Pharmaceutical composition for treating multiple organ injuries as well as preparation method and application of pharmaceutical composition

Abstract

The invention provides a pharmaceutical composition for treating injury of multiple organs, which is composed of oroxylin glucuronide and protocatechuic acid, and the mass ratio of the oroxylin glucuronide to the protocatechuic acid is (25-93.3): (6.7-75). The invention also provides a preparation method of the pharmaceutical composition. The invention also provides an application of the composition in preparation of an Nrf2 agonist. The invention also provides application of the composition in preparation of medicines for treating multiple organ injuries, wherein the multiple organ injuries include but are not limited to injuries of organs such as liver, kidney, heart and lung. The traditional Chinese medicine composition has remarkable anti-oxidation and anti-inflammatory effects, is particularly suitable for treating multiple organ injuries caused by various reasons, and can effectively protect the functional integrity of multiple organs.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to a pharmaceutical composition, specifically a pharmaceutical composition for treating multiple organ damage, a preparation method thereof and uses thereof. Background Art

[0002] Multiple organ injury (MOI), a severe complication of clinical critical illness, is commonly seen in pathological processes such as trauma, shock, infection, and major surgery. Its pathogenesis is complex, involving multiple pathological processes, including uncontrolled inflammatory responses, imbalanced oxidative stress, and abnormal apoptosis. It is characterized by high morbidity, mortality, and poor prognosis, and represents a major challenge facing modern medicine. Traditional treatments have primarily focused on supportive care and symptomatic management, but the lack of effective interventions targeting the core mechanisms of injury has hindered significant improvement in patient outcomes. In recent years, the nuclear factor E2-related factor 2 (Nrf2) pathway has become a hot topic in MOI treatment research due to its key role in regulating cellular redox homeostasis, anti-inflammatory activity, and anti-apoptosis. Activation of Nrf2 can induce the expression of phase II detoxification enzymes and antioxidant proteins (such as heme oxygenase-1 and glutathione synthetase), effectively scavenging excess reactive oxygen species, inhibiting the release of proinflammatory cytokines, and blocking apoptotic signaling pathways, thereby alleviating MOI. In-depth exploration of the endogenous protective mechanism mediated by Nrf2 will not only provide new therapeutic targets for multi-organ damage, but also bring hope for the development of safer and more effective intervention strategies.

[0003] Nrf2 is a key transcription factor that regulates the expression of numerous antioxidant and detoxification genes. Activation of Nrf2 can enhance cellular antioxidant capacity and mitigate oxidative stress damage, thereby playing a crucial role in the pathogenesis of various diseases. However, existing Nrf2 agonists still have several limitations in clinical application, such as insufficient efficacy and significant side effects. Therefore, the search for more effective and safer Nrf2 agonists is a current research hotspot.

[0004] Both melaleucatin glucuronide and protocatechuic acid have certain antioxidant and anti-inflammatory effects, but there have been no reports of combining these two ingredients to activate Nrf2. Therefore, developing a new combination to enhance Nrf2 activity has important scientific significance and clinical application value. Summary of the Invention

[0005] In response to the above-mentioned technical problems in the prior art, the present invention provides a pharmaceutical composition for treating multiple organ damage, a preparation method thereof, and use thereof. The pharmaceutical composition for treating multiple organ damage, a preparation method thereof, and use thereof are intended to solve the technical problem in the prior art that drugs in treating multiple organ damage are not effective.

[0006] The invention provides a pharmaceutical composition, which consists of melaleuca glucuronide and protocatechuic acid, wherein the mass ratio of the melaleuca glucuronide to the protocatechuic acid is (25-93.3):(6.7-75).

[0007] Furthermore, the mass ratio of the melaleuca alternifolia glucuronide to protocatechuic acid is 93.3:6.7.

[0008] Furthermore, the purity of the melaleuca alternifolia glucuronide is ≥98%, and the purity of the protocatechuic acid is ≥98%.

[0009] Furthermore, it also contains one or more pharmaceutically acceptable excipients. Relative to the total weight of the composition, the mass percentage of the melaleuca alternifolia glucuronide is 5% to 30%, and the mass percentage of the protocatechuic acid is 1% to 10%.

[0010] Specifically, the excipients are lactose, microcrystalline cellulose, magnesium stearate, etc.

[0011] Furthermore, the composition is in the form of tablets, capsules, oral solutions, injections, dialysate, inhalants, syrups or nebulized solutions.

[0012] The present invention also provides a method for preparing the above-mentioned composition, comprising the following steps:

[0013] 1) Weigh the glucuronide and protocatechuic acid of each of the melaleuca alternifolia seeds according to the mass percentage,

[0014] 2) Dissolve the melaleucatin glucuronide and protocatechuic acid in appropriate amounts of solvents to obtain a solution.

[0015] A and solution B.

[0016] 3) Mix solution A and solution B, stir evenly, and obtain a mixed solution.

[0017] 4) Drying the mixed solution to obtain dry powder.

[0018] Furthermore, the dry powder is mixed with auxiliary materials to prepare a desired dosage form, which is a tablet, capsule, oral solution, injection, dialysate, inhalant, syrup or nebulized solution.

[0019] The present invention also provides use of the above composition in preparing an Nrf2 agonist.

[0020] The present invention also provides the use of the above composition in preparing medicine for treating multiple organ damage.

[0021] The present invention also provides the use of the above composition in the preparation of a drug for treating multiple organ damage, wherein the multiple organ damage includes but is not limited to hepatitis, liver fibrosis, cirrhosis, nephritis, renal fibrosis and renal failure.

[0022] Compared with existing technologies, the present invention has significant technical effects and outstanding advantages. The melaleuca alternifolia glucuronide-protocatechuic acid composition provided by the present invention is a highly effective Nrf2 agonist with excellent antioxidant and anti-inflammatory properties. It is particularly suitable for treating cell damage to multiple organs such as the liver and kidney caused by various causes, and has significant efficacy and low side effects. In addition, the present invention also provides a simple and efficient method for preparing the composition, which is easy to industrialize and can flexibly prepare a variety of pharmaceutical dosage forms, and has broad application prospects. DETAILED DESCRIPTION

[0023] Unless otherwise defined, all technical and scientific terms used in this specification are intended to have the same meaning as that generally understood by a skilled expert in the art to which the invention belongs. Generally speaking, the nomenclature used in this specification is the nomenclature well known and commonly used in the art.

[0024] In the Examples of the present invention, the DPPH free radical scavenging effect of melaleucain glucuronide and protocatechuic acid was measured after mixing them at different weight ratios. The results showed that the mixture exhibited a superior antioxidant effect compared to using either melaleucain glucuronide or protocatechuic acid alone. Furthermore, the activation effect of melaleucain glucuronide and protocatechuic acid on Nrf2 was measured after mixing them at different weight ratios. The results showed that the mixture significantly activated Nrf2, exhibiting good antioxidant and anti-inflammatory effects.

[0025] Therefore, one aspect of the present invention relates to a pharmaceutical composition containing melaleuca glucuronide and protocatechuic acid as active ingredients. The melaleuca glucuronide of the present invention has a specific chemical structure and can be directly extracted from plants containing a large amount of melaleuca glucuronide using conventional methods in the art, or prepared from melaleuca using UGT-engineered bacteria. Extracted and refined melaleuca glucuronide can also be purchased for use. Protocatechuic acid can also be obtained commercially.

[0026] In the present invention, when the above-mentioned melaleuca glucuronide and protocatechuic acid are mixed, it is beneficial to maximize the antioxidant and anti-inflammatory effects of melaleuca glucuronide and protocatechuic acid respectively, while meeting the stability of the composition.

[0027] Furthermore, the present invention is characterized in that the weight percentage of the melaleucalyptus glucuronide is 5% to 30% relative to the total weight of the composition, and the weight percentage of the protocatechuic acid is 1% to 10% relative to the total weight of the composition. When the sum of the two active ingredients is less than 6%, the inhibitory effect on oxidative stress and inflammation is weak; when it exceeds 40%, the composition may have solubility or stability issues.

[0028] The pharmaceutical composition of the present invention can be formulated into tablets, capsules, oral solutions, injections, dialysates, inhalants, syrups, nebulized solutions, and the like, but the dosage forms are not particularly limited thereto. Furthermore, the composition of the present invention may contain excipients commonly used in the pharmaceutical field, such as fillers, disintegrants, lubricants, wetting agents, flavoring agents, and preservatives. These excipients are added in amounts commonly used in the pharmaceutical field. The composition of the present invention may also contain substances that promote drug absorption to enhance the therapeutic effect.

[0029] In addition to the above-mentioned substances, the pharmaceutical compositions of the present invention may preferably contain other ingredients that can enhance the main effects, such as other antioxidants, anti-inflammatory agents, etc., within the range that does not affect the main effects. The mixing amounts of the above-mentioned ingredients can be easily selected by those skilled in the art within the range that does not affect the purpose and effects of the present invention. The mixing amounts are 0.01-5% by weight, preferably 0.01-3% by weight, relative to the total weight of the composition.

[0030] While the above details certain aspects of the present invention, it should be understood by those skilled in the art that these specific techniques are merely preferred embodiments and the scope of the present invention is not limited thereto. Therefore, the scope of the present invention is defined by the appended claims and their equivalents.

[0031] [Reference Example 1]

[0032] The melaleuca alternifolia glucuronide used to test the effect of the composition of the present invention was purchased from Shanghai Topu Biopharmaceutical Technology Co., Ltd.

[0033] [Reference Example 2]

[0034] The protocatechuic acid used to test the effect of the composition of the present invention was purchased from Shanghai Bid Pharmaceutical Technology Co., Ltd.

[0035] [Reference Example 3]

[0036] The Nrf2 luciferase reporter gene plasmid used to test the effect of the composition of the present invention was purchased from Jiman Biotechnology (Shanghai) Co., Ltd.

[0037] [Preparation of Examples 1-3 and Comparative Examples 1-3]

[0038] The melaleuca glucuronide of Reference Example 1 and the protocatechuic acid of Reference Example 2 were mixed at the weight ratios shown in Table 1 below, and Examples 1 to 3 were prepared with the same total weight.

[0039] In addition, as a comparative example of the above-mentioned Examples 1-3, the melaleuca glucuronide in Reference Example 1 and the protocatechuic acid in Reference Example 2 were mixed in the weight ratio shown in Table 1 below to prepare Comparative Examples 1-3 with the same total weight as that of Examples 1-3.

[0040] [Table 1] (Unit: weight %)

[0041]

[0042]

[0043] [Test Example 1] Nrf2 activation test

[0044] In order to observe the Nrf2 activation effect of Examples 1-3, which is a mixture of melaleucatin glucuronide and protocatechuic acid, the Nrf2 activation effects of Comparative Examples 1-3 were measured and compared using the Nrf2 luciferase reporter gene method.

[0045] HEK293T cells stably transfected with the Nrf2 luciferase reporter gene plasmid were added to a 96-well microtiter plate containing DMEM medium containing 10% fetal bovine serum, with 10,000 cells per well, and cultured until the coverage reached about 90%. After culturing in serum-free DMEM medium for 24 hours, Examples 1-3 and Comparative Examples 1-3, dissolved in serum-free DMEM medium at the same 10 μM concentration, were treated for 24 hours. Luciferase expression was detected according to the requirements of the luciferase reporter gene test kit. The analysis results are shown in Table 2 below, where the induction fold represents the multiple of the luciferase expression level relative to the solvent control group.

[0046]

Table 2

[0047]

[0048] As can be seen from Table 2, the induction multiples of Examples 1-3 were all higher than that of the solvent control group, indicating that the mixture of melaleucatin glucuronide and protocatechuic acid can activate Nrf2. Specifically, the induction multiple of Example 1 was 1.5, the induction multiple of Example 2 was 1.7, and the induction multiple of Example 3 was 2.1, indicating that the mixture has a certain activation effect on Nrf2.

[0049] Looking at the comparative examples again, the induction fold of comparative example 1 is 1.2, the induction fold of comparative example 2 is 1.4, and the induction fold of comparative example 3 is 1.8. Compared with comparative examples 1-3, the induction folds of Examples 1 and 2 are higher than those of comparative examples 1 and 2, and the induction fold of Example 3 is higher than that of comparative example 1 and 2, and is similar to that of comparative example 3. This shows that the mixture of Examples 1-3 has certain advantages in activating Nrf2, especially in the case of using melaleuca alternifolia glucuronide or protocatechuic acid alone as represented by comparative examples 1 and 2, the activation effect of Examples 1-3 is more significant.

[0050] [Test Example 2] Antioxidant performance test

[0051] In order to verify the antioxidant effect of the mixture of melaleuca alternifolia glucuronide and protocatechuic acid (Examples 1-3), a DPPH free radical scavenging experiment was used to test and compare with Comparative Examples 1-3.

[0052] The experimental steps are as follows: 190 μL of 100 μM (in ethanol) DPPH solution was prepared with the same initial concentrations as Examples 1-3, Comparative Examples 1-3, and the synthetic antioxidant - water-soluble vitamin E (Trolox, positive control group), and diluted to final reaction concentrations of 500 μM, 250 μM, 125 μM, 62.5 μM, 31.25 μM, and 15.63 μM, respectively. 10 μL was added to each of the reaction solutions. After reacting at 37°C for 30 minutes, the absorbance was measured at 540 nm. IC was calculated. 50 The value is the sample concentration required to reduce the absorbance of DPPH radical by 50%.

[0053]

Table 3

[0054]

[0055] It can be seen from the experimental data in Table 3 that this experiment aims to verify the antioxidant effect of the mixture of melaleuca alternifolia glucuronide and protocatechuic acid (Examples 1-3) through DPPH free radical scavenging experiments, and compare it with the use of melaleuca alternifolia glucuronide alone, the use of protocatechuic acid alone, and Comparative Examples 1-3.

[0056] The experimental results showed that the IC of water-soluble vitamin E (positive control group) 50 The value was 53.3 μM, which represents the level of a known strong antioxidant. 50 The values were 231.5μM, 103.7μM and 58.5μM respectively. 50 The value is the lowest, indicating that it has the strongest antioxidant effect, even better than the known antioxidant water-soluble vitamin E.

[0057] Let's look at the comparative example. The IC of comparative example 1 50 The value was 287.5 μM, and the IC 50 The value was 255.8 μM, and the IC of Comparative Example 3 50 The value is 39.2μM. Compared with Comparative Examples 1-3, the IC values of Examples 2 and 3 are 50 The values are lower than those of Comparative Examples 1 and 2. The IC values of Example 3 are 50 The value is lower than that of Comparative Example 3. This shows that the mixture of Examples 1-3 has certain advantages in antioxidant properties, especially when melaleuca alternifolia glucuronide or protocatechuic acid is used alone as represented by Comparative Examples 1 and 2, the antioxidant effects of Examples 1-3 are more significant.

[0058] [Test Example 3] Cell protection performance test

[0059] In order to observe the protective effect of Examples 1-3, a mixture of melaleucatin glucuronide and protocatechuic acid, on liver and kidney cell damage, the protective ability of each was compared and measured with that of Comparative Examples 1-3.

[0060] (1) Hepatocyte injury model: Human hepatocytes (HepG2 cells) were seeded at a density of 10,000 cells per well in a 96-well microtiter plate containing DMEM medium with 10% fetal bovine serum and cultured until the cell coverage reached approximately 90%. Subsequently, the cells were placed in serum-free DMEM medium and cultured for 24 hours. Afterwards, the cells were pretreated with Examples 1-3 and Comparative Examples 1-3 dissolved in serum-free DMEM medium at the same concentration of 10 μM for 6 hours. After the pretreatment was completed, 40 μM cisplatin was added to each group and the treatment was continued for 24 hours to induce cell damage. The cell survival rate was determined by MTT assay, and the results were expressed as a percentage relative to the cell survival level of the untreated group.

[0061] (2) Renal cell injury model: Human kidney cells (HK-2 cells) were seeded at a density of 10,000 cells per well in a 96-well microtiter plate containing DMEM medium with 10% fetal bovine serum and cultured until the cell coverage reached approximately 90%. Subsequently, the cells were placed in serum-free DMEM medium and cultured for 24 hours. The cells were then pretreated with Examples 1-3 and Comparative Examples 1-3 dissolved in serum-free DMEM medium at the same concentration of 10 μM for 6 hours. After the pretreatment was completed, 40 μM cisplatin was added to each group for 24 hours to induce cell damage. The cell survival rate was determined by MTT assay, and the results were expressed as a percentage relative to the cell survival level of the untreated group.

[0062]

Table 4

[0063]

[0064] The experimental results in Table 5 highlight the significant protective effect of the synergistic effect of melaleuca glucuronide and protocatechuic acid on cisplatin-induced liver and kidney cell damage. In the liver cell injury model, the cell survival rate of the cisplatin-treated group was only 28.6%, while the cell survival rates of Examples 1-3 (combinations of melaleuca glucuronide and protocatechuic acid in different ratios) were 49.8%, 60.1% and 68.8%, respectively, which were significantly higher than those of the cisplatin-treated group. In particular, Example 3 (melaleuca glucuronide 93.3% and protocatechuic acid 6.7%) had a cell survival rate that was 40.2 percentage points higher than that of the cisplatin-treated group, showing excellent protective ability. In contrast, the cell survival rates of Comparative Example 1 (using only protocatechuic acid) and Comparative Example 2 (using only melaleuca glucuronide) were 34.0% and 39.7%, respectively, which were much lower than those of Example 3, indicating that the protective effect of a single compound is limited. The cell survival rate of Comparative Example 3 (93.3% of melaleucain glucuronide and 6.7% of protocatechuic acid) was 64.5%, which is close to that of Example 3, but Example 3 is more optimized in terms of component ratio, and the protective effect is more stable and has better dose dependence. In the renal cell injury model, the cell survival rate of the cisplatin-treated group was only 24.5%, while the cell survival rates of Examples 1-3 were 45.0%, 54.7% and 64.7%, respectively, which were also significantly higher than that of the cisplatin-treated group, and as the proportion of melaleucain glucuronide increased, the protective effect gradually increased. The cell survival rates of Comparative Examples 1 and 2 were 30.0% and 34.8%, respectively, and the protective effect was poor, while the cell survival rate of Comparative Example 3 was 61.0%, close to Example 3. This further confirms the synergistic effect of melaleucain glucuronide and protocatechuic acid, among which Example 3 showed the best protective effect in both cell models, which was significantly better than using melaleucain glucuronide or protocatechuic acid alone.

[0065] As described above, melaleucatin glucuronide and protocatechuic acid synergistically demonstrate excellent Nrf2 activation, antioxidant activity, and protection against cisplatin-induced liver and kidney cell damage. Those skilled in the art will appreciate that this synergistic effect is superior to either alone. However, the scope of the present invention is not limited thereto. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

[0066] The pharmaceutical compositions of the present invention can be used in a variety of dosage forms, including but not limited to tablets, capsules, oral solutions, injections, dialysates, inhalants, syrups and nebulized solutions, etc. The essential scope of the present invention should be determined according to the claims and their equivalents.

[0067] The reference ratios of various dosage forms are as follows:

[0068]

Dosage Form Example 1

[0069]

[0070]

Dosage Form Example 2

[0071]

[0072] [Dosage Form Example 3] Oral solution (taking 100 mL as an example)

[0073]

[0074]

[0075] [Dosage Form Example 4] Injection (taking 10 mL as an example)

[0076]

[0077] [Dosage Form Example 5] Dialysis fluid (taking 1L as an example)

[0078]

[0079] [Dosage Form Example 6] Inhalation (taking 10 mL as an example)

[0080]

[0081] [Dosage Form Example 7] Syrup (taking 100 mL as an example)

[0082]

[0083]

[0084] [Dosage Form Example 8] Nebulized solution (taking 10 mL as an example)

[0085]

Claims

1. A pharmaceutical composition, characterized in that The mass ratio of the melaleuca alternifolia glucuronide and protocatechuic acid is (25-93.3): (6.7-75).

2. A pharmaceutical composition according to claim 1, characterized in that The mass ratio of the melaleuca alternifolia glucuronide to protocatechuic acid is 93.3:6.

7.

3. A pharmaceutical composition according to claim 1, characterized in that The purity of the melaleuca alternifolia glucuronide is greater than or equal to 98%, and the purity of the protocatechuic acid is greater than or equal to 98%.

4. A pharmaceutical composition according to claim 1, characterized in that The composition further contains one or more pharmaceutically acceptable excipients. Relative to the total weight of the composition, the mass percentage of the melaleuca alternifolia glucuronide is 5% to 30%, and the mass percentage of the protocatechuic acid is 1% to 10%.

5. A pharmaceutical composition according to claim 1, characterized in that The composition is in the form of tablets, capsules, oral solutions, injections, dialysates, inhalants, syrups or nebulized solutions.

6. The method for preparing the pharmaceutical composition according to claim 1, characterized in that The following steps are involved: 1) Weigh the glucuronide and protocatechuic acid of each of the melaleuca alternifolia seeds according to the mass percentage, 2) Dissolve the melaleuca glucuronide and protocatechuic acid in appropriate amounts of solvents to obtain solution A and solution B, respectively. 3) Mix solution A and solution B, stir evenly, and obtain a mixed solution. 4) Drying the mixed solution to obtain dry powder.

7. The method for preparing a pharmaceutical composition according to claim 5, characterized in that: The dry powder is mixed with auxiliary materials to prepare the desired dosage form, which is tablets, capsules, oral liquids, injections, dialysate, inhalants, syrups or nebulized solutions.

8. Use of the composition according to claim 1 in the preparation of an Nrf2 agonist.

9. Use of the composition according to claim 1 in preparing a medicament for treating multiple organ damage.

10. Use of the composition according to claim 1 in preparing a medicament for treating multiple organ damage.

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