CN109419797B - Application of euphorbia factor L2 in preparation of medicine for treating rheumatoid arthritis - Google Patents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
- A61K31/235—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids having an aromatic ring attached to a carboxyl group
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/47—Euphorbiaceae (Spurge family), e.g. Ricinus (castorbean)
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Abstract
The invention discloses a separation preparation method of Euphorbia Factor L2(Euphorbia Factor L2) and a new application thereof in treating rheumatoid arthritis, belonging to the field of traditional Chinese medicines. The euphorbia factor L2 is a diterpene alcohol ester compound extracted from a Chinese medicinal material Semen Euphorbiae lathyris (Semen Euphorbiae lathyris), shows good treatment effect on a mouse model of inflammatory arthritis induced by serum transfer, shows that the euphorbia factor L2 has exact effect of resisting osteoarthritis diseases such as rheumatoid arthritis, and can be combined with auxiliary materials to prepare anti-inflammatory diseases such as rheumatoid arthritis or be combined with other medicaments for treating rheumatoid arthritis.
Description
Technical Field
The invention relates to application of an active ingredient euphorbia factor L2 of Chinese medicine moleplant seed in preparing a medicine for treating rheumatoid arthritis, belonging to the field of Chinese medicines.
Background
Rheumatoid Arthritis (RA) is a chronic, autoimmune disease characterized by synovitis that is common clinically. The world health organization data indicates that the global incidence of RA is about 1%, which seriously harms human health and reduces the quality of life. The major pathological changes in RA include synovitis and the destruction of articular bone and cartilage, the latter being the direct cause of progressive rigidity, deformity, dysfunction and even disability of the joints. The treatment of RA is currently mainly aimed at relieving pain, controlling inflammation, improving joint function and protecting organs, and the main measures include drug therapy, immune purification and surgical treatment, among which drug therapy is the most important and most widely used method. Clinically commonly used drugs include non-steroidal anti-inflammatory drugs (e.g., diclofenac), glucocorticoid drugs (e.g., prednisone), immunosuppressive agents (e.g., methotrexate), and biologics (e.g., tollizumab). However, the use of these drugs inevitably causes side effects, non-steroidal anti-inflammatory drugs cause gastrointestinal discomfort and ulcers, hormones cause osteoporosis, opportunistic infections, and the like, and immunosuppressive agents and biological agents comprehensively suppress immunity and easily cause patient infections. Therefore, there is a need for a safe and effective drug for controlling inflammation and protecting joint bones and cartilage.
Euphorbia lathyris L is a dried mature seed of Euphorbiaceae plant Euphorbia lathyris L, and is one of traditional Chinese medicinal materials in China. It is warm in nature and pungent in flavor, has the actions of expelling water, relieving swelling, removing blood stasis and dissipating nodulation, and is mainly used for edema, fullness and distention, phlegm-fluid retention, persistent stagnation, abdominal mass, amenorrhea, scabies, tinea, sore, snake bite and wart. In addition, the moleplant seed is also used as a folk traditional Chinese medicine prescription for treating rheumatic bone pain or used externally for treating traumatic injury and rheumatic arthralgia. Euphorbiae Lathyridis semen contains 40-50% of fatty oil, diterpene ester, coumarin, flavonoid and other compounds, wherein diterpene component is its main active ingredient. Euphorbia Factor L2(Euphorbia Factor L2, EFL2) extracted from Euphorbia lathyris L is diterpene alcohol ester compound with molecular formula C38H42O9Molecular weight is 642.283, and its structure is as follows:
recent pharmacological studies show that the methanol extract of euphorbia lathyris can block the generation of osteoclast caused by RNAKL by inhibiting p38 phosphorylation; the methanol extract of the plant Euphorbia hirta L of the same genus has the function of immunoregulation on T lymphocytes, and can inhibit IFN-gamma, TNF-alpha and IL-2 secretion of Th1 cells; methanol extracts of the plants Erysipelothrix (Euphora thymoli L.) and Erysipelothrix (Euphora coroperi L.) of the same genus also have the effect of inhibiting the release of Nitric Oxide (NO), tumor necrosis factor (TNF-a) and interleukin-6 (IL-6) from Lipopolysaccharide (LPS) -induced BV2 cells and RAW264.7 macrophages. The above information suggests that euphorbia may have anti-inflammatory and immunomodulatory effects, and have potential therapeutic effects in treating rheumatoid arthritis. However, no studies on the application of euphorbia factor L2 as an antirheumatic in animal disease models or clinical applications have been found.
Disclosure of Invention
The invention provides a new application of euphorbia factor L2, namely an application in preparing a medicament for treating rheumatoid arthritis.
Euphorbia factor L2 is diterpene alcohol ester compound extracted from Euphorbia lathyris L.
The preparation method of euphorbia factor L2 in the invention comprises the following steps:
weighing 10kg of semen euphorbiae decoction pieces, adding 10 times of ethanol, heating, refluxing and extracting for two times, each time for 2 hours, filtering, combining the extracting solutions, and concentrating under reduced pressure until no alcohol smell exists; adding appropriate amount of water into the extract to obtain suspension, and sequentially extracting the suspension with petroleum ether, ethyl acetate and n-butanol. The petroleum ether fraction was subjected to silica gel column chromatography, and gradient elution with petroleum ether-ethyl acetate (50: 1-1: 50) gave 3 fractions (Fr.1-Fr.3), respectively. Fr.2(361g) was subjected to silica gel column chromatography and gradient elution with petroleum ether-acetone (20: 1 → 5: 1) to give 8 sites (A1toA8), respectively. A3(32g) was chromatographed on silica gel using a petroleum ether-acetone (20: 1 → 10: 1) gradient to give the compound euphorbia factor L2(520 mg).
Pharmacodynamic experiments prove that the injection of the euphorbia factor L2(40mg/kg) into the abdominal cavity can obviously inhibit the inflammation of joints of mice (a recognized experimental animal model for rheumatoid arthritis) with arthritis induced by serum transfer, reduce the generation of inflammatory factors and inflammatory mediators and prevent the destruction of bone, and the euphorbia factor L2 can be used for treating inflammatory diseases such as rheumatoid arthritis and the like.
Drawings
FIG. 1 is a graph showing the effect of euphorbia factor L2 on joint scores in mice as a model of serum metastasis-induced arthritis
FIG. 2 is a graph showing the effect of euphorbia factor L2 on ankle swelling in serum transfer-induced arthritis model mice
FIG. 3 is a Micro-CT image of normal mouse ankle joint
FIG. 4 is a Micro-CT image of mouse ankle joint of a serum transfer induced arthritis model
FIG. 5 is a Micro-CT image of ankle joint of mice as a model of arthritis induced by serum transfer in euphorbia factor L2 treatment group
FIG. 6 is a graph showing the effect of euphorbia factor L2 on ankle relative bone volume in a serum transfer-induced arthritis model mouse
FIG. 7 is an HE staining light microscope image of a normal mouse joint tissue section (25X)
FIG. 8 is a photograph of a serum transfer induced arthritis model mouse articular histopathological section HE stained light microscope (25X)
FIG. 9 is a photograph of a mouse joint histopathological section HE stained by euphorbia factor L2 (25X) for serum metastasis-induced arthritis model
FIG. 10 shows the effect of euphorbia factor L2 on infiltration of inflammatory cells of joints in mice model arthritis induced by serum transfer
FIG. 11 shows the effect of euphorbia factor L2 on serum transfer induced joint synovial hyperplasia in arthritis model mice
FIG. 12 is a graph showing the effect of euphorbia factor L2 on the release of peripheral blood cytokines from mice in a serum transfer-induced arthritis model
Detailed Description
The invention will be better understood from the following examples. However, the contents of the embodiments are described only for illustrating the present invention, and should not be construed as limiting the present invention described in detail in the claims.
Example 1: pharmacodynamic study of euphorbia factor L2 on treatment of rheumatoid arthritis
1. Experimental methods
Arthritis-susceptible K/BxN model mice were obtained by crossing K/B mice with NOD/Lt mice. Collecting peripheral blood of K/BxN mouse of 6-8 weeks old, centrifuging at 4000rpm for 15min, and preparing serum. 36 male C57BL/6N mice, 8 weeks old, were divided randomly into three groups: normal control group, model group and euphorbia factor L2 treatment group. A Serum-induced arthritis model (SIA) was established by intraperitoneal injection of 150. mu. l K/BxN mouse Serum, and the injection day was recorded as d 0 days. Injection of euphorbia factor L2(40mg/kg) into abdominal cavity is started 1 day before molding, and administration is continued every day until the end of experiment. The thickness of the hind paw and ankle joint of the mouse is measured daily to evaluate the development of arthritis of the mouse, and the degree of joint swelling is scored by a double-blind method according to the following criteria: 0 is unchanged; 1-red swelling of the foot or enlargement of the articular nodules; 2-mild swelling and erythema of the foot; 3 points-marked swelling and erythema of the foot; 4-point-severe swelling or strong straight deformity of the foot. The sum of the clinical scores for the four feet was taken as the arthritis score for each mouse, with the highest score for each mouse being 16. Mice were sacrificed on day 14 post-sensitization and treated as follows: 1) Micro-CT examination was performed on the right ankle joint of the mouse and the relative Bone Volume (Bone Volume/Tissue Volume, BV/TV) was counted. 2) Taking the left knee joint of the mouse, fixing with formaldehyde, decalcifying, embedding in paraffin (5 μm), staining with hematoxylin-eosin (H & E), and sealing with neutral resin. Inflammatory cell infiltration and synovial hyperplasia in joint histopathological changes were evaluated according to the following scoring system: normal score 0; 1 is light; 2 is medium; grade 3 is severe. The highest integral of joint pathology was 3 points per mouse. 3) Collecting peripheral blood of the mouse, standing for 2h, centrifuging at 2000rpm for 15min, and collecting serum. The serum cytokines were detected by reference to the CBA method of BD: mu.l of serum sample or standard was incubated with 50. mu.l of lcappingure bead, 50. mu.l of Detection buffer for 2h, followed by addition of 1ml washing buffer and centrifugation at 200g for 5 min. The supernatant was discarded and 300. mu.l of washing buffer was added for resuspension. Then detecting the expression of cytokines TNF-alpha, IL-6 and IL-17A in the serum.
2. Results of the experiment
(1) Effect of Euphorbia factor L2 on clinical score in serum-induced arthritis model mice
As can be seen from FIGS. 1 and 2, the thickness of the joints and the score of the joints of the mice in the model group are significantly increased compared with those in the control group, and the difference is statistically significant (P < 0.05). And the Knoxia factor L2 treated mice have a significant reduction in joint thickness and joint score (P < 0.05) compared with the model mice. The result shows that the euphorbia factor L2 can obviously reduce the swelling degree of the joints of mice of a serum transfer induced arthritis model and reduce the clinical score of the joints.
(2) Joint imaging detection
As can be seen from FIGS. 3 to 5, the ankle destruction of the model mice was significantly lower than that of the control mice, while the ankle destruction of the euphorbia factor L2-treated mice was significantly lower than that of the model mice, and was not significantly different from that of the control mice. As can be seen from FIG. 6, the ankle relative bone volume (BV/TV) of the model group mice was significantly decreased compared to the control group, while the Knoxia factor L2-treated group was significantly increased compared to the ankle relative bone volume of the model group mice, and the difference was statistically significant (P < 0.05). The results suggest that the euphorbia factor L2 treatment group is effective in protecting the integrity of the joint bone and reducing bone loss and destruction.
(3) Joint pathology detection
As shown in FIGS. 7 to 9, the cartilage of the knee joint of the normal group of mice was covered with 1to 3 layers of normal synovial cells on both sides, and the articular cartilage and bone were not damaged. The synovium of the joints of the mice in the model group has 1-3 layers of hyperplasia into 9-10 layers, and the joints are infiltrated by multiple inflammatory cells. And the synovial hyperplasia of joints of mice in the euphorbia factor L2 treatment group is obviously reduced, and the inflammatory cell infiltration area is obviously reduced compared with that of the mice in the model group. As can be seen from FIG. 10, the arthritis cell infiltration and synovial hyperplasia scores of the mice in the model group are obviously increased compared with those of the normal control group, and the difference has statistical significance (P is less than 0.05). Compared with the model group mice, the euphorbia factor L2 treatment group mice have obviously reduced arthritis cell infiltration scores and synovial hyperplasia scores and have statistical difference (P < 0.05). The results show that the euphorbia factor L2 can effectively inhibit joint synovial hyperplasia, inflammatory cell infiltration and joint cartilage and bone destruction, and has the function of protecting joints.
(4) Cytokine detection
As shown in FIG. 12, the levels of TNF-. alpha.in the sera of three groups of mice were similar and not significantly different (P > 0.05). And the IL-6 and IL-17A levels in the serum of the mice in the SIA group are obviously increased and are respectively 3 times and 2 times of those of the mice in the normal control group. Compared with the SIA group, the euphorbia factor L2 can obviously reduce the level of IL-6 and IL-17A in serum of SIA mice, and the difference has statistical significance (P is less than 0.05). The above results indicate that euphorbia factor L2 exerts an anti-inflammatory effect by reducing the levels of IL-6 and IL-17A in serum of arthritic mice, while having no effect on the secretion of TNF-alpha in serum.
Claims (1)
1. Application of euphorbia factor L2 in preparing medicine for treating rheumatoid arthritis is provided.
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Citations (2)
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| CN105198751A (en) * | 2014-11-28 | 2015-12-30 | 天津耀宇生物技术有限公司 | Method for preparing diterpene ester compound euphorbia factor L2 |
| KR101747775B1 (en) * | 2016-07-20 | 2017-06-16 | 이화여자대학교 산학협력단 | Composition for prevention or treatment of bone disease containing Euphorbia Factor L1 or pharmaceutically acceptable salts thereof as an active ingredient |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105198751A (en) * | 2014-11-28 | 2015-12-30 | 天津耀宇生物技术有限公司 | Method for preparing diterpene ester compound euphorbia factor L2 |
| KR101747775B1 (en) * | 2016-07-20 | 2017-06-16 | 이화여자대학교 산학협력단 | Composition for prevention or treatment of bone disease containing Euphorbia Factor L1 or pharmaceutically acceptable salts thereof as an active ingredient |
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| Title |
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| Euphorbia factor L2 induces apoptosis in A549 cells through the mitochondrial pathway;Lin MT et al.;《Acta Pharmaceutica Sinica B》;20170131;第7卷(第1期);第59-64页 * |
| 千金子化学成分研究进展;刘玉婷等;《中国实验方剂学杂志》;20170731;第23卷(第13期);第220-225页 * |
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