Compound, composition and application of compound and composition in preparation of product for resisting canine microsporum

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 for resisting canine microsporum. The compound has a structure as shown in a formula I, II or III. The obtained composition comprises a combination of any two or three of a compound with a structure as shown in a formula I, a compound with a structure as shown in a formula II and a compound with a structure as shown in a formula III. Researches show that the compound or the composition disclosed by the invention has the effect of resisting canine microsporum; therefore, the compound serving as an active ingredient has important application value in preparing medicines, pet foods, sterilizing detergents or cat litter which have the effects of resisting microsporum canis and preventing and / or treating tinea of cats.

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 preparing products with anti-microsporum canis effect. BACKGROUND

[0002] Ringworm is a common pet skin disease, mainly caused by fungal infection such as microsporum canis. Microsporum canis is an animal-friendly fungus, which usually parasitizes on animals. When the resistance of cats decreases, the fungus will multiply in large quantities, causing ringworm. Ringworm is contagious and can be transmitted through direct or indirect contact, such as contact with combs, bedding, etc. of sick cats, and can also be transmitted through environmental pollutants. In a humid and warm environment, the fungus can survive for up to 18 months. The typical symptoms of ringworm include round or oval patches of hair loss on the skin, which are gray, dry and rough, and may be accompanied by thickening and hair loss, sometimes with erythema and scab, and the cat may feel itchy on the affected area, which may cause secondary bacterial infection by scratching, and in severe cases, the skin of the affected area may be red, swollen, and exude fluid.

[0003] Therefore, it is of important application value to develop a compound or composition with anti-microsporum canis effect for preventing and treating ringworm. SUMMARY

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

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

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

[0007]

[0008]

[0009] The present application further provides a composition comprising any two or three of the combination of the compound with the structure shown in formula I, the compound with the structure shown in formula II and the compound with the structure shown in formula III.

[0010]

[0011] Preferably, the composition comprises the compound with the structure shown in formula I, the compound with the structure shown in formula II and the compound with the structure shown in formula III.

[0012] Preferably, the mass ratio of the compound of formula I, the compound of formula II and the compound of formula III is 1: (1-150): (1-150).

[0013] Further preferably, the mass ratio of the compound of formula I, the compound of formula II and the compound of formula III is 1: (1-50): (1-50).

[0014] Most preferably, the mass ratio of the compound of formula I, the compound of formula II and the compound of formula III is 1:5.5:18.3.

[0015] The present application also provides use of the above-mentioned compound or composition in the preparation of a product having antibacterial effect.

[0016] Preferably, the antibacterial effect is specifically against Microsporum canis.

[0017] The present application also provides use of the above-mentioned compound or composition in the preparation of a product having the effect of preventing and / or treating cat ringworm.

[0018] Preferably, the effect of preventing and / or treating cat ringworm is specifically achieved by resisting Microsporum canis.

[0019] Preferably, the product is a medicine, pet food, disinfectant detergent or cat litter.

[0020] Beneficial effects: the present application provides a brand new compound and composition; research shows that the compound of formula I, the compound of formula II, the compound of formula III and their combination have the effect of resisting Microsporum canis. Especially, the combination of the compound of formula I, the compound of formula II and the compound of formula III can produce a synergistic effect of resisting Microsporum canis, which is much higher than that of a single compound of formula I, a single compound of formula II or a single compound of formula III; and the effect of resisting Microsporum canis is comparable to that of the positive control drug clotrimazole, which has very excellent effect of resisting Microsporum canis.

[0021] Since the compound or composition of the present application has the effect of resisting Microsporum canis, it has important application value to use it as an active ingredient to prepare a medicine, pet food, disinfectant detergent or cat litter having the effect of resisting Microsporum canis and the effect of preventing and / or treating cat ringworm. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 HR-ESI-MS spectrum of the compound of formula I.

[0023] Figure 2 DEPT NMR spectrum of the compound having the structure of Formula I. 1 H NMR spectrum.

[0024] Figure 3 DEPT NMR spectrum of the compound having the structure of Formula I.

[0025] Figure 4 DEPT NMR spectrum of the compound having the structure of Formula I. 13 C NMR spectrum.

[0026] Figure 5 HR-ESI-MS spectrum of the compound having the structure of Formula II.

[0027] Figure 6 H NMR spectrum of the compound having the structure of Formula II. 1 H NMR spectrum.

[0028] Figure 7 C NMR spectrum of the compound having the structure of Formula II. 13 C NMR spectrum.

[0029] Figure 8 DEPT-135 NMR spectrum of the compound having the structure of Formula II.

[0030] Figure 9 HR ESIMS spectrum of the compound having the structure of Formula III.

[0031] Figure 10 H NMR spectrum of the compound having the structure of Formula III. 1 H NMR spectrum.

[0032] Figure 11 C NMR spectrum of the compound having the structure of Formula III. 13 C NMR spectrum.

[0033] Figure 12 DEPT-135 NMR spectrum of the compound having the structure of Formula III. DETAILED DESCRIPTION

[0034] The present application is further explained in connection with the specific embodiments below, which do not limit the present application in any form.

[0035] Preparation of bacteriostatic active substance of pericarpium citri reticulatae

[0036] (1) 2 kg of pericarpium citri reticulatae was mixed with 3 L of ethanol aqueous solution (volume fraction of 30%) and extracted by ultrasonic for 30 min. The supernatant was collected after centrifugation for 10 min and freeze-dried to obtain 133.1 g of freeze-dried substance.

[0037] (2) Take 10 g of the freeze-dried product, dissolve it in 10 mL of water, and then load it onto a Sephadex G-15 column (specifications: 115*5 cm) for chromatography, followed by elution with a 30% ethanol aqueous solution; from the start of elution, collect every 1 / 3 column volume of eluate as one fraction; collect 9 fractions in succession; after each fraction is concentrated to remove ethanol and freeze-dried, obtain the active fraction of the column chromatography; and evaluate the in vitro anti-c Microsporum canis proliferation activity of the 9 active fraction fractions.

[0038] (3) The fraction with the best anti-c Microsporum canis proliferation activity (fraction-4, MIC 13.5 μg / mL) is separated by high-performance liquid chromatography to obtain the active substance of dried tangerine or orange peel that inhibits bacteria. The specific conditions of the high-performance liquid chromatography are as follows: a C18 column (10*100 mm, 5 μm, Waters, USA) is used as the chromatographic column; the mobile phase A is water, and the mobile phase B is methanol; the elution mode is gradient elution, wherein the gradient elution program is as follows: 0-10 min 35% B, 11-20 min 60% B; the injection volume is 100 μL; the detection wavelength is 230 nm; the flow rate is 3.0 mL / min; the column temperature is 18°C; and the eluate corresponding to the chromatographic peak with a retention time of 13.31 min is collected, concentrated, and freeze-dried to obtain the active substance of dried tangerine or orange peel that inhibits bacteria (i.e., the compound with the structure shown in Formula I).

[0039] The structure of the active substance of dried tangerine or orange peel that inhibits bacteria is as follows: colorless oil, HR-ESI-MS ( Figure 1 ) quasi-molecular ion peak m / z 611.3743 [M+Na] + (calcd for C 34 H 52 O8Na, 611.3707), indicating that the molecular formula is C 34 H 52 O8, the molecular weight is 588, and the unsaturation degree is 9. 1 H NMR (400 MHz, CDCl3, Figure 2 ) spectrum shows that the compound contains 7 methyl proton signals [δ H 1.74 (3H, s), 1.27 (3H, d, J = 4.3 Hz), 1.26 (3H, s), 1.15 (3H, s), 1.14 (3H, d, J = 4.8 Hz), 0.86 (3H, d, J = 4.5 Hz), 0.85 (3H, t, J = 5.3 Hz)], 2 olefinic hydrogen proton signals [δ H 7.54 (1H, s), 5.66 (1H, d, J = 5.3 Hz)], and 3 signals of protons on oxygen-containing carbons [δ H5.36 (IH, d, J = 7.5 Hz), 4.02 (IH, d, J = 9.9 Hz), 3.95 (IH, d, J = 9.9 Hz)]. DEPT-135 and 13 C NMR (100 MHz, CDC13, Figure 3 , Figure 4 ) spectrum showed that the compound had 34 carbon signals, including 9 quaternary carbons, 8 methine carbons, 10 methylene carbons and 7 methyl carbons. The chemical shifts were δ C 209.2, 176.9, 176.6 were the carbon signals of carbonyl groups, the chemical shifts were δ C 161.0, 140.8, 133.1, 129.5 were the carbon signals of double bond olefinic carbons, the chemical shifts were δ C 78.5, 76.9, 73.9, 68.2, 65.4 were the carbon signals of oxygenated carbons, the chemical shifts were δ C 24.1, 19.3, 19.1, 17.1, 14.6, 14.3, 10.3 were the carbon signals of methyl groups. Combined with the unsaturation, 1 HNMR data, the compound was deduced to be a diterpene with 4-ring nucleus and 2 ester groups. Among them, the chemical shifts were δ C 176.6, 34.5, 19.3, 19.1 were the carbon signals of isobutyryl group, the chemical shifts were δ C 176.9, 34.6, 32.1, 29.6, 29.5, 29.4, 29.3, 24.7, 22.9, 14.3 were the carbon signals of decanoyl group. Finally, the above pericarpium citri reticulatae antibacterial active substance was identified as a compound with the structure shown in formula I.

[0040] Preparation of clove antibacterial active substance

[0041] (1) 1.65 kg of clove powder was mixed with 2 L of ethanol aqueous solution (55% by volume) and ultrasonically extracted for 45 min. The supernatant was collected after centrifugation for 10 min and freeze-dried to obtain 98.4 g of freeze-dried material.

[0042] (2) 8.5 g of the freeze-dried material was dissolved in 10 mL of methanol and then loaded onto a silica gel column (300-400 mesh) for column chromatography (size: 85*7.3 cm), followed by elution with a mixture of ethyl acetate / methanol (7:3 by volume). From the beginning of elution, every 1 / 2 column volume of eluate was collected as a fraction. Four fractions were continuously collected. Each fraction was concentrated to remove the eluent and freeze-dried to obtain the column chromatography active fraction. The in vitro anti-c Microsporum canis proliferation activity of the four active fraction was evaluated.

[0043] (3) The best fraction (fraction-2, MIC 21.4 μg / mL) for inhibiting the proliferation of Microsporum canis was isolated by high performance liquid chromatography (HPLC), and the antibacterial active substance of clove was obtained. The HPLC conditions were as follows: a C18 column (10×100 mm, 5 μm, Waters, USA) was used; the mobile phase A was water and the mobile phase B was methanol; the elution was gradient elution, wherein the gradient elution program was as follows: 0-10 min 25% B, 11-20 min 50% B; the injection volume was 100 μL; the detection wavelength was 260 nm; the flow rate was 3.0 mL / min; and the column temperature was 18°C; the eluent corresponding to the chromatographic peak with a retention time of 10.01 min was collected, concentrated and lyophilized to obtain the antibacterial active substance of clove (i.e., a compound with the structure shown in formula II).

[0044] The structure of the antibacterial active substance of clove was analyzed as follows: a light yellow oil, high resolution mass spectrometry (HR-ESI-MS, Figure 5 ) showed a quasi-molecular ion peak m / z 357.1678 [M+Na] + (C 19 H 26 O5Na, the theoretical calculation value: 357.1672) determined the molecular formula as C 19 H 26 O5, and the unsaturation degree was 7. 1 H NMR (400 MHz, CDCl3, Figure 6 ) spectrum showed 24 proton signals. δ H 14.19 (1H, s) indicated that there was 1 hydroxyl hydrogen signal on a benzene ring; the compound also had 2 groups of olefinic proton signals δ H 6.52 (1H, overlapped), 6.64 (1H, dd, J = 16.2, 7.0 Hz) and δ H 6.51 (1H, overlapped), 5.62 (1H, d, J = 10.0 Hz); 6 methyl proton signals [δ H 3.77 (3H, s), 2.68 (3H, s), 1.48 (6H, s), 1.09 (6H, d, J = 6.5 Hz)], of which 2 were oxygen-containing methyl signals [δ H 3.77 (3H, s), 2.68 (3H, s)]. 13 C NMR (100 MHz, CDCl3, Figure 7 ) spectrum showed 19 carbon signals, and the DEPT-135 spectrum ( Figure 8 ) showed that they were 8 quaternary carbons, 5 methylenes and 6 methyls. In the low field region, there was 1 ketone carbonyl signal at δ C 203.6; δ C 142.6, 115.7 and δC 128, 116.9 are 2 pairs of double bond carbon signals; δ C 163.9, 157.9, 157.3, 110.7, 108.6, 107.1, are presumed to be benzene ring carbon signals. It is presumed that the compound is an acyl phloroglucinol compound. Further analysis of the chemical shift at C-1" position is an olefin carbon signal δ C 115.7, while the chemical shift at C-3" position is δ C 33.2. In combination with the above analysis, the compound is finally identified as a compound having the structure shown in Formula II.

[0045] Preparation method of cinnamomum cassia antibacterial active substance

[0046] (1) 1 kg of cinnamomum cassia crushed material was mixed with 2.5 L of ethanol aqueous solution (volume fraction 35%), ultrasonic extraction was performed for 40 min, the supernatant was collected after centrifugation for 10 min, and freeze-drying was performed to obtain 89.1 g of freeze-dried material.

[0047] (2) 12 g of the freeze-dried material was dissolved with 15 mL of water, and then column chromatography was performed on a macroporous resin (D101) column (specification: 60*10 cm), followed by elution with 35% ethanol aqueous solution; from the beginning of elution, the eluate of every 1 / 2 column volume was collected as one fraction; 8 fractions were continuously collected; after concentration to remove ethanol and water and freeze-drying, the column chromatography active fractions were obtained; the 8 active fraction fractions were subjected to in vitro anti-microsporum canis proliferation activity evaluation.

[0048] (3) The fraction with the best inhibition of microsporum canis proliferation activity (fraction-7, MIC 19.2 μg / mL) was separated by high performance liquid chromatography to obtain the cinnamomum cassia antibacterial active substance; 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: 0-10 min 22% B, 11-20 min 52% B; the injection amount was 100 μL; the detection wavelength was 310 nm; the flow rate was 3.0 mL / min; the column temperature was 18°C; the eluate corresponding to the chromatographic peak with a retention time of 9.55 min was collected, concentrated and freeze-dried to obtain the cinnamomum cassia antibacterial active substance (i.e. the compound having the structure shown in Formula III).

[0049] The structure analysis of the cinnamomum cassia antibacterial active substance is as follows: brownish red oil, the m / z of high resolution mass spectrometry (HR-ESI-MS, Figure 9 ) was 959.6231 [M+Na] + (calcd for C 56 H 88 NaO 11,959.6219), the molecular formula of the compound was determined as C 56 H 88 O 11 The unsaturation degree of the compound is 13. 1 H NMR (600 MHz, CDCl3, Figure 10 ) spectrum shows one low-field methine proton signal [δ H 5.09 (t, J = 3.5 Hz)], one methylene signal [δ H 3.17, 2.62 (each 1H, d, J = 19.7 Hz)], four singlet methyl signals [2.11, 1.98, 1.40, 1.34 (each 3H, s)], and seven overlapped methyl signals [δ H 1.18 (9H, overlapped); δ H 0.85 (12H, overlapped)]. 13 CNMR (150 MHz, CDCl3, Figure 11 ) spectrum shows 56 carbon signals, which, combined with its DEPT-135 spectrum ( Figure 12 ) analysis, can be assigned to 16 quaternary carbons, 7 methine carbons, 22 methylene carbons and 11 methyl carbons, including four carbonyl signals (δ C 192.0, 184.1, 183.8 x 2), one pair of double bond and one benzene ring signal (δ C 150.3, 147.2, 140.9, 136.0, 131.3, 116.2, 110.8, 109.3), three oxygen-bearing quaternary carbon signals (δ C 97.2, 75.3, 75.1), and one oxygen-bearing methine signal (δ C 63.7). Combined with the above analysis, the compound is finally identified as a compound having the structure shown in Formula III.

[0050] Preparation of bacteriostatic active material composition

[0051] The bacteriostatic active material of Pericarpium Citri Reticulatae prepared according to the methods of Examples 1, 2 and 3 is mixed with the bacteriostatic active material of Syzygium aromaticum and the bacteriostatic active material of Cinnamomum cassia in a mass ratio of 1:5.5:18.3 to obtain the bacteriostatic active material composition of the present application.

[0052] Experimental evaluation of bacteriostatic activity of bacteriostatic active material and its composition

[0053] The bacteriostatic active material in the present experimental example refers to the bacteriostatic active material prepared according to the methods described in Examples 1, 2 and 3.

[0054] The bacteriostatic active substance composition in the present experimental example refers to the bacteriostatic active substance composition (referred to as composition) prepared according to the method described in Example 4.

[0055] Minimum inhibitory concentration (MIC) determination of the bacteriostatic active substance or its composition:

[0056] Preparation of bacterial suspension: the Microsporum canis was inoculated in PDA culture medium, cultured at 28℃ for 14 days, and then prepared into bacterial suspension with sterile normal saline, adjusted to 4x10 6 CFU / mL.

[0057] Dissolution and dilution of the bacteriostatic active substance and its composition: the bacteriostatic active substance or its composition was respectively dissolved in an appropriate amount of water, and then a series of concentration gradient dilutions were carried out to obtain solutions of the bacteriostatic active substance or its composition with different concentrations.

[0058] Sample addition: different concentrations of the bacteriostatic active substance or its composition solution and an appropriate amount of bacterial suspension were added to the 96-well plate, 100 μL of the extract solution and 100 μL of the bacterial suspension were added to each well.

[0059] Culture and result observation: the 96-well plate was cultured at 27℃ for 7 days, the growth of bacteria in each well was observed, the well without the bacteriostatic active substance or its composition was used as a blank control, and the turbidity change of each well was recorded. The minimum concentration of the bacteriostatic active substance or its composition that can completely inhibit the growth of bacteria is the minimum inhibitory concentration (MIC).

[0060] Minimum fungicidal concentration (MFC) determination of the bacteriostatic active substance or its composition:

[0061] Dilution and coating: the culture after incubation of the bacteriostatic active substance or its composition with MIC concentration and higher than MIC concentration with Microsporum canis was appropriately diluted, an appropriate amount of the diluent was coated on the PDA culture medium plate, and multiple plates were coated for each concentration.

[0062] Culture and result observation: the plates were cultured at 27℃ for 7 days, and the growth of bacterial colonies was observed. The minimum concentration of the bacteriostatic active substance or its composition without bacterial colony growth is the MFC.

[0063] The experimental results are shown in Table 1. The MIC of the pericarpium citri reticulatae bacteriostatic active substance, the syzygium aromaticum bacteriostatic active substance, the cinnamomum cassia bacteriostatic active substance, and the combination thereof is 0.095 mg / mL, 0.112 mg / mL, 0.051 mg / mL, and 0.026 mg / mL, respectively, as compared with the positive control drug clotrimazole group (MIC is 0.021 mg / mL). The pericarpium citri reticulatae bacteriostatic active substance, the syzygium aromaticum bacteriostatic active substance, the cinnamomum cassia bacteriostatic active substance, and the combination thereof also have excellent bacteriostatic activity. In addition, as compared with the pericarpium citri reticulatae bacteriostatic active substance, the syzygium aromaticum bacteriostatic active substance, and the cinnamomum cassia bacteriostatic active substance, the combination thereof has more excellent bacteriostatic activity. It is speculated that the pericarpium citri reticulatae bacteriostatic active substance, the syzygium aromaticum bacteriostatic active substance, and the cinnamomum cassia bacteriostatic active substance have a synergistic effect on the anti-microsporum canis effect, and can better inhibit the proliferation of microsporum canis.

[0064] Table 1. MIC of pericarpium citri reticulatae bacteriostatic active substance, syzygium aromaticum bacteriostatic active substance, cinnamomum cassia bacteriostatic active substance, and combination thereof against microsporum canis proliferation

[0065]

[0066] Further analysis of the experimental results shows (Table 2) that the MFC of the pericarpium citri reticulatae bacteriostatic active substance, the syzygium aromaticum bacteriostatic active substance, the cinnamomum cassia bacteriostatic active substance, and the combination thereof is 0.113 mg / mL, 0.157 mg / mL, 0.131 mg / mL, and 0.042 mg / mL, respectively, as compared with the same positive control drug clotrimazole group, and the MFC thereof is 0.043 mg / mL. The pericarpium citri reticulatae bacteriostatic active substance, the syzygium aromaticum bacteriostatic active substance, the cinnamomum cassia bacteriostatic active substance, and the combination thereof also have excellent bactericidal activity. In addition, as compared with the pericarpium citri reticulatae bacteriostatic active substance, the syzygium aromaticum bacteriostatic active substance, and the cinnamomum cassia bacteriostatic active substance, the combination thereof has more excellent bactericidal activity. It is speculated that the pericarpium citri reticulatae bacteriostatic active substance, the syzygium aromaticum bacteriostatic active substance, and the cinnamomum cassia bacteriostatic active substance have a synergistic effect on the anti-microsporum canis effect, and can better kill microsporum canis.

[0067] Table 2. MFC of pericarpium citri reticulatae bacteriostatic active substance, syzygium aromaticum bacteriostatic active substance, cinnamomum cassia bacteriostatic active substance, and combination thereof against microsporum canis

[0068]

Claims

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

2. A composition, characterized in that A combination of any two or three of the compound represented by formula I, the compound represented by formula II, and the compound represented by formula III; 3. The composition according to claim 2, characterized in that The invention comprises a compound having a structure represented by formula I, a compound having a structure represented by formula II and a compound having a structure represented by formula III.

4. The composition according to claim 3, characterized in that The mass ratio of the compound represented by formula I, the compound represented by formula II and the compound represented by formula III is 1:(1-150):(1-150).

5. The composition according to claim 3, characterized in that The mass ratio of the compound represented by formula I, the compound represented by formula II and the compound represented by formula III is 1:(1-50):(1-50).

6. The composition according to claim 3, characterized in that The mass ratio of the compound represented by formula I, the compound represented by formula II and the compound represented by formula III is 1:5.5:18.

3.

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-Microsporum canis.

9. 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 the effect of preventing and / or treating ringworm in cats.

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

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