CN115715799A - Composition for preventing or treating fungal infection, preparation method and application thereof - Google Patents
Composition for preventing or treating fungal infection, preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to the field of veterinary medicine, in particular to a composition for preventing or treating pet skin fungal infection, and a preparation, a preparation method and an application thereof. The composition and the preparation thereof can safely and effectively prevent or treat fungal skin infection of animals, have remarkable curative effect, no toxic or side effect and no drug resistance.
Description
Technical Field
The invention relates to the field of veterinary medicine, in particular to a composition for preventing or treating dermatophyte infection of pets such as dogs and cats, and a preparation, a preparation method and application thereof.
Background
The dermatomycosis of pets such as dogs and cats is also called dermatophytosis, which is a fungal disease that fungi (mainly microsporomyces gypseum, microsporomyces canis, trichophyton mentagrophytes, etc.) infect epidermis and its accessory structures (hair, horn, paw). It is one of the main diseases affecting the health of dogs and cats. The disease not only affects the appearance of dogs and cats, but also can cause pruritus of dogs and cats and can cause pain when the disease is severe.
At present, most antifungal medicines used in the pet medical industry and the pet market are chemical antifungal products, such as terbinafine, itraconazole, chlorhexidine acetate compound preparation and the like. The medicines have long treatment course, repeated disease conditions and certain toxic and side effects, are mainly manifested by gastrointestinal discomfort, occasional erythema, red swelling, nervous symptoms and the like, are not beneficial to wide and long-term use, and are easy to generate drug resistance. The general diagnosis and treatment means of veterinary clinical treatment on skin diseases with fungal infection is oral administration or injection of antifungal drugs, and the treatment effect is achieved by combining the use of in vitro antifungal drugs, wherein the oral administration or injection of antifungal drugs has certain liver and kidney toxicity on animals, and is particularly serious in toxic and side effects of pets with weakened immunity, and pets, diabetes or liver patients. However, the above antifungal drugs for external use need to be worn by pets in cooperation with elizabethan rings (hoods) when used, so that toxic and side effects and weakened curative effects of the drugs caused by animals licking affected parts are avoided, most animals (especially cats) have psychology of mental depression, diet obstruction and resistance when wearing the elizabethan rings, and meanwhile, the antifungal drugs for external use with chemical drug components such as hormones cannot be used for a long time and have the risk of drug resistance. In addition, for puppies and geriatric dogs, because of low immunity, mixed infection of bacteria and fungi generally exists, dermatitis can be caused by staphylococcus aureus infection, and long-term latent skin infection enables the skin surface to be in an immune suppression state, so that the difficulty of fungal treatment is further increased. Therefore, the search for broad-spectrum, highly effective, low-toxicity antibacterial components capable of simultaneously solving the problem of mixed infection of bacteria and fungi has become a well-recognized important research direction.
The antifungal externally applied medicine (chemical medicine, etc.) in the market at present mainly effectively inhibits and synthesizes the cell walls of pathogenic fungi, so that the cell walls of the pathogenic fungi and bacteria are damaged to a certain extent, the pathogenic fungi swell or crack and die, and in the actual use process, the antifungal externally applied medicine mainly effectively influences the propagating or growing pathogenic fungi. However, when the proliferation and subculture growth of drug-resistant pathogenic bacteria occurs, the drug is at risk of drug resistance when used continuously.
In the current report, the pythium oligandrum oospore preparation is interfered by various environmental factors and the like in the application process, the applied pythium oligandrum oospore can not play a role after germination in an environment suitable for germination and growth, the disease course is a dynamic change process, and the problem can not be solved by the simple pythium oligandrum oospore preparation when secondary bacterial infection and other conditions occur. And the pythium oligandrum fermentation broth preparation is not reported to be successfully applied to animal fungal infection diseases, only the successful application of fungal infection on plant bodies is reported, and the problems of other symptoms such as pruritus, dandruff, redness and swelling and inflammatory exudation generated after animal fungal infection cannot be solved by the application of the pure pythium oligandrum fermentation broth.
Patent application CN 110225759A-preparation with viable fungal parasitic microorganism Pythium oligandrum (Pythium oligandrum) for treating dermatophytosis and yeast infection on skin and mucosa, method for determining cell viability of microbial Pythium oligandrum and method for applying the preparation "discloses a preparation containing fungal parasitic microorganism Pythium oligandrum for treating dermatophytosis and yeast infection on skin and mucosa, the antibacterial active ingredient of the preparation is viable cells of microbial Pythium oligandrum, wherein the viable cells of microbial Pythium olidrum comprise dormant oospores, cysts zoospores and viable polynuclear mycelia. The patent application uses resting oospores of pythium oligandrum as an antibacterial active ingredient, and achieves certain clinical effect in treating experimental dermatomycosis of guinea pigs. However, since animal dermatomycosis is a dynamic pathological process, and in most cases is not a single fungal infection, and may be a mixed dermatosis accompanied by bacterial (such as staphylococcus aureus) infection, there is a need for further improvement in the treatment of animal dermatomycosis.
Patent application "CN 102655754A-antifungal mixture comprising the fungal organism pythium oligandrum" discloses an antifungal mixture comprising the fungal organism pythium oligandrum. The antifungal active components of the mixture are Pythium oligandrum living cells, including zoospores and zoosporangium. The antifungal action mechanism is that the pathogenic fungi are killed by the heavy parasitism of the viable cell oospore of the pythium oligandrum after germination, and meanwhile, the pythium oligandrum produces various enzymes for decomposing host organisms after parasitizing the pathogenic fungi to achieve the antifungal effect. This patent application discloses several groups of formulation components using viable cells of pythium oligandrum as antifungal active ingredient in combination with several conventional adjuvants, which can be used in human and veterinary medicine, cosmetic industry, etc., for maintaining cleanliness and for eliminating pathogenic fungi and microorganisms on objects in contact with humans or animals. Since the surface environment of human and animal bodies is essentially different from the living environment of Pythium oligandrum as an earth-colonizing fungus, whether the Pythium oligandrum living oospore can successfully colonize on the body surfaces of human and animal bodies and play a corresponding heavy parasitic role in the patent application needs to be further verified. Meanwhile, the pythium oligandrum is taken as a microorganism of obligate heavy parasitic pathogenic fungi, and no document reports that the pythium oligandrum can play a corresponding role in heavy parasitism or resistance to staphylococcus aureus.
The method combines the course of the animal body fungal dermatosis and the micro-ecological environment of the body surface skin, solves the potential bacterial infection problem of the skin under the premise of treating the fungal infection of the skin, and simultaneously relieves various diseases caused by the mixed fungal dermatosis so as to improve the overall treatment effect of the fungal dermatosis.
The existing pet dog and cat antifungal products only solve the problem of fungal infection, and the problem of skin immunity reduction caused by recessive infection of staphylococcus aureus cannot be considered from the perspective of a skin microbial ecosystem, so that repeated outbreak of fungal infection is easily caused. In preventing or treating animal dermatophyte infection, an antifungal product which is safe, effective, stable in curative effect, free of toxic and side effects on animal organisms and capable of being used for daily skin care without generating antibacterial drug resistance is urgently needed.
Disclosure of Invention
Aiming at the technical current situation, the invention provides a composition capable of simultaneously controlling staphylococcus aureus and other common pathogenic fungi infection of pets, a preparation method and an application thereof.
The composition for preventing or treating fungal infection comprises a pythium oligandrum active component and an active component for resisting staphylococcus aureus, wherein the pythium oligandrum active component is pythium oligandrum oospore powder, pythium oligandrum fermentation supernatant or a combination of the pythium oligandrum oospore powder and the pythium oligandrum fermentation supernatant; wherein the anti-staphylococcus aureus active component is endolysin or lysozyme.
In the present invention, as one of the embodiments, the composition comprises a combination of pythium oligandrum oospore powder and endolysin or lysozyme; a combination of pythium oligandrum fermentation supernatant with endolysin or lysozyme; or comprises the combination of pythium oligandrum oospore powder and pythium oligandrum fermentation supernatant with endolysin or lysozyme;
in the present invention, as one embodiment, the composition has a mass ratio of the pythium oligandrum active component to the anti-staphylococcus aureus active component of 2.4 to 3.4:1. 2.5: 1. 2.6: 1. 2.7: 1. 2.8: 1. 2.9: 1. 3.0: 1. 3.1: 1. 3.2: 1. 3.3:1 or 3.4:1, mass ratio.
In the present invention, as one of the embodiments, the pythium oligandrum fermentation supernatant, endolysin or lysozyme is in the form of a lyophilized powder.
According to the invention, the pythium oligandrum oospore powder or the pythium oligandrum fermentation supernatant can inhibit fungal spore germination and fungal hypha growth of pet skin infection, and even die the fungal spore germination and fungal hypha growth; the endolysin or lysozyme can kill or inhibit staphylococcus aureus, solves the problem of bacterial recessive infection in the pet-suffering dermatosis, thereby improving the immunity of the pet-suffering skin, and enables the effective antifungal component of the pythium oligandrum to play a better antifungal role, and the endolysin or the lysozyme and the active component play a good synergistic effect in solving the problem of the pet-suffering mixed type fungal dermatosis.
In the present invention, as one embodiment, the composition further comprises a traditional Chinese medicine component, wherein the traditional Chinese medicine component is selected from orange peel powder, paeonol, syringic acid or dried alum, or a combination of two or more of the traditional Chinese medicine component and the dried alum; preferably, the Chinese medicinal components are selected from the group consisting of orange peel powder, paeonol, syringic acid and dried alum.
In the present invention, as one embodiment, the pythium oligandrum active ingredient in the composition is anti-staphylococcus aureus active ingredient: the mass ratio of the traditional Chinese medicine components is 2.4-3.4: 1:1.
in the invention, the traditional Chinese medicine components play roles in assisting in solving and relieving various symptoms of pruritus, desquamation and the like caused by fungal infection of a patient and a pet, so that the disease course and the skin ecology are accelerated to recover, and a further synergistic effect is played.
The invention also provides a preparation containing the composition, and the preparation comprises the following components: based on the weight percentage in the formulation,
the usage amount of the pythium oligandrum oospore powder or the pythium oligandrum fermentation supernatant freeze-dried powder or the pythium oligandrum oospore powder or the combination of the pythium oligandrum oospore powder and the pythium oligandrum oospore powder is 5-40%, preferably 10-25%, and optimally 12-17%;
the dosage of the endolysin or the lysozyme or the combination of the endolysin and the lysozyme is 0.5 to 20 percent, preferably 0.5 to 10 percent, and optimally 5 percent;
the dosage of the traditional Chinese medicine components is 0-20%, preferably 3-10%, and optimally 5%.
In the present invention, as one embodiment, in the preparation, the traditional Chinese medicine components comprise, by weight percentage in the preparation:
the amount of the orange peel powder is 0 to 5 percent, preferably 3 percent;
the amount of paeonol is 0 to 5 percent, preferably 0.5 percent;
the amount of syringic acid is 0-5%, preferably 1%;
the amount of dried alum is 0% to 5%, preferably 0.5%.
In the invention, as one embodiment, in the preparation, the 5% of traditional Chinese medicine components comprise, by weight percentage in the preparation, 3% of orange peel powder, 0.5% of paeonol, 1% of syringic acid and 0.5% of dried alum.
In the present invention, as one embodiment, the preparation further comprises an adjuvant selected from a buffer, an adsorbent or a humectant, or a combination of two or more thereof; preferably the adjuvant is selected from the group consisting of a buffer, an adsorbent and a humectant; .
In the present invention, as one embodiment, the preparation is preferably a tablet, a capsule, or a powder, a liquid, or the like.
The invention, as one of its embodiments, is in the formulation ， The buffer is selected from anhydrous citric acid, sodium carbonate or sodium bicarbonate or the combination of two or more of the above; preferably citric acid, sodium carbonate and sodium bicarbonate; to be atThe amount of the buffer is preferably 0.5 to 45 percent by weight of the preparation; further preferably 5 to 30 percent of anhydrous citric acid, preferably 23 percent; 5% -30%, preferably 18% of sodium bicarbonate; sodium carbonate 0.5-15%, preferably 2%.
In the present invention, as one embodiment, the adsorbent in the formulation is selected from silica, and the silica is used in an amount of 0.5% to 20%, preferably 8%, by weight in the formulation.
In one embodiment of the present invention, in the preparation, the humectant is selected from PEG6000, sorbitol or a combination thereof; preferably a combination of PEG6000 and sorbitol; the humectant accounts for 0.5 to 30 percent of the weight of the preparation; as an embodiment, the amount of PEG6000 is between 0.5% and 20%, preferably 3%; as an embodiment, the amount of sorbitol is between 5% and 30%, preferably 24%.
The invention also provides a preparation method of pythium oligandrum oospore powder, which is obtained by the following method:
A. reviving the pythium oligandrum strain by a PDA (personal digital assistant) plate, storing the pythium oligandrum strain in a slope, and placing the slope at 2-8 ℃ for later use;
B. b, activating and culturing the pythium oligandrum strain in the step A in a PDA culture medium for 3-5 days, transferring the pythium oligandrum strain to liquid culture solution PD for shaking culture for 3-5 days, and transferring the shaking seed solution into a fermentation tank for enlarged culture;
C. after carrying out amplification culture for 7-10 days to produce spores, collecting pythium oligandrum mycelium and oospore, and carrying out spray drying to obtain pythium oligandrum oospore powder.
The invention also provides a preparation method of the pythium oligandrum fermentation supernatant freeze-dried powder, which is obtained by the following method:
A. reviving the pythium oligandrum strain by a PDA (personal digital assistant) plate, storing the pythium oligandrum strain in a slope, and placing the slope at 2-8 ℃ for later use;
B. b, activating and culturing the pythium oligandrum strain in the step A in a PDA culture medium for 3-5 days, transferring the pythium oligandrum strain to liquid culture solution PD for shaking culture for 3-5 days, and transferring the shaking seed solution into a fermentation tank for enlarged culture;
C. after the enlarged culture is carried out for 7-10 days, collecting fermentation liquor, centrifuging and carrying out suction filtration to obtain pythium oligandrum fermentation supernatant;
D. freeze-drying the collected pythium oligandrum fermentation supernatant by a vacuum freeze-drying machine, charging nitrogen gas and collecting the freeze-dried powder to obtain the pythium oligandrum fermentation supernatant freeze-dried powder.
The present invention also provides a method for preparing a formulation for preventing or treating fungal infection, the method comprising: mixing Pythium oligandrum oospore powder and/or Pythium oligandrum fermentation supernatant freeze-dried powder, endolysin or lysozyme freeze-dried powder, optional traditional Chinese medicine components and auxiliary materials in proportion by a homogenizer, and tabletting in a dry low-temperature environment to obtain the Pythium oligandrum tablet.
The application of the pythium oligandrum antifungal compound preparation tablet comprises the following steps:
when in use, 1 tablet (3 g) per 10KG is dissolved in 150-200ml water, and is applied or sprayed on affected parts. The preparation is administered 1-2 times daily for 7-14 days.
The invention has the following technical effects:
the pythium oligandrum oospore powder or the pythium oligandrum fermentation supernatant freeze-dried powder and the staphylococcus aureus resistant active component in the composition are compounded to have the synergistic antifungal infection resistance effect, and the anti-inflammation and itching-relieving traditional Chinese medicine components are added to further enhance the antifungal effect.
1. The oospore powder of the pythium oligandrum strain or the freeze-dried powder of the pythium oligandrum fermentation supernatant has certain inhibiting effect on the germination and growth of hyphae and spores of pathogenic fungi such as microsporum gypseum, trichophyton mentagrophytes, microsporum canis and the like of dogs and cats, and can prevent or treat dermatophyte infection of pets. The pythium oligandrum oospore powder or the pythium oligandrum fermentation supernatant freeze-dried powder and the endolysin or the lysozyme are prepared into an external antibacterial preparation according to a certain proportion, and the antifungal infection effect is enhanced by regulating a skin microecosystem to achieve the synergistic effect. The pythium oligandrum egg powder, the pythium oligandrum fermentation supernatant freeze-dried powder, the endolysin and the lysozyme are safe and have no drug resistance, and can safely and effectively prevent or treat fungal skin infection of pets.
2. The pythium oligandrum oospore powder is compounded with an active component for resisting staphylococcus aureus, namely endolysin or lysozyme, and compared with singly applied pythium oligandrum oospore powder, the average medication period for pet recovery is shortened by about 4 days; the freeze-dried powder of the pythium oligandrum fermentation supernatant is compounded with the endolysin or lysozyme, the average medication period of the sick pet recovery is 7.2 days, and compared with the freeze-dried powder of the pythium oligandrum fermentation supernatant, the medication time is shortened by about 3 days. The observation of the integral state of the skin in the treatment process of each case shows that the case applying the antifungal compound preparation obviously relieves the pathological symptoms, shortens the course of disease and improves the body states such as the skin hirsutum after recovery. The results show that after the endolysin or the lysozyme inhibits staphylococcus aureus, the microecosystem of the skin is improved, the immunity of the skin is improved, the curative effect of the pythium oligandrum antifungal active ingredients is further promoted, and the synergistic effect is achieved.
3. Traditional Chinese medicine components for diminishing inflammation and relieving itching are added into the compound preparation, and the compound preparation has auxiliary synergistic effect on the antifungal active components of the pythium oligandrum, improves the symptoms of itching, scratching and the like of patients in the treatment process, and further enhances the antifungal curative effect of the compound preparation.
Drawings
FIG. 1: measuring the bacteriostatic activity of the pythium oligandrum oospore powder by a plate confrontation method;
FIG. 2 is a schematic diagram: the fermented supernatant of Pythium oligandrum has the effect of inhibiting spore germination and growth of pathogenic fungi such as microsporum gypseum, trichophyton mentagrophytes and microsporum canis.
Detailed Description
The following examples serve to further illustrate the invention, but do not in any way limit the effective scope of the invention.
Example 1 preparation of Pythium oligandrum oospore powder/Pythium oligandrum fermentation supernatant lyophilized powder and in vitro bacteriostasis verification thereof
1. Preparation of pythium oligandrum oospore powder:
A. after the pythium oligandrum strain is revived by a PDA plate, the pythium oligandrum strain is stored in a slant and is placed at the temperature of 2-8 ℃ for standby.
B. And B, performing activated culture on the pythium oligandrum strain in the step A in a PDA (PDA dextrose agar) culture medium, performing dark culture at 28 ℃ for 3 days, cutting a plate into mycelium blocks by a block cutting method, inoculating the mycelium blocks into a liquid culture solution PD for shake flask culture, performing shake flask culture at 25-28 ℃ and 190-220rpm for 3-5 days, and transferring the shake flask seed solution into a large-scale fermentation tank for expanded culture.
C. After carrying out amplification culture for 7-8 days to produce spores, centrifuging to collect pythium oligandrum mycelium and oospore, and carrying out spray drying to obtain pythium oligandrum oospore powder.
Determination of the rate of active spores in pythium oligandrum oospore powder: sampling 1g of the prepared pythium oligandrum oospore powder to prepare spore suspension, and taking 1ml (1 × 10) 7 Seed/g) is inoculated in a PD liquid culture medium, shaking culture is carried out for 12h at the temperature of 28 ℃, the germination rate of spores is sampled and checked, the germination is regarded as that when the length of a microscopic germ tube is more than or equal to the diameter of the spores, and the viable spore rate (more than or equal to 85 percent) is calculated.
Preparing pythium oligandrum fermentation supernatant freeze-dried powder:
A. after the pythium oligandrum strain is revived by a PDA plate, the pythium oligandrum strain is stored in a slant and is placed at the temperature of 2-8 ℃ for standby.
B. And B, carrying out activated culture on the pythium oligandrum strain in the step A in a PDA culture medium, carrying out dark culture for 3 days at the temperature of 25-28 ℃, cutting pythium oligandrum hypha blocks by a block cutting method, transferring the pythium oligandrum hypha blocks into a liquid culture solution PD for shake flask culture, carrying out shake flask culture for 3-5 days at the temperature of 25-28 ℃ at 190-220rpm/min, and transferring shake flask seed solution into a large fermentation tank for expanded culture.
C. After the enlarged culture is carried out for 8 days, collecting fermentation liquor, centrifuging for 15min at 10000rpm/min of a centrifugal machine, and obtaining pythium oligandrum fermentation supernatant after suction filtration.
D. And (3) freeze-drying the collected pythium oligandrum fermentation supernatant for 48 hours by a vacuum freeze-drying machine, filling nitrogen and collecting freeze-dried powder to obtain the pythium oligandrum fermentation supernatant freeze-dried powder.
2. In-vitro bacteriostasis verification of pythium oligandrum oospore powder and pythium oligandrum fermentation supernatant freeze-dried powder:
1) In-vitro bacteriostasis verification of pythium oligandrum oospore powder
By mixing pythium oligandrum oospore powder 1g (1X 10) 7 The count/g and the viable spore rate are more than or equal to 85 percent), the mixture is activated by a flat plate and then inoculated to the flat plate inoculated with pathogenic indicating bacteria (microsporidia gypsea and the like) in advance, the inhibition effect of the pythium oligandrum bacterial colony on the bacterial colony of the indicating bacteria is calculated by a flat plate confronting method, the pathogenic indicating bacteria grow normally on the flat plate of the pathogenic indicating bacteria which are not inoculated with pythium oligandrum oospore powder by the contrast of the flat plate inoculated with the pathogenic indicating bacteria alone, and the average diameter of the bacterial colony is 7.5cm; and the growth of the pathogenic indicator bacterium colony is inhibited on the pathogenic indicator bacterium plate inoculated with the pythium oligandrum oospore powder, and the average diameter of the colony is 2.5cm.
The pathogenic fungi used as indicator bacteria are: microsporidia gypseum, trichophyton mentagrophytes, microsporidia canis.
( Note: x is the average colony diameter value of the indicator bacterium in the control group; y is the average colony diameter value of the confronting group after the growth of the indicator bacteria is inhibited. )
The plate confronting method is used for measuring the bacteriostatic activity of the pythium oligandrum oospore powder, and is specifically shown in figure 1:
wherein the left picture in figure 1 is the culture of Pythium oligandrum and pathogenic fungi in opposition, pythium oligandrum is taken as a dominant bacterium and obviously covers the colony of the pathogenic fungi, and the growth of the colony of the pathogenic fungi is inhibited; the pathogenic fungal growth was not affected in the right control.
2) In-vitro bacteriostasis verification of pythium oligandrum fermentation supernatant freeze-dried powder
1g of pythium oligandrum fermentation supernatant freeze-dried powder is redissolved and then is uniformly mixed with pathogenic fungus spore suspension, the mixture is catalyzed and reacted for 6 to 24H at the temperature of 2 to 8 ℃, and then the treated suspension is inoculated onto a PDA (personal digital assistant) plate according to different catalytic and reaction time to culture and observe the germination and growth conditions of pathogenic fungi, so that the pythium oligandrum fermentation supernatant has a certain inhibiting effect on the germination and growth of pathogenic fungus spores. The pathogenic fungus spore suspension treated by the pythium oligandrum fermentation supernatant can not normally germinate and grow, while the untreated pathogenic fungus spore suspension of the control group can normally germinate and grow.
The pathogenic fungus spore suspension used as the indicator bacterium is as follows: microsporidia gypseum, trichophyton mentagrophytes, microsporidia canis, and the like.
( Note: x is the average viable count in a 1ml sample; y is the mean viable count reduced in 1ml samples. )
The pythium oligandrum fermentation supernatant has an inhibiting effect on spore germination and growth of pathogenic fungi, namely microsporum gypseum, trichophyton mentagrophytes, microsporum canis and the like, and is shown in figure 2:
wherein in fig. 2: the left graph pathogenic fungi spore is cultured after being treated by the pythium oligandrum fermentation supernatant, so that the germination and growth of the pathogenic fungi spore are inhibited; the pathogenic fungus spores of the right control picture are cultured after being treated by sterile water, so that the germination and the growth of the pathogenic fungus spores are not influenced, and corresponding colony morphology is normally grown.
Example 2 treatment of dermatological cases of fungal infection with Pythium oligandrum oospore powder and endolysin Complex formulations
Preparation 1: pythium oligandrum oospore powder is used as an antifungal component, wherein each ml of suspension contains 30-50 pythium oligandrum living cell oospores after the pythium oligandrum oospore powder is redissolved. Mixing with adjuvants at a certain ratio to make into antifungal preparation, which comprises Pythium oligandrum oospore powder, anhydrous citric acid, sodium bicarbonate, silicon dioxide, PEG6000, sodium carbonate, and sorbitol.
Group a patients had pet administration of formulation 1:
the group had a skin disease confirmed to be infected with fungi (microsporidia canis, etc.) at the time of hospital visit to pets, of which 2 male cats, 3 female cats, 2 male dogs, and 3 female dogs were examined. The average weight of the cats is 2.15kg, the average weight of the dogs is 7.58kg, the average age is 6.3 months, and no other disease history exists. In the clinical treatment experiment process, commercial food is quantitatively fed, drinking water is not limited, and conventional immunity is realized. Before clinical treatment experiments, physical examination including body weight, body temperature, respiration, heartbeat, appetite, body surface hair condition and the like is respectively carried out on various patients and pets, and blood routine and blood sugar are measured.
The pathological changes of the affected part, such as erythra, dandruff, depilation, broken hair, hairless, pustular, dryness and wetness, etc. are observed every day. Clinical symptoms and lesions were recorded before (basal) and after administration of formulation 1. The affected part is inspected by woodworker's lamp every day, and the affected tissue is scraped for fungus culture and identification. The application of formulation 1 was continuously stopped upon observing improvement.
Preparation 2: the pythium oligandrum oospore powder is used as an antifungal component, and is prepared into a compound antifungal preparation with endolysin and auxiliary materials according to a certain proportion, and the final antifungal compound preparation comprises the following components: pythium oligandrum oospore powder, endolysin, anhydrous citric acid, sodium bicarbonate, silicon dioxide, PEG6000, sodium carbonate and sorbitol.
Group B patients had pet administration of formulation 2:
the group had a skin disease confirmed to be infected with fungi (microsporobacter canis or microsporomyces gypseum, etc.) at the time of diagnosis for pet hospitals, of which 2 male cats, 3 female cats, 2 male dogs, and 3 female dogs were examined. Average weight of cats is 2.58kg, average weight of dogs is 8.25kg, average age is 5.8 months, and no other disease history exists. In the clinical treatment experiment process, commercial food is quantitatively fed, drinking water is not limited, and conventional immunity is realized. Before clinical treatment experiments, physical examination including body weight, body temperature, respiration, heartbeat, appetite, body surface hair condition and the like is respectively carried out on various patients and pets, and blood routine and blood sugar are measured.
Pathological changes of the affected part, such as erythema, dandruff, depilation, broken hair, hairless, pustular, dryness and wetness, are observed every day. Clinical symptoms and lesions were recorded before (basal) and after administration of formulation 2. The affected part is inspected by woodworker's lamp every day, and the affected tissue is scraped for fungus culture and identification. The disease was continuously observed to improve and the administration of formulation 2 was stopped.
The application method comprises the following steps: the preparation applied in the embodiment is dissolved in water, the suspension after full dissolution is taken to be smeared or sprayed on the affected part and then is naturally dried, 1 to 2 times a day, and the application is stopped after the disease condition is improved.
As a result:
association of clinical State changes after Pet administration of formulation 1 in group A
Correlation of clinical status changes following Pet-afflicted administration of formulation 2 in group B
From the data in example 2, it can be seen that:
the treatment period for recovery of group A pet-administered formulation 1 was 13.2 days on average, and no infected fungal hyphae were detected by woodworker light examination and microbiological examination after treatment. The patient suffers from pruritus scratching in the treatment process, elizabeth rings are worn to prevent scratching cooperation treatment, and the growth of the healed hairs is slow. The single application of the pythium oligandrum oospore powder can achieve the antifungal curative effect, but the application cannot achieve the obvious improvement effect on the clinical sign change condition, and meanwhile, the treatment course is basically consistent with that of the traditional antifungal medicine or product, and no significant difference exists (P is more than 0.05).
The drug cycle of the healing of the patients in group B who suffered from pet by the preparation 2 is 8.9 days on average, the mental states of the patients suffered from pet are good in the treatment process, inflammatory exudates are remarkably reduced, the patients suffered from pet occasionally scratch, and the hair growth is quickly recovered after healing. The compound preparation is proved to have obviously improved whole physical signs of pets after the compound preparation of pythium oligandrum oospore powder and endolysin is applied, and the treatment course is shortened to some extent, thus the compound preparation is proved to solve the problem of bacterial infection, improve the skin micro-ecosystem and improve the immunity of the skin in the treatment process of skin diseases with fungal infection, thereby enhancing the antifungal effect of the pythium oligandrum oospore powder. The result shows that the integral curative effect of the antifungal is improved after the pythium oligandrum oospore powder is compounded with the endolysin.
Example 3 application of Pythium oligandrum fermentation supernatant lyophilized powder to treat dermatosis cases with fungal infection and application of Pythium oligandrum fermentation supernatant lyophilized powder and endolysin composite preparation to treat dermatosis cases with fungal infection
Preparation 3: the freeze-dried powder of the fermented supernatant of the pythium oligandrum is used as an antifungal active component and is prepared into an antifungal preparation with auxiliary materials according to a certain proportion, and the antifungal preparation comprises the following components: pythium oligandrum fermentation supernatant freeze-dried powder, anhydrous citric acid, sodium bicarbonate, silicon dioxide, PEG6000, sodium carbonate and sorbitol.
Group C patients had pet application formulation 3:
the group had skin diseases which were confirmed to be infected with fungi (Microsporum canis or Trichophyton mentagrophytes, etc.) at the time of pet hospital visit, wherein 2 male cats, 3 female cats, 2 male dogs and 3 female dogs were present. The average weight of the cats is 3.12kg, the average weight of the dogs is 7.05kg, the average age is 6.4 months, and no other disease history exists. In the clinical treatment experiment process, commercial food is quantitatively fed, drinking water is not limited, and conventional immunity is realized. Before clinical treatment experiments, physical examination including weight, body temperature, respiration, heartbeat, appetite, body surface hair condition and the like is respectively carried out on each pet patient, and blood routine and blood sugar are measured.
Pathological changes of the affected part, such as erythema, dandruff, depilation, broken hair, hairless, pustular, dryness and wetness, are observed every day. Clinical symptoms and lesions were recorded before (basal) and after administration of formulation 3. The affected part is inspected by woodworker's lamp every day, and the affected tissue is scraped for fungus culture and identification. The application of formulation 3 was continuously stopped upon observing improvement.
Preparation 4: taking pythium oligandrum fermentation supernatant freeze-dried powder as an antifungal component, and preparing the antifungal component, endolysin and auxiliary materials into a composite antifungal preparation according to a certain proportion, wherein the antifungal composite preparation comprises the following components: pythium oligandrum fermentation supernatant freeze-dried powder, endolysin, anhydrous citric acid, sodium bicarbonate, silicon dioxide, PEG6000, sodium carbonate and sorbitol.
Group D patients administered agent 4:
the group had pets who had been diagnosed with fungal (microsporobacter canis, etc.) infected skin at the time of hospital visits for pets, 2 male cats, 3 female cats, 2 male dogs, and 3 female dogs. The average weight of cats is 2.96kg, the average weight of dogs is 6.95kg, the average age is 6.4 months, and no other disease history exists. In the clinical treatment experiment process, commercial food is quantitatively fed, drinking water is not limited, and conventional immunity is realized. Before clinical treatment experiments, physical examination including weight, body temperature, respiration, heartbeat, appetite, body surface hair condition and the like is respectively carried out on each pet patient, and blood routine and blood sugar are measured.
The pathological changes of the affected part, such as erythra, dandruff, depilation, broken hair, hairless, pustular, dryness and wetness, etc. are observed every day. Clinical symptoms and lesions were recorded before (basal) and after administration of formulation 4. The affected part is inspected by woodworker's lamp every day, and the affected tissue is scraped for fungus culture and identification. The disease was continuously observed to improve and the administration of formulation 4 was stopped.
The application method comprises the following steps: the preparation applied in the embodiment is dissolved in water, the suspension after full dissolution is taken to be smeared or sprayed on the affected part and then is naturally dried, 1 to 2 times a day, and the application is stopped after the disease condition is improved.
As a result:
relevance of clinical status changes following administration of formulation 3 to group C pets
Relevance of clinical status changes following Pet-suffering administration of formulation 4 in group D
From the data of example 3, it can be seen that:
the treatment period of recovery after the patient in group C applied the preparation 3 is 10.4 days on average, after treatment, wushi lamp examination has no fluorescence reaction, before treatment, bacterial infection such as depilation and skin ulcer caused by scratching due to pet itching is improved, and after treatment, inflammatory secretion is reduced. The antifungal effect can be achieved by independently applying the pythium oligandrum fermentation supernatant freeze-dried powder, a certain improvement effect is achieved on the clinical sign change condition, the treatment course is correspondingly shortened, and the antifungal effect of the pythium oligandrum fermentation supernatant is superior to that of pythium oligandrum oospore powder.
And the administration period of the recovery after the D group of patients apply the preparation 4 is 7.3 days on average, inflammatory exudates at the affected part are obviously reduced in the treatment process, the patients do not feel uncomfortable, and the hair growth is quickly recovered after the recovery. After the compound preparation of pythium oligandrum fermentation supernatant freeze-dried powder and endolysin is applied, the clinical state of pets suffering from the pythium oligandrum fermentation supernatant freeze-dried powder is obviously improved, inflammatory secretions are reduced, the treatment course is shortened, and the application of the compound preparation of pythium oligandrum fermentation supernatant freeze-dried powder and endolysin is proved, so that the antifungal effect of the pythium oligandrum fermentation supernatant freeze-dried powder is greatly improved, the micro-ecological system of the skin on the body surface of the pets suffering from the pythium oligandrum is improved, the autoimmunity of the skin is improved, and the overall curative effect is enhanced.
Example 4 treatment of dermatological disease cases with fungal infection with a combination of Pythium oligandrum fermentation supernatant lyophilized powder and endolysin and Chinese medicinal ingredients
Preparation 5: the pythium oligandrum fermentation supernatant freeze-dried powder is used as an antifungal active component, and is prepared into a compound antifungal preparation with endolysin, traditional Chinese medicine components and auxiliary materials according to a certain proportion, and the final antifungal compound preparation comprises the following components: freeze-dried powder of pythium oligandrum fermentation supernatant, endolysin, traditional Chinese medicine components (orange peel powder, paeonol, syringic acid and dried alum), anhydrous citric acid, sodium bicarbonate, silicon dioxide, PEG6000, sodium carbonate and sorbitol.
Group E patients had pet administration of formulation 5:
the group had a skin disease confirmed to be infected with fungi (microsporobacter canis, etc.) at the time of hospital visit to the pet, of which 2 male cats, 3 female cats, 4 male dogs, and 1 female dog. Average weight of 3.25kg for cats, 8.05kg for dogs, average age of 8.3 months, no history of other diseases. In the clinical treatment experiment process, commercial food is quantitatively fed, drinking water is not limited, and conventional immunity is realized. Before clinical treatment experiments, physical examination including body weight, body temperature, respiration, heartbeat, appetite, body surface hair condition and the like is respectively carried out on various patients and pets, and blood routine and blood sugar are measured.
The pathological changes of the affected part, such as erythra, dandruff, depilation, broken hair, hairless, pustular, dryness and wetness, etc. are observed every day. Clinical symptoms and lesions were recorded before (basal) and after administration of formulation 5. The affected part is inspected by woodworker's lamp every day, and the affected tissue is scraped for fungus culture and identification. The application of formulation 5 was continuously stopped upon observing improvement.
The application method comprises the following steps: the preparation applied in the embodiment is dissolved in water, the suspension after full dissolution is taken to be smeared or sprayed on the affected part and then is naturally dried, 1 to 2 times a day, and the application is stopped after the disease condition is improved.
As a result:
correlation of clinical status changes following Pet administration of formulation 5 in group E
From the data in example 4, it can be seen that:
the administration period of the recovery of group E suffering from pet after the preparation 5 is applied is 7.2 days on average, the clinical symptoms of suffering from pet are obviously improved, inflammatory exudates are reduced, no obvious itching scratching phenomenon of the suffering pet is seen after the preparation is applied for two days, pathogenic fungi and pathogenic bacteria (staphylococcus aureus) infected by the affected part after recovery are negative through microbiological examination, a skin micro-ecosystem is improved and recovered, and the disease does not relapse within 3 months after subsequent tracking. The compound preparation has better curative effect than that of independently applying the pythium oligandrum fermentation supernatant freeze-dried powder preparation, can be used for daily care, is not easy to generate drug resistance after being used for a long time, and improves the immunity of the skin on the body surface of a pet.
* Note: evaluation criteria for results of test using formulation of example of clinical animal administration
And (3) curing: after the preparation of the embodiment is applied, fungus culture shows that the negative conversion rate of dermatophytes (microsporum canis/microsporum gypseum/trichophyton mentagrophytes and the like) is more than or equal to 95 percent, simultaneously inflammatory secretion is obviously disappeared, and the affected part has no peculiar smell, has no erythra, obviously disappears dander, pustule and the like, and the star which is the symptom of pruritus disappears.
The effect is shown: after the preparation of the embodiment is applied, the fungus culture shows that the negative turning rate of the dermatophytes is more than or equal to 80 percent, simultaneously, inflammatory secretion obviously disappears, and the affected part has no peculiar smell, red rash, scurf, pustule and the like obviously disappears, and the pruritus symptom obviously disappears.
The method has the following advantages: after the preparation of the embodiment is applied, the fungus culture shows that the negative turning rate of the dermatophyte is more than or equal to 60 percent, meanwhile, the inflammatory secretion is reduced, the affected part has no peculiar smell, red rash, dandruff, pustule and the like, and the pruritus symptom is relieved.
And (4) invalidation: after the preparation of the embodiment is applied, the fungus culture shows that the negative turning rate of the dermatophytes is less than 60 percent, meanwhile, inflammatory secretion still exists, peculiar smell, erythra, dandruff, pustule and the like are not reduced at the affected part, and the pruritus symptom is still obvious.
Negative conversion rate (%) for dermatopathogenic fungus/bacteria isolation: sampling each pet suffering from the pet every day, and performing microbial culture and identification. Samples of the affected area hair were collected at 5 sites, namely, the face, the external auricle, the forelimb radius, the chest, waist and back joints and the tail. And (4) judging the negative conversion rate of pathogenic fungi/bacteria by observing the culture result of the hairy microorganisms.
Clinical signs integration: the symptoms of the clinical fungal dermatosis comprise pruritus, pimple, erythema, keratinization, scale and the like, and the infection degree of the clinical symptoms is evaluated by an integration method, and the evaluation criteria are as follows
0= none, no obvious clinical symptoms appear on the whole body, and the performance is good;
1= light, with light skin lesions and low lesion density, lesions appearing only in a small range;
2= medium, small areas of the skin are lost throughout the body, with symptoms appearing in greater degrees and densities or in greater areas throughout the body;
3= heavy, high degree and density of skin lesions or extensive skin lesions throughout the body.
Integral reduction index of clinical symptom signs = (total integral before treatment-total integral after treatment)/total integral before treatment = 100%.
The formulations administered in the examples were judged for their therapeutic effect on fungi as follows:
and (3) curing: during the application of the formulation of the examples and 2 weeks after discontinuation, the fungus disappeared in vitro, the clinical symptoms of the skin (erythema, pruritus, depilation, pustules, scales, erosion and keratosis) disappeared as a result of the fungal infection, the animal signs returned to normal and the microbiological examination was negative.
The effect is shown: during the application of the preparation of the example and 2 weeks after withdrawal, the fungus in vitro had substantially disappeared, the clinical symptoms of the skin caused by fungal infection (erythema, pruritus, depilation, pustules, scales, erosion and keratosis) > 60%, the animal signs had substantially returned to normal and the microbiological examination was negative.
Improvement: during the period of administration of the formulation of the example and 2 weeks after withdrawal, the number of fungi was checked by doctor blade at 20% to 60%, signs of the animals were improved but not restored to normal, and the microbiological examination was positive.
And (4) invalidation: during the administration of the example formulation and 2 weeks off, the number of fungi in vitro decreased, < 20% or decreased, or even increased, signs were more severe, and microbiological checks were positive.
Total effective rate (%) = (cure number + number of significant effect)/total number of treated animals 100%
* Note: although the above examples do not combine lysozyme, it is assumed that lysozyme has the same effect against staphylococcus aureus as endolysin, and thus has the same efficacy when combined with pythium oligandrum active ingredient.
Claims (14)
1. A composition for use in the prevention or treatment of a fungal infection, wherein the composition comprises a pythium oligandrum active component and an anti-staphylococcus aureus active component, wherein the pythium oligandrum active component is pythium oligandrum oospore powder, pythium oligandrum fermentation supernatant or a combination of both; wherein the anti-staphylococcus aureus active component is endolysin or lysozyme.
2. The composition of claim 1, wherein the composition comprises pythium oligandrum oospore powder in combination with endolysin or lysozyme; a combination of pythium oligandrum fermentation supernatant with endolysin or lysozyme; or comprises Pythium oligandrum oospore powder and Pythium oligandrum fermentation supernatant combined with endolysin or lysozyme;
wherein, the pythium oligandrum fermentation supernatant, the endolysin or the lysozyme are preferably in the form of freeze-dried powder.
3. The composition as claimed in claim 1, wherein the mass ratio of the Pythium oligandrum active component to the anti-Staphylococcus aureus active component in the composition is 2.4-3.4.
4. The composition of claim 1, further comprising a herbal ingredient selected from the group consisting of orange peel powder, paeonol, syringic acid, and dried alum, or a combination of two or more thereof; preferably, the Chinese medicinal materials are selected from the group consisting of pericarpium Citri Tangerinae powder, paeonol, syringic acid and dried Alumen; preferably, the active components of pythium oligandrum comprise anti-staphylococcus aureus active components: the mass ratio of the traditional Chinese medicine components is 2.4-3.4: 1:1.
5. a formulation comprising a composition according to any one of claims 1 to 2, wherein said formulation comprises: based on the weight percentage in the formulation,
the amount of the pythium oligandrum oospore powder or the pythium oligandrum fermentation supernatant freeze-dried powder or the combination of the pythium oligandrum oospore powder and the pythium oligandrum fermentation supernatant freeze-dried powder is 5-40%, preferably 10-25%, and most preferably 12-17%;
the content of the endolysin or lysozyme is 0.5 to 20 percent, preferably 0.5 to 10 percent, and the best 5 percent;
the amount of the traditional Chinese medicine components is 0-20%, preferably 3-10%, and optimally 5%.
6. The preparation of claim 5, wherein the traditional Chinese medicine components comprise, in weight percent in the preparation:
the amount of the orange peel powder is 0 to 5 percent, preferably 3 percent;
the amount of paeonol is 0 to 5 percent, preferably 0.5 percent;
the amount of syringic acid is 0-5%, preferably 1%; and
the amount of dried alum is 0% to 5%, preferably 0.5%.
7. The preparation of claim 6, wherein the 5% of the herbal ingredients in the preparation comprise, by weight percentage in the preparation, 3% of orange peel powder, 0.5% of paeonol, 1% of syringic acid and 0.5% of dried alum.
8. The formulation of claim 5, further comprising an adjuvant selected from a buffer, an adsorbent or a humectant, or a combination of two or more thereof; preferably the adjuvant is selected from the group consisting of buffers, adsorbents, and humectants; preferably, the formulation is a tablet, capsule, powder, or aqueous formulation.
9. The formulation of claim 8, wherein said formulation is ， The buffer is selected from anhydrous citric acid, sodium carbonate or sodium bicarbonate or the combination of two or more of the above; preferably a combination of citric acid, sodium carbonate and sodium bicarbonate; the amount of the buffer is preferably 0.5 to 45 percent by weight of the preparation; further preferably, the consumption of the anhydrous citric acid is 5-30 percent, and the best consumption is 23 percent; the dosage of the sodium bicarbonate is 5 to 30 percent, preferably 18 percent; the dosage of the sodium carbonate is 0.5 to 15 percent, and the best is 2 percent.
10. A formulation according to claim 8, wherein the adsorbent is selected from silica in an amount of 0.5% to 20%, preferably 8%, by weight of the formulation.
11. The formulation of claim 8, wherein the humectant is selected from PEG6000, sorbitol, or a combination thereof; preferably a combination of PEG6000 and sorbitol; the dosage of the humectant is 0.5 to 30 percent in percentage by weight in the preparation; preferably, the use amount of the PEG6000 is 0.5-20 percent, and the best is 3 percent; the dosage of the sorbierite is 5 to 30 percent, preferably 24 percent.
12. The composition of claim 1, wherein the pythium oligandrum oospore powder is obtained by the following method:
A. reviving the pythium oligandrum strain by a PDA (personal digital assistant) plate, storing the pythium oligandrum strain in a slope, and placing the slope at 2-8 ℃ for later use;
B. b, activating and culturing the pythium oligandrum strain in the step A in a PDA culture medium for 3-5 days, transferring the pythium oligandrum strain to liquid culture solution PD for shaking culture for 3-5 days, and transferring the shaking seed solution into a fermentation tank for enlarged culture;
C. after carrying out amplification culture for 7-10 days to produce spores, collecting pythium oligandrum mycelium and oospore, and carrying out spray drying to obtain pythium oligandrum oospore powder.
13. The composition of claim 1, wherein the pythium oligandrum fermentation supernatant lyophilized powder is obtained by the following method:
A. reviving the pythium oligandrum strain by a PDA (personal digital assistant) plate, storing the pythium oligandrum strain in a slope, and placing the slope at 2-8 ℃ for later use;
B. b, activating and culturing the pythium oligandrum strain in the step A in a PDA culture medium for 3-5 days, transferring the pythium oligandrum strain to liquid culture solution PD for shaking culture for 3-5 days, and transferring the shaking seed solution to a fermentation tank for amplification culture;
C. after the enlarged culture is carried out for 7-10 days, collecting fermentation liquor, centrifuging and carrying out suction filtration to obtain pythium oligandrum fermentation supernatant;
D. freeze-drying the collected pythium oligandrum fermentation supernatant by a vacuum freeze dryer, charging nitrogen gas and collecting freeze-dried powder to obtain the pythium oligandrum fermentation supernatant freeze-dried powder.
14. A method of preparing a formulation for use in the prevention or treatment of a fungal infection according to claims 5 to 11, the method comprising: mixing Pythium oligandrum oospore powder and/or Pythium oligandrum fermentation supernatant lyophilized powder, endolysin or lysozyme lyophilized powder, optional traditional Chinese medicine components and auxiliary materials in proportion by a homogenizer, and tabletting under a dry low-temperature environment to obtain the Pythium oligandrum tablet.
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