Background technology
China's history of TCM is long, and Chinese medicinal material industry is one of China's conventional industries.Chinese medicine enterprise, when producing a large amount of Chinese patent medicines, can produce a considerable amount of medicinal residues, and medicinal residues are generally wet stock, and very easily corrupt, taste is smelly different.The form that early stage Chinese medicine slag is processed mainly comprises landfill, burning, fixed area stacking etc.These processing methods not only exhaust substantial contribution, have more caused the wasting of resources and serious pollution, and how processing Chinese medicine slag has become a problem demanding prompt solution.So, utilize biotechnology to process Chinese medicine dreg, to reaching, turn waste into wealth, the utilization rate that improves natural resources of Chinese medicinal materials enjoys people's concern.In medicinal residues, not only contain the nascent materials such as rich in protein, cellulose, and due to present extraction process more single (decocting boils), part active component in these Chinese medicines after extracting, decocting still remains in medicinal residues, these residual nascent and secondary substances make medicinal residues have very high recycle value, therefore, medicinal residues start to be subject to people's attention as a kind of resource, have become one of important content of modernization of cmm.
Along with the application of the carrying out of application, bone marrow and the organ transplantation of a large amount of broad ectrum antibiotic, glucocorticoid and immunosuppressant, Catheter Interventional Therapy, particularly acquired immune deficiency syndrome (AIDS) popular, the fungal infection diseases such as candidemia and skin, mucosa increase gradually.The fungal infection incidence rate constantly rises, except the quantity of encountered pathogenic bacteria infection is on the increase, some rare fungal infection have report also the time, the situation be on the rise for mycosis, research to antifungal drug is also increasing, but curative effect antifungal drug high, that side effect is low is few at present.Therefore, research and development colleges and universities, nontoxic, cheap, apply antifungal drug easily and have profound significance.
Flos Trollii is the dry flower and alabastrum of ranunculaceae plant Flos Trollii (Trollius chinensis Bunge), is distributed widely in the areas such as southwest, northwest, North China, northeast.Flos Trollii has long medicinal history, among the peoplely with boiled water, infuses and works as tea, has the effect of heat-clearing and toxic substances removing.Order, domestic have tens of drugmakers to produce Flos Trollii Chinese medicine, the preparations such as globeflower capsule, Tabellae Trollii, tropaeolum oral liquid, Chinese globe flower injection are arranged, clinically be mainly used in treating the inflammation such as flu, fever, chronic tonsillitis, acute myringitis, urinary tract infection.According to incompletely statistics, the whole nation utilizes the medicinal residues of Flos Trollii pharmacy generation that millions of tons is arranged every year, and the overwhelming majority goes out of use, and not only contaminated environment, and pharmaceutical factory also will consume a large amount of expenses for processing medicinal residues.Therefore how the Flos Trollii medicinal residues are taken full advantage of, turn waste into wealth, of far-reaching significance.
Summary of the invention
The Flos Trollii medicinal residues fermented product and the preparation technology thereof that the purpose of this invention is to provide a kind of antifungic action, technological process is simple, with low cost, application is convenient, effective, solves the problems referred to above that exist in background technology.
The object of the present invention is achieved like this: a kind of Flos Trollii medicinal residues fermented product preparation technology of antifungic action comprises following processing step:
1. dried Flos Trollii medicinal residues are ground into to powder;
2. the yeast activation and proliferation is cultivated, and the enrichment liquid body of getting after autoclaving is cultivated based in sterile chamber, under gnotobasis, adds Angel dry yeast 5-10 gram/100ml, puts 35-37 ℃ of constant incubator and cultivates 5-7 h;
3. activating value-added yeast liquid, to add the quality volume fraction be 40-60% Flos Trollii medicinal residues powder, 4% peptone, 4% glucose, after mix homogeneously, continues fermentation;
4. fermentation condition is 30-37 ℃ of cultivation temperature, incubation time 72-96 hours;
5. after fermentation ends, fermented product is added to dehydrated alcohol, make concentration of alcohol reach 60-80%, 70 ℃ of extraction 2-4h in extractor, by extracting solution 1500-2500 rev/min of centrifugal 15-25 minute; Get supernatant and on Rotary Evaporators, reclaim ethanol, the 70 ℃ of water-baths of raffinate after recovery ethanol become paste, and 80 ℃ of baking boxs are dried, and namely obtain Flos Trollii medicinal residues fermented product.
Described Flos Trollii medicinal residues fermented product extraction ratio is 4-8%, namely after 100 gram Flos Trollii medicinal residues fermentations, obtains 4-8 gram Flos Trollii medicinal residues fermented product with ethanol extraction, and every gram Flos Trollii medicinal residues fermented product quite contains medicinal residues 12.5-25g.
Described enrichment liquid body culture medium ratio is: peptone 40 g, yeast soak powder 20 g, glucose 40 g, are placed in 1000 ml distilled water.
A kind of Flos Trollii medicinal residues fermented product of antifungic action, dried Flos Trollii medicinal residues are ground into to powder as raw material, the Angel Yeast bacterium of take is fermented bacterium, add simultaneously the necessary peptone of Yeast Growth, glucose nutritional labeling, extract Flos Trollii medicinal residues fermented product, described Flos Trollii medicinal residues fermented product is the ethanol extraction of fermentation product, and dry rear every gram Flos Trollii medicinal residues fermented product quite contains medicinal residues 12.5-25g.
Flos Trollii medicinal residues fermented product is made different dosage form as required, as antifungal drug, for the inside and outside fungal infection treatment of the body of human body and the disease treatment of poultry fungal infection.By after described Flos Trollii medicinal residues fermented product abrasive flour, can be made into the dosage forms such as tablet, capsule or granule, for oral medication whole body fungal infection disease; Also can make cream, ointment, lotion or spray, the fungal infection such as treatment skin, mucosa.
With different approaches fungal infection model in Flos Trollii medicinal residues fermented product treatment animal body, observe the therapeutic effect of the inside and outside fungal infection of its animal body, observed simultaneously its toxicity.Result shows that Flos Trollii medicinal residues fermented product has good therapeutic effect to the laboratory animal of fungal infection, does not find any toxic and side effects; External direct antifungal experiment, result show that causing a disease of clinical separation reached to the drug resistance fungal bacterial strain good inhibition or killing effect, and its antifungal concentration is starkly lower than conventional Folium Artemisiae Argyi water decoction and ethanol extraction.The present invention holds antifungal drug for clinical or domestic animal provides a kind of new selection, have with low cost, application is convenient, the benefit high.
Good effect of the present invention is: (1) Flos Trollii medicinal residues aboundresources, Flos Trollii is extracted to the medicinal residues after medicinal ingredient and garbage is administered and resource takes full advantage of effective combination, the fundamental state policy that meets sustainable development and recycling economy, all useful to country, enterprise, consumer, have popularizing application prospect, can produce economic benefit and social benefit; (2) Flos Trollii medicinal residues fermentation medical value is high, and it is that decocting boils the extract effective ingredient that pharmacy corporation is produced the Flos Trollii preparation overwhelming majority, and the part effective ingredient remains in medicinal residues.Using microbe of the present invention fermentation, can make some compositions further transform through the effect of microbial enzyme, and some effective ingredient can further extract with ethanol on the other hand, for recycling and the new purposes of Flos Trollii resource provides scientific basis; (3) preparation technology is simple: this method technological process is simple, does not need specific apparatus and equipment, and cost is low, with short production cycle, is suitable for suitability for industrialized production; (4) application is convenient, successful: the said preparation consumption is little, potency ratio is high, store and application all facilitates.And can obviously increase the therapeutic effect of fungal infection laboratory animal; (5) reduce and pollute, increase economic benefit: Flos Trollii medicinal residues water content is higher, very easily corrupt, can cause serious pollution to environment, and in the garbage of medicinal residues, still contain protein, polysaccharide, various trace element and vitamin and a certain amount of composition that utilizes, in time it is fully utilized, not only can the contaminated solution problem, also can bring certain economic benefit, have broad prospects.
The specific embodiment
By the following examples, the invention will be further described.
A kind of Flos Trollii medicinal residues fermented product preparation technology of antifungic action comprises following processing step:
1. dried Flos Trollii medicinal residues are ground into to powder;
2. the yeast activation and proliferation is cultivated, and the enrichment liquid body of getting after autoclaving is cultivated based in sterile chamber, under gnotobasis, adds Angel dry yeast 5-10 gram/100ml, puts 35-37 ℃ of constant incubator and cultivates 5-7 h;
3. activating value-added yeast liquid, to add the quality volume fraction be 40-60% Flos Trollii medicinal residues powder, 4% peptone, 4% glucose, after mix homogeneously, continues fermentation;
4. fermentation condition is 30-37 ℃ of cultivation temperature, incubation time 72-96 hours;
5. after fermentation ends, fermented product is added to dehydrated alcohol, make concentration of alcohol reach 60-80%, 70 ℃ of extraction 2-4h in extractor, by extracting solution 1500-2500 rev/min of centrifugal 15-25 minute; Get supernatant and on Rotary Evaporators, reclaim ethanol, the 70 ℃ of water-baths of raffinate after recovery ethanol become paste, and 80 ℃ of baking boxs are dried, and namely obtain Flos Trollii medicinal residues fermented product.
In an embodiment, the present invention is fermented the Flos Trollii medicinal residues by above-mentioned preparation technology, has carried out within the limits prescribed the preparation of several schemes, and its tunning extract has been carried out to the in vitro and in vivo zoopery, all obtains consistent useful result.
Embodiment mono-:
1. the yeast activation and proliferation is cultivated: enrichment liquid body culture medium: yeast=100ml:5g, 35 ℃ of constant incubators are cultivated 7 h.
2. activate yeast and Flos Trollii medicinal residues ratio: activation yeast liquid: Flos Trollii medicinal residues=100ml:40g.
3. fermentation condition is: 30 ℃ of temperature, 96 hours time.
4. fermented product extracting method: fermented product adds dehydrated alcohol, makes concentration of alcohol reach 60%, 70 ℃ of extraction 2h, 1500 rev/mins of centrifugal 25min of extracting solution.Supernatant reclaims ethanol, and the 70 ℃ of water-baths of raffinate after recovery ethanol become paste, and 80 ℃ of baking boxs are dried, and namely obtain Flos Trollii medicinal residues fermented product extract.
Embodiment bis-:
1. the yeast activation and proliferation is cultivated: enrichment liquid body culture medium: yeast=100ml:8g, 36 ℃ of constant incubators are cultivated 6 h.
2. activate yeast and Flos Trollii medicinal residues ratio: activation yeast liquid: Flos Trollii medicinal residues=100ml:50g.
3. fermentation condition is: 34 ℃ of temperature, 84 hours time.
4. fermented product extracting method: fermented product adds dehydrated alcohol, makes concentration of alcohol reach 70%, 70 ℃ of extraction 4h, 2000 rev/mins of centrifugal 20min of extracting solution.Supernatant reclaims ethanol, and the 70 ℃ of water-baths of raffinate after recovery ethanol become paste, and 80 ℃ of baking boxs are dried, and namely obtain Flos Trollii medicinal residues fermented product extract.
Embodiment tri-:
1. the yeast activation and proliferation is cultivated: YPD fluid medium: yeast=100ml:10g, 37 ℃ of constant incubators are cultivated 5 h.
2. activate yeast and Flos Trollii medicinal residues ratio: activation yeast liquid: Flos Trollii medicinal residues=100ml:60g.
3. fermentation condition is: 37 ℃ of temperature, 72 hours time.
4. fermented product extracting method: fermented product adds dehydrated alcohol, makes concentration of alcohol reach 80%, 70 ℃ of extraction 3h, 2500 rev/mins of centrifugal 15min of extracting solution.Supernatant reclaims ethanol, and the 70 ℃ of water-baths of raffinate after recovery ethanol become paste, and 80 ℃ of baking boxs are dried, and namely obtain Flos Trollii medicinal residues fermented product extract.
By embodiment mono-, two, three grind into powders, make respectively capsule, tablet, particulate oral in animal body, testing, make cream, ointment, lotion external and carry out mucocutaneous antifungal experiment.
The experiment of In Vitro Anti fungus
Adopt test tube method and Plating to observe Flos Trollii medicinal residues fermented product extract (sample) to Candida albicans and microsporum canis antifungal activity in vitro, and, with the positive contrast medicine of fluconazol, measure minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC); The results are shown in Table 1.
The demonstration of table 1 result, sample is almost close with fluconazol to inhibitory action and the killing action of Candida albicans, only differs a gradient; Sample is consistent with fluconazol with killing action to the inhibitory action of microsporum canis.Its minimum antifungal concentration MIC and antifungal concentration are all lower.
MIC and the MBC of table 1. sample to Candida albicans and microsporum canis
2. oral antifungal experiment
The healthy kunming mice of SPF level, male and female half and half are selected in experiment.At first with cyclophosphamide, cause immune function of mice low, use afterwards Candida albicans 10
7/ ml bacterium liquid tail vein injection (0.2ml/ is only), the injection fungus is each dosage form of mouse stomach sample simultaneously, by 100,50, tri-dosage groups of 25mg/kg, 20 every group, every day gavage once, continuous 7 days, observe the dead mouse situation of respectively organizing.The results are shown in Table 2.
The impact (n=20) of table 2 sample on candida albicans infection mice dying rate (%)
The demonstration of table 2 result, blank group mice is without death, the mortality of animals of model control group is 95.0%(P<0.01); Each sample group mouse death rate is along with sample dosage increases, and its mortality rate reduces, similar to fluconazol, illustrates that sample has obvious antifungic action, has protective effect to the fungal infection dead mouse.
The experiment of topical application antifungal
In the Kunming mouse intravaginal, inject Candida albicans suspension (5 * 10
6Individual/as ml) to build the vaginitis model, with sample emulsifiable paste, cream, lotion and the treatment of 10% JIEERYIN XIYE, successive administration 7 days, did smear for microscopic examination to the vaginal douche of mice on the 8th day respectively, and measure the fungus carrying capacity.The results are shown in Table 3.
Table 3 sample is to mouse vagina irrigating solution fungus carrying capacity (* 10
3Individual/ml) comparison
(`c ± s, n=20)
With model group, compare *
P<0.05, * *
P<0.01
Table 3 result shows, each sample topical application all has inhibitory action, also can obviously improve the symptoms such as vagina redness of mice Candida albicans, the curative effect of dialogue monilial vaginitis is similar to JIEERYIN, shows that sample dialogue monilial vaginitis has obvious therapeutical effect.
The present invention, by In Vitro Anti fungus and animal experimental observation, has obtained good effect, draws to draw a conclusion:
1. the present invention develops the Flos Trollii drug resource fully effectively, and the environmental pollution that not only can avoid discarded medicinal residues to bring has also solved a large amount of expenses of processing medicinal residues simultaneously, makes Flos Trollii Chinese medicine take full advantage of, and medicinal residues are turned waste into wealth, and are significant.
2. Flos Trollii medicinal residues fermented product extract of the present invention has obvious antifungic action, and whole body fungal infection and local fungal infection are all had to good therapeutical effect, can be used for the medicine of fungal infection.