Compound, composition and application of compound and composition in preparation of product with anti-candida effect

Abstract

The invention relates to the technical field of biological medicines, and particularly discloses a compound, a composition and application of the compound and the composition in preparation of a product with an anti-candida effect. The compound has a structure as shown in a formula I or II; the composition comprises a compound with a structure as shown in a formula I, a compound with a structure as shown in a formula II and a dandelion extract. Researches show that the compound or the composition disclosed by the invention has an anti-candida effect; therefore, when being used as an active ingredient for preparing drugs, pet foods, sterilizing detergents or cat litter with an anti-candida effect, the compound has important application value.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a compound, a composition and application thereof in preparation of a product with anti-candida effect. BACKGROUND

[0002] Ringworm and candida infection are common fungal skin diseases of pet cats, which pose a great threat to the health of cats and cause trouble to pet owners. Ringworm is mainly caused by Microsporum canis and Trichophyton mentagrophytes, while candida infection is mainly caused by Candida albicans, both of which can cause redness, hair loss, itching and other symptoms on the skin of cats, and are contagious. At present, the treatment of ringworm and candida infection in clinic mainly relies on antifungal drugs. Itraconazole, for example, can inhibit the synthesis of ergosterol in the cell membrane of fungi, thereby playing an antifungal role, and is a first-line oral drug for treating ringworm; terbinafine can inhibit the synthesis of fungal cell wall and has good effect on ringworm and candida infection, and can be prepared into cream, spray and other external dosage forms. In addition, amphotericin B solution is also commonly used for the treatment of ringworm with large area of skin lesions, and has strong antibacterial effect on Candida albicans. However, long-term use of these chemical drugs may produce drug resistance, and some drugs may have certain impact on the liver function of cats.

[0003] Therefore, it is of important application value to develop a compound or composition with anti-candida effect. SUMMARY

[0004] In order to overcome at least one of the technical problems in the prior art, the present application first provides a compound, a composition and application thereof in preparation of a product with anti-candida effect.

[0005] The technical scheme of the present application is as follows:

[0006] The present application first provides a compound, wherein the compound has the structure shown in Formula I or II:

[0007]

[0008]

[0009] The present application further provides a composition comprising a compound having the structure shown in Formula I and a compound having the structure shown in Formula II.

[0010]

[0011] Preferably, the composition further comprises a dandelion extract.

[0012] Preferably, in the composition, the mass ratio of the dandelion extract to the compound represented by formula I and the compound represented by formula II is 100:(1-50):(1-50).

[0013] Preferably, in the composition, the mass ratio of the dandelion extract to the compound with the structure represented by formula I and the compound with the structure represented by formula II is 100:(1-10):(1-10).

[0014] Preferably, in the composition, the mass ratio of the dandelion extract to the compound represented by formula I and the compound represented by formula II is 100:2.02:1.67.

[0015] Preferably, in the composition, the dandelion extract is obtained by extraction with ethanol and separation with macroporous resin.

[0016] The present invention also provides an application of the compound or composition in preparing a product with antibacterial effect.

[0017] Preferably, the antibacterial agent is specifically anti-Candida.

[0018] Preferably, the product is medicine, pet food, disinfectant detergent or cat litter.

[0019] Beneficial Effects: The present invention provides novel compounds and compositions. Studies have shown that compounds of Formula I, Formula II, and compositions thereof with dandelion extract exhibit anti-Candida activity. In particular, the composition obtained by combining the compounds of Formula I, Formula II, and dandelion extract exhibits synergistic anti-Candida activity that is significantly greater than that of either the compound of Formula I or Formula II alone, demonstrating excellent anti-Microsporum canis activity.

[0020] Since the compound or composition of the present invention has an anti-Candida effect, using it as an active ingredient to prepare medicines, pet food, disinfectant detergents or cat litter with an anti-Candida effect has important application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 HR-ESI-MS spectrum of the compound represented by formula I.

[0022] Figure 2 The compound of the structure shown in formula I 1 H NMR spectrum.

[0023] Figure 3 The compound of the structure shown in formula I 13 C NMR spectrum.

[0024] Figure 4 DEPT-135 NMR spectrum of the compound shown as structure I.

[0025] Figure 5 HR ESIMS spectrum of the compound shown as structure II.

[0026] Figure 6 H NMR spectrum of the compound shown as structure II. 1 H NMR spectrum of the compound shown as structure II.

[0027] Figure 7 C NMR spectrum of the compound shown as structure II. 13 C NMR spectrum of the compound shown as structure II.

[0028] Figure 8 DEPT-135 NMR spectrum of the compound shown as structure II. DETAILED DESCRIPTION

[0029] The present application is further explained with reference to the specific examples below, which do not limit the application in any form.

[0030] Example 1 Preparation of dandelion extract

[0031] (1) 1 kg of dandelion powder was mixed with 1.5 L of ethanol aqueous solution (25% by volume fraction), and percolation extraction was performed for 24 h. The supernatant of the extract was collected and freeze-dried to obtain 83.5 g of dandelion ethanol extract.

[0032] (2) 30 g of the dandelion ethanol extract was dissolved in 18 mL of water, and then loaded onto a macroporous resin (HP-20 type) column (specification: 75*12 cm) for column chromatography. Then, 1 column volume of ethanol aqueous solution with a volume fraction of 0%, 1%, 5%, 15%, and 25% was used for elution in sequence. From the beginning of elution, 1 / 2 column volume of eluate was collected as one fraction. Ten fractions were collected in sequence. Each fraction was concentrated to remove ethanol and water, and then freeze-dried to obtain a column chromatography active part. The 7th fraction was concentrated and dried to obtain the dandelion extract.

[0033] Example 2 Preparation of compound

[0034] (1) 1 kg of dandelion powder was mixed with 1.5 L of ethanol aqueous solution (25% by volume fraction), and percolation extraction was performed for 24 h. The supernatant of the extract was collected and freeze-dried to obtain 83.5 g of dandelion ethanol extract.

[0035] (2) Taraxacum ethanol extract 30 g was dissolved in 18 mL of water and then loaded onto a macroporous resin (HP-20 type) column (specification: 75*12 cm) for column chromatography, followed by elution with 1 column volume of ethanol aqueous solution with a volume fraction of 0%, 1%, 5%, 15%, and 25%, respectively; from the start of elution, 1 / 2 column volume of eluent was collected as one fraction; 10 fractions were continuously collected; each fraction was concentrated to remove ethanol and water and then freeze-dried to obtain a column chromatography active part; the 10 active part fractions were subjected to Candida albicans proliferation inhibition activity evaluation.

[0036] (3) The active substance 8.5 g of the best fraction (fraction-7) with Candida albicans proliferation inhibition activity was dissolved in 5 mL of ethyl acetate and then loaded onto a silica gel column (200-300 mesh, 150 g) for column chromatography (specification: 80*6.9 cm), followed by elution and separation with petroleum ether / ethyl acetate mixed solution (volume ratio of 75:25, 50:50, and 15:85); from the start of elution, 1 / 2 column volume of eluent was collected as one fraction; 6 fractions (fractions-1 to 6) were continuously collected; each fraction was concentrated to remove the eluent and then freeze-dried to obtain a column chromatography active part; the 6 fractions were subjected to Candida albicans proliferation inhibition activity evaluation.

[0037] (4) The best fraction (fraction-5, 0.86 g) with Candida albicans proliferation inhibition activity was separated by high performance liquid chromatography to obtain compounds-1 to 6. The high performance liquid chromatography had the following specific conditions: a C18 column (10*100 mm, 5 μm, Waters, USA) was used as the chromatographic column; the mobile phase A was water and the mobile phase B was methanol; the elution mode was gradient elution, wherein the gradient elution program was 35% B for 0-10 min and 60% B for 11-21 min; the injection amount was 100 μL; the detection wavelength was 230 nm; the flow rate was 3.0 mL / min; the column temperature was 18°C; the eluent containing the six chromatographic peaks with the highest content was collected in sequence, and compounds-1 to 6 were obtained after concentration and freeze-drying. Compounds-1 to 6 were subjected to Candida albicans proliferation inhibition activity evaluation, and compounds-3 and-4 with excellent Candida albicans proliferation inhibition activity were identified for structure and named as Taraxacum bacteriostatic active substance-α and Taraxacum bacteriostatic active substance-β, respectively, in the present application.

[0038] The structure analysis of compound-3 is as follows: white powder, HR-ESI-MS (m / z 539.2640 [M+Na]) Figure 1 (m / z 539.2640 [M+Na]) + (calcd for C 29 H 40 O8Na, 539.2615), and the molecular formula was determined as C 29 H 40 O8, the molecular weight was 516, and the unsaturation degree was 10. 1H NMR (400 MHz, CDC13) Figure 2 ) spectrum showed that the compound had 8 methyl proton signals [δ H 1.81 (3H, s), 1.77 (3H, s), 1.74 (3H, s), 1.26 (3H, s), 1.19 (3H, s), 1.18 (3H, d, J = 6.1 Hz), 1.13 (3H, d, J = 7.5 Hz), 0.86 (3H, d, J = 6.6 Hz)], 2 olefinic hydrogen proton signals [δ H 7.57 (1H, s), 5.81 (1H, s)], 3 oxygen-bonded carbon proton signals [δ H 5.42 (1H, d, J = 10.5 Hz), 4.03 (1H, d, J = 12.9 Hz), 4.00 (1H, d, J = 12.9 Hz)]. 13 C NMR Figure 3 ) and DEPT-135 (100 MHz, CDC13) Figure 4 ) spectrum showed that the compound had 29 carbon signals, δ C 209.1, 179.5, 167.7 of the 3 carbon signals were carbonyl carbon signals, δ C 160.9, 140.4, 137.5, 132.8, 129.4, 128.5 of the 6 carbon signals were double bond olefinic carbon signals, δ C 78.3, 76.7, 73.7, 68.1, 65.4 of the 5 carbon signals were oxygen-bonded carbon signals, δ C 23.9, 18.6, 18.7, 16.9, 14.4, 14.4, 12.3, 10.1 of the 8 carbon signals were methyl carbon signals, combined with the unsaturation, 1 H NMR data, it was speculated that the compound was a diterpene with a 4-ring nucleus, and had 2 ester groups. Among them, δ C 167.7, 137.5, 128.5, 14.4, 12.3 of the 5 carbon signals were tiglyl group carbon signals, δ C 179.5, 34.2, 18.7, 18.6 of the 4 carbon signals were isobutyryl group carbon signals. The compound 1 H NMR, 13 C NMR data are shown in Table 1, and the compound was finally identified as a compound with the structure shown in Formula I, which is named as dandelion antibacterial active substance-α in the present application.

[0039] Table 1 1 H and 13 C NMR data (δ, 400 MHz, in CDC13)

[0040]

[0041]

[0042] The structural analysis of compound-4 is as follows: white powder, HR-ESI-MS ( Figure 5 )m / z 541.2794[M+Na] + (calcd for C 29 H 42 O8Na, 541.2772), its molecular formula is determined to be C 29 H 42 O8, molecular weight is 518, and unsaturation is 9. 1 HNMR (400 MHz, CDCl3) Figure 6 ) spectrum showed that the compound contained 8 methyl proton signals [δ H 1.74 (3H, s), 1.24 (3H, s), 1.19 (3H, s), 1.16 (3H, d, J = 6.0 Hz), 1.15 (3H, d, J = 6.8 Hz), 1.13 (3H, d, J = 6.2 Hz), 0.91 (3H, d, J = 7.6 Hz), 0.87 (3H, d, J = 6.4 Hz)], 2 olefinic proton signals [δ H 7.57 (1H, s), 5.75 (1H, s)], the proton signals on the three oxygen-linked carbons [δ H 5.40(1H,d,J=10.4Hz), 4.03(1H,d,J=13.2Hz), 3.96(1H,d,J=13.2Hz)]. 13 C NMR ( Figure 7 ) and DEPT-135 (100MHz, CDCl3) ( Figure 8 ) spectrum showed that the compound had 29 carbon signals, among which δ C The three carbon signals at 210.4, 180.8, and 177.5 are carbonyl carbon signals, and δ C The four carbon signals of 162.3 141.9, 134.4, and 130.9 are double bond olefin carbon signals, δ C The five carbon signals of 79.8, 77.6, 75.1, 69.4, and 66.7 are oxygen-linked carbon signals. C The eight carbon signals of 25.3, 18.8, 18.5, 18.3, 15.8, 13.0, 11.5, and 10.9 are methyl carbon signals. 1 H NMR data suggest that the compound is a diterpene with a 4-ring nucleus and 2 ester groups. CThe five carbon signals at 177.5, 44.4, 28.2, 18.8, 11.5 are the carbon signals of (2-methyl)butyryl group. δ C The four carbon signals at 180.8, 35.7, 18.5, 18.3 are the carbon signals of isobutyryl group. The compound 1 H NMR, 13 C NMR data are shown in Table 2. In combination with the above analysis, the compound is finally identified as a compound having the structure shown in Formula II, which is named as Taraxacum officinale bacteriostatic active substance-β in the present application.

[0043] Table 2 1 H and 13 C NMR data (δ, 400 MHz, in CDCl3)

[0044]

[0045]

[0046] Preparation of a composition having Candida proliferation inhibiting activity

[0047] The Taraxacum officinale extract prepared according to the method of Example 1 is mixed with the Taraxacum officinale bacteriostatic active substance-α and the Taraxacum officinale bacteriostatic active substance-β prepared according to Example 2 at a mass ratio of 100:2.02:1.67 to obtain a composition having Candida proliferation inhibiting activity.

[0048] Evaluation of the bacteriostatic activity of a composition having Candida proliferation inhibiting activity

[0049] The Taraxacum officinale extract and the Taraxacum officinale bacteriostatic active substance-α and the Taraxacum officinale bacteriostatic active substance-β in this experimental example refer to those prepared according to the methods described in Examples 1 and 2, respectively.

[0050] The composition having Candida proliferation inhibiting activity in this experimental example (hereinafter referred to as the composition) refers to that prepared according to the method described in Example 3.

[0051] Determination of the minimum concentration (MIC) of the Taraxacum officinale extract, the Taraxacum officinale bacteriostatic active substance-α, the Taraxacum officinale bacteriostatic active substance-β or the composition thereof for inhibiting the proliferation of Candida (C. albicans ATCC 10231):

[0052] ①, 96-well plate preparation: under sterile conditions, different concentrations of the Taraxacum officinale extract, the Taraxacum officinale bacteriostatic active substance-α, the Taraxacum officinale bacteriostatic active substance-β or the dilution solution of the composition thereof are added to the corresponding wells of a 96-well plate, 100 μL of the dilution solution is added to each well.

[0053] ②, Candida albicans inoculation: Candida albicans culture was diluted with sterile saline to 0.5x10 6 CFU / mL, and then 100 μL of the bacterial solution was added to each well.

[0054] ③, culture: the 96-well plate was placed in a 35℃ incubator for 24 hours.

[0055] ④, determine MIC: after the end of the culture, the turbidity of each well was observed with a microplate reader. The minimum natural product concentration that completely inhibited the growth of Candida albicans was the MIC value.

[0056] Minimum fungicidal concentration (MFC) determination of dandelion extract and dandelion antibacterial active substance-α, dandelion antibacterial active substance-β or their combination:

[0057] ①, dilution and coating: a certain amount of culture (10 μL) was inoculated from the test solution at MIC concentration and higher concentrations into solid SDA medium, and multiple plates were inoculated at each concentration. Then incubate at 35℃ for 5 days, and observe the growth of colonies. The minimum concentration that can kill 99.9% of Candida albicans is the MFC value

[0058] Positive and negative controls were performed simultaneously in each experiment. The positive control was selected as an antifungal drug (amphotericin B), and the negative control was a dandelion extract culture system without Candida albicans, to ensure the reliability of the experimental results.

[0059] The experimental results are shown in Table 3. The MIC of dandelion extract, dandelion antibacterial active substance-α, dandelion antibacterial active substance-β and their combination was 69.7 μg / mL, 32.2 μg / mL, 28.3 μg / mL and 14.9 μg / mL, respectively, compared with the positive control drug amphotericin B group (MIC: 2.55 μg / mL). The dandelion extract, dandelion antibacterial active substance-α, dandelion antibacterial active substance-β and their combination provided by the present application have excellent Candida albicans proliferation inhibition activity. Compared with dandelion extract, dandelion antibacterial active substance-α and dandelion antibacterial active substance-β have more excellent antibacterial activity. Similarly, compared with dandelion extract, dandelion antibacterial active substance-α and dandelion antibacterial active substance-β, the combination has more excellent Candida albicans proliferation inhibition activity. We speculate that dandelion extract, dandelion antibacterial active substance-α and dandelion antibacterial active substance-β have a synergistic effect, and when combined together, they can better inhibit the proliferation of Candida albicans.

[0060] Table 3 Candida albicans proliferation inhibition activity (MIC) of dandelion extract, dandelion antibacterial active substance-α, dandelion antibacterial active substance-β and their combination

[0061]

[0062] Further experimental results show (Table 4) that the MFCs of the dandelion extract, dandelion antibacterial active substance-α, dandelion antibacterial active substance-β and the combination thereof are 85.9 μg / mL, 46.9 μg / mL, 39.4 μg / mL and 19.6 μg / mL respectively; compared with the positive control drug amphotericin B (MFC: 3.47 μg / mL), the dandelion extract, dandelion antibacterial active substance-α, dandelion antibacterial active substance-β and the combination thereof also have excellent bactericidal activity. Compared with the dandelion extract, the dandelion antibacterial active substance-α and the dandelion antibacterial active substance-β have more excellent bactericidal activity. Similarly, compared with the dandelion extract, the dandelion antibacterial active substance-α and the dandelion antibacterial active substance-β, the combination thereof has more excellent Candida proliferation killing activity. We speculate that the dandelion extract, dandelion antibacterial active substance-α and dandelion antibacterial active substance-β have a synergistic effect, and when combined together, they can better kill Candida.

[0063] Table 4 Candida killing activity (MFC) of the dandelion extract, dandelion antibacterial active substance-α, dandelion antibacterial active substance-β and the combination thereof

[0064]

Claims

1. A compound, characterized in that The compound has the structure shown in Formula I or II:

2. A composition, characterized in that A compound comprising a structure represented by formula I and a compound comprising a structure represented by formula II; 3. The composition according to claim 2, characterized in that Also contains dandelion extract.

4. The composition according to claim 3, characterized in that In the composition, the mass ratio of the dandelion extract to the compound with the structure represented by formula I and the compound with the structure represented by formula II is 100:(1-50):(1-50).

5. The composition according to claim 4, characterized in that In the composition, the mass ratio of the dandelion extract to the compound with the structure represented by formula I and the compound with the structure represented by formula II is 100:(1-10):(1-10).

6. The composition according to claim 4, characterized in that In the composition, the mass ratio of the dandelion extract to the compound with the structure represented by formula I and the compound with the structure represented by formula II is 100:2.02:1.

67.

7. Use of the compound according to claim 1 or the composition according to any one of claims 2 to 6 in the preparation of a product having an antibacterial effect.

8. The use according to claim 7, characterized in that The antibacterial agent is specifically anti-Candida.

9. The use according to any one of claims 7 or 8, characterized in that The product is medicine, pet food, disinfectant detergent or cat litter.

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