刺山柑提取物对试验鼠抗炎镇痛、微循环和实验性关节炎的作用
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摘要
本研究以新疆吐鲁番野生刺山柑果实为原料，通过正交设计和树脂吸附-洗提试验，优化刺山柑中黄酮类物质的提取方法（试验一），并将提取物制成试验用生药（配制成1g生药/mL供试品，其总黄酮含量为2.4%（干物质计）），以研究其对试验鼠抗炎镇痛（试验二）、微循环、（试验三）、免疫调节（试验四）和佐剂性关节炎的作用（试验五）。在试验二和试验三，将正常小鼠分为5组，即生理盐水组、低剂量刺山柑提取物（CSE）组（1.25mg/10g体重/天）、中剂量CSE组（2.5mg/10g体重/天）、高剂量CSE组（5.0mg/10g体重/天）和阿司匹林（2.5mg/10g体重/天）组，连续灌胃7天后进行试验。结果表明，与生理盐水组比较，各组小鼠对醋酸致扭体反应分别减少23.3%（P＜0.01）、39.7%（P＜0.01）、68.5%（P＜0.01）和52.1%（P＜0.01），对电热板痛阈分别提高83.5%（P＜0.01）、88.6%（P＜0.01）、95.6%（P＜0.01）和59.8%（P＜0.01），对二甲苯致小鼠耳廓肿胀度分别减少10.59%（P＜0.01）、24.92%（P＜0.01）、37.9%（P＜0.01）和22.4%（P＜0.01），对蛋清致小鼠足趾肿胀度分别减少17.6%（P＞0.05）、29.4%（P＜0.05）、38.2%（P＜0.01）和32.4%（P＜0.01）；正常小鼠耳廓细动脉管径分别扩张2.1%（P＞0.05）、9.0%（P＜0.05）、14.5%（P＜0.01）和23.3%（P＜0.01），细静脉管径分别扩张3.0%（P＞0.05）、6.5%（P＜0.01）、11.7%（P＜0.01）和18.2%（P＜0.01），耳廓毛细血管开放数目分别增加10.8%（P＜0.05）、17.0%（P＜0.01）、22.7%（P＜0.01）和32.1%（P＜0.01）；注射肾上腺素血瘀小鼠的耳廓细动脉管径分别扩张9.1%（P＜0.01）、18.4%（P＜0.01）、22.8%（P＜0.01）和30.6%（P＜0.01），细静脉管径分别扩张5.1%（P＜0.01）、7.6%（P＜0.01）、8.6%（P＜0.01）和10.7%（P＜0.01），耳廓毛细血管开放数目分别增加7.8%（P＜0.01）、18.6%（P＜0.01）、24.0%（P＜0.01）和28.4（P＜0.01）%。在试验四，制备正常和类风湿性关节炎小鼠脾细胞滤液培养，分别分为5个处理组，即生理盐水组、低剂量CSE组（2.5ppm/孔）、中剂量CSE组（5ppm/孔）、高剂量CSE组（10ppm/孔）和麝香组（5ppm/孔）组，在正常小鼠细胞，与生理盐水组比，脾脏淋巴细胞的增殖率（OD570）分别增加7.6%（P＜0.05）、23%（P＜0.01）、65%（P＜0.01），麝香组减少15%（P＜0.01），在类风湿性关节炎小鼠细胞，与生理盐水组比，脾脏淋巴细胞的增殖率（OD570）分别减少24%（P＜0.01）、50%（P＜0.01）、67%（P＜0.01）和18%（P＜0.01）。在试验五，将佐剂性关节炎大鼠分为5组，即模型组、低剂量刺山柑提取物（CSE）组（1.25mg/10g体重/天）、中剂量CSE组（2.5mg/10g体重/天）、高剂量CSE组（5.0mg/10g体重/天）和阿司匹林（2.5mg/10g体重/天）组，结果表明，与模型组比，各组大鼠足趾的18小时肿胀度分别降低14%（P＜0.01）、19%（P＜0.01）、33%（P＜0.01）和6.6%（P＜0.05），5天肿胀度分别降低15%（P＜0.01）、27%（P＜0.01）、36%（P＜0.01）和7.2%（P＜0.05），病理评分如炎细胞浸润程度分别降低14%（P＜0.01）、43%（P＜0.01）、47%（P＜0.01）和9.2%（P＞0.05），足趾炎症组织PGE2含量（OD278）分别降低6%（P＜0.05）、18%（P＜0.01）、30%（P＜0.01）和9%（P＜0.05），血清中IL-1含量分别降低7.3%（P＜0.05）、35%（P＜0.01）、60%（P＜0.01）和6.7%（P＜0.05），IL-6分别降低13%（P＜0.01）、38%（P＜0.01）、46%（P＜0.01）和5.6%（P＞0.05），TNF-α分别降低1.0%（P＞0.05）、4.2%（P＜0.05）、18%（P＜0.01）和-0.3%（P＞0.05），血细胞沉降率分别降低4.6%（P＜0.05）、12.5%（P＜0.05）、16.8%（P＜0.05）和7.8%（P＜0.05），血清中类风湿因子浓度分别降低7.3%（P＜0.05）、19%（P＜0.01）、40%（P＜0.01）和8.6%（P＜0.01）。以上试验表明，刺山柑提取物具有抗炎镇痛、免疫调节和促进微循环的作用，且与剂量相关，但其促进微循环作用的程度，不及阿司匹林，然而，刺山柑提取物却有较好的抗肿胀作用，特别是对佐剂性关节炎大鼠，还能够较好的减轻滑膜组织的病理变化，降低病变组织或血液中的PGE2、 IL-1、IL-6、TNF-α和类风湿因子的浓度，和降低血细胞沉降率，因此对关节炎可能会有较好的治疗作用。
The study adopts the orthogonal design and resin adsorption-elution test, taking thefruit of Capparis spinosa L as raw material,optimizing and extracting flavonoids in capparisspinosa(test1), and making the extract into test crude drug (the flavonoids content is2.4%(drymatter)) to study the effect of drug on muridaes in terms of anti-inflammation and analgesia(test2), microcirculation(test3), immunoregulation (test4) and adjuvant arthritis (RA)(test5).In the test2and test3, the normal mice are divided into5groups, namely, normal salinegroup, low-dose Capparis spinosa L extract (CSE) group (1.25mg/10g weight/day), mediumdose CSE group (2.5mg/10g weight/day), high dose CSE group(5.0mg/10g weight/day) andaspirin(APC)(2.5mg/10g weight/day) group, continuous lavage7days to test. Themeasurement is conducted after lavage7days in a row. The results showed, compared withnormal saline group,the ratio of acetic acid torsion body in each other group of mice isreduced by23.3%(P <0.01),39.7%(P <0.01),68.5%(P <0.01) and52.1%(P <0.01), thehot plate pain threshold is increased by83.5%(P <0.01),88.6%(P <0.01),95.6%(P <0.01)and59.8%(P <0.01), the mouse xylene-induced auricle swelling degree is reduced by10.59%(P <0.01),24.92%(P <0.01),37.9%(P <0.01) and22.4%(P <0.01), the degree oftoes swelling induced by egg white were reduced by17.6%(P <0.01),29.4%(P<0.01),38.2%(P <0.01) and32.4%(P <0.01); The normal mice auricle venule arteriole expands by2.1%, respectively(P <0.01)9.0%(P <0.01),14.5%(P <0.01) and23.3%(P <0.01), venulearteriole expands by3.0%(P <0.01),6.5%(P <0.01),11.7%(P <0.01)and18.2%(P <0.01),auricle capillary open numbers increase by10.8%(P <0.05),17.0%(P <0.01),22.7%(P <0.01)and32.1%(P <0.01)respectively; Auricle arteriole diameter of blood stasis mice causedby injection of epinephrine expands by9.1%(P <0.01),18.4%(P <0.01),22.8%(P <0.01)and30.6%(P <0.01)respectively, venule diameter expands by5.1%(P <0.01),7.6%(P <0.01),8.6%(P <0.01) and10.7%(P <0.01) respectively, auricle capillary open numbersincrease by7.8%(P <0.01),18.6%(P <0.01),24.0%(P <0.01)and28.4(P <0.01)%respectively. In test4, the mice are divided into five treatment groups in terms of preparingnormal and rheumatoid arthritis (RA) mice spleen cells with filtrate culture, namely, normalsaline group, low-dose CSE group (2.5ppm/hole),medium dose CSE group(5ppm/hole), high dose CSE group (10ppm/hole) and moschus group(5ppm/hole) group.About normal micecells, compared with normal saline group, proliferation rate of spleen lymphocyte (OD570)increases by7.6%(P <0.01),23%(P <0.01),65%(P <0.01), moschus group reduces by15%(P <0.01), about rheumatoid arthritis mice cells, compared with normal saline group,proliferation rate of spleen lymphocyte (OD570) is reduced by24%(P <0.01),50%(P <0.01),67%(P <0.01)and18%(P <0.01)respectively. In test5, the rats with adjuvant arthritis (RA)are divided into5groups, namely, model group, low dose CSE group (1.25mg/10gweight/day), medium dose CSE group (2.5mg/10g weight/day), high dose CSE group(5.0mg/10g weight/day) and aspirin (2.5mg/10g weight/day) group.
The rusults of the study show that the degree of toes swelling within18hours in eachgroup is reduced by14%(P <0.01)、19%(P <0.01)、33%(P <0.01) and6.6%(P <0.01)respectively; the degree of toes swelling within5days is reduced by15%(P <0.01)、27%(P <0.01)、36%(P <0.01) and7.2%(P <0.05); the degree of histopathologic score likeinflammatory cell infiltration is reduced by14%(P <0.01)、43%(P <0.01)、47%(P <0.01)and9.2%（P＞0.05）, the composition of PGE2（OD278）in inflammation tissue of toe isreduced by6%(P <0.05)、18%(P <0.01)、30%(P <0.01)and9%(P <0.05), the compositionof IL-1in serum is reduced by7.3%(P <0.05)、35%(P <0.01)、60%(P <0.05)and6.7%(P<0.05), IL-6is reduced by13%(P <0.01)、38%(P <0.01)、46%(P <0.01)and5.6%（P＞0.05）, TNF-α is reduced by1.0%（P＞0.05）、4.2%(P <0.05)、18%(P <0.01) and-0.3%（P＞0.05）, erythrocyte sedimentation rate(ESR) is reduced by4.6%(P <0.05）、12.5%(P<0.05)、16.8%(P <0.05) and7.8%(P <0.05), rheumatoid factor in serum is reduced by7.3%(P <0.05)、19%(P <0.05)、40%(P <0.05)and8.6%(P <0.05)respectively. Theexperiment shows that Capparis spinosa L extract has the following effects,such asanti-inflammatory and analgesia, immune regulation and improvement of microcirculation,and dose related, but the function of improvement of microcirculation is less than aspirin.However, Capparis spinosa L extract has good anti swelling effect, especially for adjuvantarthritis rats, and it can reduce pathological changes of synovial tissue, pathologicalpathological changes of tissues, the concentration of PGE2、IL-1、IL-6、TNF-α and rheumatoidfactor in blood concentration, and sedimentation rate, so it may have better therapeutic actionto arthrophlogosis.
The study adopts the orthogonal design and resin adsorption-elution test, taking thefruit of Capparis spinosa L as raw material,optimizing and extracting flavonoids in capparisspinosa(test1), and making the extract into test crude drug (the flavonoids content is2.4%(drymatter)) to study the effect of drug on muridaes in terms of anti-inflammation and analgesia(test2), microcirculation(test3), immunoregulation (test4) and adjuvant arthritis (RA)(test5).In the test2and test3, the normal mice are divided into5groups, namely, normal salinegroup, low-dose Capparis spinosa L extract (CSE) group (1.25mg/10g weight/day), mediumdose CSE group (2.5mg/10g weight/day), high dose CSE group(5.0mg/10g weight/day) andaspirin(APC)(2.5mg/10g weight/day) group, continuous lavage7days to test. Themeasurement is conducted after lavage7days in a row. The results showed, compared withnormal saline group,the ratio of acetic acid torsion body in each other group of mice isreduced by23.3%(P <0.01),39.7%(P <0.01),68.5%(P <0.01) and52.1%(P <0.01), thehot plate pain threshold is increased by83.5%(P <0.01),88.6%(P <0.01),95.6%(P <0.01)and59.8%(P <0.01), the mouse xylene-induced auricle swelling degree is reduced by10.59%(P <0.01),24.92%(P <0.01),37.9%(P <0.01) and22.4%(P <0.01), the degree oftoes swelling induced by egg white were reduced by17.6%(P <0.01),29.4%(P<0.01),38.2%(P <0.01) and32.4%(P <0.01); The normal mice auricle venule arteriole expands by2.1%, respectively(P <0.01)9.0%(P <0.01),14.5%(P <0.01) and23.3%(P <0.01), venulearteriole expands by3.0%(P <0.01),6.5%(P <0.01),11.7%(P <0.01)and18.2%(P <0.01),auricle capillary open numbers increase by10.8%(P <0.05),17.0%(P <0.01),22.7%(P <0.01)and32.1%(P <0.01)respectively; Auricle arteriole diameter of blood stasis mice causedby injection of epinephrine expands by9.1%(P <0.01),18.4%(P <0.01),22.8%(P <0.01)and30.6%(P <0.01)respectively, venule diameter expands by5.1%(P <0.01),7.6%(P <0.01),8.6%(P <0.01) and10.7%(P <0.01) respectively, auricle capillary open numbersincrease by7.8%(P <0.01),18.6%(P <0.01),24.0%(P <0.01)and28.4(P <0.01)%respectively. In test4, the mice are divided into five treatment groups in terms of preparingnormal and rheumatoid arthritis (RA) mice spleen cells with filtrate culture, namely, normalsaline group, low-dose CSE group (2.5ppm/hole),medium dose CSE group(5ppm/hole), high dose CSE group (10ppm/hole) and moschus group(5ppm/hole) group.About normal micecells, compared with normal saline group, proliferation rate of spleen lymphocyte (OD570)increases by7.6%(P <0.01),23%(P <0.01),65%(P <0.01), moschus group reduces by15%(P <0.01), about rheumatoid arthritis mice cells, compared with normal saline group,proliferation rate of spleen lymphocyte (OD570) is reduced by24%(P <0.01),50%(P <0.01),67%(P <0.01)and18%(P <0.01)respectively. In test5, the rats with adjuvant arthritis (RA)are divided into5groups, namely, model group, low dose CSE group (1.25mg/10gweight/day), medium dose CSE group (2.5mg/10g weight/day), high dose CSE group(5.0mg/10g weight/day) and aspirin (2.5mg/10g weight/day) group.
The rusults of the study show that the degree of toes swelling within18hours in eachgroup is reduced by14%(P <0.01)、19%(P <0.01)、33%(P <0.01) and6.6%(P <0.01)respectively; the degree of toes swelling within5days is reduced by15%(P <0.01)、27%(P <0.01)、36%(P <0.01) and7.2%(P <0.05); the degree of histopathologic score likeinflammatory cell infiltration is reduced by14%(P <0.01)、43%(P <0.01)、47%(P <0.01)and9.2%（P＞0.05）, the composition of PGE2（OD278）in inflammation tissue of toe isreduced by6%(P <0.05)、18%(P <0.01)、30%(P <0.01)and9%(P <0.05), the compositionof IL-1in serum is reduced by7.3%(P <0.05)、35%(P <0.01)、60%(P <0.05)and6.7%(P<0.05), IL-6is reduced by13%(P <0.01)、38%(P <0.01)、46%(P <0.01)and5.6%（P＞0.05）, TNF-α is reduced by1.0%（P＞0.05）、4.2%(P <0.05)、18%(P <0.01) and-0.3%（P＞0.05）, erythrocyte sedimentation rate(ESR) is reduced by4.6%(P <0.05）、12.5%(P<0.05)、16.8%(P <0.05) and7.8%(P <0.05), rheumatoid factor in serum is reduced by7.3%(P <0.05)、19%(P <0.05)、40%(P <0.05)and8.6%(P <0.05)respectively. Theexperiment shows that Capparis spinosa L extract has the following effects,such asanti-inflammatory and analgesia, immune regulation and improvement of microcirculation,and dose related, but the function of improvement of microcirculation is less than aspirin.However, Capparis spinosa L extract has good anti swelling effect, especially for adjuvantarthritis rats, and it can reduce pathological changes of synovial tissue, pathologicalpathological changes of tissues, the concentration of PGE2、IL-1、IL-6、TNF-α and rheumatoidfactor in blood concentration, and sedimentation rate, so it may have better therapeutic actionto arthrophlogosis.
引文
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