CN110075147B - Application of Jingdongshan orange total alkaloids - Google Patents
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Abstract
The invention discloses an application of Jingdongshan orange total alkaloids, namely the application of Jingdongshan orange total alkaloids in preparing medicines for preventing or/and treating inflammatory diseases, belonging to the technical field of natural medicine development or medicines; the method comprises the steps of taking the overground part of Jingdong mountain orange as a raw material, ultrasonically extracting the raw material by using a methanol aqueous solution, and treating the raw material by using acid and alkali to obtain a mountain orange total alkaloid part; the invention evaluates the anti-inflammatory activity of the Jingdong orange total alkaloids by an RAW264.7 cell inflammation model induced by LPS, and verifies the in-vivo anti-inflammatory activity by a mouse ear swelling and paw swelling model; the experimental result proves that the Jingdong mountain orange total alkaloids have certain improvement effect on inflammation, can play an anti-inflammatory effect in vivo, can prevent and treat diseases related to inflammation, and has the advantages of easily available plant raw materials, low price and simple alkaloid extraction process; the Jingdongshan orange total alkaloids have good development and application prospects in preparation of medicines for preventing and treating inflammatory diseases.
Description
Technical Field
The invention relates to application of Jingdongshan orange total alkaloids in preparation of medicines for preventing and treating inflammation and related diseases caused by the inflammation, and belongs to the technical field of medicines.
Background
Inflammatory responses are a pathological process that is common in the clinic and are a protective immune response triggered by noxious stimuli and specific conditions such as infection, toxins and tissue damage. However, chronic inflammation is closely associated with the pathogenesis of diseases including atherosclerosis, obesity, asthma, arthritis, cancer, and autoimmune diseases.
Epidemiological demonstrations indicate that more than 25% of cancers are caused by chronic infection or other types of inflammation, and that many factors, such as microbial infection, autoimmune diseases, and other inflammatory diseases (e.g., prostate cancer-related prostatitis), can contribute to the shift of inflammation to cancer. Induction of genomic instability, vascular proliferation, changes in epigenetic status and accelerated cell proliferation all contribute to inflammation-induced carcinogenesis. Excess Reactive oxygen and Reactive nitrogen species (ROS/RNS), abnormally expressed inflammatory cytokines and chemokines, Cyclooxygenase-2 (COX-2) and Nuclear factor-kB (NF-kB) are influencing factors that induce inflammatory carcinogenesis at the molecular level, and inflammation effects neoplastic transformation by regulating the expression of oncogenes and tumor suppressor genes. However, most of the existing drugs with anti-inflammatory, antipyretic and analgesic effects are mainly steroids (steroids) and non-steroids (e.g. aspirin), but most of the drugs cannot eradicate the cause of disease, cannot prevent the development of the course of disease and the occurrence of complications, only have symptomatic treatment effect and have certain side effects. Therefore, the development of a safe medicament for effectively relieving inflammation from natural products has profound significance.
Jingdong mountain orange (1)MelodinuskhasianusHook, f.) belongs to the genus Citrus sinensis of Apocynaceae, and is mainly produced in Yunnan and Guizhou, and is grown in the wet valley forest at an elevation of 1600-2900 m. The whole plant of Jingdongshan orange can be used as medicinal material, has the effects of eliminating internal heat, relieving spasm, dissipating stasis and the like, and is commonly used for traumatic injury and fracture; the Jingdong fructus Citri Junoris is rich in monoterpene indole alkaloid compounds, terpenoid compounds, sterol compounds and volatile oil, and has good regulation and control effect on physiological function of organism; at present, the research on the prevention and treatment of relevant diseases caused by inflammation of the Jingdongshan orange total alkaloids at home and abroad is not reported in patent documents.
Disclosure of Invention
The invention provides a new application of Jingdong mountain orange total alkaloids in medicine development, namely the Jingdong mountain orange total alkaloids are applied to the development of preparing new medicines for preventing or/and treating inflammation and related diseases caused by the inflammation.
The total alkaloids are prepared by ultrasonically extracting pulverized branches and leaves of Jingdongshan orange with 80-90 vol% methanol water solution for 20-30min, filtering, repeatedly ultrasonically extracting the residue for 2-3 times, mixing filtrates, and evaporating for concentration to obtain extract; dissolving the extract in 0.2-0.4% hydrochloric acid aqueous solution, adjusting pH to 2-3, extracting with ethyl acetate, collecting water layer, adding 4-6% ammonia water, adjusting pH to 9-10, extracting with ethyl acetate, collecting ethyl acetate layer, evaporating, concentrating, and lyophilizing.
The medicine for preventing or/and treating inflammation takes the total alkaloids of the Jingdongshan orange as an active ingredient, is used for preparing an anti-inflammatory medicine, and can be added with one or more auxiliary materials acceptable in pharmaceutical preparations or compounded with other active ingredients to play a role of synergistic anti-inflammatory.
The invention is dedicated to the experimental study of the prevention and treatment of inflammation and related diseases caused by the inflammation of the Jingdong mountain orange total alkaloids, and the in vitro and in vivo experiments prove that the Jingdong mountain orange total alkaloids have obvious prevention and treatment effects on the inflammation.
The inventor carries out a series of researches on the prevention and treatment of inflammation and other related diseases of the Jingdongshan orange total alkaloids, and proves that the Jingdongshan orange total alkaloids have the function of preventing inflammation, so that the Jingdongshan orange total alkaloids can be used for preparing and developing new drugs for preventing and treating inflammation and related diseases caused by inflammation; the Jingdongshan orange total alkaloids used for preparing the medicaments for resisting inflammation and related diseases caused by inflammation have the following advantages: the Jingdong mountain oranges are widely distributed in southern Yunnan and Guizhou places in China, and the raw materials are low in price and easy to obtain; the Jingdongshan orange plant is used as a folk medicinal plant, and the whole plant can be used as a medicinal plant; toxicology experimental study shows that the Jingdong mountain orange belongs to a low-toxicity medicinal plant, and is safe and harmless at low concentration; the process flow for extracting and preparing the Jingdongshan orange total alkaloids is simple; at present, related medicaments for treating inflammation are clinically available, and most of the medicaments have certain side effects; however, as a disease with high incidence and more complications, inflammation develops safe drugs from natural products, has wide market demand, considerable prospect and great social and economic effects.
Compared with the prior art, the invention has the following advantages:
1. the invention explores new medical application of the Jingdongshan orange total alkaloids, develops a new research field and expands the value of folk medicinal plants;
2. experiments show that the Jingdong mountain orange total alkaloids can effectively suppress inflammation at a lower dose, are natural plant raw materials and have high safety;
3. the raw materials of the Jingdongshan orange are easy to obtain, the price is low, and the alkaloid extraction process is simple; the Jingdongshan orange total alkaloids have good development and application prospects in preparation of medicines for preventing and treating related diseases caused by inflammation.
Drawings
FIG. 1 shows the toxicity measurement results of Jingdong mountain orange total alkaloids to RAW264.7 cells;
FIG. 2 shows the effect of total alkaloids from Jingdong mountain orange on NO removal in RAW264.7 cells induced by LPS;
FIG. 3 shows the effect of Jingdong mountain orange total alkaloids on the expression of inflammatory factor IL-6 in RAW264.7 cells induced by LPS;
FIG. 4 shows the effect of Jingdong mountain orange total alkaloids on the expression of inflammatory factor TNF-alpha in RAW264.7 cells induced by LPS;
FIG. 5 shows the effect of Jingdongshan orange total alkaloids on xylene-induced ear swelling;
FIG. 6 is H & E staining pattern (H & E X200) of pathological section of ear tissue after xylene-induced ear swelling of mice and dry prognosis of Jingdongshan orange total alkaloids, wherein A is control group; b is a model group; c is dexamethasone group; d is a dosage group of 50mg/kg of total alkaloids of the citrus sinensis; e is a dosage group of 100mg/kg of total alkaloids of the citrus sinensis; f is the group of 200mg/kg dosage of the total alkaloids of the citrus sinensis;
FIG. 7 shows the effect of Jingdong mountain orange total alkaloids on Cara gum-induced paw swelling in mice;
FIG. 8 is a H & E staining pattern (H & E X200) of pathological section of mouse paw after carrageenan-induced paw swelling and dry prognosis of Jingdongshan orange total alkaloids, wherein A is a control group; b is a model group; c is indomethacin group; d is a dosage group of 50mg/kg of total alkaloids of the citrus sinensis; e is a dosage group of 100mg/kg of total alkaloids of the citrus sinensis; f is the group with the dosage of 200mg/kg of the total alkaloids of the citrus aurantium.
Detailed Description
The invention is further illustrated below in conjunction with anti-inflammatory results data at the RAW264.7 cell level and at the in vivo animal level; the examples of these experiments are only for illustrating the present invention and are not intended to limit the scope of the present invention; after reading the description of the present invention, those skilled in the art will be able to make various changes, modifications and alterations equivalent to the embodiments of the present invention without specific indications, which are made from conventional commercial products or reagents prepared by conventional methods, and without specific indications, which are made by conventional methods.
Example 1: preparation of Jingdongshan orange total alkaloid
Collecting aboveground parts of Jingdongshan oranges from Guizhou Bijie, naturally drying and powdering to obtain Jingdongshan orange samples; weighing 100g of Jingdong mountain orange sample, performing ultrasonic extraction for 30min by using a methanol water solution with the volume concentration of 90% according to the proportion that the material-liquid ratio g: mL =1:10, filtering, performing ultrasonic extraction on filter residues for 3 times, combining filtrates, and performing evaporation concentration at 45 ℃ to obtain an extract; dissolving the extract in 0.3 vol% hydrochloric acid water solution, adjusting pH to 2-3, extracting with ethyl acetate, collecting water layer, adding 5 vol% ammonia water, adjusting pH to 9-10, extracting with ethyl acetate, collecting ethyl acetate layer, evaporating, concentrating, and lyophilizing to obtain Total Alkaloids (TAMK).
Example 2: evaluation of anti-inflammatory Activity at RAW264.7 cell level
Establishing a cell inflammation model by using RAW264.7 induced by LPS, and measuring the expression conditions of the total alkaloids of the Jingdong mountain orange on NO and related inflammatory factors in RAW264.7 cells to evaluate the anti-inflammatory activity of the total alkaloids of the Jingdong mountain orange;
1. cell culture and nontoxic dose screening
(1) RAW264.7 cells were seeded in DMEM containing a mixed antibiotic of 10% Fetal Bovine Serum (FBS), 1% penicillin (100U/mL) and streptomycin (100 mg/mL) and cultured in a humidified environment of 5% CO2 and 95% air at 37 ℃;
(2) determining toxicity of Jingdong fructus Citri Junoris total alkaloids on RAW264.7 cell by MTT method, i.e. culturing RAW264.7 cell at a ratio of 1 × 104Culturing in a 96-well plate for 24 h, sucking out the culture medium, adding Jingdongshan orange total alkaloids with different concentrations into the sample group, replacing the culture medium for the blank group, continuously culturing for 20h, sucking out the supernatant after the culture is completed, cleaning for 2 times by PBS, adding 150 mu L of 0.5mg/mL MTT reagent into each well, sucking out the MTT reagent after the culture is performed for 4h, adding 150 mu LDMSO, fully shaking for 10min in the dark, and measuring the light absorption value at 490 nm; according toFormula (II): cell viability = sample/blank x 100%, safe dose was calculated.
2. Evaluation of anti-inflammatory Activity at RAW264.7 cell level
Cultured RAW264.7 cells at 2X 105And (3) connecting the plates/hole in a 6-hole plate, culturing for 24 h, adding different concentrations of Jingdong mountain orange total alkaloids into each hole, continuously culturing for 2h, sucking the culture medium after the culture is finished, cleaning for 2 times by PBS, adding 1 mu g/mL LPS (lipopolysaccharide) again, continuously culturing for 22 h, and taking the supernatant after the culture is finished to measure the expression conditions of NO and related inflammatory factors.
3. Results
3.1 toxicity results of Jingdongshan orange Total alkaloid on RAW264.7 cells
As shown in figure 1, under 50 mug/mL, the total alkaloids of Jingdongshan orange have certain toxicity to RAW264.7 cells, and the cell survival rate is 74.06 +/-5.36, while when the concentrations are 3.125, 6.25, 12.5 and 25 mug/mL, the total alkaloids of Jingdongshan orange have no obvious toxicity to RAW264.7 cells, and the cell survival rates are all more than 90%, so that 3.125, 6.25, 12.5 and 25 mug/mL are selected as experimental concentrations in later experiments to continue the evaluation of the anti-inflammatory activity.
3.2 Activity of Jingdongshan orange Total alkaloid on NO scavenging induced by LPS
NO is released by macrophages and is toxic to bacteria. Due to the effect of NO on the phenotype of inflammatory cells, LPS-stimulated macrophage production of NO is used as a typical marker for common inflammatory responses; as shown in FIG. 2, NO production was significantly increased after LPS treatment compared to the blank group (p<0.05). However, after treatment of the citrus aurantium total alkaloids with different concentrations, the release amount of NO in cells is obviously reduced, and compared with a model group, the citrus aurantium total alkaloids have obvious difference and show certain dose dependence, and the clearing effect is most obvious at the concentration of 25 mug/mL.
3.3 Effect of Jingdongshan orange Total alkaloid on LPS-induced production of relevant inflammatory factors in RAW264.7 cells
As shown in figures 3 and 4, the expression level of proinflammatory factors IL-6 and TNF-alpha in RAW264.7 cells is remarkably increased after LPS inductionp<0.05). However, different concentrations of Citrus aurantium Total alkaloidAfter treatment, the expression conditions of IL-6 and TNF-alpha in cells are obviously inhibited and show a certain dose dependence, and the data show that the total alkaloids of the Jingdongshan orange can exert the anti-inflammatory activity by inhibiting the expression of inflammatory factors.
Example 3: establishing mouse ear swelling model to evaluate anti-inflammatory activity of Jingdongshan orange total alkaloids
Applying xylene for establishing a mouse ear swelling model, and evaluating the anti-inflammatory activity of the Jingdongshan orange total alkaloids by measuring the weight change of mouse ears before and after application;
1. establishment and activity evaluation of mouse ear swelling model
The male Kunming mice are all Kunming mice (18-22 g) with the age of 4-5 weeks, each group comprises 8 mice, and the experimental groups are as follows:
group K: control Group (intragastric with distilled water, K), Group M: model Group (distilled water gavage, M), Group DEX: positive control Group (dexamethasone 5 mg/kg), Group TAMKL: the low dose Group of the Jingdong mountain orange total alkaloids is 50mg/kg, Group TAMKM: the dosage of the Jingdong mountain orange total alkaloids is 100 mg/kg. Group TAMKH: the high-dose group of the Jingdongshan orange total alkaloids is 200 mg/kg.
Mice were acclimated for seven days in standard environment after purchase and were fed on standard feed. Before the first administration, all animals are fasted (without water supply) for 12h, and then the mice are subjected to gavage with the total alkaloid samples of the Jingdongshan orange with different concentrations and dexamethasone according to the weight proportion every day for 14 days; after 14 days of feeding, all mice were fasted for 12H, each mouse was coated with 30. mu.L of xylene on the left ear, the same amount of physiological saline on the right ear, and after 30min, the mice were euthanized with 10% chloral hydrate, and then a portion of the ears of the mice were weighed out using a 6.8mm cork drill, and then immersed in 10% formalin solution for subsequent H & E staining for histopathological analysis.
2. Results
As shown in figure 5, the cloisonne orange total alkaloids weaken the mouse ear swelling induced by xylene in a dose-dependent manner, the swelling degree of a model group is 146% + -8.25, the swelling degree is reduced to 19.48% + -2.14 after dexamethasone intervention, and the mouse ear swelling degree is only 12.87% + -3.41 after the high-dose cloisonne orange total alkaloids dry prognosis, so that the mouse ear swelling degree is basically recovered to be normal; FIG. 6 is a histopathological analysis showing intact ear tissue morphology, uniform cell distribution, and no inflammatory cell infiltration in control mice; in the model group, tissues are obviously swollen, and a large amount of inflammatory cells are infiltrated, however, after the total alkaloids of the Jingdongshan orange with different concentrations are used for prognosis, the ear swelling degree of a mouse is obviously weakened, the inflammatory cell infiltration is reduced, the ear tissue swelling is obviously recovered under high dose, and the effect is superior to that of dexamethasone; therefore, the obtained Jingdongshan orange total alkaloids can obviously improve mouse ear swelling caused by dimethylbenzene and have better effect on dermatitis.
Example 4: establishing a mouse paw swelling model to evaluate the anti-inflammatory activity of the Jingdongshan orange total alkaloids
Injecting carrageenan into feet to establish a mouse paw swelling model, and evaluating the anti-inflammatory activity of the Jingdongshan orange by comparing the swelling degree of the model group with that of the Jingdongshan orange total alkaloid treatment group;
1. establishment and activity evaluation of mouse paw swelling model
The male Kunming mice are all Kunming mice (18-22 g) with the age of 4-5 weeks, each group comprises 8 mice, and the experimental groups are as follows:
group K: control Group (intragastric with distilled water, K), Group M: model Group (distilled water gavage, M), Group IM: positive control Group (indomethacin 5 mg/kg), Group TAMKL: the low dose Group of the Jingdong mountain orange total alkaloids is 50mg/kg, Group TAMKM: the dosage group in the Jingdongshan orange total alkaloids is 100 mg/kg; group TAMKH: the high-dose group of the Jingdongshan orange total alkaloids is 200 mg/kg.
Mice were acclimated for seven days in standard environment after purchase and were fed on standard feed. Before the first administration, all animals are fasted (without water) for 12h, and then the mice are subjected to intragastric gavage on different dose samples and indomethacin according to the weight ratio every day for 14 days; injecting 100 μ L of 1% carrageenan prepared at present into the left sole of each mouse 60min after the last intragastric administration; the paw volume measured before injection was taken as Vo, and the paw volumes at 0.5 h, 1 h, 2h, 4h, 6h, 18h after injection were recorded as Vt with a vernier caliper, and the degree of paw swelling was calculated as follows: foot swelling (%) = (Vt-Vo)/Vo 100%;
after 18H, the mice are anesthetized with 10% chloral hydrate and euthanized, the paw tissues of the mice are taken down, EDTA is added for decalcification, H & E staining is carried out after the decalcification is finished, and histopathological analysis is carried out.
2. Results
The blank mice did not undergo any conditional intervention, and the degrees of paw swelling were all 0, so the results were not shown in fig. 7 and 8; as shown in fig. 7, after injecting carrageenan, the paw swelling degree of the model group mice is continuously increased and is not reduced within 6 h; however, the claw parts of the mice in the group intervened by the total alkaloids of the Jingdongshan orange gradually swell within 2h, and the swelling degree of the claw parts of the mice after 2h shows a reduced trend, and has a significant difference compared with a model group; the swelling reduction degree of the high-dose group is better than that of indometacin, and the high-dose group almost recovers to be normal after 18 h; the histopathological results are shown in fig. 8, no significant damage was seen in the control group (fig. 8A); the capillaries were normal with a very low number of monocytes, suggesting no inflammatory activity. However, after carrageenan injection, the mouse paw was swollen with significant inflammatory cell infiltration and moderate to severe bleeding, i.e. the paw tissue produced inflammatory lesions (fig. 8B); however, after indometacin and samples (TAMK 50mg/kg, 100mg/kg and 200 mg/kg) of Jingdongshan orange with different concentrations are dried, the infiltration of inflammatory cells of paw tissues of mice is reduced, the bleeding condition is weakened, and inflammatory injury is obviously recovered. The total alkaloids of the Jingdong citrus reticulata blanco weaken the mouse paw swelling induced by the carrageenan in a dose-dependent mode.
In summary, the following steps: the total alkaloids of the Jingdong mountain orange can effectively inhibit inflammation, thereby effectively preventing and treating related diseases caused by inflammation. Therefore, the Jingdong mountain orange total alkaloids can be used for preventing and treating inflammation and related diseases caused by the inflammation.
Claims (2)
1. Application of Jingdongshan orange total alkaloids in preparing medicine for preventing or/and treating inflammatory diseases;
the total alkaloids are prepared by ultrasonically extracting crushed branches and leaves of Jingdongshan orange with methanol water solution, filtering, repeatedly ultrasonically extracting filter residues for 2-3 times, mixing filtrates, and evaporating and concentrating to obtain extract; dissolving the extract in 0.2-0.4% hydrochloric acid aqueous solution, adjusting pH to 2-3, extracting with ethyl acetate, collecting water layer, adding 4-6% ammonia water, adjusting pH to 9-10, extracting with ethyl acetate, collecting ethyl acetate layer, evaporating, concentrating, and lyophilizing.
2. Use according to claim 1, characterized in that: ultrasonically extracting the crushed branches and leaves of the Jingdong mountain orange for 20-30min by using a methanol water solution with the volume concentration of 80-90%.
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| Melokhanines A-J, Bioactive Monoterpenoid Indole Alkaloids with Diverse Skeletons from Melodinus khasianus;Gui-Guang Cheng等;《Journal of Natural Products》;20161231;第2158-2166页 * |
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