentrating disappear in core, and do not change the structure of its effective ingredient, the Chinese medicine cattle shellfish the formed nuclear extract that disappears is deep processing to Chinese crude drug, be new Chinese medicine form prepared by application modern advanced, embody the technological progress of Chinese medicine, meet the requirement of the modernization of Chinese medicine; Decrease drug administration amount, improve patient's compliance, simultaneously also this extract can be increased suitable adjuvant and make the common dosage forms such as granule, capsule, be convenient to that medicine carries, storage etc.

The patent No. is that the Chinese invention patent of 200710048778.X discloses one and is used for the treatment of Chinese medicine lungy, its crude drug formula is made up of Bulbus Fritillariae Uninbracteatae 3-5 part, Pseudobulbus Bletillae (Rhizoma Bletillae) 5-10 part, Fructus Arctii 3-5 part, Radix Platycodonis 3-5 part, Oryza glutinosa 3-10 part and Herba Houttuyniae 3-5 part, the mixing of crude drug grinds is made.

The patent No. is that the Chinese invention patent of 200710048778.X has following weak point: first, because this patent of invention just cleans each Chinese crude drug, air-dry, and pulverize, then mix, packaging forms, and causes taking dose large, the poor compliance of patient; Secondly, some effective ingredient is not easy to disengage in the gastrointestinal tract; 3rd, although Chinese crude drug cleans, do not carry out aseptic process, the antibacterial contained does not get rid of the probability causing the tuberculosis patient of hypoimmunity to infect.

Because this Chinese Herbs is totally good, in conjunction with chemotherapeutics coupling, better effects if, therefore, this patent inventor has set up the R&D team of this patent, improves this patent and develops, and uses of the new technology and achieves the new effect of this patent.

The present inventor deep ploughs in the research of tubercular drugs for a long time, although find that some medicines are from broadly can treating or preventing tuberculosis, but but difference is very large in their aspects of improving the patient's condition that caused by tuberculosis, therefore there is larger limitation to the treatment of the larger patient crowd of each side patient's condition degree difference.Disclosed in No. 200710048778.Xth, Chinese patent, Chinese medicine is no exception.

The present inventor finds, it is better to the improvement effect of the liver disease caused by tuberculosis, but then general, even invalid to the improvement effect of the pulmonary lesion caused by tuberculosis and aspects such as (losing weight) of becoming thin.For this patent drug weak point in patient's condition improvement, usually adopt with other drug coupling with complementation.

Therefore, there is provided one to improve patient's compliance, particularly effectively can improve symptom of becoming thin, there is antibacterial, sterilization curative effect, improve the disappear method of nuclear extract and Synthesis and applications thereof of the Antituberculous Chinese Traditional Medicine cattle shellfish of immunity of organisms and just become the technical barrier that this technical field is badly in need of solving.

Summary of the invention

An object of the present invention is to provide one can improve patient's compliance, particularly effectively can improve symptom of becoming thin, have antibacterial, sterilization curative effect, improve the cattle shellfish of immunity of organisms and to disappear nuclear extract.

Foregoing invention object of the present invention reaches by the following technical programs:

A kind of Antituberculous Chinese Traditional Medicine cattle shellfish disappears nuclear extract, it is characterized in that: comprise following preparation process:

(1) crude drug compatibility;

(2) alcohol extraction and/or water extraction;

(3) preparation of extract.

Concrete scheme is as follows:

Scheme one (alcohol water extract):

(1) crude drug compatibility

Bulbus Fritillariae Uninbracteatae 3-5 weight portion, Pseudobulbus Bletillae (Rhizoma Bletillae) 5-10 weight portion, Fructus Arctii 3-5 weight portion, Radix Platycodonis 3-5 weight portion, Oryza glutinosa 3-10 weight portion, Herba Houttuyniae 3-5 weight portion;

(2) alcohol extraction

By step (1) described formula, take Herba Houttuyniae, Radix Platycodonis and Bulbus Fritillariae Uninbracteatae, add the ethanol of medical material gross weight 6 ~ 10 times, soak after 0.5 ~ 1 hour, reflux, extract 1 ~ 2 time, each 1 ~ 2 hour, filter, obtain filtering residue and extracting solution, extracting solution decompression recycling ethanol is also concentrated, obtains alcohol extraction thick paste, for subsequent use;

(3) water extraction

By step (1) described formula, take Pseudobulbus Bletillae (Rhizoma Bletillae), Fructus Arctii and Oryza glutinosa, and mix with step (2) gained filtering residue, then, add the water of medical material gross weight 6 ~ 10 times, soak after 0.5 ~ 1 hour, heated and boiled (keeping micro-to boil), extract 2 ~ 3 times, each 1 ~ 3 hour, filter, obtain aqueous extract, extracting solution concentrating under reduced pressure, obtains water extraction thick paste;

(4) preparation of extract

Step (2) gained alcohol extraction thick paste and step (3) gained water extraction thick paste merge, and drying under reduced pressure, obtains and get dry extract, and pulverizes and sieves, and obtains alcohol water extract.

Wherein, described crude drug compatibility is as follows: Bulbus Fritillariae Uninbracteatae 4 weight portion, Pseudobulbus Bletillae (Rhizoma Bletillae) 8.5 weight portion, Fructus Arctii 4 weight portion, Radix Platycodonis 4 weight portion, Oryza glutinosa 5.5 weight portion, Herba Houttuyniae 4 weight portion.

Wherein, the concentration of ethanol described in described step (2) is 50% ~ 90% (volume %).

Wherein, described in described step (1), Bulbus Fritillariae Uninbracteatae is Bulbus Fritillariae Cirrhosae.

Scheme two (water extract):

(1) crude drug compatibility

(2) water extraction

By step (1) described formula, take Herba Houttuyniae, Radix Platycodonis, Bulbus Fritillariae Uninbracteatae, Pseudobulbus Bletillae (Rhizoma Bletillae), Fructus Arctii and Oryza glutinosa, add the water of medical material gross weight 6 ~ 12 times, soak after 0.5 ~ 1 hour, heated and boiled (keeping micro-to boil), extracts 2 ~ 3 times, each 1 ~ 3 hour, filter, obtain aqueous extract;

(3) preparation of extract

Step (2) gained aqueous extract concentrating under reduced pressure becomes thick paste, drying under reduced pressure, obtains and gets dry extract, pulverize and sieve, obtain water extract.

Scheme three (alcohol extract):

(1) crude drug compatibility

(2) alcohol extraction

By step (1) described formula, take Herba Houttuyniae, Radix Platycodonis, Bulbus Fritillariae Uninbracteatae, Pseudobulbus Bletillae (Rhizoma Bletillae), Fructus Arctii and Oryza glutinosa, add the ethanol of medical material gross weight 6 ~ 10 times, soak after 0.5 ~ 1 hour, reflux, extract 2 ~ 3 times, each 1 ~ 2 hour, filter, obtain alcohol extract;

(3) extract preparation

Step (2) gained alcohol extract decompression recycling ethanol is also condensed into thick paste, drying under reduced pressure, makes dry cream, pulverizes and sieves, obtain alcohol extract.

Wherein, the concentration of ethanol is 50% ~ 90% (volume %).

Another object of the present invention be to provide above-mentioned Antituberculous Chinese Traditional Medicine cattle shellfish disappear core three kinds of extracts preparation treatment tuberculosis in application.

A kind of Antituberculous Chinese Traditional Medicine cattle shellfish disappear nuclear extract preparation treatment tuberculosis in application.

Beneficial effect:

It is how to play tuberculosis effect that No. 200710048778.Xth, Chinese patent does not understand Chinese medicine disclosed in it, inventor herein has found the cattle shellfish nuclear extract that disappears how to play tuberculosis effect, illustrating its mechanism of action, preventing from becoming thin as put on weight; Alleviate the damage of lung, liver, spleen; Reduce the quantity of antibacterial in internal organs; Cattle shellfish disappear nuclear extract not only to mycobacterium tuberculosis also to respiratory tract common bacteria have antibacterial, sterilization curative effect; To immunoregulatory pathways, there is rise effect.

The present inventor studies the display cattle shellfish core three kinds of extracts that disappear all has obvious inhibitory action to bacill calmette-guerin and mycobacterium tuberculosis type strain in vitro, wherein alcohol extract fungistatic effect is best, and three kinds of extracts also all have certain inhibitory action to substance of medicines-resistant branched tubercle bacillus.The cattle shellfish core three kinds of extracts that disappear all have inhibitory action in various degree to the pathogen of the common medicaments insensitive of the respiratory tracts such as staphylococcus aureus, staphylococcus epidermidis, Acinetobacter bauamnnii, Pseudomonas aeruginosa, enterococcus faecalis, escherichia coli, bacillus cloacae, Klebsiella Pneumoniae or drug resistance in vitro, and wherein alcohol extract fungistatic effect is best.The cattle shellfish core three kinds of extracts that disappear all have therapeutic effect in various degree to mice tuberculosis model in vivo, can suppress or kill mycobacterium tuberculosis, alleviating tuberculosis degree, improve general status.

Extract prepared by the present invention, for tuberculotherapy, can effectively improve patient's compliance, particularly effectively can improve symptom of becoming thin.

Therefore, cattle shellfish provided by the invention disappears core three kinds of extracts, can suppress or kill the growth of inside and outside mycobacterium tuberculosis and respiratory tract encountered pathogenic bacteria, can be used for treatment lungy, and for providing experimental basis for clinical prevention, treatment tuberculosis concurrent infection.

Antituberculous Chinese Traditional Medicine cattle shellfish of the present invention disappears core three kinds of extracts: the first is alcohol water extract, and the second is water extract, and the third is alcohol extract.Three kinds of extracts are as treatment by Chinese herbs preparation, and treatment mice tuberculosis model, makes Mouse Weight than the growth all had before treatment in various degree, and compared with former medicine powder group, the body weight of mice increases; Spleen tissue bacterial population significantly reduces, and pulmonary lesion all has and alleviates in various degree, visible normal lung structure.Though its therapeutic effect is not as isoniazid, suitable with the therapeutic effect of existing commercial tuberculosis immunity therapeutic preparation microcaloire vaccine inoculation liquid, can be used for the medicine preparing treatment tuberculosis patient.Therefore, the present invention will have broad application prospects in therapeutic alliance lungy.

The present inventor is with long-term experience, on the basis of Chinese medicine disclosed in No. 200710048778.Xth, Chinese patent, develop Chinese medicine extract, for the improvement of the various patient's condition caused by tuberculosis, have his own strong points, can apply alone or in combination to cure the larger patient crowd of each side patient's condition degree difference comprehensively; These Chinese medicine extract doses are little, improve patient's compliance, and the corresponding volume also reducing its pharmaceutical preparation, medicine of being more convenient for carries, stores.

Antituberculous Chinese Traditional Medicine cattle shellfish prepared by the present invention disappears nuclear extract, and tuberculosis infection Mouse Weight can be made to increase, and pulmonary lesion alleviates, and suppresses the growth of mycobacterium tuberculosis in body, can be used for the treatment of tuberculosis infection.

Disappear nuclear extract and cattle shellfish of Antituberculous Chinese Traditional Medicine preparation cattle shellfish of the present invention disappears compared with the full medicine powder of core, directedly obtains and effective ingredient that Antituberculous Chinese Traditional Medicine cattle shellfish of concentrating disappears in core, does not change the structure of its effective ingredient; By the deep processing to Chinese crude drug, form new Chinese medicine form, embody the technological progress of Chinese medicine, meet the requirement of the modernization of Chinese medicine; Decrease drug administration amount, improve patient's compliance; Simultaneously also this extract can be increased suitable adjuvant and make the common dosage forms such as granule, capsule, be convenient to that medicine carries, storage etc.

Below in conjunction with the drawings and specific embodiments, the present invention will be further described, but and do not mean that limiting the scope of the invention.As do not specialized part, can with reference to those skilled in the art the prior art such as " pharmaceutics " be familiar with and the Experimental Standardization of SFDA and the operation instruction of guide and reagent used and equipment.

Accompanying drawing explanation

Fig. 1 is 7 days fixing rear mouse lung general pathologies of formaldehyde after Application Example 3 of the present invention modeling.

Fig. 2 is the 7 days fixing rear mouse lung tissue pathology of formaldehyde after Application Example 3 of the present invention modeling.

Fig. 3-1 to Fig. 3-7 is that Application Example 3 of the present invention treats the 12 weeks fixing rear mouse lung tissue pathology of formaldehyde, wherein, Fig. 3-1 is blank group, Fig. 3-2 is microcaloire group, Fig. 3-3 is isoniazid group, and Fig. 3-4 to disappear core No. I group for cattle shellfish, and Fig. 3-5 to disappear core No. II group for cattle shellfish, Fig. 3-6 to disappear core No. III group for cattle shellfish, and Fig. 3-7 to disappear core No. IV group for cattle shellfish.

Detailed description of the invention

Embodiment 1: cattle shellfish disappears the preparation of No. II, core (alcohol water extract)

(1) crude drug compatibility:

Herba Houttuyniae 800g, Radix Platycodonis 800g, Bulbus Fritillariae Cirrhosae 800g, Pseudobulbus Bletillae (Rhizoma Bletillae) 1700g, Fructus Arctii 800g, Oryza glutinosa 1100g;

(2) alcohol extraction

Take Herba Houttuyniae 800g, Radix Platycodonis 800g, Bulbus Fritillariae Cirrhosae 800g, put into container, the concentration adding medical material gross weight 8 times is the ethanol of 50%, soak after 30 minutes, reflux, extract 1.5 hours, filtered while hot, obtains filtrate and filtering residue, and it is the ethanol of 50% that filtering residue adds medical material gross weight 6 times of concentration again, reflux, extracts 1 hour, filtered while hot, obtain filtrate and filtering residue, merge 2 filtrates, decompression recycling ethanol is also concentrated, obtain alcohol extraction thick paste, for subsequent use;

(3) water extraction

By step (1) described formula, take Pseudobulbus Bletillae (Rhizoma Bletillae), Fructus Arctii and Oryza glutinosa, and mix with step (2) gained filtering residue, add the water of medical material gross weight 10 times, soak after 30 minutes, heated and boiled (keeping boiling), extracts 1.5 hours, filtered while hot, obtain filtrate and filtering residue, filtering residue adds the water of medical material gross weight 8 times again, heated and boiled (keeping boiling), extract 1 hour, filtered while hot, obtains filtrate and filtering residue, merges 2 filtrates, concentrating under reduced pressure, obtains water extraction thick paste;

(4) extract preparation

Step (2) gained alcohol extraction thick paste and step (3) gained water extraction thick paste merge, and drying under reduced pressure, obtains and get dry extract, and pulverize, and cross 80 mesh sieves, and get Niu Bei disappears core No. II alcohol water extract.

Embodiment 2: the preparation of alcohol water extract

(1) crude drug compatibility:

(2) alcohol extraction

Take Herba Houttuyniae 800g, Radix Platycodonis 800g, Bulbus Fritillariae Cirrhosae 800g, put into container, the concentration adding medical material gross weight 8 times is the ethanol of 50%, soaks after 30 minutes, reflux, extract 1.5 hours, filtered while hot, obtains filtrate and filtering residue, and decompression filtrate recycling ethanol is also concentrated, obtain alcohol extraction thick paste, for subsequent use;

(3) water extraction

By step (1) described formula, take Pseudobulbus Bletillae (Rhizoma Bletillae), Fructus Arctii and Oryza glutinosa, and mix with step (2) gained filtering residue, add the water of medical material gross weight 10 times, soak after 30 minutes, heated and boiled (keeping boiling), extract 1.5 hours, filtered while hot, obtains filtrate and filtering residue, obtains filtrate and filtering residue; Filtering residue adds the water of medical material gross weight 8 times again, heated and boiled (keeping boiling), and extract 1 hour, filtered while hot, obtains filtrate and filtering residue; Obtain filtrate and filtering residue, filtering residue adds the water of medical material gross weight 8 times again, heated and boiled (keeping boiling), and extract 1 hour, filtered while hot, obtains filtrate and filtering residue, and merge 3 filtrates, concentrating under reduced pressure, obtains water extraction thick paste;

(4) extract preparation

Step (2) gained alcohol extraction thick paste and step (3) gained water extraction thick paste merge, and drying under reduced pressure, obtains and get dry extract, and pulverize, and cross 80 mesh sieves, obtain alcohol water extract.

Embodiment 3 (extraction secondary): cattle shellfish disappears the preparation of core No. III (water extract)

(1) crude drug compatibility:

Bulbus Fritillariae Cirrhosae 800g, Radix Platycodonis 800g, Pseudobulbus Bletillae (Rhizoma Bletillae) 1700g, Fructus Arctii 800g, Herba Houttuyniae 800g, Oryza glutinosa 1100;

(2) water extraction:

According to step (1) described formula, weighting raw materials, puts into container, adds the water of medical material gross weight 10 times, soaks after 0.5 hour, heated and boiled (keeping micro-to boil), extracts, and filters, obtains filtrate and filtering residue; Gained filtering residue adds the water of medical material gross weight 10 times again, soaks after 0.5 hour, heated and boiled (keeping micro-to boil), extracts, and filters, obtains filtrate and filtering residue; Merge 2 filtrates, obtain aqueous extract;

(3) preparation of extract

Step (2) gained aqueous extract concentrating under reduced pressure becomes thick paste, drying under reduced pressure, obtains and gets dry extract, and pulverizes, and crosses 80 mesh sieves, and get Niu Bei disappears core No. III water extract.

Embodiment 4 (extracting three times): the preparation of water extract

(1) crude drug compatibility:

(2) water extraction:

According to step (1) described formula, weighting raw materials, puts into container, adds the water of medical material gross weight 10 times, soaks after 0.5 hour, heated and boiled (keeping micro-to boil), extracts, and filters, obtains filtrate and filtering residue; Gained filtering residue adds the water of medical material gross weight 10 times again, soaks after 0.5 hour, heated and boiled (keeping micro-to boil), extracts, and filters, obtains filtrate and filtering residue; Gained filtering residue adds the water of medical material gross weight 10 times again, soaks after 0.5 hour, heated and boiled (keeping micro-to boil), extracts, and filters, obtains filtrate and filtering residue; Merge 3 filtrates, obtain aqueous extract;

(3) preparation of extract

Step (2) gained aqueous extract concentrating under reduced pressure becomes thick paste, drying under reduced pressure, obtains and gets dry extract, and pulverizes, and crosses 80 mesh sieves, obtains water extract.

Embodiment 5: cattle shellfish disappears the preparation of core No. IV (alcohol extract)

(1) crude drug compatibility

Bulbus Fritillariae Cirrhosae 800g, Radix Platycodonis 800g, Pseudobulbus Bletillae (Rhizoma Bletillae) 1700g, Fructus Arctii 800g, Herba Houttuyniae 800g, Oryza glutinosa 1100g;

(2) alcohol extraction

Take the crude drug in the described crude drug formula of step (1), add in container, the concentration then adding medical material gross weight 8 times is the ethanol of 50%, soaks after 30 minutes, reflux, and extract 1.5 hours, filtered while hot, obtains filtrate and filtering residue; The concentration that gained filtering residue adds medical material gross weight 6 times is again the ethanol of 50%, reflux, and extract 1 hour, filtered while hot, obtains filtrate and filtering residue; Merge 2 filtrates, obtain alcohol extract;

(3) preparation of alcohol extract

Step (2) gained alcohol extract decompression recycling ethanol is condensed into alcohol extraction thick paste, and further drying under reduced pressure becomes dry cream, pulverizes, and crosses 80 mesh sieves, and get Niu Bei disappears core No. IV alcohol extract.

Embodiment 6 (extracting three times): the preparation of alcohol extract

(1) crude drug compatibility

(2) alcohol extraction

Take the crude drug in the described crude drug formula of step (1), add in container, the concentration then adding medical material gross weight 8 times is the ethanol of 50%, soaks after 30 minutes, reflux, and extract 1.5 hours, filtered while hot, obtains filtrate and filtering residue; The concentration that gained filtering residue adds medical material gross weight 6 times is again the ethanol of 50%, reflux, and extract 1 hour, filtered while hot, obtains filtrate and filtering residue; The concentration that gained filtering residue adds medical material gross weight 6 times is again the ethanol of 50%, reflux, and extract 1 hour, filtered while hot, obtains filtrate and filtering residue; Merge 3 filtrates, obtain alcohol extract;

(3) preparation of alcohol extract

Step (2) gained alcohol extract decompression recycling ethanol is condensed into alcohol extraction thick paste, and further drying under reduced pressure becomes dry cream, pulverizes, and crosses 80 mesh sieves, obtains alcohol extract.

Comparative example: former medicine powder

By Bulbus Fritillariae Cirrhosae 800g, Radix Platycodonis 800g, Pseudobulbus Bletillae (Rhizoma Bletillae) 1700g, Fructus Arctii 800g, Herba Houttuyniae 800g, Oryza glutinosa 1100g, mixing, powder is made in grinding, and get Niu Bei disappears No. I, core.

Application Example 1: cattle shellfish disappears and checks the In Vitro Bacteriostasis of mycobacterium tuberculosis

One, material

(1) strain and source

1, bacill calmette-guerin (BCG): this laboratory preserves bacterial strain;

2, mycobacterium tuberculosis type strain (H37Rv): this laboratory preserves bacterial strain;

3, substance of medicines-resistant branched tubercle bacillus No.13485 (resistance to rifampicin, resistance to isoniazid, resistance to ethambutol): clinical separation strain.

(2) main agents

1, isoniazid: specification: 0.1g/ sheet, producer: Shenyang Hongqi Pharmaceutical Co., Ltd., adult's effective clinical dosage 0.3g/d;

2, cattle shellfish disappears No. I, core (comparative example, former medicine powder): adult's clinical administration effective dose is 20g/d;

3, cattle shellfish disappears No. II, core (embodiment 1, alcohol water extract): adult's clinical administration effective dose is 12g/d;

4, cattle shellfish disappears No. III, core (embodiment 3, water extract): adult's clinical administration effective dose is 10g/d;

5, cattle shellfish disappears No. IV, core (embodiment 5, alcohol extract): adult's clinical administration effective dose is 4.5g/d;

6, potassium dihydrogen phosphate (KH ₂pO ₄): molecular weight: 136.09, purity: analytical pure, specification: 500g, producer: Beijing chemical reagents corporation;

7, sodium hydrogen phosphate (Na ₂hPO ₄12H ₂o): molecular weight: 358.14, purity: analytical pure, specification: 500g, producer: Beijing chemical reagents corporation;

8, glycerol (glycerol Glycerol): molecular weight: 92.09, purity: analytical pure, specification: 500ml, producer: Xilong Chemical Co., Ltd;

9, magnesium citrate: molecular weight: 703.4, purity: analytical pure, specification: 500g, producer: Solution on Chemical Reagents in Shanghai company;

10, magnesium sulfate (MgSO ₄): molecular weight: 246.47, purity: analytical pure, specification: 500g, producer: Xi Long chemical plant, Shantou, Guangdong city;

11, monosodium glutamate: purity: sodium glutamate content >=99%, specification: 400g, producer: Shenyang Fructus Mume monosodium glutamate limited company;

12, starch: specification: 300g, producer: sky, Beijing benefit trading trade company limited;

Peacock green: molecular weight: 364.92, purity: analytical pure, specification: 100g, producer: Xi Long Chemical Co., Ltd. of Shantou City;

13, tween 80: Amresco, molecular weight 1300, specification: 500ml, producer: Beijing Jie Hui Bioisystech Co., Ltd;

14, fresh ovum gallinaceum liquid;

15, sodium hydroxide (NaOH): molecular weight: 40, purity: analytical pure, specification: 500g, producer: Beijing Reagent Company;

16, sodium chloride (NaCl): molecular weight: 58.44, purity: analytical pure, specification: 500g, producer: Beijing chemical reagents corporation;

17, Amphotericin B for injection: specification: 25mg/ bottle powder, producer: North China Pharmacuetical Group New Drug Research & Development Co., Ltd.

(3) preparation of reagents

The preparation of 1,0.5% Tween 80-normal saline: the 0.9%NaCL 80ml getting sterilizing, adds 0.5ml tween 80, supplements 0.9%NaCL to 100ml, subpackage 10 bottles, 121 DEG C of autoclavings;

2, the preparation of 2% peacock green: take 10g malachite green oxalate, adds 500ml distilled water, 121 DEG C of autoclavings;

3, the preparation of antituberculotics solution and dilution, specifically in table 1.

The preparation of table 1 antituberculotics solution and dilution

Note: INH sterilized water for injection configures; The cattle shellfish core that disappears directly adds culture medium

Two, experimental technique

Appropriate BCG or Mycobacterium tuberculosis H37Rv reference culture or MDR-M.tb clinical separation strain are inoculated in not pastille or containing in the modified Russell medium of variable concentrations medicine, by Absolute concentration method, Study of Traditional Chinese Medicine cattle shellfish disappears and checks mycobacterium tuberculosis In Vitro Bacteriostasis.

1. the preparation of culture medium

First prepare culture fluid, concrete compound method is in table 2.

The preparation of Roche (L ǒ wenstein-Jensen, L-J) culture medium improved by table 2

Composition	Consumption
		KH ₂PO ₄	2.4g
MgSO ₄·7H ₂O	0.24g
		Fructus Citri Limoniae (citron) sour magnesium	0.6g
Sodium glutamate	3.6g
		Glycerol	12ml
Distilled water	600ml
		Potato starch	30g
Fresh ovum gallinaceum liquid	1000ml
		2% malachite green solution	20ml

After each salt component dissolves, add potato starch, mixing, boiling water bath boils and within 1 hour, (frequently shakes, anti-grumeleuse) high pressure in the pasty state after cold, adds the fresh ovum gallinaceum liquid filtered through antiseptic gauze, mixing, then add 2% malachite green solution 20ml, fully mix.Blank tube culture medium is prepared: mixed by above-mentioned mixed liquor, and aseptic subpackaged 7ml/ pipe, 85 DEG C solidify 50min.INH pastille culture medium is prepared: the INH solution that every 100ml basal medium adds 1ml preparation, diluted, mixing, and aseptic subpackaged 7ml/ pipe, 85 DEG C solidify 50min.Cattle shellfish disappears the preparation of core pastille culture medium: every 100ml basal medium adds the cattle shellfish weighed up and to disappear core, and grinding, mixing, aseptic subpackaged 7ml/ pipe, 85 DEG C solidify 50min.

2. the preparation of bacterium liquid

With the fresh mycobacterium tuberculosis of inoculating loop scraping continuous culture 2-3 week cell age on modified Russell medium, 10mg is taken with microbalance, be placed in the sterile centrifugation tube of 2ml 0.5%Tween 80-normal saline solution, uniform bacteria suspension is carefully ground to form with sterile grinder, again with physiological saline solution dilution, be made into the bacteria suspension of 1mg/ml.

3. inoculate, cultivate and observe

The bacteria suspension 10 of 1mg/ml is doubly diluted to 10-2mg/ml, accurately draws bacterium liquid 0.1ml with 100ul sample injector and be inoculated in pastille culture medium respectively and without on medicine modified Russell medium inclined-plane, often pipe quantity of microorganism inoculated is 10-3mg.Postvaccinal culture tube is placed in 37 DEG C of incubation 3-4 week observed results.

4. report the test mode

Report contrasts and colony growth situation in pastille culture medium in the following manner:

Mycobacteria is cultivated negative: inclined-plane is without colony growth;

Mycobacteria is cultivated positive (1+): colony growth accounts for 1/4 of inclined-plane area;

Mycobacteria is cultivated positive (2+): colony growth accounts for 1/2 of inclined-plane area;

Mycobacteria is cultivated positive (3+): colony growth accounts for 3/4 of inclined-plane area;

Mycobacteria is cultivated positive (4+): colony growth is covered with whole inclined-plane;

When in medium slant, clump count is less than 20, report clump count.

Three, experimental result

(1) cattle shellfish disappears and checks bacill calmette-guerin In vitro chemo-drug sensitive test result

Repeat experiment 2 times, result is similar, and representative result is as follows:

1, cattle shellfish core No. II minimum bactericidal concentration that disappear is 50mg/ml, and cattle shellfish core No. III minimum bactericidal concentration that disappear is 25mg/ml, and cattle shellfish core No. IV minimum bactericidal concentration that disappear is 12.5mg/ml, and concrete outcome is in table 3.

When 2, cultivating 3 weeks, blank group visible bacill calmette-guerin growth (average 34 CFU); The each concentration pipe of isoniazid group is showed no bacterial growth; Disappear No. II, core, No. III minimum inhibitory concentration of cattle shellfish is 12.5mg/ml; Cattle shellfish core No. IV minimum inhibitory concentration that disappear is 10mg/ml, and concrete outcome is in table 3.

When 3, cultivating 4 weeks, blank group visible bacill calmette-guerin growth (average CFU is 2+); The each concentration pipe of isoniazid group is showed no bacterial growth; Cattle shellfish core No. II minimum inhibitory concentration that disappear is 25mg/ml; Cattle shellfish core No. III minimum inhibitory concentration that disappear is 20mg/ml; Cattle shellfish core No. IV minimum inhibitory concentration that disappear is 10mg/ml, and concrete outcome is in table 3.Table 3 cattle shellfish disappears and checks the In vitro chemo-drug sensitive test result of bacill calmette-guerin

(2) cattle shellfish disappears and checks the In vitro chemo-drug sensitive test result of mycobacterium tuberculosis (H37Rv)

Repeat experiment 2 times, result is similar, and representative drug sensitivity tests is as follows:

1, disappear No. II, core, No. III minimum bactericidal concentration of cattle shellfish is 100mg/ml, and cattle shellfish core No. IV minimum bactericidal concentration that disappear is 25mg/ml, and concrete outcome is in table 4.

When 2, cultivating 3 weeks, blank group visible bacill calmette-guerin growth (average 34 CFUs); The each concentration pipe of isoniazid group is showed no bacterial growth; Disappear No. II, core, No. III minimum inhibitory concentration of cattle shellfish is 50mg/ml; Cattle shellfish core No. IV minimum inhibitory concentration that disappear is 12.5mg/ml, and concrete outcome is in table 4.

When 3, cultivating 4 weeks, blank group visible bacill calmette-guerin growth (average CFUs is 2+); The each concentration pipe of isoniazid group is showed no bacterial growth; Disappear No. II, core, No. III minimum inhibitory concentration of cattle shellfish is 50mg/ml; Cattle shellfish core No. IV minimum inhibitory concentration that disappear is 25mg/ml, and concrete outcome is in table 4.

Table 4 cattle shellfish disappears and checks the In vitro chemo-drug sensitive test result of H37Rv

(3) cattle shellfish disappears and checks substance of medicines-resistant branched tubercle bacillus (No.13458) In vitro chemo-drug sensitive test result

When cultivating 4 weeks, the visible No.13458 of blank group grows (average CFU is 4+); Isoniazid group each concentration Guan Junjian bacterial growth (average CFU is 4+); Cattle shellfish No. II, core, cattle shellfish No. III, core and cattle shellfish core No. IV minimum inhibitory concentration that disappear that disappear that disappear is 200mg/ml, and concrete outcome is in table 5.

When table 5 cultivates 4 weeks, cattle shellfish disappears and checks the In vitro chemo-drug sensitive test result of substance of medicines-resistant branched tubercle bacillus

Application Example 2: cattle shellfish disappears and checks the In Vitro Bacteriostasis of the common fastbacteria of respiratory tract

One, material

(1) strain and source

3 strain Acinetobacter bauamnniis, 3 strain staphylococcus aureuses, 2 staphylococcus epidermidis, 2 strain Klebsiella Pneumoniaes, 2 strain escherichia colis, 2 Enterococcus faecalis, 1 Pseudomonas aeruginosa strain, 1 strain bacillus cloacae clinical separation strain all derive from the Chinese People's Liberation Army the 3rd O nine hospital laboratory.Concrete antimicrobial spectrum is specifically in table 6.

The common fastbacteria antimicrobial spectrum of table 6 respiratory tract

Note: R represents drug resistance, and S represents medicaments insensitive, and M represents and is in intermediate state to medicine, and N represents and do not do.

(2) main agents

1, amikacin sulfate injection: specification: 2ml:0.2g (200,000 unit), producer: bactrim Pharmaceutical Group Plc of Shandong side;

2, injection liquid of gentamicin: specification: 2ml:8 ten thousand unit, producer: Tianjin Pharmaceutical Jiaozuo Co., Ltd.;

3, Co-trimoxazole Tablet: specification: 0.4g, packaging: 200, producer: garden, Zhejiang pharmaceutcal corporation, Ltd;

4, cattle shellfish disappears No. II, core (embodiment 1, alcohol water extract): adult's clinical administration effective dose is 12g/d;

5, cattle shellfish disappears No. III, core (embodiment 3, water extract): adult's clinical administration effective dose is 10g/d;

6, cattle shellfish disappears No. IV, core (embodiment 5, alcohol extract): adult's clinical administration effective dose is 4.5g/d;

7, sodium chloride: molecular weight: 58.44, purity: analytical pure, specification: 500g, producer: Beijing chemical reagents corporation;

8, glycerol (Glycerol): molecular weight: 92.09, purity: analytical pure, specification: 500ml, producer: Xilong Chemical Co., Ltd;

9, beef extract powder (LAB-LEMCO ' POWDER): specification 500g, producer: OXOID;

10, agar (Agar): specification: 500g, producer: Japan B iotopped;

11, peptone: specification: 250g, producer: the extensive and profound in meaning star Bioisystech Co., Ltd in Beijing;

12, caseinic acid hydrolysate (Casein Hydrol Ysate): specification: 500g, producer: OXOID;

13, Maxwell opacity tube: specification: 6/1 bottle, producer: Beijing Zhe Cheng company limited;

14, potato starch: specification: 300g, producer: sky, Beijing benefit trading trade company limited.

(3) preparation of reagents

The preparation of drug solution and dilution, specifically in table 7.

The preparation of table 7 antituberculotics solution and dilution

Medicine	Medicine final concentration in culture medium	Add culture medium prodrug
			Bactrim	0.01、0.1、1、10、20、100μg/ml	1、10、100、1000、2000、10000μg/ml
Amikacin	0.01、0.1、1、10、100μg/ml	1、10、100、1000、10000μg/ml
			Gentamycin	0.01、0.1、1、10、100μg/ml	1、10、100、1000、10000μg/ml
Cattle shellfish disappears core (II, III, IV)	20、40、80、160mg/ml	2、4、8、16g

Note: bactrim, amikacin, gentamycin sterilized water for injection configure

Two, experimental technique

Appropriate respiratory tract common bacteria is inoculated in not pastille or containing in the culture medium of variable concentrations medicine, observes cattle shellfish by MH drug sensitive experiment and disappear and check the impact of respiratory tract common bacteria growth and breeding.

(1) preparation of MH culture medium

The preparation of MH agar culture medium, concrete compound method is in table 8.

The preparation of table 8 MH agar culture medium

Composition	Dosage
		Beef extract powder	2g
Caseinic acid hydrolysate	17.5g
		Starch	1.5g
Agar	17g
		Distilled water	1000ml

Above-mentioned each composition mixing, is adjusted to pH (7.2-7.4), is cooled to about 50 DEG C after high pressure steam sterilization 121 DEG C of 15min.Prepared by blank cultures: incline above-mentioned mixing liquid to lithographic plate, agar plate thickness 4mm.Bactrim, amikacin and gentamycin pastille culture medium are prepared: the solution that every 100ml basal medium adds 1ml (milliliter) preparation, diluted, mixing, and sterile working, inclines the culture medium of mixing to lithographic plate, agar plate thickness 4mm.Cattle shellfish disappears the preparation of core pastille culture medium: every 100ml basal medium adds the cattle shellfish weighed up and to disappear core, and grinding, mixing, sterile working, inclines the culture medium of mixing to lithographic plate, agar plate thickness 4mm.

(2) preparation of bacterium liquid

On the flat board of clinical separation strain, a picking 4-5 diameter is about the colony inoculation of 1mm in 3ml (milliliter) MH meat soup culture tube, and 35 DEG C of joltings hatch 5 hours.Than turbid standard, (concentration is equivalent to 1.5 × 10 in bacterium liquid 3-5ml normal saline corrected concentrations to 0.5 Maxwell after growth ⁸then use normal saline dilution 1:10 (bacteria containing amount about 1.5 × 10 CFU/ml), ⁷cFU/ml).Bacterium liquid after dilution should (in 15min) inoculation as early as possible.

(3) inoculate, cultivate and observe

Prepare bacterium liquid 2 μ l with the sample injector absorption of 2.5 μ l (microlitre) and be inoculated in agar plate surface, often some bacterium number is about 10 ⁴cFU, forming diameter is the bacterial plaque of 5-8mm.Good rearmounted 35 DEG C of inoculation hatches 16-20 hour (methicillin-resistant Staphylococci, Vancomycin-resistant Enterococcus incubation time should expire 24h), observed result.

(4) report the test

Flat board is placed on dead color, no-reflection body surface and judges test endpoint, with the lowest concentration of drug of bacteria growing inhibiting for MIC.

Three, result

1. the drug sensitive experiment result display of staphylococcus aureus 3 strain drug resistance separated strain: the visible staphylococcus aureus growth of blank group; Gentamycin minimum inhibitory concentration is 1-100 μ g/ml; Bactrim minimum inhibitory concentration is 0.1-10 μ g/ml; Amikacin minimum inhibitory concentration is 1-100 μ g/ml; Disappear No. II, core and No. III minimum inhibitory concentration of cattle shellfish is 160mg/ml; Cattle shellfish disappears core No. IV minimum inhibitory concentration for≤40mg/ml, and concrete outcome is in table 9.

2. the drug sensitive experiment result display of Acinetobacter bauamnnii 3 strain drug resistance separated strain: the visible Acinetobacter bauamnnii growth of blank group; Gentamycin minimum inhibitory concentration is 10 μ g/ml; Bactrim minimum inhibitory concentration is 1-10 μ g/ml; Amikacin minimum inhibitory concentration is 10 μ g/ml; Cattle shellfish core No. II minimum inhibitory concentration that disappear is 160mg/ml; Cattle shellfish core No. III minimum inhibitory concentration that disappear is 80-160mg/ml; Cattle shellfish core No. IV minimum inhibitory concentration that disappear is 40mg/ml, and concrete outcome is in table 9.

3. the drug sensitive experiment result display of staphylococcus epidermidis 2 strain drug resistance separated strain: the visible staphylococcus epidermidis growth of blank group; Gentamycin minimum inhibitory concentration is 1 μ g/ml; Bactrim minimum inhibitory concentration is 0.1-20 μ g/ml; Amikacin minimum inhibitory concentration is 0.1-100 μ g/ml; Cattle shellfish core No. II minimum inhibitory concentration that disappear is 40mg/ml; Cattle shellfish core No. III minimum inhibitory concentration that disappear is 80mg/ml; Cattle shellfish disappears core No. IV minimum inhibitory concentration≤20mg/ml, and concrete outcome is in table 9.

4. the drug sensitive experiment result display of escherichia coli 2 strain drug resistance separated strain: the visible escherichia coli growth of blank group; Gentamycin minimum inhibitory concentration is 100 μ g/ml; The all long bacterium of each concentration of bactrim; Amikacin minimum inhibitory concentration is 10 μ g/ml; Cattle shellfish No. II, core and cattle shellfish core No. III minimum inhibitory concentration that disappear that disappear is 160mg/ml; Cattle shellfish core No. IV minimum inhibitory concentration that disappear is 80mg/ml, and concrete outcome is in table 9.

5. the drug sensitive experiment result display of enterococcus faecalis 2 strain drug resistance separated strain: the visible enterococcus faecalis growth of blank group; Gentamycin minimum inhibitory concentration≤0.01 μ g/ml; Bactrim minimum inhibitory concentration≤1 μ g/ml; Amikacin minimum inhibitory concentration is 100 μ g/ml; Cattle shellfish No. II, core and cattle shellfish core No. III minimum inhibitory concentration that disappear that disappear is 160mg/ml; Cattle shellfish core No. IV minimum inhibitory concentration that disappear is 40-80mg/ml, and concrete outcome is in table 9.

6. the drug sensitive experiment result display of Klebsiella Pneumoniae 2 strain drug resistance separated strain: the visible Klebsiella Pneumoniae growth of blank group; Gentamycin minimum inhibitory concentration is 1 μ g/ml; Bactrim minimum inhibitory concentration is 1-20 μ g/ml; Disappear No. II, core and cattle shellfish of cattle shellfish disappears all long bacterium of core No. III each concentration; Cattle shellfish core No. IV minimum inhibitory concentration that disappear is 40mg/ml, and concrete outcome is in table 9.

7. the drug sensitive experiment result display of antibiotic resitance of P. aeruginosa separated strain: the visible P. aeruginosa growth of blank group; Gentamycin minimum inhibitory concentration is 10 μ g/ml; Bactrim minimum inhibitory concentration is 20 μ g/ml; Amikacin minimum inhibitory concentration is 10 μ g/ml; Disappear No. II, core and cattle shellfish of cattle shellfish disappears core No. III minimum inhibitory concentration 160mg/ml; Cattle shellfish core No. IV minimum inhibitory concentration that disappear is 40mg/ml, and concrete outcome is in table 9.

8. the drug sensitive experiment result display of enterobacter cloacae drug resistance separated strain: the visible enterobacter cloacae growth of blank group; Gentamycin minimum inhibitory concentration is 100 μ g/ml; The all long bacterium of each concentration of bactrim; Amikacin minimum inhibitory concentration is 100 μ g/ml; Disappear No. II, core and cattle shellfish of cattle shellfish disappears all long bacterium of core No. III each concentration; Cattle shellfish core No. IV minimum inhibitory concentration that disappear is 80mg/ml, and concrete outcome is in table 9.

Table 9 cattle shellfish disappears and checks the drug sensitive test result of the common drug-resistant bacteria clinical separation strain of respiratory tract

Note: the not long bacterium of-representative ,+representing long bacterium, N representative is not done

Application Example 3: be used for the treatment of mice tuberculosis model

The core three kinds of extracts that disappeared by Antituberculous Chinese Traditional Medicine cattle shellfish prepared by the embodiment of the present invention 1,3 and 5 are used for the treatment of mice tuberculosis model, using it as treatment preparation, obtain good therapeutic effect.Mice therapeutic process is as follows:

One, experiment material and method

(1) experiment material

1. strain, mice and source

(1) Mycobacterium tuberculosis H37Rv type strain: tuberculosis prevention and treatment key lab of the entire PLA preserves bacterial strain;

(2) body weight buying the quality certification [licence is numbered SCXK (capital) 2012-0001] from Beijing Vital River Experimental Animals Technology Co., Ltd. is the female BAl BIc/c mice 130 in 6-8 age in week of 17-19g, is no more than 2g with batch test weight differences.

2. main agents

(1) cattle shellfish disappears No. I, core (comparative example): adult's clinical administration effective dose is 20g/d;

(2) cattle shellfish disappears No. II, core (embodiment 1, alcohol water extract): adult's clinical administration effective dose is 12g/d;

(3) cattle shellfish disappears No. III, core (embodiment 3, water extract): adult's clinical administration effective dose is 10g/d;

(4) cattle shellfish disappears No. IV, core (embodiment 5, alcohol extract): adult's clinical administration effective dose is 4.5g/d;

(5) isoniazid: specification: 0.1g/ sheet, producer: Shenyang Hongqi Pharmaceutical Co., Ltd., adult's effective clinical dosage 0.3g/d;

(6) microcaloire vaccine (injection mycobacterium vaccae): specification: 22.5 μ g/ prop up, producer: Longkema Pharmaceutical Co., Ltd., Anhui Prov.;

3. main agents preparation

(1) cattle shellfish disappears No. I, core: take 0.28g cattle shellfish and to disappear No. I, core, add the high distilled water pressed through of 10.5ml, every day boils, and gives mouse stomach after cooling.

(2) cattle shellfish disappears No. II, core: take 0.16g cattle shellfish and to disappear No. II, core, add the high distilled water pressed through of 10ml, every day boils, and gives mouse stomach after cooling.

(3) cattle shellfish disappears No. III, core: take 0.14g cattle shellfish and to disappear No. III, core, add the high distilled water pressed through of 10.5ml, every day boils, and gives mouse stomach after cooling.

(4) cattle shellfish disappears No. IV, core: take 0.06g cattle shellfish and to disappear No. IV, core, add the high distilled water pressed through of 10ml, every day boils, and gives mouse stomach after cooling.

(5) isoniazid preparation: 0.04g adds with the high distilled water pressed through of 100ml, fully mixes, to 4 DEG C of Refrigerator stores.

The preparation of (6) 22.5 μ g/ml microcaloires: get 22.5 μ g/ and prop up microcaloire 18, add 1.8ml sterilized water for injection, mixing.

(2) experimental technique

1. the foundation of mice tuberculosis model

Every mice is through tail vein injection 5 × 10 ⁵the Mycobacterium tuberculosis H37Rv type strain bacteria suspension of CFU, preparation mice tuberculosis model.

2. experiment grouping

After mouse weights, be divided into 15 groups equably at random, often organize 8.Treatment within 7th day, is started after infecting.

(1) model group: kill Mus on the 7th day after infecting;

(1) blank group: every mice gavage 0.5ml every day distilled water, gavage 5 days (Mon-Fri) is to killing Mus weekly;

(2) microcaloire treatment group: every mice bilateral tibialis anterior slowly injects 22.5 μ g/100 μ l at every turn, injection in every 2 weeks 1 time, totally 3 times;

(3) isoniazid treatment group: every mice gavage 0.4mg/0.5ml every day isoniazid, gavage 5 days (Mon-Fri) is to killing Mus weekly;

(4) cattle shellfish disappears core No. I group: every former medicine powder of mice gavage 13.3mg/0.5ml every day, and gavage 5 days (Mon-Fri) is to killing Mus weekly;

(5) cattle shellfish disappears core No. II group: every mice gavage 8mg/0.5ml every day alcohol water extract, and gavage 5 days (Mon-Fri) is to killing Mus weekly;

(6) cattle shellfish disappears core No. III group: every full liquid medicine extract of mice gavage 6.7mg/0.5ml every day, and gavage 5 days (Mon-Fri) is to killing Mus weekly;

(7) cattle shellfish disappears core No. IV group: every full medicine ethanol extraction of mice gavage 3mg/0.5ml every day, gavage 5 days (Mon-Fri) is to killing Mus weekly.

3. zootomy

Whether 7 days after infection, 8 tuberculosis model group sacrifice were also dissected, successfully set up to understand tuberculosis model.Treatment 12 weeks, often organize mice and all put to death and dissect, to disappear the therapeutic effect of core to understand cattle shellfish.

4. observation index

(1) general state: observe the mental status of mice, hair color and active situation etc.

(2) Mouse Weight growth pattern: weigh before treatment 1 time, weighs weekly after treatment 1 time.After treatment, body weight deducts the body weight that namely the front body weight for the treatment of treats rear growth.

(3) dead mouse number and mortality rate: record dead mouse number, calculating mortality rate, dead mice anatomic observation pulmonary lesion immediately.

(4) lung, liver, splenic organs ponderal index: precise Mouse Weight before dissecting, then accurately take lung, liver, spleen weight, calculate lung, liver, spleen ponderal index (WI)=(organ weights/Mouse Weight) × 100% calculating.

(5) lung, liver, splenomegaly body pathological observation: observe mouse lung, liver, splenic organs size, extent of disease, surface nodules, necrosis region etc., concrete evaluation criteria sees the following form 10.

Table 10 internal organs general pathology lesion degree summary sheet

1) pathologic inspection: mouse lung lobus dexter is fixed on 10% neutral formalin, specimens paraffin embedding slices, after hematoxylin/Yihong (HE) dyeing, 5 μm of serial section, Microscopic observation pathologic changes.

2) spleen colony counting: get spleen the first half and add 3ml normal saline respectively, grind to form tissue suspension with tissue grinder, spleen normal saline serial dilution becomes 10 ^-1, 10 ^-2, 10 ^-3bacteria suspension, by bacteria suspension 10 ^-1, 10 ^-2, 10 ^-3bacteria suspension 0.1ml is inoculated on the modified Russell medium containing amphotericin B, and each dilution factor inoculates 2 culture medium, smears evenly, cultivates 4 weeks in 37 DEG C.

5. statistical analysis

Application SPSS 16.0 statistical software carries out date processing, and continuous data represents with X ± S, and each group data carry out normality and homogeneity test of variance, and many group means compare employing one factor analysis of variance.P < 0.05 represents that difference has significance.

(3) experimental result

1. the foundation of mice tuberculosis model

1) 7 days lung hepatosplenomegaly body pathology is infected: mouse lung visible speckles sample pathological changes, liver, spleen silght enlargement, concrete outcome is in table 11 and Fig. 1 (this figure uses ordinary camera shooting at close range).

Table 11 infects and within 7 days, kills Mus perusal lung, liver, splenomegaly body pathological change

2) lung colony counting in 7 days is infected: model group lung colony counting result is 5.93log.

3) 7 days histopathologies are infected: mouse lung tissue pathological changes is extensively, alveolar structure destroys, protein liquid, foam cell and neutrophilic granulocyte that in alveolar space, visible volume is oozed out, alveolar septum edema, broadening with volume lymphocytic infiltration; Visible downright bad neutrophilic granulocyte chip in bronchioles tube chamber, concrete outcome is shown in Fig. 2 (this figure microscope photographing, 40 times).

2. therapeutic effect

1) Mouse Weight growth pattern:

Treat 12 weeks, each group Mouse Weight is than the growth all had before treatment in various degree.Compare with blank group, microcaloire treatment group, isoniazid treatment group, cattle shellfish No. IV, the core group Mice Body weight average that disappears significantly increases (F=1.881, P=0.037), and concrete outcome is in table 12.Disappear compared with core No. I group with cattle shellfish, it is more obvious that cattle shellfish disappears that core No. II group, cattle shellfish disappear that core No. III group and cattle shellfish disappear that the Mouse Weight of No. IV, core group increases.

12 weeks respectively group Mouse Weight growth patterns treated by table 12

Group	Mouse Weight increases (X gram ± S)	P value
			Blank group	1.21±2.02	0.037
Microcaloire treatment group	3.48±0.72	0.001
			Isoniazid treatment group	2.96±0.70	0.008
Cattle shellfish disappears core No. I group	1.78±1.45	0.40
			Cattle shellfish disappears core No. II group	2.23±1.73	0.12
Cattle shellfish disappears core No. III group	2.24±0.84	0.11
			Cattle shellfish disappears core No. IV group	3.25±0.68	0.002

2) organ weights and ponderal index thereof:

(1) lung organ index: compare with blank group, microcaloire group, isoniazid group lung organ index all significantly reduce (F=3.055, P<0.05), all the other group mouse lung organ indexs are without significant difference (P>0.05), but cattle shellfish disappears nuclear extract especially alcohol extract group all have reduction compared with blank group and former medicine powder group lung organ index, concrete outcome is in table 13.

(2) liver organ index: compare with blank group, microcaloire group, isoniazid group liver organ index all significantly reduce (F=5.493, P<0.05), all the other group Mouse Liver organ indexs are without significant difference (P>0.05), but cattle shellfish disappears the former medicine powder group of core and extract group comparatively blank group liver organ index all have reduction, especially former medicine powder group declines more, and concrete outcome is in table 13.

(3) splenic organs index: compare with blank group, microcaloire group, isoniazid group, cattle shellfish core No. I group, cattle shellfish core No. III group, cattle shellfish No. IV, the core group splenic organs index that disappears that disappears that disappears all significantly reduces (F=5.885, P<0.05), all the other group mice spleen organ indexs are without significant difference (P>0.05).Cattle shellfish the disappear splenic organs index decreased of the more former medicine powder group of No. IV, core group of No. I, core group and cattle shellfish that disappears is obvious, but cattle shellfish disappears No. II, core organize splenic organs index comparatively blank group also have reduction, concrete outcome is in table 13.

12 weeks respectively group mice organs indexes treated by table 13

3) lung general pathology changes:

Compare with blank group, isoniazid treatment group, cattle shellfish core No. IV group, cattle shellfish disappear core No. III group, the microcaloire treatment group pathological changes of core No. II group, cattle shellfish that disappear that disappear is lighter.Cattle shellfish disappears core three extract group more former medicine powder group pathological changes comparatively gently, and concrete pathological changes index results is in table 14.

12 weeks respectively group mouse lung general pathology observed results treated by table 14

4) colony counting of spleen:

Compare with blank group, core No. II group, cattle shellfish No. IV, the core group spleen colony counting that disappears that disappears of microcaloire treatment group, isoniazid treatment group, cattle shellfish all significantly reduces (F=2.311, P=0.009), all the other group mice spleen clump counts are without significant difference (P>0.05).But cattle shellfish disappears No. III, core group and former medicine powder group spleen clump count comparatively blank group decline, cattle shellfish disappear No. II, core group and No. IV organize also more former medicine powder group spleen clump count decline, concrete outcome is in table 15.

Respectively group mice spleen colony counting in 12 weeks treated by table 15

5) pathologic:

Compare with blank group, each treatment group all can alleviate lung tissue disease's variable area in various degree, and alleviate alveolar structure and destroy, alleviate pathologic lesion degree, concrete outcome is shown in Fig. 3-1 to Fig. 3-7 (this figure microscope photographing, 40 times.Wherein: Fig. 3-1 is blank group, and Fig. 3-2 is microcaloire group, Fig. 3-3 is isoniazid group, and Fig. 3-4 to disappear core No. I group for cattle shellfish, and Fig. 3-5 to disappear core No. II group for cattle shellfish, and Fig. 3-6 to disappear core No. III group for cattle shellfish, and Fig. 3-7 to disappear core No. IV group for cattle shellfish).

6) each group dead mouse situation:

Death during blank group 1 mouse infection 22 days; Death during No. I group 1 mouse infection 84 days; All the other each group is showed no dead mouse.

Above-mentioned experimental result shows that Antituberculous Chinese Traditional Medicine cattle shellfish prepared by the present invention disappears core three kinds of extracts, treatment mice tuberculosis model, tuberculosis infection Mouse Weight can be made to increase, pulmonary lesion alleviates, organs and tissues bacterial population significantly reduces, though its therapeutic effect is not as isoniazid, suitable with the therapeutic effect of existing commercial tuberculosis immunity therapeutic preparation microcaloire vaccine inoculation liquid.The cattle shellfish core three kinds of more former medicine powders of extract general curative effect that disappear are good.Therefore, the present invention will have broad application prospects in therapeutic alliance lungy.

The Antituberculous Chinese Traditional Medicine preparation cattle shellfish of the present invention core that disappears is by the practicality side of Bulbus Fritillariae Uninbracteatae, Radix Platycodonis, Pseudobulbus Bletillae (Rhizoma Bletillae), Fructus Arctii, Herba Houttuyniae, Oryza glutinosa Six-element medicine compatibility, adopt alcohol water, water or alcohol extraction, obtaining three kinds of extracts, is alcohol water extract, water extract, alcohol extract respectively.Take medical material in proportion, add ethanol and/or water, soak post-heating reflux, extract, filter, obtain extracting solution, decompression and solvent recovery is also condensed into thick paste, and drying under reduced pressure becomes dry cream, pulverizes and sieves, and to obtain final product.Crude drug is by following % by weight scope proportioning: Bulbus Fritillariae Uninbracteatae 8% ~ 23%, Pseudobulbus Bletillae (Rhizoma Bletillae) 14% ~ 40%, Fructus Arctii 8% ~ 23%, Radix Platycodonis 8% ~ 23%, Herba Houttuyniae 8% ~ 23% and Oryza glutinosa 9% ~ 37%, all can reach the technique effect of above-described embodiment.Cattle shellfish of the present invention disappears core three kinds of extracts as treatment by Chinese herbs preparation, and treatment mice tuberculosis model, makes Mouse Weight than the growth all had before treatment in various degree, also increase many than distilled water matched group and former medicine powder group body weight; Spleen tissue bacterial population can be made significantly to reduce, and pulmonary lesion all has and alleviates in various degree, visible normal lung structure, can be used for the medicine preparing treatment tuberculosis patient.

Claims

1. Antituberculous Chinese Traditional Medicine cattle shellfish disappears a nuclear extract, it is characterized in that: comprise following preparation process:

(1) crude drug compatibility;

(2) alcohol extraction and/or water extraction;

(3) preparation of extract.

2. Antituberculous Chinese Traditional Medicine cattle shellfish according to claim 1 disappears nuclear extract, it is characterized in that: described step (1) described crude drug compatibility is specific as follows:

The concrete steps of described step (2) are as follows:

1) alcohol extraction:

By step (1) described formula, take Herba Houttuyniae, Radix Platycodonis and Bulbus Fritillariae Uninbracteatae, add the ethanol of medical material gross weight 6 ~ 10 times, soak post-heating backflow in 0.5 ~ 1 hour, extract 1 ~ 2 time, each 1 ~ 2 hour, filter, obtain filtering residue and alcohol extract, alcohol extract decompression recycling ethanol is also concentrated, obtain alcohol extraction thick paste, for subsequent use;

2) water extraction:

By step (1) described formula, take Pseudobulbus Bletillae (Rhizoma Bletillae), Fructus Arctii and Oryza glutinosa, and mix with step (2) gained filtering residue, add the water of medical material gross weight 6 ~ 10 times, soak after 0.5 ~ 1 hour, heated and boiled, extracts 2 ~ 3 times, each 1 ~ 3 hour, filter, obtain aqueous extract, aqueous extract concentrating under reduced pressure, obtains water extraction thick paste;

Described in described step (3), the concrete steps of the preparation of extract are as follows:

Step 1 in step (2)) gained alcohol extraction thick paste and step 2) gained water extraction thick paste merges, and drying under reduced pressure, obtains and gets dry extract, and pulverizes and sieves, and get Niu Bei disappears core alcohol water extract.

3. Antituberculous Chinese Traditional Medicine cattle shellfish according to claim 2 disappears nuclear extract, it is characterized in that: described in described step (1), crude drug compatibility is as follows: Bulbus Fritillariae Cirrhosae 4 weight portion, Pseudobulbus Bletillae (Rhizoma Bletillae) 8.5 weight portion, Fructus Arctii 4 weight portion, Radix Platycodonis 4 weight portion, Oryza glutinosa 5.5 weight portion, Herba Houttuyniae 4 weight portion.

4. Antituberculous Chinese Traditional Medicine cattle shellfish according to claim 2 disappears nuclear extract, it is characterized in that: step 1 described in described step (2)) described in the concentration of ethanol be 50 volume % ~ 90 volume %.

5. Antituberculous Chinese Traditional Medicine cattle shellfish according to claim 1 disappears nuclear extract, it is characterized in that: described in described step (1), crude drug compatibility is as follows:

(1) crude drug compatibility

Described in described step (2), the concrete steps of water extraction are as follows:

By step (1) described formula, take Herba Houttuyniae, Radix Platycodonis, Bulbus Fritillariae Uninbracteatae, Pseudobulbus Bletillae (Rhizoma Bletillae), Fructus Arctii and Oryza glutinosa, add the water of medical material gross weight 6 ~ 12 times, soak after 0.5 ~ 1 hour, heated and boiled, extract 2 ~ 3 times, each 1 ~ 3 hour, filter, obtain aqueous extract;

Step (2) gained aqueous extract concentrating under reduced pressure becomes thick paste, drying under reduced pressure, obtains and gets dry extract, pulverize and sieve, and get Niu Bei disappears core water extract.

6. Antituberculous Chinese Traditional Medicine cattle shellfish according to claim 5 disappears nuclear extract, it is characterized in that: wherein, described crude drug compatibility is as follows: Bulbus Fritillariae Cirrhosae 4 weight portion, Pseudobulbus Bletillae (Rhizoma Bletillae) 8.5 weight portion, Fructus Arctii 4 weight portion, Radix Platycodonis 4 weight portion, Oryza glutinosa 5.5 weight portion, Herba Houttuyniae 4 weight portion.

7. Antituberculous Chinese Traditional Medicine cattle shellfish according to claim 1 disappears nuclear extract, it is characterized in that: described step (1) described crude drug compatibility is specific as follows:

Described in described step (2), the concrete steps of alcohol extraction are as follows:

The concrete steps that described in described step (3) prepared by extract are as follows:

Step (2) gained alcohol extract decompression recycling ethanol is also condensed into thick paste, drying under reduced pressure, makes dry cream, pulverizes and sieves, and get Niu Bei disappears core alcohol extract.

8. Antituberculous Chinese Traditional Medicine cattle shellfish according to claim 7 disappears nuclear extract, it is characterized in that: described crude drug compatibility is as follows: Bulbus Fritillariae Uninbracteatae 4 weight portion, Pseudobulbus Bletillae (Rhizoma Bletillae) 8.5 weight portion, Fructus Arctii 4 weight portion, Radix Platycodonis 4 weight portion, Oryza glutinosa 5.5 weight portion, Herba Houttuyniae 4 weight portion.

9. Antituberculous Chinese Traditional Medicine cattle shellfish according to claim 7 disappears nuclear extract, and it is characterized in that: wherein, the concentration of ethanol is 50 volume % ~ 90 volume %.

10. the Antituberculous Chinese Traditional Medicine cattle shellfish according to any one of claim 1-9 disappear nuclear extract preparation treatment tuberculosis in application.