CN113491740B

CN113491740B - Composition for preventing and treating candida albicans disease and medicine and feed additive containing same

Info

Publication number: CN113491740B
Application number: CN202010251799.7A
Authority: CN
Inventors: 王占新; 胡文锋; 郭罕星; 祁姣姣; 鲁俊鹏; 覃健萍; 胡学生; 房向杰; 元波
Original assignee: Bioforte Biotechnology Shenzhen Co ltd; Wens Foodstuff Group Co Ltd
Current assignee: Bioforte Biotechnology Shenzhen Co ltd; Wens Foodstuff Group Co Ltd
Priority date: 2020-04-01
Filing date: 2020-04-01
Publication date: 2022-05-27
Anticipated expiration: 2040-04-01
Also published as: CN113491740A

Abstract

The invention provides a composition for preventing and treating candida albicans, and a medicament and a feed additive containing the composition. The composition for controlling candida albicans comprises plant essential oil, wherein the plant essential oil consists of citrus oil, thyme oil, garlic oil and cinnamaldehyde; the composition for preventing and treating the candida albicans disease further comprises a plant extract, wherein the plant extract is one or more of a glossy privet fruit extract, a cherokee rose fruit extract, a radish seed extract, a coronene of phaseolus vulgaris and a macleaya cordata extract. The composition for preventing and treating the white candidiasis, provided by the invention, overcomes the problems of drug resistance and safety caused by using antifungal antibiotics such as nystatin, amphotericin B and the like, can effectively replace the function of the antibiotics in preventing and treating the white candidiasis of pigeons, is used for preparing natural medicines, feed additives and drinking water for administration, is a safe product capable of replacing the antibiotics, and has great application value in the healthy breeding of young pigeons.

Description

Composition for preventing and treating candida albicans disease and medicine and feed additive containing same

Technical Field

The invention relates to the technical field of medicines, and particularly relates to a composition for preventing and treating candida albicans, and a medicine and feed additive containing the composition.

Technical Field

Candida albicans is a common opportunistic fungus that can cause deep or superficial fungal infections in humans and animals. Mechanisms responsible for c albicans virulence include adhesion/invasion molecules, hydrolases, biofilm formation, and yeast to hyphal conversion, among others. Candida albicans can grow in the mouth, gastrointestinal tract and vagina, causing serious infection in millions of people worldwide. At present, the treatment of candidiasis of white is mainly based on the use of antifungal antibiotics such as nystatin and amphotericin B. However, the use of antibiotics in large quantities causes problems of resistance to candida albicans in humans and animals, and the remaining antibiotic residues cause secondary harm to humans and animals themselves and the surrounding environment. It is therefore essential to find effective alternatives to antibiotics for the treatment of candida albicans infections.

Because the Chinese herbal medicines have small toxic and side effects and do not generate drug resistance, etc., the Chinese herbal medicines are used for treating the candida albicans disease. For example, the Chinese patent application with the publication number of CN105456112A discloses an oral cavity washing and caring product with the function of resisting candida albicans, which contains the following active ingredients in parts by weight: 2-15% of chenopodium album leaf extract, 1-20% of one or more of lavender extract, scutellaria baicalensis extract and curcuma zedoary extract. The oral candida albicans inhibitor has a good effect of inhibiting candida albicans, and can be used for preventing or relieving oral candida diseases caused by the candida albicans.

In addition, the plant essential oil can be widely accepted by the public and researchers as a bacteriostatic product. Many researchers have studied the bacteriostatic properties of pathogenic bacteria, and it is hoped that plant essential oils can be used as good bacteriostatic agents. A large amount of long-term research work proves that the plant essential oil has bacteriostatic property and can be used as an effective bacteriostatic agent for inhibiting pathogenic bacteria such as escherichia coli, salmonella and the like.

Plant essential oils have also been tried for the control of candida albicans disease. For example, the Chinese patent with publication number CN1810947A discloses that emulsion and solid lipid nano granules prepared from garlic essential oil can be directly administered intravenously, all indexes are qualified after experiments, and the garlic oil has the effects of resisting deep fungal and bacterial infection, preventing and treating acute and chronic bacillary dysentery and enteritis, pertussis, fungal infection of lung and digestive tract, candida albicans bacteremia, cryptococcal meningitis, tuberculosis and the like.

The white candidiasis infection parts of the pigeons mainly comprise digestive tracts, skins and the like, the sick pigeons are main infection sources, and the pigeons are infected with the diseases by eating polluted feed or drinking water. The infection rate of young pigeons is higher, and adult pigeons can also be infected. Pigeon candidiasis is often sporadic, and once outbreaks occur, the mortality rate is extremely high, which can cause great loss. The human candidiasis infected sites are the oral cavity, digestive tract and reproductive organs of the human, but the mortality rate is extremely low. This can lead to a very high mortality rate if candida albicans disseminates into the blood causing candida septicemia and disseminated candidiasis.

Although plant essential oils and plant extracts can prevent and treat candida albicans of human origin, no report is made on the prevention and treatment of candida albicans of animal origin, especially candida albicans of pigeon origin. Candida albicans is common in infection and pathogenesis of the squab, causes serious loss and is a common problem in the squab breeding industry. At present, the young pigeon breeding industry uses acidifier and antibiotics to prevent and treat candida albicans infection, so that the problem of food safety can be caused. Therefore, how to effectively solve the problem of pigeon candidiasis needs to develop non-antibiotic products to prevent and treat candida albicans infection, thereby reducing economic loss of young pigeon breeding industry.

Disclosure of Invention

Aiming at the problems, the invention provides a composition for preventing and treating candida albicans, which can effectively prevent and treat the candida albicans, particularly the pigeon source candida albicans, and can be prepared into medicines, feed additives and the like for replacing antibiotics to be applied to the prevention and treatment of the candida albicans.

The technical purpose is achieved through the following scheme:

the invention provides a composition for preventing and treating candida albicans, which comprises the plant essential oil composition, wherein the plant essential oil composition consists of citrus oil, thyme oil, garlic oil and cinnamaldehyde.

Preferably, the vegetable essential oil composition consists of the following components in percentage by mass:

15 to 20 percent of citrus oil, 20 to 30 percent of thyme oil, 15 to 20 percent of garlic oil and 35 to 40 percent of cinnamaldehyde.

More preferably, the mass ratio of the citrus oil to the thyme oil to the garlic oil to the cinnamaldehyde is 1:1-1.5:1: 2.

The composition for preventing and treating the candida albicans disease further comprises a plant extract, wherein the plant extract is one or more of a glossy privet fruit extract, a cherokee rose fruit extract, a radish seed extract, a coronene of phaseolus vulgaris and a macleaya cordata extract.

The plant extract comprises the following components in percentage by mass: 10 to 15 percent of glossy privet fruit extract, 20 to 25 percent of cherokee rose fruit extract, 20 to 25 percent of radish seed extract, 25 to 30 percent of nutmeg extract and 10 to 15 percent of macleaya cordata extract

The plant essential oil composition and the plant extract comprise the following components in percentage by mass: 1: 1.

the Candida albicans is Pigeon source Candida albicans.

A drug for preventing and treating candida albicans comprises the composition for preventing and treating the candida albicans.

Preferably, the mass percentage concentration of the composition for preventing and treating the candida albicans disease in the medicine is 0.5-2%.

A feed additive comprises the composition for preventing and treating candida albicans.

Preferably, the feed additive is an acidulant.

Compared with the prior art, the invention has the following advantages and beneficial effects:

the invention provides a composition for preventing and treating candida albicans, which comprises a plant essential oil composition consisting of citrus oil, thyme oil, garlic oil and cinnamaldehyde, and also can contain one or more of glossy privet fruit extract, cherokee rose fruit extract, radish seed extract, coronene extract and macleaya cordata extract. The composition for preventing and treating the candida albicans overcomes the problems of drug resistance and safety caused by antifungal antibiotics such as nystatin, amphotericin B and the like in use, can effectively replace the function of the antibiotics in preventing and treating the candida albicans, can be applied to preparation of medicines and feed additives, and is an extremely safe product capable of replacing the antibiotics. Can be administered by feeding, drinking and other ways, and has great application prospect in the field of pigeon breeding.

Drawings

FIG. 1 shows the crop dissection lesion of a diseased pigeon after the pigeon is separated from Candida albicans and attacked for 7 days. Wherein, figure A, B, C is the craw dissection lesion map of days 1, 2 and 3 respectively.

Figure 2 shows the diseased pigeon crop dissected lesion after treatment. Wherein, panel a was treated with the composition for preventing and treating candidiasis of example 1, panel B was treated with the composition for preventing and treating candidiasis of example 2, panel C, F was treated with the composition for preventing and treating candidiasis of example 3, and panel D, E is a control group.

Detailed Description

The present invention will be further described with reference to the following embodiments.

The candida albicans used in the invention is separated from pigeons by the applicant, and is determined to be a type A, B, C candida albicans strain through physiological and biochemical identification and molecular biological identification.

Example 1

Citrus oil, thyme oil, garlic oil and cinnamaldehyde are mixed according to the mass ratio of 1: 1: 1:2 mixing into plant essential oil composition, adding sterile distilled water containing 0.5% (v/v) tween-80 as the composition for preventing and treating candida albicans, preparing into medicine with 0.5% (v/v) concentration, and uniformly oscillating with a vortex oscillator. Sterile distilled water containing 0.5% (v/v) Tween-80 was used as a control. The test strain was candida albicans type A, B, C strain. Counting viable bacteria to obtain 2 × 10⁷CFU/ml bacterial solution 50ul was added to sterile plates. Then pouring the high-pressure YPD culture medium at about 60 ℃ into a sterile plate, and uniformly shaking. Each plate is averagely divided into 4 areas, after the culture medium is solidified, sterile oxford cups with the same specification and size are taken by sterile tweezers and placed on the surface of the agar plate of each area, then 100ul of plant essential oil composition with the corresponding concentration is added into the oxford cups by a liquid-transferring gun, and the mark is made. Each plate was blanked at the 4-zone junction with a solvent other than essential oil. The cells were incubated overnight in an incubator at 37 ℃. And measuring the size of the inhibition zone by using a vernier caliper, and photographing and recording the result. The experiment was repeated three times and the average was calculated.

Example 2

Citrus oil, thyme oil, garlic oil and cinnamaldehyde are mixed according to the mass ratio of 2: 3: 2: 4: mixing to obtain plant essential oil composition, adding sterile distilled water containing 0.5% (v/v) Tween-80 as the composition for preventing and treating Candida albicans, making into medicine with concentration of 0.5% (v/v), and shaking with vortex oscillator. Sterile distilled water containing 0.5% (v/v) Tween-80 was used as a control. The test strain was candida albicans type A, B, C strain. Counting viable bacteria to obtain 2 × 10⁷CFU/ml bacterial solution 50ul was added to sterile plates. Then pouring the high-pressure YPD culture medium at about 60 ℃ into a sterile plateThe mixture is evenly mixed by medium-level shaking. Each plate is averagely divided into 4 areas, after the culture medium is solidified, sterile oxford cups with the same specification and size are taken by sterile tweezers and placed on the surface of the agar plate of each area, then 100ul of plant essential oil composition with the corresponding concentration is added into the oxford cups by a liquid-transferring gun, and the mark is made. Each plate was blanked at the 4-zone junction with a solvent other than essential oil. The cells were incubated overnight in an incubator at 37 ℃. And measuring the size of the inhibition zone by using a vernier caliper, and photographing and recording the result. The experiment was repeated three times and the average was calculated.

Example 3

Citrus oil, thyme oil, garlic oil and cinnamaldehyde are mixed according to the mass ratio of 1: 1: 1:2, mixing the glossy privet fruit, the cherokee rose fruit, the radish seed, the cardamom and the macleaya cordata extract according to the mass ratio of 1: 2: 2: 2.5: 1, mixing the components into a plant extract composition, wherein the plant essential oil composition and the plant extract composition are mixed according to the mass percentage of 1:1 the composition for preventing and treating the candida albicans is prepared. And sterile distilled water containing 0.5% (v/v) Tween-80 is added, the composition for preventing and treating candida albicans is prepared into a medicine with the concentration of 0.5% (v/v), and the medicine is uniformly oscillated by using a vortex oscillator. The experimental procedure was the same as in examples 1 and 2. Table 1 shows the results of the zone of inhibition measurements of indicator bacteria in example 1, example 2 and example 3, and comparative tests using plant essential oil alone, plant extract alone and nystatin in table 1 were performed.

TABLE 1 plant essential oil, plant extract and the results of the determination of zone of inhibition of indicator bacteria in examples 1, 2 and 3

As can be seen from table 1, the plant essential oils and plant extracts in table 1 have a certain degree of inhibitory effect on different isolated strains of candida albicans of pigeons, the compositions for preventing and treating candida albicans of examples 1, 2 and 3 have significantly improved bacteriostatic ability on different isolated strains of candida albicans of pigeons compared with the components of a single plant essential oil and plant extract, and the compositions of examples 1, 2 and 3 have a significant difference in bacteriostatic effect on different isolated strains of candida albicans of pigeons compared with nystatin, and the compositions of examples 1, 2 and 3 all have a superior bacteriostatic effect to nystatin.

Example 4

This example examined minimum inhibitory concentrations (MIC for short) of plant essential oils, plant extracts, plant essential oil compositions prepared in examples 1 and 2 and example 3 against three strains of candida albicans type A, B, C by the broth dilution method.

100ul of sterile water was added to each well of a 96-well plate, 2% (v/v) of each plant essential oil to be tested, plant extract, the composition for controlling candida albicans prepared in examples 1, 2 and 3 and nystatin were added to each initial well, 100ul of the mixture was sucked after being blown and mixed by a pipette, then added to the second well of each row, diluted sequentially to the last well in the same manner, and the excess 100ul of the last well was discarded. Then, 90ul of 2 XYPD liquid medium and 10ul of viable bacteria were added to each well to count 2X 10⁷CFU/ml bacterial solution. One sample to be tested is repeated three times, and each three columns are one sample to be tested. Column 10 is positive growth control, column 11 is tween-80 cosolvent control with the same concentration as the initial plant essential oil to observe whether the cosolvent tween-80 affects the growth of candida albicans. After incubation for 12h at 37 ℃, each well of the 96-well plate was observed for turbidity to determine whether Candida albicans was growing. Some essential oils have color, which makes it impossible to accurately judge whether Candida albicans has grown. Then, 100ul of each well was pipetted into each column and spread on YPD solid medium, and the culture was carried out at 37 ℃ for 12 hours, and the results were observed. The highest dilution concentration of this plant essential oil, plant extract and the composition for the prevention and treatment of Candida albicans prepared in examples 1, 2 and 3 on aseptically grown YPD solid plates was MI for three strains of Candida albicans type A, B, CC。

TABLE 2 MIC result table of minimum inhibitory concentration

As can be seen from table 2, the compositions for controlling candida albicans prepared in example 1, example 2 and example 3 increased the effect of inhibiting the isolation of candida albicans strain from pigeon relative to the single plant essential oil components and plant extracts. The effect of the single plant essential oil and the plant extract is not as good as that of the bacteriostatic agent of the composition for preventing and treating candida albicans prepared in example 1, example 2 and example 3, and the formulas of example 1, example 2 and example 3 have better bacteriostatic performance. Meanwhile, the formula of the examples 1, 2 and 3 has better effect on inhibiting the isolated candida albicans strain of the pigeons than nystatin, reduces the dosage required for preventing and treating the candida albicans, has higher safety compared with the nystatin and can generate better food calling.

Example 5

This example examines the effect of the composition for preventing and treating candida albicans disease on meat pigeon. 20 healthy meat pigeons of 35 days old are taken and randomly divided into 2 groups, 15 experimental groups and 5 control groups. The challenge strain is a candida albicans A type strain separated from the pigeon; selecting single colony on YPD plate of pigeon isolate strain with inoculating loop, inoculating into YPD liquid culture medium, and culturing at 37 deg.C for 18 hr; dipping a liquid culture suspension of a pigeon separated strain by using an aseptic inoculating loop, and microscopically examining cell morphology to determine that the thallus morphology is a yeast state; re-inoculating according to the inoculation amount of 2%, culturing at 37 ℃ for 18h, counting the cultured Candida albicans by a plate counting method, repeating three times for each dilution, and taking an average value after counting.

Inoculating at 2%, culturing at 37 deg.C for 18h, counting by plate, and diluting with sterilized YPD medium to 2 × 10⁷And (3) feeding the test pigeons with CFU/ml bacterial liquid, wherein the toxin counteracting dose of each pigeon is 200 ul. The control group of pigeons were fed with an equal amount of sterile water.

After inoculation, each group of pigeons are separated and fed conventionally in cages, and the breeding environment is consistent with the conditions; recording the mental state, ingestion and drinking of the pigeons every day; continuously attacking for 7 days, and after 7 days, dissecting and killing the pigeon to observe crop disease condition, and taking a picture and recording; the continuous killing and observation are carried out for 3 days. So as to verify the effectiveness of the animal challenge model.

Therapeutic agents were formulated as in examples 1, 2 and 3, and the pigeon 12 in the challenge group was divided into two groups of 6 animals each. The test groups were fed 200ul of therapeutic agent per pot. The other group served as a control group and was not fed with therapeutic agents. The pigeon crop disease is observed and photographed and recorded after the pigeon crop disease is observed.

The result of the challenge test shows that the control group has no clinical symptoms and grows normally; and the clinical symptoms of mental depression, dyspepsia, crop cannibalism and the like after the toxicity attack are shown in the test group. The diseased pigeon is dissected and killed, and the microbe is separated and identified to obtain the diseased pigeon suffering from the white candidiasis. The red arrow in the A, B, C three pictures in figure 1 indicates a pseudomembrana albicans which is identified and found to be candida albicans by separation, so the pigeon challenge test is successful.

The treatment shows that the control group can see clinical symptoms of candida albicans and still has a pseudowhite membrane; after a course of treatment, the meat pigeons are good in spirit and normal in eating and drinking water. The killing was performed one day apart to verify whether the candida albicans pseudomembrane was recovered. The result of killing the craw shows that the craw is clean and has no white pseudomembrane, and the craw is separated and identified by microorganisms to obtain the craw almost without the growth of candida albicans. The red arrows in fig. 2 indicate the distinctive features. Therefore, through the preliminary treatment test, the crop of the pigeon returns to be normal, and the growth of the pigeon is guaranteed.

The above examples are merely representative of a few embodiments of the present invention, and although the description is specific and detailed, the present invention should not be construed as limited the scope of the claims. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention.

Claims

1. The composition for preventing and treating the candidiasis comprises a plant essential oil composition and a plant extract, wherein the mass percentage of the plant essential oil composition to the plant extract is as follows: 1: 1;

the plant essential oil composition comprises the following components in percentage by mass: 15 to 20 percent of citrus oil, 20 to 30 percent of thyme oil, 15 to 20 percent of garlic oil and 35 to 40 percent of cinnamaldehyde;

the plant extract comprises the following components in percentage by mass: 10-15% of glossy privet fruit extract, 20-25% of cherokee rose fruit extract, 20-25% of radish seed extract, 25-30% of nutmeg extract and 10-20% of macleaya cordata extract.

2. The composition for preventing and treating candida albicans as claimed in claim 1, wherein the mass ratio of the citrus oil, the thyme oil, the garlic oil and the cinnamaldehyde is 1:1-1.5:1: 2.

3. The composition for preventing and treating candida albicans according to any one of claims 1-2, wherein the candida albicans is a pigeon originated candida albicans.

4. A drug for controlling candida albicans, comprising the composition for controlling candida albicans according to any one of claims 1-3.

5. A feed additive characterized by comprising the composition for controlling candida albicans as claimed in any one of claims 1 to 3.