CN111617110A - 一种林蛙油冻干粉补肾药理作用的研究方法 - Google Patents
一种林蛙油冻干粉补肾药理作用的研究方法 Download PDFInfo
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- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
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Abstract
本发明公开了一种林蛙油冻干粉补肾药理作用的研究方法,包括步研究林蛙油冻干粉对小鼠网状内皮系统炭粒廓清能力的影响、研究林蛙油冻干粉对幼年雄性小鼠的生殖附性器官精液囊、睾丸、包皮腺、免疫器官胸腺和脾脏的重量指数的影响、研究林蛙油冻干粉对肾阳虚大鼠内分泌功能的影响、研究林蛙油冻干粉对去势小鼠精液囊与包皮腺重量指数的影响、研究林蛙油冻干粉对精子数及活精子百分率的影响和依据上述研究做出整体评估,该研究方法可从肾功能影响的多方面对林蛙油冻干粉进行研究,形成系统的评价方法,对林蛙油冻干工艺的发展和冻干制品的推广具有重大意义。
Description
技术领域
本发明涉及保健食品技术领域,特别涉及一种林蛙油冻干粉补肾药理作用的研究方法。
背景技术
林蛙,又称蛤什蟆,是我国著名的集药用、滋补和美容于一体的经济蛙种。林蛙油是雌林蛙怀卵成熟期的输卵管,它含有18种氨基酸、13种无机元素、9种维生素和多种复合多肽等生物活性因子,特别富含三种性激素,即雌二醇、睾酮、孕酮。其中雌二醇和睾酮分别是雌性激素和雄性激素中生理作用最强的激素,具有显著的滋阴强肾激发免疫功能和调节机理作用。
直接从雌林蛙体内获得的林蛙油为干林蛙油,其属于油脂类物质不易被人体消化,同时也带有病毒或寄生虫卵以及浓烈的血腥味,不经过加工处理不能直接食用。现有林蛙油的加工处理方法一般是将林蛙油进行高温蒸煮(100℃),达到除腥、除菌的目的。但是,长时间高温蒸煮使得林蛙油营养流失严重,其具有功效的活性物质被破坏,如不饱和脂肪酸、维生素E、维生素C以及雌二醇等,严重影响其功效。此外,高温蒸煮只能消除绝大部分腥味,制成的林蛙油成品仍然带有一丝腥味,同时高温蒸煮后的林蛙油也不够柔软,影响其口感。因此,越来越多的技术指向林蛙油冻干的研究,冻干工艺使林蛙油具有使用方便快捷的效果,但是针对冻干后的林蛙油是否会有功能上的影响,现有技术还研究较少,且缺少系统的研究方法对林蛙油冻干粉在补肾方面的药理作用研究,阻碍了林蛙油制品的发展进程和广泛推广。
因此,如何提供一种系统的林蛙油冻干粉补肾药理作用的研究方法是本领域技术人员亟待解决的问题。
发明内容
有鉴于此,本发明提供了一种林蛙油冻干粉补肾药理作用的研究方法,以实验室常用的鼠类为研究对象,从对肾功能影响的多方面进行研究,以达到可系统综合评价林蛙油补肾作用的效果。
为了达到上述目的,本发明采用如下技术方案:
一种林蛙油冻干粉补肾药理作用的研究方法,包括以下步骤:
步骤一:研究林蛙油冻干粉对小鼠网状内皮系统炭粒廓清能力的影响
将实验用雄性KM小鼠等数分为若干组,设置空白对照组、空白模型组、实验组和对照组并进行相应的处理,之后以0.1mL/10g体重的量对所有组别小鼠静脉注射印度墨汁,注射后间隔一定时间取两次血分别测定OD1和OD2值,采血完成后处死取肝、脾分析称重,计算廓清指数和吞噬系数;
步骤二:研究林蛙油冻干粉对幼年雄性小鼠的生殖附性器官精液囊、睾丸、包皮腺、免疫器官胸腺和脾脏的重量指数的影响
将实验用雄性KM小鼠等数分为若干组,设置空白对照组、空白模型组、实验组和对照组并进行相应的处理,末次处理后24h处死称重并取胸腺、脾脏、精液囊、睾丸、包皮腺并分别称重,以器官重量与体重之比为指数计算;
步骤三:研究林蛙油冻干粉对肾阳虚大鼠内分泌功能的影响
取SD雄性大鼠等数分为若干组,设置空白对照组、空白模型组、实验组和对照组并进行相应的处理,末次给药后1h取静脉血测定血清中皮质醇、睾丸酮、肾上腺素、去甲肾上腺素的含量;
步骤四:研究林蛙油冻干粉对去势小鼠精液囊与包皮腺重量指数的影响
将实验用雄性KM小鼠深度麻醉去势处理后,等数分为若干组,设置空白对照组、实验组和对照组并进行相应的处理,连续处理10d后处死剪取各组小鼠精囊腺和包皮腺称湿重;
步骤五:研究林蛙油冻干粉对精子数及活精子百分率的影响
取SD雄性大鼠等数分为若干组,设置空白对照组、实验组和对照组并进行相应的处理,末次处理后24h,处死解剖取出附睾计算精子总数、死精子百分率和活精子百分率;
步骤六:依据步骤一到步骤五所测定数据结果综合评估。
进一步的,所述空白对照组为生理盐水处理组。
进一步的,所述实验组为林蛙油冻干粉混悬液给药组,其包括三个及以上不同剂量给药组。
进一步的,所述对照组包括林蛙油混悬液给药组与对应的现有技术药物给药组。
进一步的,所述步骤一中的印度墨汁用1%明胶液稀释4倍后于尾静脉注射。
进一步的,所述步骤一中注射后2min眼眶取血测定OD1,注射后10min眼眶取血测定OD2值,计算吞噬指数K=(lgOD1-lgOD2)/(T2-T1),其中T为时间,T1为第2min,T2为第10min。
进一步的,所述步骤一中的吞噬系数α=体重/(肝重+脾重)×K1/3。
进一步的,所述步骤五中采用台盼蓝染色法计算精子总数、死精子百分率和活精子百分率。
经由上述技术方案,与现有技术相比,本发明提供了一种林蛙油冻干粉补肾药理作用的研究方法,可从肾功能影响的多方面对林蛙油冻干粉进行研究,形成系统的评价方法,对林蛙油冻干工艺的发展和推广具有重大意义。
具体实施方式
下面将结合本发明的实施例,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
实验材料
1.受试药物
名称 林蛙油冻干粉
提供单位 黑龙江四宝生物科技股份有限公司
批号 20171101
含量 符合要求
理化性质本品为淡黄色粉末。
2.试验动物
品系 SD品系大鼠,昆明品系小鼠。
来源 青岛市实验动物和动物实验中心提供,合格证号为SCXK(鲁)20140001。
体重 见各项试验。
性别 雄性。
动物数 见各项试验。
3.主要药品及试剂
4.主要仪器
101A-1E型电热鼓风干燥箱 | 上海实验仪器有限公司 |
FA2004分析电子天平 | 上海良平仪器仪表 |
倒置显微镜 | 日本Olympus公司 |
VICTORX3酶标仪 | PerkinElmer公司 |
TDL-4型低速台式离心机 | 上海安享科学仪器厂 |
108mL手动组织匀浆器 | 天津玻璃仪器厂 |
H2050R型台式高速冷冻离心机 | 湖南湘仪实验室仪器开发有限公司 |
(1)林蛙油冻干粉对小鼠网状内皮系统炭粒廓清能力的影响
取雄性KM小鼠70只,体重18~22g。随机分成7组,每组10只。①正常对照组(生理盐水灌胃0.5mL/d)、②空白模型组(环磷酰胺30mg/kg)、③LWY-冻干粉低剂量干预组(0.07g/kg林蛙油冻干粉混悬液)、④LWY-冻干粉中剂量干预组(0.14g/kg林蛙油冻干粉混悬液)、⑤LWY-冻干粉高剂量干预组(0.28g/kg林蛙油冻干粉混悬液)、⑥LWY组(0.14g/kg林蛙油混悬液)、⑦贞芪扶正颗粒组(3.90g/kg贞芪扶正颗粒混悬液)。第②组于实验开始第1、3、5天皮下注射环磷酰胺造模(30mg/kg),于造模第一天开始①~②组灌胃生理盐水(0.5mL/d)、③~⑥组开始灌胃给药林蛙油冻干粉混悬液、⑦组灌胃给药贞芪扶正颗粒混悬液,每日1次,连续给药14天。于末次给药后24h,将所有组鼠于尾静脉注入稀释印度墨汁(用1%明胶液稀释4倍)0.1mL/10g体重,立即计时,并分别于第2、10min时眼眶取血,用微量取样器(预先用肝素浸润)吸取20μl,分别加入到2mL的0.1%碳酸钠溶液中,摇匀,在600nm波长处测定OD值,2min血样为OD1,10min血样为OD2。采血后的小鼠处死取其肝、脾,清除周围组织,电子分析天平称重,计算廓清指数K及吞噬系数α。结果见表1。
吞噬指数K=(lgOD1-lgOD2)/(T2-T1)
吞噬系数α=体重/(肝重+脾重)×K1/3
注:T为时间,T1为第2min,T2为第10min。
表1林蛙油冻干粉对小鼠炭粒廓清能力的影响(±s;n=10)
注:与正常组对比#P<0.05,##P<0.01,###P<0.001,与模型组对比*P<0.05,**P<0.01,***P<0.001,与LWY组对比〇P<0.05,〇〇P<0.01,〇〇〇P<0.001。
廓清指数表示血流中异物被巨噬细胞清除的速率,用于反映单核巨噬细胞吞噬功能。单核巨噬细胞系统在保持机体内环境稳定上是一个重要系统,因它能迅速清除多种致病物质,如病原微生物及其毒素,衰老、死亡或突变细胞、凝血块与纤溶产物、补体碎片及抗原抗体复合物等,故在多种疾病的发生、发展或预后中起很重要作用。
结果表明:与正常组相比,模型组吞噬指数和吞噬系数明显下降(P<0.01),说明造模成功;LWY-冻干粉各给药组与模型组相比,吞噬指数和吞噬系数均高于模型组,并具有显著性差异(P<0.05,P<0.01或P<0.001),说明林蛙油冻干粉对单核巨噬细胞的吞噬功能具有增强作用;且在相同剂量下林蛙油冻干粉与林蛙原油组相比,吞噬指数(K)林蛙原油组增强作用更加明显(P<0.05),吞噬系数(α)干预组的增强作用更加明显(P<0.001)。
(2)林蛙油冻干粉对幼年雄性小鼠的生殖附性器官精液囊、睾丸、包皮腺、免疫器官胸腺和脾脏的重量指数的影响
取雄性KM小鼠70只,体重18~22g。随机分成7组,每组10只。①正常对照组(生理盐水灌胃0.5mL/d)、②空白模型组(环磷酰胺30mg/kg)、③LWY-冻干粉低剂量干预组(0.07g/kg林蛙油冻干粉混悬液)、④LWY-冻干粉中剂量干预组(0.14g/kg林蛙油冻干粉混悬液)、⑤LWY-冻干粉高剂量干预组(0.28g/kg林蛙油冻干粉混悬液)、⑥LWY-组(0.14g/kg林蛙油混悬液)、⑦贞芪扶正颗粒组(3.90g/kg贞芪扶正颗粒混悬液)。第②组于实验开始第1、3、5天皮下注射环磷酰胺造模(30mg/kg),于造模第一天开始①~②组灌胃生理盐水(0.5mL/d)、③~⑥组开始灌胃给药林蛙油冻干粉混悬液、⑦组灌胃给药贞芪扶正颗粒,每日1次,连续给药14天。于末次给药后24h,处死小鼠,称体重,摘出胸腺、脾脏、精液囊、睾丸、包皮腺,清除周围组织,电子分析天平称重,以器官重量(mg)与体重(g)之比为指数。结果见表2。
注:与正常组对比#P<0.05,##P<0.01,###P<0.001,与模型组对比*P<0.05,**P<0.01,***P<0.001,与LWY组对比〇P<0.05,〇〇P<0.01,〇〇〇P<0.001。
结果表明:模型组小鼠的包皮腺、睾丸、精液囊的重量与正常组比较,重量减少且具有显著性差异(P<0.05或P<0.001),证明造模成功。模型组与LWY-冻干粉各给药组相比较,胸腺与脾脏的重量无显著性差异(P>0.05),表明林蛙油冻干粉对小鼠胸腺和脾脏等免疫器官无明显影响。LWY-冻干粉给药各组的包皮腺、睾丸、精液囊与模型组比较均增加并具有显著性差异(P<0.05、P<0.01或P<0.001),说明林蛙油冻干粉对小鼠的生殖附性器官具有增强作用。且在相同剂量下林蛙油冻干粉与林蛙原油组相比,林蛙原油组对小鼠的包皮腺、睾丸和精液囊重量增加作用更加明显(P<0.05或P<0.01),对胸腺和脾脏重量并无显著性差异。
(3)林蛙油冻干粉对肾阳虚大鼠内分泌功能的影响
取SD雄性大鼠70只,体重160~180g。随机分为7组,每组10只。①正常对照组(生理盐水灌胃1mL/d)、②空白模型组(氢化可的松15mg/kg)、③LWY-冻干粉低剂量干预组(0.05g/kg林蛙油冻干粉混悬液)、④LWY-冻干粉中剂量干预组(0.10g/kg林蛙油冻干粉混悬液)、⑤LWY-冻干粉高剂量干预组(0.20g/kg林蛙油冻干粉混悬液)、⑥LWY组(0.10g/kg林蛙油混悬液)、⑦右归胶囊组(1.458g/kg右归胶囊混悬液)。②~⑦组每日肌注氢化可的松进行造模,连续2周。给药组③~⑦组于造模同时开始灌胃给药,①~②组给予生理盐水1mL/d,持续至造模结束后第6d。①~⑦组动物均于末次给药后1h迅速(15s内)从眼眶后静脉丛取血,测定血清中皮质醇、睾丸酮、肾上腺素、去甲肾上腺素的含量。结果见表3。
注:与正常组对比#P<0.05,##P<0.01,###P<0.001,与模型组对比*P<0.05,**P<0.01,***P<0.001,与LWY组对比〇P<0.05,〇〇P<0.01,〇〇〇P<0.001。
睾丸酮是一种类固醇荷尔蒙,具有维持肌肉强度及质量、维持骨质密度及强度、提神及提升体能等作用。皮质醇是肾上腺在应激反应里产生的一种类激素,是身体在压力状态维持正常生理机能的一种物质。肾上腺素是由人体分泌出的一种激素为身体活动提供更多能量,使反应更加快速。去甲肾上腺素是在面临短期压力时分泌产生,表现与肾上腺素有些类似,作为一种压力激素,它的增加促进食物能量的储存,并能提高大脑的醒觉度。
结果表明:模型组和正常对照组比较皮质酮、睾丸酮、去甲肾上腺素、肾上腺素均有显著性降低(P<0.05或P<0.001),说明造模成功。LWY-冻干粉各给药组皮质醇、睾丸酮、去甲肾上腺素和肾上腺素比模型组均升高且有显著性差异(P<0.05,P<0.01或P<0.001)。实验结果表明林蛙油冻干粉对肾阳虚大鼠内分泌功能具有增加作用,且在相同剂量下林蛙油冻干粉与林蛙原油组相比,林蛙原油组对肾上腺素的增强作用更加明显(P<0.01),对皮质酮、睾丸酮和去甲肾上腺素含量并无显著性差异。
(4)林蛙油冻干粉对去势小鼠精液囊与包皮腺重量指数的影响
取雄性KM小鼠60只,体重25~30g。所有小鼠用水合氯醛深度麻醉,然后置于手术台上,将睾丸处皮肤用碘酒和酒精先后消毒,用手术剪剪开皮肤,长约0.5cm,用镊子分离皮下组织,将左右两侧睾丸拉出,连同附睾一并剪除,缝合创口并用黄药水对伤口进行消毒处理。将手术后的小鼠置于电暖扇周围进行保温至苏醒。为防术后感染给予小鼠青霉素钠注射液(0.46g/kg)肌肉注射3天。手术后第3d,将全部小鼠随机分为6组:①去势组(生理盐水灌胃0.5mL/d)、②LWY-冻干粉低剂量干预组(0.07g/kg林蛙油冻干粉混悬液)、③LWY-冻干粉中剂量干预组(0.14g/kg林蛙油冻干粉混悬液)、④LWY-冻干粉高剂量干预组(0.28g/kg林蛙油冻干粉混悬液)、⑤LWY组(0.14g/kg林蛙油混悬液)、⑥丙酸睾丸素组(丙酸睾丸素20mg/kg肌肉注射)。②~⑤组每日灌胃给药一次,⑥组丙酸睾丸素20mg/kg肌肉注射,隔天一次,共10d。10d后处死全部动物,剪取各组小鼠精囊腺和包皮腺,清除周围脂肪等,立即在电子分析天平上称其湿重。结果见表4。
组别 | 剂量(g/kg) | 精液囊(mg/g) | 包皮腺(mg/g) |
去势组 | - | 3.882±0.447 | 3.157±0.350 |
LWY-冻干粉组 | 0.070 | 4.239±0.261<sup>*</sup> | 3.671±0.514<sup>*</sup> |
LWY-冻干粉组 | 0.140 | 4.380±0.302<sup>**〇</sup>〇 | 3.897±0.441<sup>***</sup> |
LWY-冻干粉组 | 0.280 | 5.031±0.382<sup>***</sup> | 4.052±1.115<sup>*</sup> |
LWY组 | 0.140 | 5.384±0.548<sup>**</sup> | 4.399±0.604<sup>***</sup> |
丙酸睾丸素组 | 0.020 | 6.038±0.762<sup>***</sup> | 3.924±0.504<sup>**</sup> |
注:与正常组对比#P<0.05,##P<0.01,###P<0.001,与模型组对比*P<0.05,**P<0.01,***P<0.001,与LWY组对比〇P<0.05,〇〇P<0.01,〇〇〇P<0.001。
结果表明:LWY-冻干粉各给药组的精液囊和包皮腺均比去势组重量增加且具有显著性差异(P<0.05,P<0.01或P<0.001),说明林蛙油冻干粉能使去势小鼠精液囊和包皮腺重量显著增加。且在相同剂量下林蛙油冻干粉与林蛙原油组相比,林蛙原油组对去势小鼠的精液囊重量增加作用更加明显(P<0.01),对包皮腺重量并无显著性差异。
(5)林蛙油冻干粉对精子数及活精子百分率的影响
取雄性成年SD大鼠60只,体重200~280g,随机分成6组,分别为①空白对照组(生理盐水灌胃1mL/d)、②LWY-冻干粉低剂量组(0.05g/kg林蛙油冻干粉混悬液)、③LWY-冻干粉中剂量组(0.10g/kg林蛙油冻干粉混悬液)、④LWY-冻干粉高剂量组(0.20g/kg林蛙油冻干粉混悬液)、⑤LWY-组(0.10g/kg林蛙油冻干粉混悬液)、⑥男宝胶囊组(男宝胶囊1g/kg)。①组每天灌胃生理盐水1mL/d,②~⑥组每日灌胃给药1次,连续17d。于末次给药后24h,股动脉放血处死动物,解剖取出附睾,用剪刀在尾端十字形剪开0.5cm,置于装有生理盐10mL的粗试管中,保温37℃10min,取悬液滴于细胞计数板中,用活体染料台盼蓝对细胞进行染色,按白细胞计数方法在血细胞计数板进行计数,便于确定细胞的生活状况。台盼蓝不能透过活细胞正常完整细胞膜,故活细胞不着色。而死细胞的细胞膜通透性增高,可使染料进入细胞内而使细胞着色(蓝色)。计算精子总数、死精子百分率和活精子百分率。结果见表5。
注:与空白对照组对比*P<0.05,**P<0.01,***P<0.001,与LWY组对比〇P<0.05,〇〇P<0.01,〇〇〇P<0.001。
结果表明:LWY-冻干粉各给药组精子总数、活精子数、活精子百分率均增加,与空白组比较具有显著性差异(P<0.05,P<0.01)。与空白组比较死精子数和死精子百分率均降低,与空白组比较具有显著性差异(P<0.05,P<0.01),说明林蛙油对大鼠精子数量及活性具有增强作用,且在相同剂量下林蛙油冻干粉与林蛙原油组相比,林蛙原油组对精子总数和活精子数作用更加明显(P<0.05),对死精子数并无显著性差异。
通过上述的分步骤系统评估可以发现,林蛙油冻干粉相较于林蛙油普通制品和其余对应功能的药物组分,均具有更好的效果,因此,上述评估方法对林蛙油冻干粉的在肾脏作用方面做出了整体评价,具有较高的试剂参考意义。
本说明书中各个实施例采用递进的方式描述,每个实施例重点说明的都是与其他实施例的不同之处,各个实施例之间相同相似部分互相参见即可。
对所公开的实施例的上述说明,使本领域专业技术人员能够实现或使用本发明。对这些实施例的多种修改对本领域的专业技术人员来说将是显而易见的,本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下,在其它实施例中实现。因此,本发明将不会被限制于本文所示的这些实施例,而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。
Claims (8)
1.一种林蛙油冻干粉补肾药理作用的研究方法,其特征在于,包括以下步骤:
步骤一:研究林蛙油冻干粉对小鼠网状内皮系统炭粒廓清能力的影响
将实验用雄性KM小鼠等数分为若干组,设置空白对照组、空白模型组、实验组和对照组并进行相应的处理,之后以0.1mL/10g体重的量对所有组别小鼠静脉注射印度墨汁,注射后间隔一定时间取两次血分别测定OD1和OD2值,采血完成后处死取肝、脾分析称重,计算廓清指数和吞噬系数;
步骤二:研究林蛙油冻干粉对幼年雄性小鼠的生殖附性器官精液囊、睾丸、包皮腺、免疫器官胸腺和脾脏的重量指数的影响
将实验用雄性KM小鼠等数分为若干组,设置空白对照组、空白模型组、实验组和对照组并进行相应的处理,末次处理后24h处死称重并取胸腺、脾脏、精液囊、睾丸、包皮腺并分别称重,以器官重量与体重之比为指数计算;
步骤三:研究林蛙油冻干粉对肾阳虚大鼠内分泌功能的影响
取SD雄性大鼠等数分为若干组,设置空白对照组、空白模型组、实验组和对照组并进行相应的处理,末次给药后1h取静脉血测定血清中皮质醇、睾丸酮、肾上腺素、去甲肾上腺素的含量;
步骤四:研究林蛙油冻干粉对去势小鼠精液囊与包皮腺重量指数的影响
将实验用雄性KM小鼠深度麻醉去势处理后,等数分为若干组,设置空白对照组、实验组和对照组并进行相应的处理,连续处理10d后处死剪取各组小鼠精囊腺和包皮腺称湿重;
步骤五:研究林蛙油冻干粉对精子数及活精子百分率的影响
取SD雄性大鼠等数分为若干组,设置空白对照组、实验组和对照组并进行相应的处理,末次处理后24h,处死解剖取出附睾计算精子总数、死精子百分率和活精子百分率;
步骤六:依据步骤一到步骤五所测定数据结果综合评估。
2.根据权利要求1所述的一种林蛙油冻干粉补肾药理作用的研究方法,其特征在于,所述空白对照组为生理盐水处理组。
3.根据权利要求1所述的一种林蛙油冻干粉补肾药理作用的研究方法,其特征在于,所述实验组为林蛙油冻干粉混悬液给药组,其包括三个及以上不同剂量给药组。
4.根据权利要求1所述的一种林蛙油冻干粉补肾药理作用的研究方法,其特征在于,所述对照组包括林蛙油混悬液给药组与对应的现有技术药物给药组。
5.根据权利要求1所述的一种林蛙油冻干粉补肾药理作用的研究方法,其特征在于,所述步骤一中的印度墨汁用1%明胶液稀释4倍后于尾静脉注射。
6.根据权利要求1所述的一种林蛙油冻干粉补肾药理作用的研究方法,其特征在于,所述步骤一中注射后2min眼眶取血测定OD1,注射后10min眼眶取血测定OD2值,计算吞噬指数K=(lgOD1-lgOD2)/(T2-T1),其中T为时间,T1为第2min,T2为第10min。
7.根据权利要求1所述的一种林蛙油冻干粉补肾药理作用的研究方法,其特征在于,所述步骤一中的吞噬系数α=体重/(肝重+脾重)×K1/3。
8.根据权利要求1所述的一种林蛙油冻干粉补肾药理作用的研究方法,其特征在于,所述步骤五中采用台盼蓝染色法计算精子总数、死精子百分率和活精子百分率。
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