Berberine organic acid salt, preparation method and application thereof

Abstract

This invention belongs to the field of biomedical technology, and discloses a berberine organic acid salt, its preparation method, and its applications. Its structural formula is as follows: Wherein, X ‑ It is a monovalent organic acid anion, selected from the formula where R is H or OH, and n is 1, 3 or 5. Preparation method: (1) Under strong alkalinity, berberine hydrochloride is reacted with acetone to obtain berberine acetone; (2) The berberine acetone obtained in step (1) is dissolved in anhydrous ethanol, and under heating and reflux with stirring, it is added with X ‑ The corresponding organic acid was reacted for 6–10 h, filtered, dried, and precipitated with ethyl acetate. The precipitate was collected by filtration and dried to obtain berberine organic acid salt. The preparation route of this invention is simple, and the berberine organic acid salt prepared by this invention has significant antibacterial and cytotoxic effects.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a berberine organic acid salt, its preparation method, and its application. Background Technology

[0002] In recent years, bacterial resistance has become a major challenge in clinical treatment worldwide, necessitating the development of novel antibacterial drugs. Berberine is a major active ingredient in traditional Chinese medicines such as Coptis chinensis, possessing antibacterial, anti-inflammatory, and anti-apoptotic activities, as well as favorable pharmacological properties. However, its poor water and lipid solubility leads to low bioavailability. Therefore, the modification of berberine has become a hot topic. Most reported bioactive derivatives are obtained by attaching chain-linked fatty acids to the four rings of berberine. However, this method excessively alters the original structure of berberine, weakening or eliminating its biocompatibility. Moreover, most structural modifications are performed on the isoquinoline region of berberine, with less consideration given to the anionic portion. Furthermore, the waste liquid generated from commonly used berberine hydrochloride and berberine bisulfate production has a certain environmental impact. Therefore, the synthesis of novel berberine salts is necessary. Summary of the Invention

[0003] To overcome the shortcomings of existing technologies in modifying berberine, such as excessive structural changes and the generation of polluting waste liquid, the present invention aims to provide a berberine organic acid salt, its preparation method, and its application.

[0004] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0005] A berberine organic acid salt has the following structural formula:

[0006]

[0007] Among them, X - It is a monovalent organic acid anion, which is selected from

[0008] In the formula, R is H or OH, and n is 1, 3 or 5.

[0009] The preparation method of the berberine organic acid salt includes the following steps:

[0010] (1) Berberine hydrochloride is reacted with acetone under strong alkaline conditions to produce acetone berberine.

[0011] (2) Dissolve the acetone berberine obtained in step (1) in anhydrous ethanol, and add it to X under reflux with stirring. - The corresponding organic acid was reacted for 6-10 hours, filtered, dried, and ethyl acetate was added to precipitate. The precipitate was collected by filtration and dried to obtain berberine organic acid salt.

[0012] Preferably, in step (2), the molar ratio of acetone berberine to organic acid is 1:(1-1.5).

[0013] Preferably, in step (2), the ratio of berberine acetone to anhydrous ethanol is 1 mmol to (20-30) mL.

[0014] Preferably, in step (2), the temperature of the heating reflux is 40-60°C.

[0015] In step (1), berberine acetone can be prepared according to existing technology. The preferred preparation process of this invention is as follows: berberine hydrochloride is placed in water, heated and stirred under reflux until completely dissolved, pH is adjusted to 10-13 with inorganic alkali solution, acetone is added, heated and stirred under reflux for 1-2 hours, filtered after standing, washed and dried to obtain berberine acetone.

[0016] Preferably, in step (1), the ratio of berberine hydrochloride:water:acetone is 0.02 mmol:(80-120) mL:(4-10) mL.

[0017] Preferably, in step (1), the temperature of the heating reflux is 60-70°C.

[0018] Preferably, in step (1), the concentration of the inorganic alkali solution is 0.1 to 7 mol / L, and the inorganic alkali is sodium hydroxide or potassium hydroxide.

[0019] The berberine organic acid salt is used as an antibacterial agent or in the preparation of antitumor drugs.

[0020] Drug salts are a class of compounds composed of active pharmaceutical ingredients and organic or inorganic counterions. This invention employs an organic acid salt-forming modification strategy, replacing the original anions in berberine salts with biodegradable organic acids to form drug salts. This approach not only modifies the anions without altering the parent structure of berberine, improving the drug's biocompatibility and safety, but also avoids the toxicity of chloride ions in the original berberine hydrochloride, thereby improving the physicochemical properties of berberine and synergistically increasing its pharmacological activity.

[0021] Beneficial effects: The preparation route of this invention is simple, low-cost, convenient for post-processing, high-yield, high-purity, and the product has good physicochemical properties and good stability. Moreover, the solvent used in the preparation process is anhydrous ethanol, which will not have an adverse impact on the environment. The berberine organic acid salt prepared by this invention has significant antibacterial effects against common clinical strains such as Staphylococcus aureus, Bacillus subtilis, Candida albicans, and Streptococcus mutans, and has obvious cytotoxic effects on mouse cancer cells. It can be used as an antibacterial agent and anticancer agent in clinical applications. Attached Figure Description

[0022] Figure 1 This is a flowchart illustrating the preparation process of the present invention;

[0023] Figure 2 The compound of Example 1 1 H NMR spectrum;

[0024] Figure 3 The compound of Example 2 1 H NMR spectrum;

[0025] Figure 4 The compound of Example 3 1 H NMR spectrum;

[0026] Figure 5 The compound of Example 4 1 H NMR spectrum;

[0027] Figure 6 The compound of Example 5 1 H NMR spectrum;

[0028] Figure 7 The compound of Example 1 13 C NMR spectrum;

[0029] Figure 8 The compound of Example 2 13 C NMR spectrum;

[0030] Figure 9 The compound of Example 3 13 C NMR spectrum;

[0031] Figure 10 The compound of Example 4 13 C NMR spectrum;

[0032] Figure 11 The compound of Example 5 13 C NMR spectrum;

[0033] Figure 12 The infrared (IR) spectra of the compound in Example 1 and the two raw materials, berberine hydrochloride and syringic acid, are shown below.

[0034] Figure 13 The infrared (IR) spectra of the compound in Example 2 and the two raw materials, berberine hydrochloride and 3,5-dimethoxybenzoic acid, are shown below.

[0035] Figure 14 The infrared (IR) spectra of the compound in Example 3 and the two raw materials, berberine hydrochloride and 5-hexenoic acid, are shown below.

[0036] Figure 15The infrared (IR) spectra of the compound in Example 4 and the two raw materials, berberine hydrochloride and 8-nonenoic acid, are shown.

[0037] Figure 16 The infrared (IR) spectra of the compound in Example 5 and the two raw materials, berberine hydrochloride and 10-undecenoic acid, are shown below.

[0038] Figure 17 The graph shows the relative inhibitory activity of different concentrations of the compound from Example 1 on B16-F10 cells;

[0039] Figure 18 The graph shows the relative inhibitory activity of different concentrations of the compound from Example 2 on B16-F10 cells;

[0040] Figure 19 The graph shows the relative inhibitory activity of different concentrations of the compound from Example 3 on B16-F10 cells;

[0041] Figure 20 The graph shows the relative inhibitory activity of different concentrations of the compound from Example 4 on B16-F10 cells;

[0042] Figure 21 The graph shows the relative inhibitory activity of different concentrations of the compound from Example 5 on B16-F10 cells. Detailed Implementation

[0043] To make the present invention clearer and more explicit, the present invention will be further described in detail below. It should be understood that the specific embodiments described herein are only for explaining the present invention and are not intended to limit the present invention.

[0044] The preparation route of this invention is as follows: Figure 1 As shown, the intermediate product is acetone berberine.

[0045] Example 1: Synthesis of eugenol berberine salt

[0046]

[0047] (1) Take 0.02 mol of berberine hydrochloride and add it to 100 mL of distilled water. Reflux and stir in a round-bottom flask at 60 °C until it is completely dissolved. Add 5 mol / L sodium hydroxide solution to adjust the pH to 11-12. Stir for 0.5 h and then slowly add 6 mL of acetone. Stir for 1.5 h. A yellow flocculent precipitate will form. After standing, take the supernatant and add acetone. No precipitate will form, indicating that the reaction is over. Filter under reduced pressure to collect the precipitate. Wash with distilled water until colorless. Dry in a vacuum drying oven at 70 °C for 48 h. Remove trace water with phosphorus pentoxide to obtain berberine acetone.

[0048] (2) Dissolve 1 mmol of acetone berberine in 25 mL of anhydrous ethanol, add 1 mmol of syringic acid under reflux and stirring at 45 °C for 6 h. After the reaction is complete, remove the solvent anhydrous ethanol and the byproduct acetone by rotary evaporator. After drying for 3 h, add ethyl acetate to precipitate flocculent product. Collect and dry the product under reduced pressure to obtain a yellow powder, which is the target compound - syringic acid berberine salt.

[0049] Figure 2 The compound of Example 1 1 H NMR spectrum, 1 H NMR (600MHz, d6-DMSO): δ (ppm) 9.89 (s, 1H), 8.93 (s, 1H), 8.17 (d, J = 9.1Hz, 1H), 7.99 (d, J = 9.1Hz, 1H), 7.79 (s, 1H) ,7.12(s,2H),6.15(s,2H),5.02-4.82(m,2H),4.06(d,J=14.1Hz,6H),3.70(s,6H),3.21-3.15(m,2H),2.07(s,1H).

[0050] Figure 7 The compound of Example 1 13 C NMR spectrum, 13 C NMR (151MHz, d6-DMSO): δ (ppm) 169.19 (s), 150.86 (s), 150.28 (s), 148.14 (s), 147.0 9(s),145.88(s),144.14(s),137.94(s),136.84(s),133.46(s),131.14(s),127.18( s),124.05(s),121.88(s),120.89(s),120.64(s),108.89(s),107.07(s),105.91(s) ),102.53(s),62.38(s),57.52(s),56.14(s),55.70(s),30.92-30.79(m),26.81(s).

[0051] Figure 12 The infrared (IR) spectra of the compound in Example 1 and the two raw materials, berberine hydrochloride and syringic acid, are shown below. IR(KBr)v: 3431, 2938, 2846, 1620, 1600, 1567, 1549, 1506, 1460, 1457, 1388, 1371, 1232.

[0052] 11H NMR data showed the disappearance of the characteristic peak (δ(ppm) = 12) of hydrogen on the syringic acid-COOH group, indicating that syringic acid in the compound of Example 1 exists in an anionic state; IR spectroscopy data showed that the raw material syringic acid had peaks at 1699 and 1418 cm⁻¹. -1 The stretching vibration peak corresponding to the -COOH group disappeared in the IR spectrum of the compound in Example 1, and peaks at 1549 and 1388 cm⁻¹ appeared. -1 The new peaks at the locations correspond to COO and COO, respectively. - The symmetric and antisymmetric stretching vibration peaks, and the C=N ratio in the raw material berberine hydrochloride. + The characteristic peak shifted from 1636 to 1600 cm⁻¹ -1 The above data confirms the formation of the target compound, eugenol berberine salt.

[0053] Example 2: Synthesis of berberine salt of 3,5-dimethoxybenzoic acid

[0054]

[0055] (1) Same as step (1) in Example 1;

[0056] (2) 3,5-Dimethoxybenzoic acid was used instead of syringic acid, and the rest was the same as step (2) in Example 1.

[0057] Figure 3 The compound of Example 2 1 H NMR spectrum, 1 H NMR (600MHz, d6-DMSO): δ (ppm) 9.90 (s, 1H), 8.94 (s, 1H), 8.20 (d, J = 9.1Hz, 1H), 8.00 (d, J = 9.1Hz, 1H), 7.80 (s, 1H), 7.08 (s, 1H), 6.9 9(d,J=2.4Hz,2H),6.34(t,J=2.4Hz,1H),6.17(s,2H),4.94(t,J=6.0Hz,2H),4.08(d,J=14.3Hz,6H),3.70(s,6H),3.24-3.08(m,2H).

[0058] Figure 8 The compound of Example 2 13 C NMR spectrum, 13C NMR (151MHz, d6-DMSO): δ (ppm) 167.71 (s), 159.52 (s), 150.52 (s), 149.95 (s), 147. 82(s),145.65(s),144.33(s),143.81(s),137.60(s),133.13(s),130.81(s),126. 84(s),123.71(s),121.55(s),120.60(s),120.36(s),108.55(s),106.68(s),105. 61(s),102.22(s),100.95(s),62.05(s),57.19(s),55.32(s),55.03(s),26.49(s).

[0059] Figure 13 The infrared (IR) spectra of the compound in Example 2 and the two raw materials, berberine hydrochloride and 3,5-dimethoxybenzoic acid, are shown below. IR(KBr)v: 3416, 2945, 2847, 1632, 1599, 1569, 1505, 1459, 1389.

[0060] 1 ¹H NMR data showed the disappearance of the characteristic peak (δ(ppm) = 12) of hydrogen on the -COOH group of 3,5-dimethoxybenzoic acid, indicating that 3,5-dimethoxybenzoic acid exists in an anionic state in the compound of Example 2; IR spectroscopy data showed that the starting material 3,5-dimethoxybenzoic acid had peaks at 1690 and 1418 cm⁻¹. -1 The stretching vibration peak corresponding to the -COOH group disappeared in the IR spectrum of the compound in Example 2, and peaks at 1569 and 1389 cm⁻¹ appeared. -1 The new peaks at the locations correspond to COO and COO, respectively. - The symmetric and antisymmetric stretching vibration peaks, and the C=N ratio in the raw material berberine hydrochloride. + The characteristic peak shifted from 1636 cm⁻¹ to 1632 cm⁻¹ -1 The above data confirms the formation of the target compound, berberine salt of 3,5-dimethoxybenzoic acid.

[0061] Example 3: Synthesis of 5-Hexenoic acid berberine salt

[0062]

[0063] (1) Same as step (1) in Example 1;

[0064] (2) Use 5-hexenoic acid instead of syringic acid, and follow the same steps as in Example 1 (2).

[0065] Figure 4 The compound of Example 31 H NMR spectrum, 1 H NMR (600MHz, D2O): δ (ppm) 9.47 (s, 1H), 7.91 (s, 1H), 7.79 (d, J = 6.7Hz, 1H), 7. 56(d,J=6.7Hz,1H),6.95(s,1H),6.76(s,1H),5.97(s,2H),5.87(dt,J=6.7,6 .7,12.9Hz,1H),5.05(d,J=17.3Hz,1H),4.98(d,J=9.7Hz,1H),4.71(s,2H),4 .05(s,3H),3.95(s,3H),3.07(s,2H),2.16(s,2H),2.03(s,2H),1.62(s,2H).

[0066] Figure 9 The compound of Example 3 13 C NMR spectrum, 13 C NMR (151MHz, D2O): δ (ppm) 183.59 (s), 150.38 (s), 149.91 (s), 147.47 (s), 144.00 (s),143.03(s),139.27(s),136.98(s),132.61(s),129.73(s),126.24(s),123.2 5(s),121.05(s),119.22(s),119.02(s),114.62(s),108.35(s),104.18(s),102. 40(s),62.14(s),56.55(s),55.70(s),37.01(s),32.90(s),26.42(s),25.14(s).

[0067] Figure 14 The infrared (IR) spectra of the compound in Example 3 and the two raw materials, berberine hydrochloride and 5-hexenoic acid, are shown below. IR(KBr)v: 3416, 2944, 2843, 1630, 1599, 1566, 1505, 1458, 1383, 1355, 1335, 1315, 996, 911.

[0068] 1 1H NMR data showed the disappearance of the characteristic peak (δ(ppm) = 12) of hydrogen on the -COOH group of 5-hexenoic acid, indicating that 5-hexenoic acid exists in an anionic state in the compound of Example 3; IR spectroscopy data showed that the starting material 5-hexenoic acid had peaks at 1709 and 1414 cm⁻¹. -1 The stretching vibration peak corresponding to the -COOH group disappeared in the IR spectrum of the compound in Example 3, and peaks at 1566 and 1383 cm⁻¹ appeared.-1 The new peak at that point corresponds to COO - The symmetric and antisymmetric stretching vibration peaks, and the C=N ratio in the raw material berberine hydrochloride. + The characteristic peak shifted from 1636 cm⁻¹ to 1630 cm⁻¹ -1 The above data confirms the formation of the target compound, berberine 5-hexenoic acid salt.

[0069] Example 4: Synthesis of 8-nonenoic acid berberine salt

[0070]

[0071] (1) Same as step (1) in Example 1;

[0072] (2) Use 8-nonenoic acid instead of syringic acid, and follow the same steps as in Example 1 (2).

[0073] Figure 5 The compound of Example 4 1 H NMR spectrum, 1 H NMR (600MHz, D2O): δ (ppm) 9.51 (s, 1H), 7.93 (s, 1H), 7.82 (d, J = 8.9Hz, 1H), 7.59 (d, J = 8.9Hz, 1H), 6.97 (s, 1H), 6.7 8(s,1H),5.99(s,2H),5.86(ddt,J=6.6,6.6,10.2,16.9Hz,1H),5.01(dq,J=1.7,1.8,1.8,17.2Hz,1H),4.94(dq,J= 1.5,1.5,1.5,10.3Hz,1H),4.75(t,J=6.4,6.4Hz,2H),4.08(s,3H),3.97(s,3H),3.10(t,J=6.5,6.5Hz,2H),2.14(t ,J=7.6,7.6Hz,2H),2.02–1.93(m,2H),1.50(p,J=7.3,7.3,7.3,7.3Hz,2H),1.35–1.27(m,2H),1.28–1.21(m,4H).

[0074] Figure 10 The compound of Example 4 13 C NMR spectrum, 13C NMR (151MHz, D2O): δ (ppm) 183.93 (s), 150.50 (s), 150.00 (s), 147.58 (s), 144.09 (s), 143 .17(s),140.31(s),137.17(s),132.81(s),129.89(s),126.43(s),123.43(s),121.22(s) ),119.41(s),119.28(s),113.96(s),108.43(s),104.39(s),102.43(s),62.22(s),56.6 7(s),55.80(s),37.67(s),33.07(s),28.64(s),28.17(s),28.07(s),26.53(s),25.88(s)

[0075] Figure 15 The infrared (IR) spectra of the compound in Example 4 and the two raw materials, berberine hydrochloride and 8-nonenoic acid, are shown below. IR(KBr)v: 3423, 2925, 2853, 1630, 1599, 1569, 1507, 1459, 1385, 999, 911.

[0076] 1 1H NMR data showed the disappearance of the characteristic peak (δ(ppm) = 12) of hydrogen on the 8-nonenoic acid-COOH group, indicating that 8-nonenoic acid exists in an anionic state in the compound of Example 4; IR spectroscopy data showed that the starting material 8-nonenoic acid had peaks at 1714 and 1414 cm⁻¹. -1 The stretching vibration peak corresponding to the -COOH group disappeared in the IR spectrum of the compound in Example 4, and peaks at 1569 and 1385 cm⁻¹ appeared. -1 The new peak at that point corresponds to COO - The symmetric and antisymmetric stretching vibration peaks, and the C=N ratio in the raw material berberine hydrochloride. + The characteristic peak shifted from 1636 cm⁻¹ to 1630 cm⁻¹ -1 The above data confirms the formation of the target compound, 8-nonenoic acid berberine salt.

[0077] Example 5: Synthesis of 10-Undecenoic Acid Berberine Salt

[0078]

[0079] (1) Same as step (1) in Example 1;

[0080] (2) 10-Undecenoic acid is used instead of syringic acid, and the rest is the same as step (2) in Example 1.

[0081] Figure 6 The compound of Example 51 H NMR spectrum, 1 H NMR (600MHz, D2O): δ (ppm) 9.60 (s, 1H), 8.26 (s, 1H), 7.97 (d, J = 9.2Hz, 1H), 7.81 (d, J = 9.1Hz, 1H), 7.28 (s, 1H), 6.91 (s, 1H),6.08(s,2H),5.94(ddt,J=6.7,6.7,10.3,17.0Hz,1H),5.06(dq,J=1.7,1.7,1.7,17.3Hz,1H),4.99(ddt,J=1.3,1. 3,2.6,10.3Hz,1H),4.84(t,J=6.4,6.4Hz,2H),4.12(s,3H),4.07(s,3H),3.20(t,J=6.4,6.4Hz,2H),2.18(t,J=7.5,7. 5Hz,2H),2.06(q,J=7.1,7.1,7.2Hz,2H),1.55(q,J=7.2,7.2,7.3Hz,2H),1.37(q,J=7.0,7.0,7.0Hz,2H),1.29(s,8H).

[0082] Figure 11 The compound of Example 5 13 C NMR spectrum, 13 C NMR (151MHz, d6-DMSO): δ (ppm) 174.88 (s), 150.86 (s), 150.27 (s), 148.14 (s), 145.94 (s), 144. 14(s),139.29(s),137.91(s),133.47(s),131.12(s),127.19(s),124.01(s),121.87(s),120.9 1(s),120.69(s),115.10(s),108.88(s),105.92(s),102.55(s),62.42(s),57.55(s),55.65(s) ),34.14(s),33.63(s),29.17(d,J=3.9Hz),28.96(d,J=9.9Hz),28.71(s),26.81(s),24.95(s).

[0083] Figure 16 The infrared (IR) spectra of the compound in Example 5 and the two raw materials, berberine hydrochloride and 10-undecenoic acid, are shown below. IR(KBr)v: 3422, 2923, 2850, 1630, 1602, 1567, 1506, 1462, 1393, 977, 911.

[0084] 1 1H NMR data showed the disappearance of the characteristic peak (δ(ppm) = 12) of hydrogen on the -COOH group of 10-undecenoic acid, indicating that 10-undecenoic acid exists in an anionic state in the compound of Example 5; IR spectroscopy data showed that the starting material 10-undecenoic acid had peaks at 1715 and 1405 cm⁻¹. -1 The stretching vibration peak corresponding to the -COOH group disappeared in the IR spectrum of the compound in Example 5, and peaks at 1567 and 1393 cm⁻¹ appeared. -1 The new peak at that point corresponds to COO - The symmetric and antisymmetric stretching vibration peaks, and the C=N ratio in the raw material berberine hydrochloride. + The characteristic peak shifted from 1636 cm⁻¹ to 1630 cm⁻¹ -1 The above data confirms the formation of the target compound, 10-undecenoic acid berberine salt.

[0085] In vitro antibacterial performance test

[0086] Using a high-temperature, high-pressure steam sterilizer (Hirayama HVE-50) and a constant-temperature shaker (Crystal IS-RDV1, USA), the antibacterial effects, MIC, and MBC values ​​of the compounds in Examples 1-5 against Staphylococcus aureus (catalog number: ATCC29213), Bacillus subtilis (catalog number: ATCC6633), Candida albicans (catalog number: ATCC10231), and Streptococcus mutans (catalog number: ATCC25175) were tested. Four bacterial suspensions were prepared according to the following methods:

[0087] Staphylococcus aureus and Bacillus subtilis bacterial suspensions: Take 12 mL of bacterial culture tube, add 3 mL of LB liquid medium, select single colonies from the solid medium of Staphylococcus aureus and Bacillus subtilis respectively and add them to the liquid medium, place in a constant temperature shaker (37℃, 200 rpm) and shake for 15 h to obtain Staphylococcus aureus / Bacillus subtilis bacterial suspensions;

[0088] Candida albicans culture: Take 12 mL of bacterial culture tube, add 3 mL of PDB liquid medium, select a single colony from the Candida albicans solid medium and add it to the liquid medium, place it in a constant temperature shaker (28℃, 200 rpm) and shake for 24 h;

[0089] Streptococcus mutans culture medium: Take 12 mL of bacterial culture tube, add 3 mL of BHI liquid medium, select a single colony from the Streptococcus mutans solid medium and add it to the liquid medium, place it in a constant temperature shaker (37℃, 200 rpm) and shake for 24 h.

[0090] (I) Testing the antibacterial effect

[0091] In Examples 1-5, solutions of the compounds were prepared with methanol at a concentration of 2 mg / mL. Staphylococcus aureus, Bacillus subtilis, Candida albicans, and Streptococcus mutans were diluted to 10 μL with PBS. 6 CFU / mL, 100 μL of diluted Staphylococcus aureus and Bacillus subtilis suspensions were evenly spread onto LB solid medium, 100 μL of diluted Candida albicans suspensions were evenly spread onto PDA solid medium, and 100 μL of diluted Streptococcus mutans suspensions were evenly spread onto BHIA solid medium. Holes were punched using a 9 mm punch and Oxford cups were placed on each. 100 μL of the methanol solution of each compound from Examples 1-5 was added to each of the different mediums. The mediums were then incubated at 37°C for 24 h (corresponding to Staphylococcus aureus and Bacillus subtilis), 28°C for 48 h (corresponding to Candida albicans), and 37°C for 18 h (corresponding to Streptococcus mutans). The diameter of the inhibited bacteria was recorded by photographing.

[0092] Table 1. Diameter of inhibition zones of compounds in Examples 1-5 against different bacterial strains.

[0093]

[0094]

[0095] (II) Testing MIC and MBC values

[0096] Based on the different solubilities of the compounds, stock solutions of the compounds in Examples 1-5 were prepared with methanol at concentrations of 100 mg / mL, 100 mg / mL, 125 mg / mL, 125 mg / mL, and 125 mg / mL, respectively. Staphylococcus aureus and Bacillus subtilis cultures were diluted to 10⁻¹⁰ using LB liquid medium. 6 CFU / mL, diluted with PDB liquid medium to 10. 6 CFU / mL, diluted with BHI liquid medium to 10. 6CFU / mL. MIC was determined using the broth microdilution method. 200 μL of co-culture medium (diluted bacterial solution: original compound solution = 49:1) was added to the first well of a 96-well plate. Then, following the two-fold dilution method, 100 μL of co-culture medium was added to the second well, followed by another 100 μL of diluted bacterial solution, and so on, gradually diluting from the first well to the eleventh well (adding 100 μL of diluted bacterial solution to each well except the first one), to prepare compound-bacterial suspensions of different concentrations. Control group: A system of 20 μL methanol + 980 μL diluted bacterial solution was prepared. 200 μL of this system was added to the first well of another 96-well plate, and the two-fold dilution method was repeated. The 96-well plates were incubated at 37°C for 24 h (corresponding to Staphylococcus aureus, Bacillus subtilis, and Streptococcus mutans) or 28°C for 48 h (corresponding to Candida albicans). Images were taken and the MIC values ​​were recorded. Based on the MIC, the MBC test was performed. The first three wells without bacterial growth were selected for further coating. The mixture was incubated in a constant temperature incubator at 37°C for 18 hours. The concentration when the number of colonies was less than 5 was the MBC.

[0097] Table 2. MIC and MBC values ​​of compounds from Examples 1-5 against different strains.

[0098]

[0099] The results in Tables 1 and 2 show that the compounds synthesized in this invention exhibit varying degrees of antibacterial activity against the selected strains, effectively inhibiting their growth and demonstrating good antibacterial performance.

[0100] Cytotoxicity test

[0101] Using instruments such as a CO2 constant temperature incubator (Beijing Sangye Experimental Instrument Research Institute, model: WIGGENSWCI-180), a centrifuge (Shanghai Luxiangyi Centrifuge Instrument Co., Ltd., model: TD5), and an ELISA reader (TECAN, model: SPARK 10M), the effects of the compounds in Examples 1-5 on the activity of B16-F10 cells (mouse tumor cell line) were tested by the MTT assay. The complete culture medium described below was prepared according to a volume ratio of DMEM low sodium bicarbonate medium to fetal bovine serum of 9:1.

[0102] B16-F10 cells were cultured in complete culture medium in a 5% CO2 incubator at 37°C. B16-F10 cells in the logarithmic growth phase were harvested for cell counting at a rate of 6 × 10⁻⁶ cells / cells. 3 / wells were seeded into 96-well plates and incubated overnight in a 5% CO2, 37°C incubator. 10 mg of the solid compound powder samples obtained in Examples 1-5 were sterilized by UV irradiation for 30 min, and 400 μL of DMSO was added. The mixture was sonicated for 10 min to prepare a 25 mg / mL compound solution, which was subsequently diluted with complete culture medium. Each compound was divided into a control group and a drug salt group. The control group received 100 μL / well of complete culture medium, while the drug salt groups received 100 μL / well of diluted compound solutions at concentrations of 0.5 μg / mL, 1 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, 50 μg / mL, 100 μg / mL, 150 μg / mL, 200 μg / mL, and 250 μg / mL, respectively. After incubation for 24 h, the culture medium was aspirated, and each well was washed three times with PBS. 100 μL of a 0.5 mg / mL solution was added to each well. The complete culture medium of MTT was cultured in a 5% CO2 incubator at 37°C for 4 hours. The supernatant was discarded, and 100 μL of DMSO was added to each well. After shaking gently for 10 minutes, the absorbance at 570 nm was measured, and the relative inhibitory activity was calculated according to the following formula.

[0103] Relative inhibitory activity (%) = (absorbance of drug salt group - background absorbance) / (absorbance of control group - background absorbance) × 100%;

[0104] The background absorbance is the absorbance of the multi-well plate itself (wells without culture medium and bacteria).

[0105] IC was calculated using nonlinear regression fitting for each concentration and relative inhibitory activity. 50 .

[0106] Figure 17 The graph shows the relative inhibitory activity of different concentrations of the compound from Example 1 on B16-F10 cells; Figure 18 The graph shows the relative inhibitory activity of different concentrations of the compound from Example 2 on B16-F10 cells; Figure 19 The graph shows the relative inhibitory activity of different concentrations of the compound from Example 3 on B16-F10 cells; Figure 20 The graph shows the relative inhibitory activity of different concentrations of the compound from Example 4 on B16-F10 cells; Figure 21 The graph shows the relative inhibitory activity of different concentrations of the compound from Example 5 on B16-F10 cells.

[0107] Examples 1-5 show the effect of compounds on IC50 in B16-F10 cells. 50 See Table 3.

[0108] Table 3 shows the IC50 of compounds in Examples 1-5 on B16-F10 cells. 50

[0109] <![CDATA[IC 50 (μg / mL)]]> 24.25 19.57 51.04 46.30 41.02

[0110] Table 3 shows that B16-F10 cells were significantly inhibited by the compounds in Examples 1-5, and the IC50 of the compounds in Examples 1-5 on B16-F10 cells was [not specified]. 50 The concentrations were 24.25 μg / mL, 19.57 μg / mL, 51.04 μg / mL, 46.30 μg / mL, and 41.02 μg / mL, respectively.

Claims

1. A berberine organic acid salt, characterized in that, Its structural formula is as follows: ； Among them, X - It is a monovalent organic acid anion, which is selected from or In the formula, R is H or OH, and n is 1, 4 or 6.

2. A method for preparing berberine organic acid salt as described in claim 1, characterized in that, Includes the following steps: (1) Berberine hydrochloride is reacted with acetone under strong alkaline conditions to produce berberine acetone. (2) Dissolve the acetone berberine obtained in step (1) in anhydrous ethanol, and add it to X under reflux and stirring. - The corresponding organic acid was reacted for 6-10 h, filtered, dried, and ethyl acetate was added to precipitate. The precipitate was collected by filtration and dried to obtain berberine organic acid salt.

3. The method for preparing berberine organic acid salts as described in claim 2, characterized in that: In step (2), the molar ratio of berberine acetone to organic acid is 1: (1~1.5).

4. The method for preparing berberine organic acid salts as described in claim 2, characterized in that: In step (2), acetone berberine : anhydrous ethanol = 1 mmol : (20~30) mL.

5. The method for preparing berberine organic acid salts as described in claim 2, characterized in that: In step (2), the temperature of the heating reflux is 40~60 ℃.

6. The method for preparing berberine organic acid salts according to any one of claims 2 to 4, characterized in that, Step (1) is as follows: berberine hydrochloride is placed in water, heated and stirred under reflux until completely dissolved, pH is adjusted to 10-13 with inorganic alkali solution, acetone is added, heated and stirred under reflux for 1-2 h, filtered after standing, washed and dried to obtain berberine acetone.

7. The method for preparing berberine organic acid salts as described in claim 6, characterized in that: In step (1), the ratio of berberine hydrochloride to water to acetone is 0.02 mmol to (80~120) mL to (4~10) mL.

8. The method for preparing berberine organic acid salts as described in claim 6, characterized in that: In step (1), the temperature of the heating reflux is 60~70 ℃.

9. The method for preparing berberine organic acid salts as described in claim 6, characterized in that: In step (1), the concentration of the inorganic alkali solution is 0.1~7 mol / L, and the inorganic alkali is sodium hydroxide or potassium hydroxide.

10. The use of the berberine organic acid salt as described in claim 1 in the preparation of antitumor drugs or antibacterial drugs.

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