A plant-derived bactericidal composition from Syzygium cumini having broad-spectrum antibacterial activity and a preparation method thereof

Abstract

This invention discloses a broad-spectrum antibacterial composition derived from Syzygium sima (Syzygium sima) and its preparation method, relating to the field of biomedical technology. The composition comprises an active ingredient and pharmaceutically acceptable excipients. The active ingredient includes ursane-type triterpenoids or their pharmaceutically acceptable salts, esters, or prodrugs. The active ingredient provided by this invention exhibits potent inhibitory activity against a variety of clinically common pathogenic fungi, particularly showing significant antibacterial effects against Candida albicans, Cryptococcus neoformans, Candida glabrata, Aspergillus fumigatus, and Aspergillus niger. This compound is a natural product with an ursane-type triterpenoid skeleton, featuring a structure with α-L-rhamnose linked at C-3 and a free carboxyl group retained at C-28, overcoming the shortcomings of some existing plant-derived antibacterial agents, such as narrow antibacterial spectrum and poor efficacy against specific fungi.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a bactericidal composition derived from Syzygium simaoense with broad-spectrum antibacterial activity and its preparation method. Background Technology

[0002] *Syzygium* is a genus of plants belonging to the Myrtaceae family, comprising over 1200 species worldwide, widely distributed in tropical and subtropical regions. Many plants in this genus have a long history of traditional medicinal use, traditionally employed to treat fever, diarrhea, diabetes, and various infectious diseases. Modern pharmacological studies have shown that *Syzygium* plants are rich in terpenes, phloroglucinol, flavonoids, and other novel secondary metabolites, exhibiting significant antibacterial, anticancer, antioxidant, and protein tyrosine phosphatase 1B inhibitory activities.

[0003] Syzygium szemaoense is a fruit tree endemic to southwestern China, mainly distributed in Yunnan Province. To date, there are no patent reports, either domestically or internationally, on the systematic study of the chemical components and the evaluation of the bioactivity of this plant.

[0004] The main problems with existing plant-derived antibacterial agent technologies include:

[0005] (1) It has a narrow antibacterial spectrum and is not effective against drug-resistant bacteria;

[0006] (2) The active ingredients are unclear, making quality control difficult;

[0007] (3) Some natural products have poor water solubility and low bioavailability.

[0008] To address the aforementioned technological gaps and deficiencies in existing technologies, this application systematically isolates, structurally identifies, and evaluates the bioactivity of antibacterial active ingredients from *Syzygium sapiens*, and proposes a *Syzygium sapiens* plant-derived bactericidal composition with broad-spectrum antibacterial activity and its preparation method, thereby solving the above-mentioned problems. Summary of the Invention

[0009] The purpose of this invention is to provide a plant-derived bactericidal composition and preparation method of Syzygium simaoense with broad-spectrum antibacterial activity, in order to solve the problems of existing plant-derived antibacterial agents, such as narrow antibacterial spectrum, poor effect on drug-resistant bacteria, unclear active ingredients, difficulty in quality control, and poor water solubility and low bioavailability of some natural products.

[0010] To achieve the above objectives, the present invention provides the following technical solution:

[0011] In a first aspect, this application provides a bactericidal composition derived from Syzygium simaoense with broad-spectrum antibacterial activity, comprising an active ingredient and pharmaceutically acceptable excipients;

[0012] The active ingredient includes ursane-type triterpenoids or their pharmaceutically acceptable salts, esters, or prodrugs.

[0013] Furthermore, the active ingredient also includes at least one selected from the following compounds:

[0014] Compound 1, syzygiumursanolide A, has the structure (2R,3R,6R,17S,18S)-19(18→17)-trans-28-norus-12-ene-2,3,6,18,23-pentaol, and the molecular formula C1. 29 H 48 O5, compound 1 has a 19(18→17)-displaced spirocyclic skeleton with hydroxyl groups attached at positions C-2, C-3, C-6, C-18, and C-23;

[0015] Compound 2, syzygiumursanolide B, has the structure (2R,3R,6R,17S,18S)-19(18→17)-shift-24,28-noruso-4(23),12-diene-2,3,6,18-tetraol, with the molecular formula C. 28 H 44 O4, compound 2 has a 19(18→17)-displaced spirocyclic skeleton with hydroxyl groups attached at positions C-2, C-3, C-6, and C-18;

[0016] Compound 3, namely syzygiumursanolide C, has the structure 2α,3β,6β,23-tetrahydroxyursanolide-12,20(30)-diene-28-acid β-D-glucopyranoside, with the molecular formula C. 36 H 56 O 11 The carboxyl group at C-28 of compound 3 forms a glycoside with β-D-glucose;

[0017] Compound 4, syzygiumursanolide D, has the structure 2α,3β-dihydroxyursano-12-en-28-oic acid 3-O-α-L-rhamnoside, with the molecular formula C. 36 H 58 O8, compound 4 has α-L-rhamnose linked at the C-3 position.

[0018] Furthermore, the pharmaceutically acceptable excipients include at least one of diluents, carriers, and excipients; the dosage form of the composition is tablets, capsules, granules, powders, oral liquids, injections, lyophilized powder for injection, ointments, creams, gels, sprays, lotions, patches, or suppositories.

[0019] Furthermore, the minimum inhibitory concentration (MIC) of compound 4 against Candida albicans, Cryptococcus neoformans, Candida parapsilosis, Aspergillus fumigatus and Aspergillus niger is 6.25–25 μg / mL.

[0020] Secondly, this application provides a method for preparing a broad-spectrum antibacterial composition derived from Syzygium simaoense with the broad-spectrum antibacterial activity as described in the first aspect, comprising the following steps:

[0021] S1. Pulverize the dried leaves of Syzygium simaoense, extract them by percolation with 80% ethanol aqueous solution, collect the 80% ethanol extract, and concentrate it under reduced pressure to obtain an extract.

[0022] S2. The extract is suspended in water and sequentially extracted with petroleum ether, ethyl acetate, and n-butanol using liquid-liquid partition extraction. The ethyl acetate extract is then collected.

[0023] S3. The ethyl acetate extract was subjected to MCI gel CHP-20P column chromatography with water-methanol gradient elution, and multiple fractions were collected.

[0024] S4. The multi-components were repeatedly separated and purified by Sephadex LH-20 gel column chromatography, silica gel column chromatography, and ODS reversed-phase column chromatography to obtain compound 1, compound 2, compound 3, and compound 4.

[0025] Further, in step S3, the water-methanol gradient elution procedure includes a water:methanol volume ratio of 1:0, gradually transitioning to 0:1, and finally eluting with acetone;

[0026] In step S4, the separation and purification process involves tracking and detecting the target fraction using thin-layer chromatography.

[0027] In a second aspect, this application provides the use of the composition as described in the first aspect in the preparation of an antimicrobial drug, characterized in that the antimicrobial drug is used to inhibit or kill fungi.

[0028] Furthermore, the fungus includes at least one of Candida albicans, Cryptococcus neoformans, Candida parapsilosis, Aspergillus fumigatus, and Aspergillus niger.

[0029] Furthermore, the antibacterial drug is also used to inhibit Staphylococcus aureus, Bacillus subtilis, or Escherichia coli.

[0030] Fourthly, this application provides a bactericide for controlling fungal and bacterial infections, containing the active ingredient as described in the first aspect, and a pharmaceutically acceptable carrier;

[0031] The bactericide is used to prevent and treat infectious diseases caused by Candida albicans, Cryptococcus neoformans, Candida parapsilosis, Aspergillus fumigatus or Aspergillus niger.

[0032] Compared with existing technologies, this invention provides a broad-spectrum antibacterial composition derived from Syzygium sima and its preparation method. The active ingredient provided by this invention exhibits strong inhibitory activity against a variety of clinically common pathogenic fungi, particularly showing significant antibacterial effects against Candida albicans, Cryptococcus neoformans, Candida parapsilosis, Aspergillus fumigatus, and Aspergillus niger. This compound is a natural product with a ursane-type triterpenoid skeleton, featuring a structure with α-L-rhamnose linked at C-3 and a free carboxyl group retained at C-28, overcoming the shortcomings of some existing plant-derived antibacterial agents, such as narrow antibacterial spectrum and poor efficacy against specific fungi.

[0033] When the active ingredient of this invention is used in combination with other ursane-type triterpenoids from Syzygium sima, it exhibits a significant synergistic inhibitory effect against drug-resistant fungi (such as fluconazole-resistant Candida albicans). This synergistic effect can significantly reduce the dosage of each individual drug, improving the therapeutic effect against drug-resistant strains while helping to reduce the toxic side effects of the drugs, providing a new and effective strategy for the clinical treatment of drug-resistant fungal infections. Attached Figure Description

[0034] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this invention. For those skilled in the art, other drawings can be obtained based on these drawings.

[0035] Figure 1 A structural diagram of compound 1 provided in an embodiment of the present invention;

[0036] Figure 2 The structural diagram of compound 2 provided in the embodiments of the present invention;

[0037] Figure 3 The structural diagram of compound 3 provided in the embodiments of the present invention;

[0038] Figure 4 The structure diagram of compound 4 provided in the embodiments of the present invention. Detailed Implementation

[0039] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings.

[0040] As attached Figure 1 To be continued Figure 4 As shown:

[0041] Example 1:

[0042] Extraction and separation of active compounds from Syzygium simaoense:

[0043] Experimental instruments and materials:

[0044] Plant material: Leaves of Syzygium szemaoense, collected in September 2017 from Yunnan Province, China.

[0045] Instruments: Rotary evaporator (BUCHI R-100), MCI gel CHP-20P column (Mitsubishi Chemical), Sephadex LH-20 column (GE Healthcare), silica gel column chromatography (Qingdao Ocean Chemical), ODS reversed-phase column chromatography (YMC), nuclear magnetic resonance spectrometer (Bruker Advance III-600MHz), high-resolution mass spectrometer (Bruker MicroTOF-Q).

[0046] Extraction and separation steps:

[0047] Take 5.0 kg of dried Syzygium simaoense leaves, crush them, and extract them by percolation with 80% ethanol aqueous solution (10 L × 4) at room temperature for 72 hours.

[0048] The 80% ethanol extracts were combined and concentrated under reduced pressure to obtain 510.0 g of extract. The extract was suspended in water and subjected to liquid-liquid partition extraction with petroleum ether, ethyl acetate, and n-butanol, five times each.

[0049] 120.0 g of ethyl acetate extract was collected and subjected to MCI gel CHP-20P column chromatography, eluted with a water-methanol gradient (1:0 → 0:1, v / v), and finally eluted with acetone to obtain 6 fractions (Fr.AF).

[0050] The 100% methanol eluent (Fr. A, 5.4 g) was repeatedly separated and purified by Sephadex LH-20 gel column chromatography (methanol), silica gel column chromatography (dichloromethane-methanol 10:1 → 0:1), and ODS reversed-phase column chromatography (methanol-water 90:10) to obtain 6.2 mg of compound syzygiumursanolide D (i.e., compound 4), with the structure shown below. Figure 1 As shown.

[0051] High-resolution mass spectrometry, one-dimensional and two-dimensional nuclear magnetic resonance spectroscopy (…) 1 H-NMR, 13 C-NMR, COSY, HSQC, HMBC, NOESY) and GC-MS analysis after acid hydrolysis confirmed that the compound is 2α,3β-dihydroxyurs-12-en-28-oic acid 3-O-α-L-pyranoside.

[0052] The 80% methanol eluent (Fr. B, 10.7 g) was separated by silica gel column chromatography (dichloromethane-methanol 40:1 to 0:1, v / v) to obtain six subfractions (Fr. B1-6). Fr. B3 (301.0 mg) was separated by silica gel column chromatography (dichloromethane-methanol 20:1 to 10:1, v / v), and then separated by Sephadex LH-20 column chromatography (100% methanol) to give compound 2 (8.5 mg), namely syzygiumursanolide B, with the structure shown below. Figure 2 As shown.

[0053] Fr.B4 (4.0 g) was separated by silica gel column chromatography (dichloromethane-methanol 15:1 to 5:1, v / v) and further purified by ODS column chromatography (methanol-water 80:20) to give compound 1 (4.2 mg), namely syzygiumursanolide A, with the structure shown below. Figure 3 As shown.

[0054] The 60% methanol eluent (Fr. C, 19.1 g) was passed through a silica gel column (dichloromethane-methanol 10:1 to 0:1, v / v) to obtain five subfractions (Fr. C1-5). Fr. C3 (1.4 g) was passed sequentially through a Sephadex LH-20 column and an ODS column (methanol-water 70:30) to obtain compound 3 (46.8 mg), namely syzygiumursanolide C, with the structure shown below. Figure 4 As shown.

[0055] Example 2:

[0056] Evaluation of the antifungal activity of compounds 1, 2, 3, and 4:

[0057] 1. Experimental materials:

[0058] Test strains: Candida albicans ATCC MYA-2876, Cryptococcus neoformans ATCC 208821, Candida parapsilosis ATCC 22019, Aspergillus fumigatus CCTCC AF 93,048, and Aspergillus niger CCTCC AF 93,021.

[0059] Positive control drug: Amphotericin B (Sigma-Aldrich).

[0060] Experimental instruments: 96-well plate (Corning), microplate reader (BioTek Instruments, model: Synergy H1), constant temperature incubator (Shanghai Yiheng, model: DHP-9082).

[0061] 2. Experimental Method (Micro-broth Dilution Method):

[0062] The test compound syzygiumursanolide AD (prepared in Example 1) was dissolved in DMSO and diluted with RPMI-1640 medium to prepare a series of concentration gradients: 100, 50, 25, 12.5, 6.25, 3.125, and 1.56 μg / mL.

[0063] Add 100 μL of 2×10⁻⁶ ppm solution to each well of a 96-well plate. 3 A fungal suspension of CFU / mL was prepared, followed by the addition of 1 μL of sample solutions of different concentrations.

[0064] Fungal culture conditions: Candida albicans, Cryptococcus neoformans, Candida parapsilosis, and Aspergillus fumigatus were cultured at 28°C for 48 hours; Aspergillus niger was cultured at 37°C for 48 hours.

[0065] After incubation, add 10 μL of MTT (5 mg / mL) to each well and continue incubation for 4 hours. Remove the supernatant, add 150 μL of DMSO, and measure the absorbance (OD value) at 490 nm using a microplate reader.

[0066] The minimum inhibitory concentration (MIC) is defined as the lowest concentration of a test sample that can completely inhibit the visible growth of microorganisms. The experiment was repeated three times.

[0067] 3. The experimental results are shown in Table 1 below:

[0068] Table 1. Minimum inhibitory concentrations (MIC μg / mL) of syzygium ursanolide AD against five pathogenic fungi.

[0069] strains Compound 1 Compound 2 Compound 3 Compound 4 Comparison Staphylococcus aureus 50 25 >100 50 <![CDATA[0.5 a ]]> Bacillus subtilis 50 25 >100 50 <![CDATA[0.25 a ]]> E. coli 50 25 >100 50 <![CDATA[0.25 a ]]> Candida albicans 100 >100 >100 12.5 <![CDATA[2.0 b ]]> Cryptococcus neoformans 50 >100 >100 6.25 <![CDATA[2.0 b ]]> Candida parapsilosis >100 >100 >100 12.5 <![CDATA[2.0 b ]]> Aspergillus fumigatus 100 >100 >100 12.5 <![CDATA[2.0 b <!-- 4 -->]]> Aspergillus niger >100 >100 >100 25 <![CDATA[2.0 b ]]>

[0070] Wherein, a: ciprofloxacin. b: amphotericin B.

[0071] 4. Conclusion:

[0072] Experimental results showed that compounds 1, 2, 3, and 4 all exhibited significant inhibitory activity against a variety of common clinical pathogenic fungi. Among them, compound 4 showed the strongest antifungal activity against a group of fungi, with a minimum inhibitory concentration (MIC) ranging from 6.25 to 25 μg / mL. This compound showed broad-spectrum antifungal activity and has the potential to be developed into a novel antifungal drug.

[0073] As can be seen from the above, the active ingredient provided by this invention has strong inhibitory activity against a variety of clinically common pathogenic fungi, especially showing significant antibacterial effects against Candida albicans, Cryptococcus neoformans, Candida parapsilosis, Aspergillus fumigatus, and Aspergillus niger. This compound is a natural product with a ursane-type triterpenoid skeleton, featuring a structure with α-L-rhamnose linked at C-3 and a free carboxyl group retained at C-28, overcoming the shortcomings of some existing plant-derived antibacterial agents, such as narrow antibacterial spectrum and poor efficacy against specific fungi.

[0074] When the active ingredient of this invention is used in combination with other ursane-type triterpenoids from Syzygium sima, it exhibits a significant synergistic inhibitory effect against drug-resistant fungi (such as fluconazole-resistant Candida albicans). This synergistic effect can significantly reduce the dosage of each individual drug, improving the therapeutic effect against drug-resistant strains while helping to reduce the toxic side effects of the drugs, providing a new and effective strategy for the clinical treatment of drug-resistant fungal infections.

[0075] The foregoing has only described certain exemplary embodiments of the present invention by way of illustration. Undoubtedly, those skilled in the art can modify the described embodiments in various ways without departing from the spirit and scope of the present invention. Therefore, the foregoing drawings and descriptions are illustrative in nature and should not be construed as limiting the scope of protection of the claims of the present invention.

Claims

1. A fungicide composition derived from Syzygium simaoense with broad-spectrum antibacterial activity, characterized in that, The composition comprises an active ingredient and pharmaceutically acceptable excipients; The active ingredient includes ursane-type triterpenoids or their pharmaceutically acceptable salts, esters, or prodrugs.

2. The bactericidal composition derived from Syzygium simaoense with broad-spectrum antibacterial activity according to claim 1, characterized in that, The active ingredient is selected from at least one of the following compounds: Compound 1, syzygiumursanolide A, has the structure (2R,3R,6R,17S,18S)-19(18→17)-trans-28-norus-12-ene-2,3,6,18,23-pentaol, and the molecular formula C1. 29 H 48 O5, compound 1 has a 19(18→17)-displaced spirocyclic skeleton with hydroxyl groups attached at positions C-2, C-3, C-6, C-18, and C-23; Compound 2, syzygiumursanolide B, has the structure (2R,3R,6R,17S,18S)-19(18→17)-shift-24,28-noruso-4(23),12-diene-2,3,6,18-tetraol, with the molecular formula C. 28 H 44 O4, compound 2 has a 19(18→17)-displaced spirocyclic skeleton with hydroxyl groups attached at positions C-2, C-3, C-6, and C-18; Compound 3, namely syzygiumursanolide C, has the structure 2α,3β,6β,23-tetrahydroxyursanolide-12,20(30)-diene-28-acid β-D-glucopyranoside, with the molecular formula C. 36 H 56 O 11 The carboxyl group at C-28 of compound 3 forms a glycoside with β-D-glucose; Compound 4, syzygiumursanolide D, has the structure 2α,3β-dihydroxyurs-12-en-28-oic acid 3-O-α-L-rhamnopyranoside, with the molecular formula C. 36 H 58 O8, compound 4 has α-L-rhamnose linked at the C-3 position.

3. The bactericidal composition derived from Syzygium simaoense with broad-spectrum antibacterial activity according to claim 1, characterized in that, The pharmaceutically acceptable excipients include at least one of diluents, carriers, and excipients; the dosage form of the composition is tablets, capsules, granules, powders, oral liquids, injections, lyophilized powder for injection, ointments, creams, gels, sprays, lotions, patches, or suppositories.

4. The bactericidal composition derived from Syzygium simaoense with broad-spectrum antibacterial activity according to claim 2, characterized in that, The minimum inhibitory concentration of compound 4 against Candida albicans, Cryptococcus neoformans, Candida parapsilosis, Aspergillus fumigatus, and Aspergillus niger is 6.25–25 μg / mL.

5. A method for preparing a fungicide composition derived from Syzygium sima and possessing broad-spectrum antibacterial activity as described in any one of claims 1-4, characterized in that, Includes the following steps: S1. Pulverize the dried leaves of Syzygium simaoense, extract them by percolation with 80% ethanol aqueous solution, collect the 80% ethanol extract, and concentrate it under reduced pressure to obtain an extract. S2. The extract is suspended in water and sequentially extracted with petroleum ether, ethyl acetate, and n-butanol using liquid-liquid partition extraction. The ethyl acetate extract is then collected. S3. The ethyl acetate extract was subjected to MCI gel CHP-20P column chromatography with water-methanol gradient elution, and multiple fractions were collected. S4. The multi-components were repeatedly separated and purified by Sephadex LH-20 gel column chromatography, silica gel column chromatography, and ODS reversed-phase column chromatography to obtain compound 1, compound 2, compound 3, and compound 4.

6. The bactericidal composition derived from Syzygium simaoense with broad-spectrum antibacterial activity and its preparation method according to claim 5, characterized in that, In step S3, the water-methanol gradient elution procedure includes a water:methanol volume ratio of 1:0, gradually transitioning to 0:1, and finally eluting with acetone. In step S4, the separation and purification process involves tracking and detecting the target fraction using thin-layer chromatography.

7. The use of the composition according to claims 1-4 in the preparation of antibacterial drugs, characterized in that, The antibacterial drug is used to inhibit or kill fungi.

8. The application according to claim 7, characterized in that, The fungi include at least one of Candida albicans, Cryptococcus neoformans, Candida parapsilosis, Aspergillus fumigatus, and Aspergillus niger.

9. The application according to claim 7, characterized in that, The antibacterial drug is also used to inhibit Staphylococcus aureus, Bacillus subtilis, or Escherichia coli.

10. A bactericide for controlling fungal and bacterial infections, characterized in that, Contains the active ingredient as described in claim 1 or 2, and a pharmaceutically acceptable carrier; The bactericide is used to prevent and treat infectious diseases caused by Candida albicans, Cryptococcus neoformans, Candida parapsilosis, Aspergillus fumigatus or Aspergillus niger.

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