Preparation method of antibacterial compound, composition containing taxus chinensis var mairei and application

Abstract

The invention belongs to the technical field of daily necessities and biological medicines, and particularly relates to a preparation method of an antibacterial compound, a composition containing taxus chinensis var mairei and application. According to the present invention, the yield of the antibacterial compound is improved by 5.6 times or more by using the co-culture technology of the Amylocatopsis sp. 26-4 and the T. pulmonis TP-B0596, and the compound has broad-spectrum antibacterial activity, and can be used as the preservative in chemical daily necessities.

Description

Technical Field

[0001] This invention belongs to the fields of daily necessities and biomedicine, specifically relating to a method for preparing an antibacterial compound and a composition containing Taxus wallichiana and its application. Background Technology

[0002] Cosmetics are a widely used consumer product in people's daily lives. With social progress and increased public health awareness, consumers have placed higher demands on the safety of cosmetics, and relevant regulatory standards are becoming increasingly stringent. According to the "Cosmetic Safety Technical Specifications" (2015 edition), cosmetics must undergo microbial limit testing, mainly including five indicators: total bacterial count, thermotolerant coliforms, Staphylococcus aureus, Pseudomonas aeruginosa, and molds and yeasts. Among them, the total bacterial count limit for eye, lip, and children's cosmetics is ≤500 CFU / g (ml), and for other types of products it is ≤1000 CFU / g (ml), and all pathogenic bacteria must not be detected.

[0003] In recent years, the southern yew has gradually attracted attention in the daily chemical industry due to its various bioactive components, and has been applied in skin care and hair care products. For example, patent CN109200057A discloses the application potential of polysaccharides extracted from yew fruit in whitening and anti-aging; patent CN103417430A reports the application value of a hair care shampoo containing yew extract in promoting microcirculation in the dermis and hair follicles of the scalp.

[0004] However, because cosmetics are rich in moisture and nutrients, they are susceptible to microbial contamination. Preservatives are typically added to ensure product safety within its shelf life. While traditional preservatives are effective at inhibiting bacteria, some ingredients have raised concerns about potential irritation or safety issues, prompting the industry to seek safer and more efficient solutions.

[0005] Therefore, developing a novel preservative system with broad-spectrum antibacterial activity and good safety is of great significance in the fields of cosmetics and medical technology. Summary of the Invention

[0006] To overcome the shortcomings of existing technologies, this invention provides a composition containing Taxus chinensis extract, a preparation method, and its applications. This invention, through co-culturing Amycolatopsis sp. 26-4 and T. pulmonis TP-B0596, increases the yield of antibacterial compounds by more than 5.6 times, and the compound exhibits broad-spectrum antibacterial activity, making it suitable for use as a preservative in cosmetics.

[0007] The present invention solves the above-mentioned technical problems through the following technical solutions.

[0008] This invention provides a method for preparing an antibacterial compound, which is obtained through the following steps: S1, Amycolatopsis sp. 26-4 was cultured in seed medium to obtain seed solution 1; S2, T. pulmonis TP-B0596 was cultured in seed culture medium to obtain seed solution 2; S3, simultaneously inoculate the seed liquid 1 and the seed liquid 2 into the fermentation medium for fermentation to obtain fermentation liquid; S4, the fermentation broth is filtered, extracted with organic solvents, concentrated, and purified to obtain the antibacterial compound; wherein, the antibacterial compound is... .

[0009] In this invention, in S1 and S2, the culture temperature is 25-35℃ (e.g., 28-30℃, or 28℃ or 30℃), and the culture time is 1-10 days (e.g., 4 days).

[0010] In this invention, during the cultivation process in S1 and S2, the stirring speed is 50-300 rpm (e.g., 180 rpm).

[0011] In this invention, in S1 and S2, the seed culture medium contains glucose, malt extract and yeast extract.

[0012] In this invention, the concentration of glucose in the seed culture medium is 2.0-8.0 g / L (e.g., 4.0 g / L); the concentration of malt extract is 5.0-15.0 g / L (e.g., 10.0 g / L); and the concentration of yeast extract is 2.0-8.0 g / L (e.g., 4.0 g / L).

[0013] In this invention, in step S3, the fermentation temperature is 20-40℃ (e.g., 28℃); the fermentation time is 1-10 days (e.g., 5 days).

[0014] In this invention, during the fermentation process in S3, the stirring speed of the fermentation medium is 50-300 rpm (e.g., 180 rpm).

[0015] In this invention, in step S3, the fermentation culture medium comprises oat flakes, FeSO4·7H2O, MnCl2·4H2O, and ZnSO4·7H2O.

[0016] In this invention, the concentration of oat flakes in the fermentation medium is 10.0-30.0 g / L (e.g., 20.0 g / L), the concentration of FeSO4·7H2O is 0.5-3.0 mg / L (e.g., 1.0 mg / L), the concentration of MnCl2·4H2O is 0.5-3.0 mg / L (e.g., 1.0 mg / L), and the concentration of ZnSO4·7H2O is 0.5-3.0 mg / L (e.g., 1.0 mg / L).

[0017] This invention provides the application of a compound in cosmetics, wherein the compound is... .

[0018] In this invention, the daily necessities are cosmetics; preferably hair cosmetics; more preferably shampoos.

[0019] This invention also provides the use of a compound in the preparation of antibacterial drugs, wherein the compound is... .

[0020] In this invention, the bacteria are one or more of the following: thermotolerant Escherichia coli, Staphylococcus aureus, Bacillus subtilis, and Pseudomonas aeruginosa.

[0021] This invention also provides a composition containing *Taxus wallichiana*, comprising plant extracts, surfactants, fragrances, thickeners, chelating agents, pH adjusters, colorants, and preservatives; wherein the plant extracts comprise *Taxus wallichiana* branch and leaf extracts; and the preservatives are... .

[0022] In this invention, the plant extracts include one or more of the following: Taxus chinensis branch and leaf extract, Dictamnus dasycarpus root bark extract, Astragalus membranaceus raw extract, Saposhnikovia divaricata extract, ginseng extract, Angelica sinensis extract, and processed Polygonum multiflorum extract.

[0023] In this invention, the plant extract is composed of extracts of Taxus wallichiana branches and leaves, Dictamnus dasycarpus root bark, Astragalus membranaceus, Saposhnikovia divaricata, ginseng, Angelica sinensis, and Polygonum multiflorum.

[0024] In this invention, the plant extract is prepared by the following method: 10-40 parts by weight of Taxus chinensis branches and leaves, 8-16 parts by weight of Dictamnus dasycarpus root bark, 5-15 parts by weight of Astragalus membranaceus extract, 5-15 parts by weight of Saposhnikovia divaricata, 3-12 parts by weight of ginseng, 3-12 parts by weight of Angelica sinensis, and 3-12 parts by weight of processed Polygonum multiflorum are decocted in a solvent; preferably, the solvent is water or ethanol.

[0025] In this invention, the surfactant is one or more of sodium lauryl ether sulfate, cocamide DEA, ammonium lauryl sulfate, cocamidopropyl betaine, and hydroxypropyl guar hydroxypropyltrimethylammonium chloride.

[0026] In this invention, the thickener is PEG-250 distearate.

[0027] In this invention, the chelating agent is described.

[0028] In this invention, the pH adjuster is disodium citrate.

[0029] The reagents and raw materials used in this invention are all commercially available.

[0030] Compared with the prior art, the beneficial effects of the present invention are: the present invention increases the yield of antibacterial compounds by more than 5.6 times through the co-culture technology of Amycolatopsis sp. 26-4 and T. pulmonis TP-B0596, and provides strong support for further analysis of the biological activity and other properties of the compounds.

[0031] This invention discovers that cyclic lipopeptide compounds have excellent broad-spectrum antibacterial activity, exhibiting excellent inhibitory effects against Escherichia coli, Staphylococcus aureus, Bacillus subtilis, and Pseudomonas aeruginosa, especially against Pseudomonas aeruginosa. Attached Figure Description

[0032] Figure 1 The image shows the 1H NMR spectrum of compound 1 in Example 1.

[0033] Figure 2 The image shows the carbon NMR spectrum of compound 1 in Example 1.

[0034] Figure 3 The image shows the COSY NMR spectrum of compound 1 in Example 1.

[0035] Figure 4 The image shows the HSQC NMR spectrum of compound 1 in Example 1.

[0036] Figure 5 The image shows the HMBC NMR spectrum of compound 1 in Example 1.

[0037] Figure 6 This is a high-resolution mass spectrum of compound 1 in Example 1. Detailed Implementation

[0038] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0039] The Amycolatopsis sp. 26-4 and T. pulmonis TP-B0596 used in this invention were cultured and isolated using the methods described in the literature (Pan C, Kuranaga T, Cao X, et al. Amycolapeptins A and B, cyclic nonadepsipeptides produced by combined-culture of Amycolatopsis sp. and Tsukamurella pulmonis. The Journal of Organic Chemistry, 2021, 86(2): 1843-1849; Pan C, Kuranaga T, Liu C, et al. Thioamycolamides A–E, sulfur-containing cyclic lipopeptides produced by the rare Actinomycete Amycolatopsissp. Organic letters, 2020, 22(8): 3014-3017.).

[0040] Example 1: Amycolatopsis sp. 26-4 fermented alone Amycolatopsis sp. 26-4 was cultured for 4 days in a 500 mL Erlenmeyer flask containing 200 mL of seed culture medium (ISP2: glucose 4.0 g / L, malt extract 10.0 g / L, yeast extract 4.0 g / L) at 28 °C and 180 rpm. Then, 1 mL of each seed culture was inoculated into a 1 L Erlenmeyer flask containing 400 mL of fermentation medium (ISP3: oat flakes 20.0 g / L, FeSO4·7H2O 1.0 mg / L, MnCl2·4H2O 1.0 mg / L, ZnSO4·7H2O 1.0 mg / L) and cultured at 28 °C and 180 rpm with shaking for 5 days.

[0041] Extraction and Separation: After filtration of 10 L of fermentation broth, the extract was extracted with EtOAc (2 × 10 L) and concentrated under reduced pressure to obtain 0.73 g of crude extract. The crude extract was then subjected to silica gel column chromatography with a gradient elution of n-hexane / EtOAc (5%, 10%, 20%, 40%, 100% EtOAc) and 100% MeOH to obtain 8 fractions. Each fraction was dissolved in methanol and centrifuged at 8000 rpm for 5 min. The fourth fraction was purified by semi-preparative HPLC (Cosmosil ODS SP100, Φ10 × 250 mm, MeCN:H2O = 40:60, 2 mL / min) to obtain compound 1 (1, 0.12 mg, tR 22.0 min).

[0042] Example 2: T. pulmonis TP-B0596 fermented alone T. pulmonis TP-B0596 was cultured for 4 days in a 500 mL Erlenmeyer flask containing 200 mL of seed culture medium (ISP2: glucose 4.0 g / L, malt extract 10.0 g / L, yeast extract 4.0 g / L) at 28℃ and 180 rpm. Then, 1 mL of each seed culture was inoculated into a 1 L Erlenmeyer flask containing 400 mL of fermentation medium (ISP3: oat flakes 20.0 g / L, FeSO4·7H2O 1.0 mg / L, MnCl2·4H2O 1.0 mg / L, ZnSO4·7H2O 1.0 mg / L) and cultured at 28℃ and 180 rpm with shaking for 5 days.

[0043] Extraction and separation: After filtration of 10 L of fermentation broth, it was extracted with EtOAc (2×10 L), concentrated under reduced pressure to obtain an extract, and analyzed by gradient elution analysis by high performance liquid chromatography (Cosmosil ODS SP100, Φ4.6×250 mm, 20%-100% MeCN 30 min, 1 mL / min). It was found that the extract did not contain compound 1.

[0044] Example 3: Amycolatopsis sp. 26-4 and T. pulmonis TP-B0596 co-culture S1: Amycolatopsis sp. 26-4 was cultured for 4 days at 28°C and 180 rpm in a 500 mL Erlenmeyer flask containing 200 mL of seed culture medium (ISP2: glucose 4.0 g / L, malt extract 10.0 g / L, yeast extract 4.0 g / L).

[0045] S2: T. pulmonis TP-B0596 was cultured for 4 days in a 500 mL Erlenmeyer flask containing 200 mL of seed culture medium (ISP2: glucose 4.0 g / L, malt extract 10.0 g / L, yeast extract 4.0 g / L) at 28℃ and 180 rpm.

[0046] S3: Inoculate 1 mL of each seed culture into a 1 L Erlenmeyer flask containing 400 mL of fermentation medium (ISP3: 20.0 g / L oat flakes, 1.0 mg / L FeSO4·7H2O, 1.0 mg / L MnCl2·4H2O, 1.0 mg / L ZnSO4·7H2O) and culture at 28℃ and 180 rpm for 5 days.

[0047] S4: Extraction and Separation: After filtration of 10 L of fermentation broth, the extract was extracted with EtOAc (2 × 10 L) and concentrated under reduced pressure to obtain 0.73 g of crude extract. The crude extract was then subjected to silica gel column chromatography with a gradient elution of n-hexane / EtOAc (5%, 10%, 20%, 40%, 100% EtOAc) and 100% MeOH to obtain 8 fractions. Each fraction was dissolved in methanol and centrifuged at 8000 rpm for 5 min. The fourth fraction was purified by semi-preparative HPLC (Cosmosil ODS SP100, Φ10 × 250 mm, MeCN:H2O = 40:60, 2 mL / min) to obtain compound 1 (1, 0.68 mg, tR 22.0 min).

[0048] Example 4: Following the method of Example 3, the only difference was the culture medium formulation. Both the seed culture medium and the fermentation culture medium used were ISP2 medium (glucose 4.0 g / L, malt extract 10.0 g / L, yeast extract 4.0 g / L). The extract was obtained by the same extraction method and analyzed by gradient elution analysis using high performance liquid chromatography (Cosmosil ODS SP100, Φ4.6×250 mm, 20%-100% MeCN 30 min, 1 mL / min). Compound 1 (0.46 mg, tR 22.0 min) was found.

[0049] Example 5: Following the method of Example 3, the only difference was the culture temperature. The seed culture and fermentation culture temperatures were adjusted to 20°C, 30°C, and 40°C, respectively. Extracts were obtained using the same extraction method. High-performance liquid chromatography gradient elution analysis (Cosmosil ODS SP100, Φ4.6×250 mm, 20%-100% MeCN 30 min, 1 mL / min) revealed that the yield of compound 1 was basically the same at 30°C and 28°C. However, at 20°C and 40°C, the growth of the strain was limited, and compound 1 was not detected.

[0050] Compound 1 prepared in Examples 1, 3, 4, and 5 was combined and its structure was characterized. The structural information of compound 1 is as follows: White powder; [α] 2 D –94.4 (c 0.06, CH3OH); UV / vis (CH3OH) λmax (log ε) 205 (4.61), 252 (3.77) nm; IR vmax 3296, 2971, 2928, 2855, 1653, 1544, 1014 cm -1 ;CD (c1.04 × 10 -3 mol / L, MeOH) λmax (Δε): 222 (12.9), 254 (–10.8) nm; 1 H NMR (such as) Figure 1 (as shown) and 13 C NMR (such as Figure 2 The data is shown in Table 1. .

[0051] HRMS (ESI) C 23 H 34 N3O4S2 + [M + H] + Calculated value: 480.1985, measured value: 480.1976; HRMS experimental results are as follows: Figure 6 As shown.

[0052] Based on the above experimental results, the structural formula of compound 1 is derived as follows: .

[0053] The specific analysis is as follows: Compound 1 is a white powder, and its molecular formula (C10) is given by HRMS. 23 H 34 N3O4S2 + (m / z480.1976 [M + H]) +, calculated 480.1985); through 1D and 2D NMR (COSY (e.g. Figure 3 As shown), HSQC (such as) Figure 4 As shown), HMBC (as shown) Figure 5 As shown) Analytical planar structure: 1 ¹H NMR shows one methyl group, eight methylene groups, five methine groups, five aromatic protons, two amino groups, and two hydroxyl groups; COSY correlations of H13 / H12 / H11 indicate the presence of a 1-substituted benzene ring; HMBC correlations of H9 with C10 and C11 confirm the C10–C9 linkage; COSY and HMBC correlations of H9 / H8 / 8-NH indicate that this fragment is a phenylalanine unit; HMBC correlations of 8-NH with C16 position the carboxyl group adjacent to C16; the aliphatic chain portion is established by COSY and HMBC correlations of H17–H21; C1 is linked to C18 via a heteroatom. The linkages of C1–C2–C3–3-OH and 2-NH–C4 are confirmed by both COSY and HMBC correlations; the C5–C6–C7 segment forms a thiazoline ring. Comparison with previously reported NMR data for thioamycolamide D showed that the two compounds were essentially identical, differing only in the presence of an additional hydroxyl group at position 22. Furthermore, compound 1 was isolated from the same strain as thioamycolamide D, and its main skeleton NMR data were almost identical, suggesting that they may originate from similar biosynthetic pathways. The remaining spectroscopic features of compound 1 were highly consistent with those of thioamycolamide D, indicating that the absolute configuration of its chiral center was consistent with this series.

[0054] Therefore, the planar structure of compound 1 is confirmed. .

[0055] Results and Discussion: This study analyzed the latent secondary metabolites of *Amycolatopsis* sp. 26-4 using a co-culture technique. This technique utilizes algorithms to compare the MS / MS similarity of different precursor molecules, clustering structurally similar compounds into "molecular clusters" to visualize the component relationships in complex mixtures. For the EtOAc extract co-cultured with *Amycolatopsis* sp. 26-4 and *T. pulmonis* TP-B0596, a series of thioamycolamide derivatives were discovered, with molecular ion m / z values ​​of 452.16, 466.36, and 480.20, respectively. Compound 1 was obtained through scaled-up fermentation and mass spectrometry-guided separation.

[0056] Example 6: Evaluation of the antibacterial activity of compound 1: The conventional broth dilution method was used, and the experimental steps are as follows: 1) Using sterile broth medium, the test strain was diluted to 10⁻¹⁰ using a turbidimetric method. 6 CFU / mL available; 2) Using a prepared bacterial broth medium, compound 1 was serially diluted according to a concentration gradient, specifically starting from 256 μg / mL, to obtain bacterial cultures of compound 1 at concentrations of 256 μg / mL, 128 μg / mL, 64 μg / mL, 32 μg / mL, 16 μg / mL, 8 μg / mL, 4 μg / mL, 2 μg / mL, 1 μg / mL, 0.5 μg / mL, 0.25 μg / mL, and 0.125 μg / mL. Tetracycline was set as a positive control group, and methanol as a negative control group. 3) Inoculate 0.2 mL into each well of a 96-well plate and incubate at 30°C for 24 hours; 4) Judgment criteria: sterile is clear, and bacterial is turbid. The minimum drug concentration that completely inhibits the growth of the bacterial strain, as observed by the naked eye, is the MIC value.

[0057] Based on the general methods described above, *Escherichia coli* [CMCC (B) 44102], *Staphylococcus aureus* [CMCC (B) 26003], *Bacillus subtilis* [CMCC (B) 63501], and *Pseudomonas aeruginosa* [CMCC(B) 10104] were tested, with tetracycline as a positive control. The experimental results are shown in Table 2. .

[0058] As shown in Table 2, the MICs of compound 1 against the three bacteria were 64, 32, 32, and 0.5 μg / mL, respectively. In particular, it exhibited significantly superior antibacterial activity against *Pseudomonas aeruginosa* compared to tetracycline. This indicates that compound 1 possesses broad-spectrum antibacterial activity.

[0059] Example 7: The formula for hair-nourishing shampoo 1 is shown in Table 3. ; The raw materials used in the plant extracts are shown in Table 4. .

[0060] Weigh all components according to the proportions in Table 4, soak them in deionized water for 30 minutes, drain the water, and then add them to the extraction tank at a weight ratio of 1:5 for the traditional Chinese medicine and ethanol solution. Heat and reflux for 1.5 hours.

[0061] The formula of hair shampoo 2 is the same as that of hair shampoo 1 and its preparation method. The only difference is that no preservative compound 1 is added.

[0062] Evaluation of the anti-corrosion (antibacterial) properties of compound 1 in hair shampoo.

[0063] Pseudomonas aeruginosa suspension [CMCC(B) 10104] was inoculated into culture dishes using hair care shampoos 1 and 2 respectively. After 72 hours, it was observed that hair care shampoos 1 did not deteriorate, while hair care shampoos 2 showed obvious deterioration.

[0064] Experimental results show that the compounds provided by this invention can be used in cosmetics as preservatives or antibacterial agents.

Claims

1. The application of a compound as a preservative in daily consumer goods, wherein, The compound is .

2. The application as described in claim 1, wherein the daily necessities are cosmetics.

3. The use of a compound in the preparation of an antibacterial drug, wherein, The compound is .

4. A method for preparing an antibacterial compound, characterized in that, It is prepared through the following steps: S1, Amycolatopsis sp. 26-4 was cultured in seed medium to obtain seed solution 1; S2, T. pulmonis TP-B0596 was cultured in seed culture medium to obtain seed solution 2; S3, simultaneously inoculate the seed liquid 1 and the seed liquid 2 into the fermentation medium for fermentation to obtain fermentation liquid; S4, the fermentation broth is filtered, extracted with organic solvents, concentrated, and purified to obtain an antibacterial compound; wherein, the antibacterial compound is... .

5. A composition containing *Taxus wallichiana*, comprising plant extracts, surfactants, fragrances, thickeners, chelating agents, pH adjusters, colorants, and preservatives; wherein, The plant extract includes extracts from the branches and leaves of the southern yew tree; the preservative is... .

6. The composition containing *Taxus wallichiana* as described in claim 5, characterized in that, The plant extract was obtained by decocting the branches and leaves of Taxus wallichiana, Dictamnus dasycarpus root bark, Astragalus membranaceus, Saposhnikovia divaricata, ginseng, Angelica sinensis, and Polygonum multiflorum.

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