Low-toxicity cortex dictamni extract as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of medicines, and particularly relates to a low-toxicity cortex dictamni extract, a preparation method, a pharmaceutical composition and application. Specifically, according to the low-toxicity cortex dictamni extract, the dictamnine content is lower than 50 ppm, and the fraxinellone content is lower than 250 ppm. The hepatotoxicity of the extract is greatly reduced, and the extract has a good effect of inhibiting release of histamine and beta-Hex, so that the extract can be used for preparing medicines for treating allergic or immune inflammation related diseases. The invention provides the densefruit pittany root-bark extract which is safer and more effective and the preparation method thereof, so that the densefruit pittany root-bark extract has a good application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of medicine, and particularly relates to a low-toxic Dictamnus cortex extract, a preparation method and application thereof. Background Art

[0002] Atopic dermatitis (eczema) is a complex, chronic inflammatory skin disease characterized by dry skin, chronic eczematous lesions, intense itching, and susceptibility to infection. Some patients also have other atopic diseases such as allergic asthma, allergic rhinitis, and allergic conjunctivitis. Mast cells play an important role in the pathogenesis of atopic dermatitis. They are important immune cells that can store and release a variety of bioactive molecules, including histamine, leukotrienes, and other inflammatory mediators. These mediators can cause vasodilation, increase vascular permeability, attract immune cells to sites of inflammation, and participate in the generation of pain and itching. Because mast cells have the ability to resist pathogens and regulate the immune system's inflammatory response, the development of innovative therapeutic strategies targeting mast cells is expected to reduce allergic reactions and immune inflammatory responses, thereby improving patients' symptoms and quality of life.

[0003] Dictamnus dasycarpus is the dried root bark of the Rutaceae herb Dictamnus dasycarpus. The Compendium of Materia Medica records that Dictamnus dasycarpus is "cold and beneficial, bitter and dry in nature, and is an important medicine for various jaundice, rheumatism, and rheumatism." Clinically, it is mainly used to treat a variety of skin diseases such as damp-heat sores, eczema, rubella, scabies, and scabies. However, in actual application, it was found that the intake of Dictamnus dasycarpus or its related compound preparations can cause drug-induced liver injury [Liu Xinyue, Chen Lele, Sun Peng, et al., Chinese Journal of New Drugs, 2023, 32(8)]. Dictamnus dasycarpus contains a variety of furan components such as limonin and furan alkaloids. It is generally believed that the furan components in Dictamnus dasycarpus are the toxic components that cause liver injury (Huang Linyan, Zunyi Medical University Thesis, 2020; Fan QY, et al. Molecules, 2018, 23(10): 2486). Some studies suggest that the furan compounds distillersine and tautomer are the primary active ingredients in Dictamnus cortex that exert its anti-dermatitis effects (Yang Niuniu et al., Journal of Nanjing Medical University (Natural Science Edition), 2023, 43, 1613). This suggests that distillersine and tautomer are both toxic components that cause liver damage and effective ingredients for treating atopic dermatitis. Given this research background, obtaining a Dictamnus cortex extract with high efficacy and low toxicity for treating atopic dermatitis remains a major challenge in modern Dictamnus cortex research.

[0004] The inventors discovered that a Dictamni extract, even after removing distilled water daffodil and tauranga, still exhibits significant antihistamine and anti-β-Hex release activities, with activity far greater than that of components containing distilled water daffodil and tauranga. Therefore, it can be used to treat allergies and atopic dermatitis. Because it removes toxic components that cause liver damage while retaining active ingredients with anti-allergic and atopic dermatitis properties, this extract offers the technical advantage of high efficiency and low toxicity. This extract has broad application prospects in the preparation of low-toxic single-ingredient Dictamni extracts and their pharmaceuticals, as well as compound extracts and their pharmaceuticals containing Dictamni, and is of great significance for improving the safety of traditional Chinese medicines. Summary of the Invention

[0005] The technical problem of the present invention is to provide a low-toxic Dictamnus cortex extract, a preparation method thereof, and use thereof in treating allergic diseases and immune inflammatory diseases.

[0006] This method differs from traditional methods for extracting Dictamnus cortex. Its unique feature is that, through extraction, alcohol precipitation, and adsorption, it specifically removes toxic components in Dictamnus cortex that cause liver damage, while retaining its active ingredients. This patent fully guarantees the safety and effectiveness of Dictamnus cortex in clinical use and has significant application prospects.

[0007] In order to solve the technical problems of the present invention, the present invention provides the following technical solutions:

[0008] The first aspect of the technical solution of the present invention is to provide a low-toxic Dictamnus cortex extract, characterized in that it is extracted and prepared from Dictamnus cortex, wherein the dioscorene content is below 50 ppm and the galactone content is below 250 ppm.

[0009] The content of daunoline and truncatine can be determined using commonly used methods in the art, including but not limited to liquid chromatography, gas chromatography, and capillary electrophoresis chromatography, with liquid chromatography being preferred. Detectors include but are not limited to ultraviolet (UV) detectors, diode array detectors, evaporative light detectors, hydrogen flame detectors, and mass spectrometry detectors, with UV and mass spectrometry detectors being preferred, and mass spectrometry detectors being even more preferred.

[0010] The second aspect of the technical solution of the present invention is to provide a method for preparing a low-toxic dioscorea alkali extract, which is characterized by comprising the following steps:

[0011] 1) Extracting the Dictamnus cortex with water, alcohol or an alcohol-water solution in any proportion and concentrating the extract. The alcohol is preferably ethanol or methanol, more preferably ethanol, more preferably 0-50% ethanol-water solution, and more preferably an aqueous solution.

[0012] The extraction method can be various methods known in the art such as heating, soaking, and percolation. Preferably, heating extraction is used.

[0013] The concentration can be carried out under reduced pressure or at normal pressure. Preferably, the method of under reduced pressure is used, and usually 100 g of the medicinal material is concentrated to 100 ml.

[0014] 2) adding an extraction solvent to the Dictamnus cortex extract in step 1), concentrating the aqueous phase after extraction to obtain a Dictamnus cortex extract with low toxicity.

[0015] The extraction solvent is an organic solvent that is immiscible with water. Organic solvents include, but are not limited to, n-butanol, ethyl acetate, chloroform, dichloromethane, and ether. Furthermore, n-butanol or ethyl acetate is preferably used for extraction.

[0016] Alternatively, a precipitation solvent is added to the Dictamnus cortex extract in step 1), and the precipitate is separated to obtain a Dictamnus cortex extract with low toxicity.

[0017] The precipitation solvent is an organic solvent miscible with water, including but not limited to ethanol, methanol, and acetone. Preferably, the organic solvent is ethanol.

[0018] The final concentration of the precipitation solvent is 50% to 95%, preferably 75% to 95%.

[0019] Alternatively, an adsorbent is added to the Dictamnus cortex extract in step 1), the adsorbent is separated, and then the adsorbent is washed with a 50% to 95% ethanol aqueous solution. The washing solution is combined with the unadsorbed solution and concentrated to obtain a low-toxic Dictamnus cortex extract.

[0020] The adsorbent includes, but is not limited to, reverse-phase adsorbents such as macroporous adsorption resins, microporous adsorption resins, and C1-C18 bonded silica gels. Macroporous resins are preferred. Washing is preferably performed with a 50% to 80% ethanol aqueous solution. Furthermore, the macroporous adsorption resins include, but are not limited to, D101, AB-8, XAD16, HP-20, SP700, and SP800 series.

[0021] A third aspect of this technical solution provides a pharmaceutical composition comprising the low-toxicity Dictamni cortex extract provided in the first aspect and any other drug. The other drug includes, but is not limited to, traditional Chinese medicine, chemical drugs, and biological drugs. Preferably, the composition is combined with any other traditional Chinese medicine to form a traditional Chinese medicine compound, including traditional Chinese medicine powders, traditional Chinese medicine extracts, and traditional Chinese medicine granules. The content of the low-toxicity Dictamni cortex extract in the compound is 0.1% to 99.9%.

[0022] A fourth aspect of the present technical solution provides a pharmaceutical composition. The pharmaceutical composition comprises the low-toxicity Dictamni cortex extract of the first aspect or the pharmaceutical composition of the third aspect and a pharmaceutically acceptable carrier or excipient. The pharmaceutical composition may be in the form of tablets, granules, capsules, powders, pills, suppositories, patches, films, ointments, gels, pastes, drop pills, oral liquids, or suspensions. The pharmaceutical composition may be formulated as a conventional formulation, a sustained-release formulation, a controlled-release formulation, a targeted formulation, or various microparticle delivery systems.

[0023] The fifth aspect of the present technical solution provides the use of the low-toxic Dictamnus cortex extract provided by the first aspect or the pharmaceutical composition provided by the third and fourth aspects in the preparation of drugs for treating allergic or immune inflammatory-related diseases.

[0024] The allergic diseases include: allergic dermatitis, allergic rhinitis, allergic asthma, allergic conjunctivitis, allergic purpura, and allergies caused by food, drugs and environmental factors, etc. Furthermore, allergic dermatitis includes drug rash, contact dermatitis, eczema, urticaria, skin scratches, etc.

[0025] The immune inflammatory diseases include: specific skin diseases, autoimmune diseases, autoinflammatory diseases (such as tumor necrosis factor receptor-associated periodic syndrome), etc. Further, specific skin diseases include atopic dermatitis, urticaria, contact dermatitis, psoriasis, rosacea, etc.; autoimmune diseases include rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, scleroderma, Sjögren's syndrome, ankylosing spondylitis, psoriatic arthritis, thyroiditis, etc.

[0026] Beneficial technical effects:

[0027] The present invention specifically removes the hepatotoxic components in the Dictamnus cortex, retains the effective components, and proves through pharmacological activity experiments and toxicity experiments that the prepared low-toxic Dictamnus cortex extract can greatly reduce its hepatotoxicity and enhance its safety in clinical use while ensuring effectiveness, thus having very important application value. DETAILED DESCRIPTION

[0028] The following examples and pharmacological activity experiments are used to further illustrate the present invention, but they do not mean any limitation of the present invention.

[0029] Comparative Example:

[0030] Add 50 g of Dictamnus cortex to 500 ml of water, soak overnight, heat and extract twice, each time for 30 min, filter, combine the extracts, concentrate under reduced pressure at 50°C, and dry.

[0031] Example 1

[0032] 50g of Dictamnus cortex was added to 500ml of 50% ethanol and heated for extraction twice, each time for 60 minutes. The extracts were filtered, combined, concentrated until alcohol-free, and suspended in 50ml of water. 50ml of ethyl acetate was added and extracted three times. The aqueous phase was concentrated and dried.

[0033] Example 2

[0034] Add 50g of Dictamnus cortex to 500ml of water, soak overnight, and heat extract twice for 30 minutes each time. Filter, combine the extracts, and concentrate under reduced pressure at 60°C to about 50ml. Add 50ml of n-butanol and extract twice. Concentrate the aqueous phase and dry it.

[0035] Example 3

[0036] 50g of Dictamnus cortex was extracted with 400ml, 300ml, and 300ml of water, respectively, by heating three times for 30 minutes each time. The extracts were filtered, combined, and concentrated to approximately 50ml. Anhydrous ethanol (approximately 20 times the volume) was added to the aqueous extract to a final concentration of approximately 95%. The precipitate was dried.

[0037] Example 4

[0038] Add 50g of Dictamnus cortex to 500ml of water and soak overnight. Heat extract twice for 30 minutes each time. Filter, combine the extracts, and concentrate under reduced pressure at 50°C to approximately 50ml. Add 9 times the volume of anhydrous ethanol to the aqueous extract to a final concentration of approximately 90%. Dry the precipitate.

[0039] Example 5

[0040] Add 50g of Dictamnus cortex to 500ml of water and heat extract twice, 60 minutes each time. Filter, combine the extracts, and concentrate under reduced pressure at 60°C to approximately 50ml. Add 4 times the volume of 95% ethanol to the aqueous extract to a final concentration of approximately 75%. Retain the precipitate and dry it.

[0041] Example 6

[0042] Add 50g of Dictamnus cortex to 500ml of water and soak overnight. Heat extract twice for 60 minutes each time, filter, and combine the extracts. Add 50ml of macroporous adsorption resin HP-20 to the extract and adsorb overnight. After filtering, load the resin onto a column and wash the resin with 200ml of 70% ethanol. Combine the unadsorbed liquid and the washing solution, concentrate under reduced pressure at 60°C, and dry.

[0043] Table 1 Relative attenuation extraction rate of Examples 1 to 6 and Comparative Example 1*

[0044]

[0045] *Relative attenuation extraction rate = Example weight / Comparative example weight×100%

[0046] Component analysis experiments and pharmacological experiments:

[0047] Component Analysis:

[0048] Instrument: ACQUITY H-CLASS QDA LC / MS

[0049] Chromatographic conditions: Mobile phase: 0.5% formic acid aqueous solution (A)-methanol (B); Flow rate: 0.3 ml / min; Gradient: 5% B (0 min) → 5% B (2 min) → 80% B (16 min) → 80% B (18 min); Column: CAPCELL PAK-MGⅡC18 (100 × 2.1 mm, 2.0 μm, Osaka soda)

[0050] Mass spectrometry conditions: Scan range: 100-800 m / z; Probe temperature: 400°C; Cone voltage: 15 V (ES+). Selected ions (SIR mode): 200, 233, 455.

[0051] Experimental results: UPLC-MS selected ion detection was used to determine the content of toxic components. The results are shown in Table 2.

[0052] Table 2 Toxic component content in Dictamnus cortex extract (ppm)

[0053]

[0054] Effect Example 1 Inhibits the release of His and β-Hex from mast cells

[0055] Instruments: CO2 incubator (Thermo Scientific); small desktop refrigerated centrifuge (Sigma); fluorescence microplate reader (TriStar 2 S LB 942), clean bench (Beijing Yatailong Instrument Technology Co., Ltd.).

[0056] Reagents: MEM medium (Invitrogen); DMSO (Sigma); anti-DNP-IgE (Biosearch Technologies); Triton X-100 (Dingguo); DNP-BSA (Sigma); 4-nitrophenyl-N-acetyl-β-D-aminoglycosidase (Sigma); Na2CO3 (Beijing Chemical Plant); NaHCO3 (Beijing Chemical Plant); citric acid (Beijing Chemical Plant); hydrochloric acid (Beijing Chemical Plant).

[0057] Preparation of test solution: Dissolve the sample in 1 / 1000 DMSO and dilute to 0.5 mg / ml in culture medium.

[0058] Cell line: Rat basophil leukemia cell line (RBL-2H3), purchased from the Cell Resource Center of Shanghai Institutes for Biological Studies, Chinese Academy of Sciences, cultured in MEM supplemented with 15% fetal bovine serum and 100 U MI -1 Penicillin, 100 U MI -1 Streptomycin, culture in a 37°C, 5% CO2 incubator.

[0059] Cell grouping and dosing: RBL-2H3 cells in the logarithmic growth phase were seeded in 96-well plates at a density of 2 × 104 cells / well. Zero-adjustment, normal, model, lysis, and various dosing groups were set up. MEM complete medium was added to the normal and lysis groups. 200 μL of anti-DNP-IgE prepared in complete medium to a final concentration of 750 ng / mL was added to the model group. The dosing groups were incubated with 200 μL of anti-DNP-IgE containing various drug concentrations and a final concentration of 750 ng / mL, respectively, for 22 hours. The plates were then washed. 200 μL of blank modified benchtop buffer was added to the zero-adjustment and normal groups. 200 μL of 1% Triton X-100 lysis buffer was added to the lysis group. DNP-BSA prepared in modified benchtop buffer to a final concentration of 1 μg / mL was added to the model and various dosing groups. After 2 hours of incubation, the supernatants were collected and the release rates of His and β-Hex were measured.

[0060] Determination of His release rate: 100 μL of supernatant from each well was placed in a black opaque 96-well plate. 20 μL of 1 mol / L NaOH was added to each well, followed by 20 μL of 1% o-phthalaldehyde substrate. The plates were incubated at 37°C for 15 min. The reaction was terminated by adding 20 μL of 1 mol / L hydrochloric acid. The A value of each group was immediately measured using a microplate reader at Em / Ex = 350 / 460, and the His release rate was calculated.

[0061] His release rate = (A 样品上清 -A 调零 ) / (A 裂解 -A 调零 )

[0062] β-Hex release rate determination: 50 μL of the supernatant was added to a new 96-well plate, 50 μL of 4-nitrophenyl-N-acetyl-β-D-aminoglycosidase (1.70 mg / mL) was added to each well, and the plates were incubated at 37°C for 1.5 h. 150 μL of Na2CO3 / NaHCO3 stop solution was added to each well. The A value of each group at 405 nm was measured using a microplate reader, and the β-Hex release rate was calculated.

[0063] β-Hex release rate = (A 样品上清 -A 调零 ) / (A 裂解 -A 调零 )

[0064] Data Statistics and Analysis: Data are expressed as mean ± standard deviation. All results were analyzed by analysis of variance (ANOVA) using IBM SPSS Statistics 26 software. Significance was assessed at ±5% (P < 0.05). The results are shown in Table 3.

[0065] Effect Example 2: Acute toxicity experiment in zebrafish

[0066] Instruments: Z560 aquatic organism rearing system (Aquaneering, USA), BP3100P / BS210 Sartorius electronic balance (Sartorius, USA) and SZX-ILLK2002 aquatic organism microscope (Olympus, Japan)

[0067] Reagents: Calcium chloride (CaCl2) (10005861 Sinopharm Group, China); Sodium chloride (NaCl) (10019318 Sinopharm Group, China); Potassium chloride (KCl) (10016318 Sinopharm Group, China); Magnesium sulfate (MgSO4) (20025118 Sinopharm Group, China)

[0068] Preparation of 1×E3 culture medium: Weigh appropriate amounts of reagents and dissolve them in double-distilled water until completely dissolved. Prepare 10×E3 embryo culture medium with a final concentration of 12.7 mg / L KCl, 36.6 mg / L CaCl2, 39.6 mg / L MgSO4, and 292.7 mg / L NaCl. Dilute 10-fold (1×E3) with double-distilled water before each use and aerate until saturated.

[0069] Preparation of test sample solution: Weigh an appropriate amount of test sample and prepare a concentration gradient solution within the range of 500-5000 mg / L using oxygenated 1×E3 culture medium.

[0070] Experimental Animals: Wild-type AB zebrafish were purchased from Shanghai Feixi Biotechnology Co., Ltd. and maintained in an Aquaneering aquatic system (water temperature 28 ± 1°C, pH 7.0 ± 0.5, conductivity 800-900 μs / cm). All parameters were controlled within the experimental requirements. The lighting conditions in the fish room were 14 h light / 10 h dark, and brine shrimp were fed in the morning and evening.

[0071] Zebrafish Embryo Acquisition and Culture: 1 hour before the end of the first day's photoperiod, separate adult zebrafish in a transparent mating tank with a 1:2 ratio of male to female. Pair at least five tanks to minimize genetic variation. On the second day of photoperiod, mix male and female zebrafish for mating and spawning. Collect eggs approximately 1.5 hours after mating. Rinse eggs with 1× E3 medium and transfer to 188 mm glass aquariums containing 1 L of fresh, oxygenated 1× E3 medium (200-300 eggs per tank). Replace with fresh, oxygenated 1× E3 medium at 24 hpf and promptly remove any dead embryos.

[0072] Toxicity test in zebrafish at 72 hpf: At 48 hpf, healthy, non-deformed zebrafish embryos of the same developmental stage were selected and plated in twelve-well plates. Ten eggs were placed in each well of the twelve-well plate. 2 mL of fresh, oxygenated 1×E3 culture medium was added and cultured until 72 hpf. At 72 hpf, the zebrafish were dosed, the 1×E3 culture medium was removed, and fresh, oxygenated 1×E3 culture medium containing the corresponding concentration of the test solution was added. Ten eggs were placed in three parallel wells for each dose. The total volume of the test solution in each well was 2 mL. The dose concentrations are shown in Table 2. 48 hours after dosing (120 hpf), the mortality rate of each experimental group was observed and recorded under a microscope.

[0073] Data statistics and analysis: IBM SPSS Statistics 21 statistical software was used for data processing and LD 50 The statistical calculation results are shown in Table 3.

[0074] Table 3 Activity and toxicity of Dictamnus cortex extracts

[0075]

[0076] **P<0.01, compared with the model group

[0077] In the anti-His release experiment, it was found that the active extracts of Examples 1 to 5 all had the effect of reducing His release, among which Example 2 was the most obvious. In the β-Hex release rate determination experiment, it was found that Examples 1, 2, 3, and 5 had different degrees of reducing β-Hex release, among which Example 3 had the most obvious reducing effect. In summary, Example 2 had the best effect of inhibiting the release of β-Hex and His at the same time. In the zebrafish acute toxicity evaluation experiment, the LD of Examples 1 to 5 was 50 The Dictamnus cortex extract of the present invention greatly reduces its toxicity while maintaining good activity, thus enhancing the safety of clinical medication and having very important clinical application value.

Claims

1. A low-toxicity Dictamnus cortex extract, characterized in that The extract is prepared by extracting from the bark of Dictamnus chinensis, wherein the content of dittanyine is lower than 50ppm and the content of tautomerone is lower than 250ppm.

2. The low-toxicity Dictamnus cortex extract according to claim 1, wherein The preparation method of removing the toxic components divaricate and truncatine is as follows: The Dictamnus cortex is extracted 2-3 times with an alcohol-water solution, and the extracts are combined; after the extracts are concentrated, they are extracted 2-3 times with an organic solvent immiscible with water, and the aqueous phase is concentrated to obtain a final product. The organic solvent immiscible with water includes n-butanol, ethyl acetate, chloroform, dichloromethane, and ether. or after the extract is concentrated, a precipitation solvent miscible with water is added to a final concentration of 50% to 95%, and the precipitate is separated and dried to obtain the precipitate, wherein the precipitation solvent miscible with water includes ethanol, methanol, and acetone; Alternatively, a reverse phase adsorbent is added to the extract, separated and washed with a 50-95% alcohol aqueous solution, and the unadsorbed liquid and the washing liquid are combined and concentrated to obtain the product. The reverse phase adsorbent includes macroporous adsorption resin, microporous adsorption resin, and C1-C18 bonded silica gel.

3. According to the preparation method of claim 2, the extraction solvent is preferably 0-50% ethanol; the extraction solvent is preferably ethyl acetate or n-butanol; the precipitation solvent is preferably ethanol, and the final concentration is preferably 75%-95%; the adsorbent is preferably a macroporous adsorption resin, and is preferably washed with a 50-80% ethanol aqueous solution.

4. A pharmaceutical composition, characterized in that The pharmaceutical composition contains the low-toxicity Dictamni cortex extract of claim 1 and any other drugs, wherein the any other drugs include traditional Chinese medicine, chemical medicine, and biological medicine.

5. A pharmaceutical composition, characterized in that The pharmaceutical composition contains the low-toxicity Dictamni cortex extract of claim 1 or the pharmaceutical composition of claim 4 and a pharmaceutically acceptable carrier or excipient.

6. The pharmaceutical composition according to claim 5, characterized in that The dosage forms of the pharmaceutical composition include tablets, granules, capsules, powders, pills, suppositories, patches, films, ointments, gels, pastes, pills, oral solutions or suspensions.

7. Use of the low-toxicity Dictamnus cortex extract of claim 1 or the pharmaceutical composition of any one of claims 4 to 6 in the preparation of a medicament for treating allergic diseases or immune inflammatory diseases.

8. The use according to claim 7, wherein the allergic diseases include allergic dermatitis, allergic rhinitis, allergic asthma, allergic conjunctivitis, Henoch-Schonlein purpura, and allergies caused by food, drugs and environmental factors.

9. The use according to claim 7, wherein the immune inflammatory disease comprises specific skin diseases, autoimmune diseases, and autoinflammatory diseases.

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