Anti-hepatitis active compositions, processes for their preparation and use

Abstract

This invention discloses an anti-hepatitis active composition, its preparation method, and its application. The anti-hepatitis active composition, by mass percentage, comprises 70.3±0.4% cucurbitacin B, 26.1±0.2% isocucurbitacin B, and 3.6±0.3% cucurbitacin E. Using widely available sunflower seed shells as raw material, this invention obtains a high-purity, quality-controllable anti-hepatitis active composition by optimizing the extraction solvent, eluent composition, and chromatographic detection methods, ensuring the uniformity and controllability of TCs quality. Compared with cucurbitacin tablets, the anti-hepatitis active composition of this invention has a higher median lethal dose (LD50) and a NOAEL of 15 mg / kg BW, exhibiting higher safety.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical chemistry technology, and relates to an anti-hepatitis active composition, its preparation method, and its application. Background Technology

[0002] The shell of the pine melon seed is the mature fruit shell of the pine melon plant (Cucurbita stenoptera), a member of the Cucurbitaceae family. It is commonly used in traditional Chinese medicine to treat jaundice hepatitis (Yang Jingsheng, initially called Jiangcuo. Diqing Tibetan Medicine [M]. Yunnan Nationalities Publishing House, 1987.). Chemical analysis revealed that it is rich in total cucurbitacins (TCs). Cucurbitacins are a class of tetracyclic triterpenoids widely found in Cucurbitaceae plants, possessing various pharmacological activities such as antitumor, anti-inflammatory, antioxidant, and neuroprotective effects (Varela C, Melim C, Neves BG, et al. Cucurbitacins as potential anticancer agents: new insights on molecular mechanisms [J]. Journal of Translational Medicine, 2022, 20(1): 1-16.). Based on its excellent pharmacological activity, a traditional Chinese medicine, cucurbitacin tablets, was developed from the stalk of the Chinese medicinal melon in the 1980s. Its main components are cucurbitacins (the nominal main components are cucurbitacin B and cucurbitacin E), which are mainly used as adjuvant treatment for hepatitis and primary liver cancer. However, there is very little information about cucurbitacin tablets. Due to the limitations of the technology at the time, there were omissions in the analysis, separation and identification of the main components, and even a lack of necessary safety evaluation data. At the same time, the content of its main components will vary due to the influence of the raw materials and batches, thus affecting the stability of the efficacy (Yang Liling, Wu Tie. Effect of different powdering degree on the extraction rate of three different monomers of ginsenosides [J]. Journal of Changchun University of Traditional Chinese Medicine, 2009, 25(05):669-671. DOI:10.13463 / j.cnki.cczyy.2009.05.066.).

[0003] Currently, there are no studies, either domestically or internationally, on the preparation, chromatographic analysis optimization, and content determination of TCs in wavy sunflower seed shells, nor are there any comparative studies on the efficacy and safety of TCs and cucurbitacin tablets. Summary of the Invention

[0004] One of the objectives of this invention is to provide an anti-hepatitis active composition, which, by mass percentage, comprises the following components: cucurbitacin B 70.3±0.4%, isocucurbitacin B 26.1±0.2% and cucurbitacin E 3.6±0.3%.

[0005] A second objective of this invention is to provide a method for preparing the above-mentioned anti-hepatitis active composition, comprising the following steps:

[0006] Step 1: Crush the dried pine nut shells into powder, then extract the coarse powder with ethanol by reflux 2-3 times, 1-2 hours each time. Combine the filtrates and concentrate under reduced pressure to obtain the ethanol extract.

[0007] Step 2: The ethanol extract was suspended in water and extracted sequentially with petroleum ether and ethyl acetate. The ethyl acetate phase was then eluted with a gradient of petroleum ether-acetone at a volume ratio of 8:2 to 4:6 on a silica gel column. Thin-layer chromatography (TLC) was used with dichloromethane-acetone at a volume ratio of 25:1 as the developing solvent to detect each fraction.

[0008] Step 3: Combine the steps from step 2, using a 254 nm UV lamp to apply R... f The fraction with a concentration of 0.15–0.3 was concentrated under reduced pressure and dried under vacuum to obtain an anti-hepatitis active composition consisting of cucurbitacin B 70.3±0.4%, isocucurbitacin B 26.1±0.2% and cucurbitacin E 3.6±0.3%.

[0009] Preferably, in step 1, the shells of the sunflower seeds are crushed to 30 mesh, and the mass-to-volume ratio of the coarse sunflower seed shell powder to ethanol is 1:10~15, kg:L.

[0010] Preferably, in step 2, the gradient volume ratios of petroleum ether and acetone are 8:2, 7:3, 6:4, 5:5, and 4:6, respectively.

[0011] A third objective of this invention is to provide the application of the above-mentioned anti-hepatitis active composition in the preparation of a drug for treating liver injury.

[0012] Compared with the prior art, the present invention has the following advantages:

[0013] (1) The anti-hepatitis active composition of the present invention has the same composition as cucurbitacin tablets, but there are significant differences between the components. Compared with cucurbitacin tablets (oral LD50 of 14±3 mg / kg BW), the anti-hepatitis active composition of the present invention has a higher median lethal dose (oral LD50 of 36.21 mg / kg BW) and a NOAEL of 15 mg / kg BW, and its safety is higher;

[0014] (2) This invention includes component separation and identification, content determination, histopathological sectioning, biochemical analysis, weight changes during drug administration, and relative liver index. 1 The anti-hepatitis effect of the composition was investigated from multiple aspects, including H NMR identification of biomarkers, LD50, and NOAEL. The results showed that the composition has a significant anti-hepatitis effect, even better than cucurbitacin tablets of the same dose.

[0015] (3) The preparation method of the anti-hepatitis active composition provided by the present invention uses the widely available shells of balsamic melon seeds as raw materials. By optimizing the extraction solvent, eluent composition and chromatographic detection, a high-purity and quality-controllable anti-hepatitis active composition is obtained, ensuring the uniformity and controllability of TCs quality. Attached Figure Description

[0016] Figure 1 High-performance liquid chromatograms and standard curves of the main components in TCs and cucurbitacin tablets.

[0017] Figure 2 The changes in body weight, relative liver index, and serum biochemistry of mice in each group were recorded.

[0018] Figure 3 Pathological sections of liver tissue from mice in each group.

[0019] Figure 4 This is a multivariate analysis diagram of serum extracts.

[0020] Figure 5 This is a multivariate analysis graph of liver extract.

[0021] Figure 6 The curve shows the mortality rate of mice versus dosage. Detailed Implementation

[0022] The present invention will be further described below with reference to specific embodiments and accompanying drawings.

[0023] Example 1

[0024] The specific steps for preparing an anti-hepatitis active composition from sunflower seed shells are as follows:

[0025] (1) The dried sunflower seed shells (7.4 kg) were crushed to 30 mesh using a multi-functional crusher. The resulting coarse powder was extracted twice by reflux with 74 L of ethanol (1:10, w / v), 2 h each time. The two filtrates were combined and concentrated under reduced pressure to obtain 390 g of ethanol extract.

[0026] (2) The ethanol extract was suspended in water and extracted sequentially with petroleum ether and ethyl acetate. The collected ethyl acetate phase (48.5 g) was eluted with a gradient of petroleum ether-acetone (volume ratios of 8:2, 7:3, 6:4, 5:5, and 4:6) on a silica gel column (8 cm × 50 cm). Thin-layer chromatography (TLC) was used to extract the ethyl acetate phase using dichloromethane-acetone (V... 二氯甲烷 V 丙酮 =25:1) is used as the developing solvent to detect each fraction.

[0027] (3) Collect R under a 254 nm UV lamp in step 2f The fraction with a concentration of 0.15–0.3 was concentrated under reduced pressure and thoroughly dried in a vacuum drying oven to obtain 3.42 g of wavy sunflower seed shells (TCs).

[0028] Example 2

[0029] The specific steps for determining the content of TCs in bougainvillea seed shells and the main components of cucurbitacin tablets are as follows:

[0030] (1) Chromatographic conditions: Chromatographic analysis was performed using an InertSustain C18 reversed-phase column (250 mm × 4.6 mm, 5 μm), with acetonitrile-water (1:1, v / v) as the mobile phase, a flow rate of 1 ml / min, a detection wavelength of 230 nm, and a column temperature of 30 °C.

[0031] (2) Content determination: Quantitative analysis was performed using the external standard method. A mixed standard solution containing cucurbitacin B (2.4 mg), isocucurbitacin B (0.64 mg), and cucurbitacin E (0.32 mg) was prepared in a 1 ml volumetric flask. The solution was continuously diluted four times using the doubly diluted method to obtain concentrations of cucurbitacin B of 0.15, 0.3, 0.6, 1.2, and 2.4 mg / mL; isocucurbitacin B of 0.04, 0.08, 0.16, 0.32, and 0.64 mg / mL; and cucurbitacin E of 0.02, 0.04, 0.08, 0.16, and 0.32 mg / mL. 10 μL of each solution was precisely pipetted and injected, and the analysis was repeated three times by HPLC. A standard curve was obtained by plotting the relationship between peak area and concentration. The content of each component in the TCs of the sunflower seed shell and the cucurbitacin tablets was calculated based on the peak area.

[0032] The main components of diced cucurbitacin tablets and cucurbitacin shells are the same, both containing cucurbitacin B, isocucurbitacin B, and cucurbitacin E. The difference lies in the content of each component. Optimized analytical and separation conditions and standard curves are shown below. Figure 1 Cucurbitacin B and isocucurbitacin B could not be completely separated by 65% ​​methanol (Fig. 1(A)a), while the two overlapping isomers could be completely separated by 50% acetonitrile (Fig. 1(A)b, (A)c, and (A)d), with a resolution R > 1.5. Therefore, isocratic elution with 50% acetonitrile was used to determine cucurbitacin B, isocucurbitacin B, and cucurbitacin E in TCs and cucurbitacin tablets (Fig. 1(B)). The contents of cucurbitacin B, isocucurbitacin B, and cucurbitacin E in TCs of bougainvillea seed shells were 70.3±0.4%, 26.1±0.2%, and 3.6±0.3%, respectively, while the contents in cucurbitacin tablets were 41.0±1.1%, 42.8±0.7%, and 16.2±0.6%, respectively. The contents of each component are shown in Table 1.

[0033] Table 1. Determination of the contents of cucurbitacin B, isocucurbitacin B, and cucurbitacin E in TCs from bougainvillea seed shells and cucurbitacin tablets (n = 3)

[0034]

[0035] Research on the components of TCs and cucurbitacin tablets has shown that previous analytical techniques contained errors in the analysis of components in cucurbitacin tablets. Through sophisticated instruments and optimized chromatographic conditions, it was discovered that cucurbitacin tablets contain not only cucurbitacin B and cucurbitacin E, but also the isomer of cucurbitacin B, isocucurbitacin B. The main difference between TCs and cucurbitacin tablets lies in the content of isocucurbitacin B. Therefore, in-depth research on isocucurbitacin B will be more helpful in revealing its pharmacological effects.

[0036] Example 3

[0037] The steps involved in investigating the effects of TCs from pine nut seed shells and cucurbitacin tablets against carbon tetrachloride-induced liver injury in mice are as follows:

[0038] (1) Experimental grouping: After acclimatization in the laboratory environment for 7 days, male ICR mice were randomly divided into 6 groups (n=10 per group): C (blank group), M (model group), P (positive control group), L (low-dose TCs group), E (medium-dose TCs group), and H (high-dose TCs group). Groups L, E, and H were administered TCs (suspended in 0.5% CMC-Na solution) by gavage for 7 consecutive days at doses of 0.1, 0.2, and 0.4 mg / kg body weight, respectively. Mice in group P were given cucurbitacin tablet suspension (equivalent to a medium dose of TCs), while groups C and M were given the same volume of 0.5% CMC-Na solution. Furthermore, the body weight of mice in each group was recorded at 9:00 AM daily. Two hours after administration on day 7, groups M, P, L, E, and H were intraperitoneally injected with 0.3% CCL4 olive oil (10 ml / kg BW), while group C received the same volume of olive oil.

[0039] (2) Histopathological evaluation: Liver tissue was randomly selected from each group, and the liver lobules were quickly fixed with 10% formalin to prepare paraffin sections with 5 μm thin sections for H&E staining. The remaining sections were stored at -80°C for other analyses.

[0040] (3) Biochemical analysis: Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) are two of the most sensitive serum biochemical markers of liver injury. They were analyzed using commercial kits according to the instructions of the commercial companies.

[0041] (4) Liver tissue and serum samples 11H NMR analysis: Frozen liver tissue samples were sliced ​​and rapidly weighed, and homogenized (5 ml / g) in a pre-cooled solvent (50% acetonitrile / 50% H2O, v / v) on an ice bath. The homogenate was centrifuged at 12000 rpm for 10 min at 4°C, and the supernatant was transferred to centrifuge tubes. Acetonitrile was removed using a nitrogen blower. The centrifuge tubes were sealed with sealing film and stored overnight at -80°C. After lyophilization, the samples were stored at -80°C for later use. For 1H NMR analysis, the dried sample was dissolved in 200 μL of 99.8% D2O phosphate buffer (0.2 M, Na2HPO4 and 0.2 M NaH2PO4, pH 7.0) containing 0.05% (w / v) TSP. The solution was vortexed for several seconds and centrifuged at 12000 rpm for 10 min. The supernatant was then transferred to 3 mm NMR tubes for analysis. 1 H NMR analysis.

[0042] The serum sample was slowly thawed on ice, then twice the volume of methanol was added. The mixture was vortexed and allowed to stand at -20°C for 20 min. After centrifugation at 13,000 rpm for 15 min at 4°C, the methanol was removed from the supernatant using a nitrogen blower. The sample was then incubated overnight at -80°C and lyophilized. Subsequent steps were the same as those for NMR analysis on liver tissue.

[0043] (5) Safety assessment of TCs (LD50 and NOAEL): LD50 and NOAEL are two important indicators for assessing drug safety. NOAEL indicates that no experimental animals showed clinical symptoms at this dose. The subjects of this study were female ICR mice (20.0±2 g). The mice were fasted but allowed free access to water overnight (12 hours). The next morning, a single dose of TCs was administered by gavage (20 ml / kg BW) and placed in metabolic cages for the next two weeks, with free access to food and water. The optimized "4-level up and down" method was used to determine the LD50. First, a preliminary experiment was conducted with dose groups of 100, 50, and 25 mg / kg BW, with one mouse in each dose group. As a result, the mice in the 100 and 50 mg / kg BW groups all died. Therefore, the four dose groups were set at 80, 40, 20, and 10 mg / kg BW, respectively, and the "4-level up and down" method was used with 3, 5, 7, and 9 mice, respectively. Prepare a 2% Tween-80 saline solution by adding 0.4 g of Tween-80 to 20 ml of physiological saline. Accurately weigh 30 mg of TCs and add them to 7.5 ml of the above solution to make an initial concentration of 4 mg / ml. Then dilute to concentrations of 2, 1, and 0.5 mg / ml. After gavage administration, observe and record the symptoms of each mouse in detail, including apathy, piloerection, rapid breathing, mild convulsions, half-closed eyes, and survival time. According to the "4-level up and down" procedure, add two more dose groups (30 and 15 mg / kg BW) to obtain more accurate LD50 and NOAEL.

[0044] The effects of TCs and cucurbitacin tablets on reducing transaminase levels and anti-hepatitis in mice were investigated by measuring changes in body weight, relative liver index, and serum biochemical analysis (ALT and AST) during drug administration. Although the body weight and growth rate of group P remained lower than those of other groups throughout the drug administration period, overall, the body weight of mice in all groups showed a continuous upward trend during the 7-day treatment period. Figure 2 a) This indicates that TCs and cucurbitacin tablets at therapeutic doses had almost no effect on feeding behavior in mice. Hepatomegaly was induced by CCl4, with the mean relative liver weight in group M being significantly higher than that in group C (P<0.01), while the relative liver weight in the TCs-treated groups and group P was significantly lower (P<0.01), and there was no significant difference between groups P and E (Figure 2b). CCl4 interfered with hepatocyte membrane structure, leading to a significant increase in serum AST and ALT levels in the CCl4 group (P<0.0001). Figure 2(Figures 2c and 2d). Following TC treatment, ALT and AST levels in groups E and H decreased significantly in a dose-dependent manner (P<0.01 and P<0.0001, respectively) (Figures 2c and 2d). Furthermore, there was no significant difference in ALT and AST levels between groups P and E. These results indicate that TCs can prevent CCl4-induced hepatomegaly in mice.

[0045] The effects of different doses of total liver cells (TCs) and cucurbitacin tablets on reducing liver inflammation were examined microscopically using liver tissue histopathological sections from each group. Histological sections showed that the lobular structure and cellular morphology of group C were normal (Figure 3). Liver tissue sections from group M, particularly around the central vein (indicated by red arrows), showed significant localized hepatocyte necrosis, loss of hepatocyte structure, and inflammatory cell infiltration. Inflammatory cell infiltration was most common in the portal areas, manifested as reduced nuclear volume, loss of cellular structure, or abnormal cell shape. Group P showed considerable improvement in hepatocyte morphology and inflammatory infiltration. Group L had lower perivascular inflammatory cell infiltration than group M. Increasing the TCs dose significantly reduced inflammatory cell infiltration and improved cell morphology, reducing the area of ​​liver tissue necrosis in a dose-dependent manner. Group E performed slightly better than group P, while group H (0.4 mg / kg bw) showed the best hepatoprotective effect.

[0046] Through liver tissue and serum 1 ¹H NMR analysis identified biomarkers of liver injury. After treatment with TCs and cucurbitacin tablets, the effects of these biomarkers on liver injury and their possible mechanisms were explored.

[0047] The major metabolic differences among the groups were investigated using the OPLS-DA model. Groups M and C were clearly separated in serum and liver samples, as shown in Figures 4 and 5. After TC treatment, groups E and H were significantly different from group M, while group H almost overlapped with group C. Group H was better separated from group M compared to group P. These results suggest that TCs reverse CCl4-induced liver injury in a dose-dependent manner.

[0048] In summary, the results indicate that, compared to cucurbitacin tablets, at the same dose concentration (0.2 mg / kg BW), TCs showed a slightly better anti-hepatotoxic effect, and at a high dose (0.4 mg / kg BW), TCs showed an even better anti-hepatotoxic effect than cucurbitacin tablets.

[0049] Example 4

[0050] The safety assessment of TCs in bougainvillea seed shells and cucurbitacin tablets (using LD50 and NOAEL as evaluation indicators) proceeds as follows:

[0051] (1) The improved "4-level up-down" method demonstrated dose-dependent mortality induced by TCs. Figure 6 In this study, all results were expressed as percentage of mouse mortality relative to the dose of TCs used. Based on these data, the oral LD50 of TCs was estimated to be 36.21 mg / kg BW using a nonlinear regression fitting procedure, which is greater than the reported LD50 of the positive control drug rutin tablets (14 ± 3 mg / kg BW).

[0052] Table 2. Mice mortality and corresponding survival time induced by different doses of TCs.

[0053]

[0054] Table 3. Proportion of mice exhibiting adverse symptoms after treatment with different doses of TCs

[0055]

[0056] (2) To determine the highest dose level at which no adverse effect was observed (NOAEL), nine mice were also treated with 15 and 10 mg / kg BW TCs, respectively. All of these mice survived (Table 2) and showed no adverse symptoms within two weeks (Table 3).

Claims

1. Antihepatitis active composition, characterized in that, consists of: cucurbitacin B 70.3±0.4%, isocucurbitacin B 26.1±0.2% and cucurbitacin E 3.6±0.3% by mass percentage; and a preparation method of the anti-hepatitis active composition, comprising the steps of: Step 1, dry the Trichosanthes Kirilowii Maxim. seed coat into coarse powder, then reflux extract the coarse powder with ethanol for 2~3 times, each time for 1~2h, combine the filtrate, and concentrate under reduced pressure to obtain ethanol extract; Step 2, suspend the ethanol extract in water, and extract with petroleum ether and ethyl acetate in turn, then gradient elute the ethyl acetate phase with petroleum ether-acetone in volume ratio of 8:2~4:6 on silica gel column, and detect each flow fraction with thin layer chromatography using dichloromethane-acetone in volume ratio of 25:1 as developing agent; Step 3, combine the flow fractions in step 2 with Rf value of 0.15~0.3 under 254 nm ultraviolet light, concentrate under reduced pressure, and vacuum dry to obtain the anti-hepatitis active composition consisting of cucurbitacin B 70.3±0.4%, isocucurbitacin B 26.1±0.2% and cucurbitacin E 3.6±0.3%.

2. The antihepatitis active composition according to claim 1, characterized in that, In step 1, the Trichosanthes Kirilowii Maxim. seed coat is crushed to 30 mesh, and the mass-volume ratio of the Trichosanthes Kirilowii Maxim. seed coat coarse powder and ethanol is 1 kg:10~15 L.

3. The antihepatitis active composition according to claim 1, characterized in that, In step 2, the gradient volume ratio of petroleum ether-acetone is 8:2, 7:3, 6:4, 5:5, 4:6 in turn.

4. The anti-hepatitis active composition according to any one of claims 1~3 in the preparation of a liver damage treatment drug.

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