基于奶牛胎衣不下基本病机—治则—疗效评价体系的中药复方制剂研究
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摘要
胎衣不下是奶牛临床上常见产科疾病之一，常继发奶牛产后子宫疾病，降低产奶量，降低产后繁殖性能。由于其病因的不确定性和复杂性，目前尚缺乏有效治疗方案。传统兽医学在防治家畜胎衣不下方面累积了丰富的证治经验，但由于传统兽医学辨证论治个体化诊疗特点，目前尚缺乏基于群体防治的有效中药制剂。本研究以奶牛胎衣不下为研究对象，基于传统兽医理论，根据目前规模化牛场该病发病特点和用药需求，构建奶牛胎衣不下的基本病机–治则–疗效评价体系，开展中兽医药防治奶牛胎衣不下的研究，旨在研制有效防治奶牛胎衣不下的中药复方制剂。
运用传统兽医学辨证分型和基于临床病例分析，建立防治奶牛胎衣不下药物疗效评价标准，构建了奶牛胎衣不下基本病机(血瘀证)–治则(活血化瘀)–疗效评价体系，以临床疗效为导向，进行有效方筛选，确立了防治奶牛胎衣不下中药制剂——“宫衣净酊”。
采用L49（3）正交设计方法，优选了“宫衣净酊”渗漉提取制备工艺，即加入10倍量70%乙醇，浸渍24h，以2mL/(min·kg)进行渗漉提取；开展薄层色谱鉴定试验研究，确定了急性子、红花、葛根及川牛膝4味主药的TLC检测条件；建立了以羟基红花黄色素A（HSYA）和葛根素为主要成分的HPLC定量检测方法；确定了制剂质量控制参数，分别为相对密度不低于0.90mg/mL；“宫衣净酊”中，HSYA含量不低于0.350mg/mL，葛根素含量不低于1.35mg/mL；pH范围在4.5~6.5；乙醇含量不高于40浓度%（v/v）。
采用急性毒性和亚慢性毒性试验方法，开展“宫衣净酊”临床前安全性评价，改良寇氏法测得“宫衣净酊”对小鼠的经口LD50为28.84g/kg·bw，以7.00g/kg·bw（相当于1/10~1/4LD50剂量）作为最高给药剂量，连续给药30天，“宫衣净酊”对受试大鼠体重及脏器系数、血液常规、生化指标及主要脏器无不良影响；综合分析，“宫衣净酊”属实际无毒性产品。
开展了“宫衣净酊”防治奶牛胎衣不下的临床疗效评价研究，结果表明，“宫衣净酊”可有效促进滞留胎衣自主排出，有效率为76.4%；与子宫灌注土霉素疗法比较，给予“宫衣净酊”治疗后，受试母牛产后首次配种时间显著提前（74.8±23.7vs.94.6±33.3days, P <0.01），空怀时间显著缩短（94.8±41.3vs.141.7±53.3days, P <0.01），首次配种怀孕率（56.9%vs.40.7%, P=0.06）和产后180天内的怀孕率（84.9%vs.72.7%, P=0.054）均有升高趋势。与健康产犊母牛比较，产后2~4h内灌服“宫衣净酊”，受试母牛产后胎衣不下发病率（3.3%vs.17.0%, P <0.01）和产褥期子宫炎发病率（6.1%vs.20.9%, P <0.01）显著降低，产后首次配种时间显著提前（71.8±18.4vs.86.4±33.5days, P <0.01），空怀时间显著缩短（89.8±32.3vs.113.6±42.9days, P <0.01），并且产后180天内的怀孕率有升高趋势（87.3%vs.80.7%, P=0.096）。表明“宫衣净酊”是防治奶牛胎衣不下的有效中药制剂。
以奶牛胎衣不下自然病例为研究对象，从自由基代谢抗氧化酶活性、炎性细胞因子水平和肝功能酶活性角度，探讨“宫衣净酊”治疗奶牛胎衣不下的作用机理，研究表明，”宫衣净酊”可有效调节胎衣不下患牛血清中GSH-Px、SOD、MPO、IL–2、TNF–α及IFN–γ水平，而对ALT和AST水平的影响不显著，表明“宫衣净酊”防治奶牛胎衣不下可能与其调节机体自由基代谢抗氧化酶活性和炎性细胞因子水平有关；并且按照推荐给药剂量和治疗程序，该制剂不会对奶牛肝脏功能造成不良影响。
采用大鼠血瘀模型和小鼠疼痛模型，开展“宫衣净酊”药理作用研究，结果表明，“宫衣净酊”可有效提高Dextran-500诱导的血瘀大鼠血浆中NO和6-keto-PGF1α水平，降低ET-1和TXB2水平，调节ET-1/NO和TXB2/6-keto-PGF1α比例平衡；抑制醋酸诱导的小鼠疼痛反应，延迟小鼠对热刺激的痛觉反应时间，并具有明显量-效和时-效关系；提示“宫衣净酊”具有改善机体血瘀状态和缓解疼痛的作用。
Retained placenta is one of the most common puerperal disorders, which usually leads certainnegative effects including reduced milk yield, increased incidence of metritis and impaired subsequentfertility. There are few effective therapeutic approaches for retained placenta due to its multifactorialetiology. It is well documented that herbal formula for prevention and control of retained placenta incows for centuries in China. However, the challenge now is the lack of certain effective herbalpreparations for prevention and control of diseases on group hierarchy under the condition of the largescale farming due to individualized diagnosis and treatment characteristics based on the syndromedifferentiation theory of traditional Chinese veterinary medicine (TCVM). According to TCVM theoryand the clinical epidemiology of diseases, the present study is to investigate an effective herbal formulapreparation against retained placenta for the requirement of clinical therapy in dairy cows on grouphierarchy, based on the theory of the basic pathogenesis-therapeutic principles-clinical effectivenessevaluation.
Based on the syndrome differentiation theory of TCVM, and the criterion of effectivenessevaluation derived from clinical case analysis of retained placenta in cows, the basic pathogenesis ofretained placenta (blood stasis syndrome)-therapeutic principles (Promoting blood circulation andremoving blood stasis)-clinical effectiveness evaluation was established for screening the herbalformula for prevention and control of retained placenta in cows. An effective herbal preparation“Gong-Yi-Jing Tincture” was developed to prevent and control of retained placenta in dairy cows basedon the clinical effectiveness.
The optimized extraction process was optimized with L49(3) orthogonal trial and the optimizedextraction condition was to use the soak the medicinal materials for24h with10times share of70%ethanol and collect the percolation liquid by2mL/(min·kg). The thin-layer chromatographic detectingconditions of Impatientis Semen, Radix Puerariae, Carthami Flos and Cyathulae Radix in Gong-Yi-JingTincture were established with TCL method, and the quantitative determination of HSYA and puerarinin Gong-Yi-Jing Tincture was established with a high-performance liquid chromatographic method. Thequality control parameters of Gong-Yi-Jing Tincture was established, including relative densities≥0.85mg/mL, the content of HSYA not less than0.350mg/mL, the content of puerarin not less than0.350mg/mL, the range of pH was4.5~6.5, ethanol content not more than40%(v/v).
To evaluate the preclinical safety of Gong-Yi-Jing Tincture for prevention and control of retainedplacenta in dairy cow, the acute&sub-acute toxicity was assessed in mice and rat. The results showedthat Gong-Yi-Jing Tincture was not to be toxic if administered orally, according to Hodge and Sternerscale, in acute toxicity tests (LD50=28.84g/kg·bw) in mice, and no negative impacts on increasedbodywight, routine hematology indexes, blood biochemistry indexes, organ coefficients and mainvisceral organ in rats given orally at dose of7g/kg·bw (equal to the1/10–1/4dose of LD50) once dailyover30-day period.
The present study has assessed the efficacy of Gong-Yi-Jing Tincture for prevention and control ofretained placenta in cows, the results demonstrated that Gong-Yi-Jing Tincture could effectivelyfacilitate expulsion of retained placenta (76.4%), advance the calving-to-first-service interval (74.8±23.7vs.94.6±33.3days; P <0.01), shorten the calving-to-conception interval (94.8±41.3vs.141.7±53.3days; P <0.01), elevate first AI conception proportion (56.9%vs.40.7%, P=0.06) and proportionof cows that were pregnant within180days postpartum (84.9%vs.72.7%, P=0.054) compared to cowsreceived oxytetracycline infusion into the uterus. The present results would support efforts to the use ofGong-Yi-Jing tincture immediately after delivery as a prophylactic strategy, which would not onlyeffectively reduced the incidence of retained placenta (3.3%vs.17.0%, P <0.01) and the risk ofoccurrence of puerperal metritis (6.1%vs.20.9%, P <0.01), but also help to advance thecalving-to-first-service interval (71.8±18.4vs.86.4±33.5days; P <0.01), shorten thecalving-to-conception interval (89.8±32.3vs.113.6±42.9days; P <0.01), and elevate the proportionof cows that were pregnant within180days postpartum (87.3%vs.80.7%, P=0.096) in cows inGong-Yi-Jing Tincture group compared to the controls. Thus, Gong-Yi-Jing Tincture treatment couldpresent effective treatment option for retained placenta in cows.
The present study has investigated the therapeutic mechanism of Gong-Yi-Jing Tincture forretained placenta in cows based on the anti-oxidant enzyme activities of free radical metabolisml,inflammatory cytokine and enzyme activity of hepatic function. The results demonstrated thatGong-Yi-Jing Tincture treatment could effectively elevate the levels of GSH-Px, SOD, MPO, IL–2,TNF–α and IFN–γ in serum of cows affected with retained placenta, while no significant impact on thelevel of ALT and AST in serum. The results suggested that the therapeutic mechanism of Gong-Yi-JingTincture for retained placenta might be associated with modulating the anti-oxidant enzyme activities offree radical metabolism and the levels of inflammatory cytokine. Moreover, Gong-Yi-Jing Tincture hadno harm to hepatic function according to the normal dosage and treatment course.
To investigate the pharmacologic action of Gong-Yi-Jing Tincture for retained placenta, the bloodstasis model in rats induced by Dextran-500and pain model in mice were established. The resultsshowed that Gong-Yi-Jing Tincture could elevate the level of NO and6-keto-PGF1α, decrease the levelof ET-1and TXB2, and modulate the dynamic equilibrium of ET-1/NO and TXB2/6-keto-PGF1αinplasma in rat with blood stasis induced by Dextran-500. In addition, Gong-Yi-Jing Tincture couldeffectively restrain the writhing response induced by intraperitoneal injection of0.7%acetic acid inmice and increase the latency to thermal stimuli, which were dose-dependent and time-dependent. Itsuggested that the pharmacologic action of Gong-Yi-Jing Tincture for retained placenta might beassociated with modulation the blood stasis status and relieving pain.
运用传统兽医学辨证分型和基于临床病例分析，建立防治奶牛胎衣不下药物疗效评价标准，构建了奶牛胎衣不下基本病机(血瘀证)–治则(活血化瘀)–疗效评价体系，以临床疗效为导向，进行有效方筛选，确立了防治奶牛胎衣不下中药制剂——“宫衣净酊”。
采用L49（3）正交设计方法，优选了“宫衣净酊”渗漉提取制备工艺，即加入10倍量70%乙醇，浸渍24h，以2mL/(min·kg)进行渗漉提取；开展薄层色谱鉴定试验研究，确定了急性子、红花、葛根及川牛膝4味主药的TLC检测条件；建立了以羟基红花黄色素A（HSYA）和葛根素为主要成分的HPLC定量检测方法；确定了制剂质量控制参数，分别为相对密度不低于0.90mg/mL；“宫衣净酊”中，HSYA含量不低于0.350mg/mL，葛根素含量不低于1.35mg/mL；pH范围在4.5~6.5；乙醇含量不高于40浓度%（v/v）。
采用急性毒性和亚慢性毒性试验方法，开展“宫衣净酊”临床前安全性评价，改良寇氏法测得“宫衣净酊”对小鼠的经口LD50为28.84g/kg·bw，以7.00g/kg·bw（相当于1/10~1/4LD50剂量）作为最高给药剂量，连续给药30天，“宫衣净酊”对受试大鼠体重及脏器系数、血液常规、生化指标及主要脏器无不良影响；综合分析，“宫衣净酊”属实际无毒性产品。
开展了“宫衣净酊”防治奶牛胎衣不下的临床疗效评价研究，结果表明，“宫衣净酊”可有效促进滞留胎衣自主排出，有效率为76.4%；与子宫灌注土霉素疗法比较，给予“宫衣净酊”治疗后，受试母牛产后首次配种时间显著提前（74.8±23.7vs.94.6±33.3days, P <0.01），空怀时间显著缩短（94.8±41.3vs.141.7±53.3days, P <0.01），首次配种怀孕率（56.9%vs.40.7%, P=0.06）和产后180天内的怀孕率（84.9%vs.72.7%, P=0.054）均有升高趋势。与健康产犊母牛比较，产后2~4h内灌服“宫衣净酊”，受试母牛产后胎衣不下发病率（3.3%vs.17.0%, P <0.01）和产褥期子宫炎发病率（6.1%vs.20.9%, P <0.01）显著降低，产后首次配种时间显著提前（71.8±18.4vs.86.4±33.5days, P <0.01），空怀时间显著缩短（89.8±32.3vs.113.6±42.9days, P <0.01），并且产后180天内的怀孕率有升高趋势（87.3%vs.80.7%, P=0.096）。表明“宫衣净酊”是防治奶牛胎衣不下的有效中药制剂。
以奶牛胎衣不下自然病例为研究对象，从自由基代谢抗氧化酶活性、炎性细胞因子水平和肝功能酶活性角度，探讨“宫衣净酊”治疗奶牛胎衣不下的作用机理，研究表明，”宫衣净酊”可有效调节胎衣不下患牛血清中GSH-Px、SOD、MPO、IL–2、TNF–α及IFN–γ水平，而对ALT和AST水平的影响不显著，表明“宫衣净酊”防治奶牛胎衣不下可能与其调节机体自由基代谢抗氧化酶活性和炎性细胞因子水平有关；并且按照推荐给药剂量和治疗程序，该制剂不会对奶牛肝脏功能造成不良影响。
采用大鼠血瘀模型和小鼠疼痛模型，开展“宫衣净酊”药理作用研究，结果表明，“宫衣净酊”可有效提高Dextran-500诱导的血瘀大鼠血浆中NO和6-keto-PGF1α水平，降低ET-1和TXB2水平，调节ET-1/NO和TXB2/6-keto-PGF1α比例平衡；抑制醋酸诱导的小鼠疼痛反应，延迟小鼠对热刺激的痛觉反应时间，并具有明显量-效和时-效关系；提示“宫衣净酊”具有改善机体血瘀状态和缓解疼痛的作用。
Retained placenta is one of the most common puerperal disorders, which usually leads certainnegative effects including reduced milk yield, increased incidence of metritis and impaired subsequentfertility. There are few effective therapeutic approaches for retained placenta due to its multifactorialetiology. It is well documented that herbal formula for prevention and control of retained placenta incows for centuries in China. However, the challenge now is the lack of certain effective herbalpreparations for prevention and control of diseases on group hierarchy under the condition of the largescale farming due to individualized diagnosis and treatment characteristics based on the syndromedifferentiation theory of traditional Chinese veterinary medicine (TCVM). According to TCVM theoryand the clinical epidemiology of diseases, the present study is to investigate an effective herbal formulapreparation against retained placenta for the requirement of clinical therapy in dairy cows on grouphierarchy, based on the theory of the basic pathogenesis-therapeutic principles-clinical effectivenessevaluation.
Based on the syndrome differentiation theory of TCVM, and the criterion of effectivenessevaluation derived from clinical case analysis of retained placenta in cows, the basic pathogenesis ofretained placenta (blood stasis syndrome)-therapeutic principles (Promoting blood circulation andremoving blood stasis)-clinical effectiveness evaluation was established for screening the herbalformula for prevention and control of retained placenta in cows. An effective herbal preparation“Gong-Yi-Jing Tincture” was developed to prevent and control of retained placenta in dairy cows basedon the clinical effectiveness.
The optimized extraction process was optimized with L49(3) orthogonal trial and the optimizedextraction condition was to use the soak the medicinal materials for24h with10times share of70%ethanol and collect the percolation liquid by2mL/(min·kg). The thin-layer chromatographic detectingconditions of Impatientis Semen, Radix Puerariae, Carthami Flos and Cyathulae Radix in Gong-Yi-JingTincture were established with TCL method, and the quantitative determination of HSYA and puerarinin Gong-Yi-Jing Tincture was established with a high-performance liquid chromatographic method. Thequality control parameters of Gong-Yi-Jing Tincture was established, including relative densities≥0.85mg/mL, the content of HSYA not less than0.350mg/mL, the content of puerarin not less than0.350mg/mL, the range of pH was4.5~6.5, ethanol content not more than40%(v/v).
To evaluate the preclinical safety of Gong-Yi-Jing Tincture for prevention and control of retainedplacenta in dairy cow, the acute&sub-acute toxicity was assessed in mice and rat. The results showedthat Gong-Yi-Jing Tincture was not to be toxic if administered orally, according to Hodge and Sternerscale, in acute toxicity tests (LD50=28.84g/kg·bw) in mice, and no negative impacts on increasedbodywight, routine hematology indexes, blood biochemistry indexes, organ coefficients and mainvisceral organ in rats given orally at dose of7g/kg·bw (equal to the1/10–1/4dose of LD50) once dailyover30-day period.
The present study has assessed the efficacy of Gong-Yi-Jing Tincture for prevention and control ofretained placenta in cows, the results demonstrated that Gong-Yi-Jing Tincture could effectivelyfacilitate expulsion of retained placenta (76.4%), advance the calving-to-first-service interval (74.8±23.7vs.94.6±33.3days; P <0.01), shorten the calving-to-conception interval (94.8±41.3vs.141.7±53.3days; P <0.01), elevate first AI conception proportion (56.9%vs.40.7%, P=0.06) and proportionof cows that were pregnant within180days postpartum (84.9%vs.72.7%, P=0.054) compared to cowsreceived oxytetracycline infusion into the uterus. The present results would support efforts to the use ofGong-Yi-Jing tincture immediately after delivery as a prophylactic strategy, which would not onlyeffectively reduced the incidence of retained placenta (3.3%vs.17.0%, P <0.01) and the risk ofoccurrence of puerperal metritis (6.1%vs.20.9%, P <0.01), but also help to advance thecalving-to-first-service interval (71.8±18.4vs.86.4±33.5days; P <0.01), shorten thecalving-to-conception interval (89.8±32.3vs.113.6±42.9days; P <0.01), and elevate the proportionof cows that were pregnant within180days postpartum (87.3%vs.80.7%, P=0.096) in cows inGong-Yi-Jing Tincture group compared to the controls. Thus, Gong-Yi-Jing Tincture treatment couldpresent effective treatment option for retained placenta in cows.
The present study has investigated the therapeutic mechanism of Gong-Yi-Jing Tincture forretained placenta in cows based on the anti-oxidant enzyme activities of free radical metabolisml,inflammatory cytokine and enzyme activity of hepatic function. The results demonstrated thatGong-Yi-Jing Tincture treatment could effectively elevate the levels of GSH-Px, SOD, MPO, IL–2,TNF–α and IFN–γ in serum of cows affected with retained placenta, while no significant impact on thelevel of ALT and AST in serum. The results suggested that the therapeutic mechanism of Gong-Yi-JingTincture for retained placenta might be associated with modulating the anti-oxidant enzyme activities offree radical metabolism and the levels of inflammatory cytokine. Moreover, Gong-Yi-Jing Tincture hadno harm to hepatic function according to the normal dosage and treatment course.
To investigate the pharmacologic action of Gong-Yi-Jing Tincture for retained placenta, the bloodstasis model in rats induced by Dextran-500and pain model in mice were established. The resultsshowed that Gong-Yi-Jing Tincture could elevate the level of NO and6-keto-PGF1α, decrease the levelof ET-1and TXB2, and modulate the dynamic equilibrium of ET-1/NO and TXB2/6-keto-PGF1αinplasma in rat with blood stasis induced by Dextran-500. In addition, Gong-Yi-Jing Tincture couldeffectively restrain the writhing response induced by intraperitoneal injection of0.7%acetic acid inmice and increase the latency to thermal stimuli, which were dose-dependent and time-dependent. Itsuggested that the pharmacologic action of Gong-Yi-Jing Tincture for retained placenta might beassociated with modulation the blood stasis status and relieving pain.
引文
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