A lotus seed alkaloid glycyrrhizic acid composition and its application

Abstract

The invention discloses a lotus seed alkaloid glycyrrhizic acid composition and application thereof. The effective ingredients mainly include lotus seed alkaloid and glycyrrhizic acid. The lotus seed alkaloid is made into a solid dispersion to improve the solubility, and then combined with glycyrrhizic acid to show stronger anti-inflammatory, antibacterial and acute lung injury therapeutic effects, thereby effectively broadening and improving the application scope of lotus seed alkaloid and glycyrrhizic acid in the medical field.

Description

Technical Field

[0001] The invention relates to the field of glycyrrhizic acid compositions, in particular to a lotus seed alkaloid glycyrrhizic acid composition and application thereof. Background Art

[0002] Traditional medicine is an important source of new molecules in modern drug development. There is great potential for exploring new drugs for the treatment of inflammatory diseases in Chinese herbal medicines or their active ingredients. Lianxin alkaloids are isoquinoline alkaloids extracted from the Chinese herbal medicine lotus seed core. They have antibacterial, antihypertensive, antiarrhythmic effects, and can block adrenal α receptors and inhibit intracellular calcium release. Lianxin alkaloids were initially shown to have antioxidant effects in the rat cerebral ischemia reperfusion model. It was also found that it could inhibit the expression of inflammatory factors TNF-α and IL-1 after rat cerebral ischemia reperfusion. The researchers further established an LPS-induced RAW264.7 macrophage model to prove its anti-inflammatory and antioxidant effects. A large number of experiments have shown that Lianxin alkaloids can reduce protein exudation, reduce pulmonary microvascular permeability, inhibit the formation of pulmonary edema, and can effectively reduce the inflammatory response of LPS-induced acute lung injury. Glycyrrhizic acid is one of the ingredients extracted from the root of licorice. It is the main active ingredient in licorice. It is composed of 18-glycyrrhetinic acid and two molecules of glucuronic acid. It has anti-inflammatory, anti-apoptotic and antiviral effects. In the LPS-stimulated macrophage model, glycyrrhizic acid can effectively inhibit the inflammatory response by inhibiting NF-κB. The researchers confirmed through experiments that glycyrrhizic acid can improve the general activity of mice with LPS-induced acute lung injury model, reduce LPS-induced pulmonary edema, reduce alveolar hemorrhage and inflammatory cell infiltration, and inhibit the development of LPS-induced pulmonary interstitial edema. Glycyrrhizic acid has a protective effect on LPS-induced acute lung injury. Summary of the invention

[0003] Purpose of the invention: In order to overcome the problems existing in the prior art, the present invention proposes a lotus seed alkaloid glycyrrhizic acid composition, which shows stronger anti-inflammatory, antibacterial and acute lung injury treatment effects by making the lotus seed alkaloid into a solid dispersion to improve the solubility and then combining it with glycyrrhizic acid.

[0004] In order to solve the above technical problems, the technical solution adopted by the present invention is: a lotus pith alkaloid glycyrrhizic acid composition, comprising the following components in parts by weight: 1-20 parts of lotus pith alkaloid solid dispersion and 1-20 parts of glycyrrhizic acid.

[0005] Furthermore, the lotus seed alkaloid solid dispersion comprises the following components by weight: 5-50% lotus seed alkaloid and the rest is carrier material.

[0006] Furthermore, the preparation method of the lotus seed alkaloid solid dispersion is as follows: mix the lotus seed alkaloid and the carrier material, dissolve them in ethanol, and concentrate them to dryness under reduced pressure at 40-70°C.

[0007] Furthermore, the extraction method of the lotus seed alkaloids is as follows: take the commercially available Hunan lotus seed heart, crush it through an 80-mesh sieve, weigh a certain amount, add 10-20 times 80% ethanol solution, soak for 10-60 minutes, ultrasonically extract for 10-40 minutes, filter, and distill under reduced pressure to obtain an extract. The extract is suspended in water, applied to a D101 macroporous adsorption resin column, first eluted with 20% ethanol, and the eluent is discarded; then eluted with 80% ethanol at pH 2-10, the eluent is collected, concentrated and spin-dried to obtain the lotus seed alkaloids.

[0008] Furthermore, the carrier material is selected from one or a combination of polyvinyl pyrrolidone (PVP) and polyethylene glycol (PEG).

[0009] Furthermore, the lotus seed alkaloid glycyrrhizic acid composition also includes other pharmaceutical excipients.

[0010] Furthermore, the other pharmaceutical excipients are one or a mixture of sodium chloride, anhydrous sodium sulfite, disodium edetate, cysteine ​​hydrochloride, glycine, and mannitol.

[0011] The invention also discloses the application of the lotus seed alkaloid glycyrrhizic acid composition, specifically the application in anti-inflammatory drugs.

[0012] The invention also discloses the application of the lotus seed alkaloid glycyrrhizic acid composition, specifically the application in antibacterial drugs.

[0013] The invention also discloses the application of the lotus seed alkaloid glycyrrhizic acid composition, specifically the application in medicine for preventing and treating acute lung injury.

[0014] Beneficial effects:

[0015] 1) In many studies on lotus seed alkaloids or glycyrrhizic acid at home and abroad, the experimenters have proved that lotus seed alkaloids or glycyrrhizic acid have therapeutic effects on acute lung injury by establishing experiments at the cellular level and animal models such as LPS-induced RAW264.7 macrophages and LPS-induced mouse acute lung injury models.

[0016] 2) The present invention prepares the lotus seed alkaloid into a solid dispersion to improve its solubility, and then combines it with glycyrrhizic acid, and establishes cell experiments and animal model experiments. The results show stronger anti-inflammatory, antibacterial and acute lung injury therapeutic effects, verifying that the combination of the lotus seed alkaloid dispersion and glycyrrhizic acid has a certain synergistic effect, effectively broadening and improving the application scope of the lotus seed alkaloid and glycyrrhizic acid in the medical field. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a schematic diagram of the dissolution test results of the lotus seed alkaloid solid dispersion prepared in Example 1-4;

[0018] Figure 2 This is a schematic diagram of the dissolution test results of the lotus seed alkaloid solid dispersion prepared in Examples 5-8. DETAILED DESCRIPTION

[0019] The present invention is further described in detail below in conjunction with embodiments:

[0020] 1. Examples 1-8 are the preparation of solid dispersions of lotus seed alkaloids.

[0021] Example 1: Accurately weigh 1 g of lotus seed alkaloids and 4 g of PVP-K30, mix them, dissolve them in ethanol 20 times the total weight of the solid, and concentrate them under reduced pressure at 55° C. to dryness.

[0022] Example 2: Accurately weigh 1 g of lotus seed alkaloids and 1 g of PVP-K30, mix them, dissolve them in ethanol 20 times the total weight of the solid, and concentrate them under reduced pressure at 55° C. to dryness.

[0023] Example 3: Accurately weigh 1 g of lotus seed alkaloids and 19 g of PVP-K30, mix them, dissolve them in ethanol 20 times the total weight of the solid, and concentrate them under reduced pressure at 55° C. to dryness.

[0024] The preparation method of Examples 4-8 is the same as that of Examples 1-3, except for the amount of lotus seed alkaloid and carrier material used.

[0025] In Example 4, the amount of lotus seed alkaloids used is 1 g, and the carrier material is PVP-K30, and the amount used is 9 g;

[0026] In Example 5, the amount of lotus seed alkaloids used is 1 g, and the carrier material is PEG-4000, and the amount used is 4 g;

[0027] In Example 6, the amount of lotus seed alkaloids used is 1 g, and the carrier material is PEG-4000, and the amount used is 1 g;

[0028] In Example 7, the amount of lotus seed alkaloids used is 1 g, and the carrier material is PEG-4000, and the amount used is 19 g;

[0029] In Example 8, the amount of lotus seed alkaloids used is 1 g, and the carrier material is PEG-4000, and the amount used is 9 g.

[0030] 2. Dissolution test experiment:

[0031] The solid dispersion of lotus seed alkaloid prepared in Examples 1-8 was tested for dissolution as follows:

[0032] Take the sample to be tested, use pH 6.8 phosphate buffer as the dissolution medium, the dissolution medium is 1L, and determine the dissolution rate according to the slurry method in the dissolution determination method (Appendix XC Method 2 of Part II of the 2010 edition of the Chinese Pharmacopoeia). The speed is 100r / min, and 7mL of samples are taken at 0, 3, 8, 10, 20, 30, and 45min respectively (the same amount of dissolution medium is immediately added after taking), pass through a 0.45μm microporous filter membrane, and take the filtrate as the test sample. Determine its absorbance value at 282nm. The cumulative dissolution percentage is calculated as follows:

[0033] q(%)=[Cq×V1+(C1+C2+…+Cq-1)×V2] / M / 1000×100%

[0034] Where q (%): cumulative dissolution percentage at the qth sampling point;

[0035] Cq: drug concentration at the qth sampling point;

[0036] V1: total volume of dissolution medium, V1 = 1000 mL;

[0037] V2: The volume of each sample, V2 = 7 mL

[0038] M: The amount of lotus seed alkaloids contained in the weighed sample.

[0039] The prepared lotus seed alkaloid dispersion was tested using this method, and the results are as follows: Figure 1 and Figure 2 As shown; from the experimental data, it can be seen that after the lotus seed alkaloids are made into solid dispersions, the cumulative dissolution rate increases significantly. Comparing the cumulative dissolution rate curves of the lotus seed alkaloid raw materials and the dispersions under different conditions, it can be seen that the maximum value is basically reached at 45 minutes; with PVP-K30 as the carrier, the dissolution performance of the dispersion is significantly better than PEG-4000; the optimal condition is lotus seed alkaloid: PVP-K30 = 1:4, that is, 20% concentration of lotus seed alkaloids.

[0040] 3. Examples 9-11 are preparations of lotus seed alkaloid glycyrrhizic acid compositions;

[0041] Embodiment 9:

[0042] 13 g of the lotus seed alkaloid solid dispersion prepared in Example 1 was accurately weighed and mixed with 17 g of glycyrrhizic acid to obtain the solid dispersion.

[0043] In Examples 10-11, the weight ratio of each component of the Chinese medicine monomer is as follows. Comparative Examples 1 and 2 are also provided, in which the same weight of lotus seed alkaloids and glycyrrhizic acid are used separately, as shown in Table 1:

[0044] Table 1 Proportions of Examples 10-11 and Comparative Examples 1-2

[0045] Ingredients (g) Example 10 Embodiment 11 Comparative Example 1 Comparative Example 2 Lotus seed alkaloid dispersion 5 25 30 0 Glycyrrhizic acid 25 5 0 30

[0046] IV. Application efficacy experiment

[0047] 1. Anti-inflammatory activity application

[0048] Experimental cells: RAW264.7 mouse cells.

[0049] Cell culture: The revived RAW264.7 cells were cultured in high-glucose DMEM containing 12% fetal bovine serum (FBS) and 1% double-antibody (100 U / mL penicillin and 100 U / mL streptomycin) and placed in a 5% CO2, 37°C incubator.

[0050] Reagent preparation:

[0051] Lotus seed alkaloid glycyrrhizic acid composition: 10 mL of DMSO was added to 1000 mg of the lotus seed alkaloid glycyrrhizic acid composition to dissolve it into a 100 mg / mL mother solution, which was then dispensed into 2 mL EP tubes and stored at -80°C for later use. During the experiment, it was diluted with PBS to the required concentration.

[0052] 1.1 Detection of inflammatory mediators such as NO and PGE2

[0053] 1) Detection of NO content:

[0054] ① RAW264.7 cells were cultured at 1×10 4 Cells / well were seeded in 96-well plates and incubated for 24 h.

[0055] ② Treat the cells with the lotus seed alkaloid glycyrrhizic acid composition (50 μg / mL) for 2 h, and then stimulate with 2 μg / mL LPS for 24 h.

[0056] ③Take the cell culture medium, centrifuge at 2000r / min for 10min, and collect the supernatant.

[0057] ④ Add 50 μL of room temperature Griess Reagent I and Griess Reagent II to each well.

[0058] ⑤ Gently shake the 96-well plate. After sufficient reaction, detect the OD value of each well at a wavelength of 540 nm.

[0059] 2) Detection of PGE2 content:

[0060] ① RAW264.7 cells were cultured at 1×10 4 Cells / well were seeded in 96-well plates and incubated for 24 h.

[0061] ② Treat the cells with the lotus seed alkaloid glycyrrhizic acid composition (50 μg / mL) for 2 h, and then stimulate with 2 μg / mL LPS for 24 h.

[0062] ③ Take the cell culture medium, centrifuge it at 2000r / min for 10min, and collect the supernatant. The concentration of PGE2 in the culture medium is detected by a specific enzyme-linked immunosorbent (ELISA).

[0063] 1.2 Detection of inflammatory cytokines such as TNF-α, IL-6, and IL-1β

[0064] ① RAW264.7 cells were cultured at 1×10 4 Cells / well were seeded in 96-well plates and incubated for 24 h.

[0065] ② Treat the cells with the lotus seed alkaloid glycyrrhizic acid composition (50 μg / mL) for 2 h, and then stimulate with 2 μg / mL LPS for 24 h.

[0066] ③ Take the cell supernatant, centrifuge at 2000r / min for 10min, and collect the culture medium. Detect the contents of inflammatory factors TNF-α, IL-6, and IL-1β according to the instructions of the ELISA kit.

[0067] 1.3 Test results

[0068] Table 2 shows the effects of the lotus seed alkaloid glycyrrhizic acid composition on the contents of inflammatory mediators NO and PEG2 in LPS-induced RAW264.7 macrophages.

[0069] Table 2 Test results of examples and comparative examples

[0070] Group NO(μM) PEG2 (pg / mL) Blank Group 1.06±0.04 4.55±0.23 LPS group 9.01±0.43 39.72±2.45 Example 9 2.98±0.15 11.93±0.57 Example 10 4.02±0.19 16.86±0.84 Embodiment 11 3.85±0.19 16.14±0.77 Comparative Example 1 4.68±0.20 17.39±0.83 Comparative Example 2 6.36±0.25 19.10±0.91

[0071] By detecting the contents of inflammatory mediators NO and PGE2, it was found that LPS treatment can significantly increase the contents of NO and PGE2, and the difference is statistically significant compared with the blank group (P < 0.05). The lotus seed alkaloid glycyrrhizic acid combination can significantly reduce the contents of NO and PGE2 after treatment, and Example 1 has the best effect, and the difference is statistically significant compared with the LPS group. The effects of the lotus seed alkaloid and glycyrrhizic acid treatment alone in Comparative Examples 1 and 2 on reducing NO and PGE2 are obviously not as good as the combined synergistic effect of Examples 9-11.

[0072] Table 4 shows the effects of the lotus seed alkaloid glycyrrhizic acid composition on the contents of inflammatory cytokines TNF-α, IL-6 and IL-1β in RAW264.7 macrophages induced by LPS.

[0073] Table 3 Test results of examples and comparative examples

[0074]

[0075]

[0076] ELISA test found that LPS treatment can significantly increase the content of TNF-α, IL-6 and IL-1β in the cell culture supernatant, and the difference is statistically significant compared with the blank group (P < 0.05); while the lotus seed alkaloid glycyrrhizic acid combination can significantly reduce the content of TNF-α, IL-6 and IL-1β in the cell culture supernatant after treatment, and the difference is statistically significant compared with the LPS group (P < 0.05). The effect of reducing the content of TNF-α, IL-6 and IL-1β in the supernatant by the treatment of lotus seed alkaloids and glycyrrhizic acid alone in Comparative Examples 1 and 2 is obviously not as good as the combined synergistic effect of Examples 9-11.

[0077] The above results indicate that the lotus seed alkaloid glycyrrhizic acid composition has an anti-inflammatory effect on LPS-induced RAW264.7 cells.

[0078] 2. Establishment of LPS-induced acute lung injury model in mice

[0079] 80 mice were randomly divided into eight groups, including a normal control group, a model group (LPS), Examples 9-11, and Comparative Examples 1-2, with 10 mice in each group. After fasting for 12 hours, the mice were injected with physiological saline, the composition prepared in the example (8 mg / kg), and dexamethasone (mg / kg) through the tail vein. One hour after administration, the mice were anesthetized with sodium pentobarbital, and LPS was dripped into the nostrils of the mice (10 μg LPS, dissolved with 50 μL PBS), and 50 μL PBS was dripped into the nostrils of the mice in the normal group. The mice were killed 12 hours later.

[0080] After the mice were killed, the trachea was exposed and a catheter was inserted into the trachea. A 1 mL syringe was used to draw 0.5 mL of PBS for lavage. The BALF was collected by lavage three times. A portion of the BALF was taken to detect the protein content using the BCA protein kit. The remaining BALF was quickly centrifuged at 4°C, 1400 r / min, and 10 min. The supernatant was stored at -20°C and the content of inflammatory factors TNF-α, IL-6, and IL-1β was detected according to the ELISA kit instructions.

[0081] Table 4 shows the effects of the lotus seed alkaloid glycyrrhizic acid composition on the protein concentration and the contents of TNF-α, IL-6, and IL-1β in BALF.

[0082] Table 4 Test results of examples and comparative examples

[0083]

[0084]

[0085] The results of detecting the protein concentration in BALF by BCA kit showed that the protein concentration in the LPS group was significantly higher than that in the normal control group, while that in the lotus seed alkaloid glycyrrhizic acid composition group was lower than that in the LPS group, and the effect of Example 1 was the best, P<0.05.

[0086] ELISA test results showed that the contents of TNF-α, IL-6 and IL-1β in BALF of LPS group were significantly higher than those of normal control group, while those of lotus seed alkaloid glycyrrhizic acid composition group were lower than those of LPS group, and Example 1 had the best effect, all P<0.05.

[0087] The above experimental data show that the lotus seed alkaloid glycyrrhizic acid composition can effectively alleviate the LPS-induced ALI inflammatory response, reduce protein exudation, and reduce the release of inflammatory cytokines.

[0088] 3. Antibacterial experiment

[0089] Preparation of culture medium: Use beef extract peptone culture medium, including 4g beef extract, 2g peptone, 2g NaCl, 400mL distilled water, pH 7.0-7.5, sterilize for 20min, cool and set aside.

[0090] The test bacteria were inoculated into sterilized culture medium, cultured at 30°C for 24 hours, and then diluted with sterile saline. The concentration of each test bacteria was approximately 107-108 CFU / mL.

[0091] The samples of Examples 9-11 and Comparative Examples 1-2 were dissolved in a certain amount of sterile water and added to the culture medium by a two-fold dilution method. The diluted test bacteria were then inoculated into each test tube and cultured in a 37°C incubator for 24 hours. The highest dilution with completely sterile growth was taken as the minimum inhibitory concentration (MIC) to examine the antibacterial effect of the composition.

[0092] The antibacterial results of the composition are shown in Table 5.

[0093] Table 5 Test results of examples and comparative examples

[0094]

[0095] The experimental results show that the lotus seed alkaloid glycyrrhizic acid composition has a good inhibitory effect on Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Bacillus subtilis, and the minimum inhibitory concentration is less than 0.25 mg / mL.

[0096] Further analysis of the efficacy experiments in the above three directions revealed that both lotus seed alkaloids and glycyrrhizic acid alone have certain anti-inflammatory, antibacterial, and acute lung injury therapeutic activities, while the combination of the two has better performance, with the best effect at the ratio of Example 9, indicating that the combination of lotus seed alkaloids and glycyrrhizic acid exhibits a synergistic effect.

[0097] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A lotus seed alkaloid glycyrrhizic acid composition for preventing or treating acute lung injury, characterized in that The invention comprises the following components in parts by weight: 13 parts of lotus seed alkaloid solid dispersion and 17 parts of glycyrrhizic acid; the lotus seed alkaloid solid dispersion comprises the following components by weight: 5-50% of lotus seed alkaloid and the rest of the components are carrier materials; the preparation method of the lotus seed alkaloid solid dispersion is as follows: the lotus seed alkaloid and the carrier material are mixed, dissolved in ethanol, and concentrated to dryness under reduced pressure at 40-70°C; the extraction method of the lotus seed alkaloid is as follows: the commercially available Hunan lotus seed core is crushed and passed through an 80-mesh sieve, a certain amount is weighed, 10-20 times of 80% ethanol solution is added, soaked for 10-60 minutes, ultrasonically extracted for 10-40 minutes, filtered, and distilled under reduced pressure to obtain an extract; the extract is suspended in water, applied to a D101 macroporous adsorption resin column, first eluted with 20% ethanol, and the eluent is discarded; then eluted with 80% ethanol at pH 2-10, the eluent is collected, concentrated and spin-dried to obtain the lotus seed alkaloid.

2. The lotus seed alkaloid glycyrrhizic acid composition for preventing or treating acute lung injury according to claim 1, characterized in that: The carrier material is selected from polyvinyl pyrrolidone (PVP) and polyethylene glycol (PEG), or a combination of the two.

3. The lotus seed alkaloid glycyrrhizic acid composition for preventing or treating acute lung injury according to claim 1, characterized in that It also includes other pharmaceutical excipients.

4. The lotus seed alkaloid glycyrrhizic acid composition for preventing or treating acute lung injury according to claim 1, characterized in that: The other pharmaceutical excipients are one or a mixture of sodium chloride, anhydrous sodium sulfite, disodium edetate, cysteine ​​hydrochloride, glycine, and mannitol.

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