CN101297692A - 樟叶越橘苷的提取及其在健康产品中的应用 - Google Patents
樟叶越橘苷的提取及其在健康产品中的应用 Download PDFInfo
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- CN101297692A CN101297692A CNA2008100584411A CN200810058441A CN101297692A CN 101297692 A CN101297692 A CN 101297692A CN A2008100584411 A CNA2008100584411 A CN A2008100584411A CN 200810058441 A CN200810058441 A CN 200810058441A CN 101297692 A CN101297692 A CN 101297692A
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Abstract
本发明是樟叶越橘苷的提取及其在健康产品中的应用。提取方法:取樟叶越橘粉碎,加水6~8倍,煎煮2~3小时,过滤,滤液用大孔吸附树脂吸附先用水洗脱,除去寡糖类成分,再用75%乙醇洗脱,收集洗脱液,浓缩至无乙醇,放置,结晶,过滤,结晶再用50%乙醇加热溶解,放置结晶,过滤,50℃以下干燥,得樟叶越橘苷;或煎煮液过滤后用乙酸乙酯或丁醇萃取,萃取液浓缩,残留物加75%乙醇溶解,放置,结晶,过滤,结晶再用50%乙醇加热溶解,放置结晶,过滤,50℃以下干燥,得樟叶越橘苷。将其制成饮料、口服液、片剂、胶囊。可以用于降血脂、胆固醇和抗血栓,肿瘤疾病的治疗,病原性微生物引起的炎症治疗等。
Description
技术领域
本发明涉及健康产品(食品、保健品及药物)技术领域,具体地说是樟叶越橘苷(1-O-对羟基苯酚-6-O-咖啡酰基葡萄糖苷,以下简称PPCG)的提取及在健康产品(食品、保健品及药物)中的应用。
背景技术
随着人类生产力的迅猛发展,越来越多的人更加关注自身机体的健康保健。从大自然获取物质基础满足这一需求仍是安全和快速的选择。特别是那些已经为人类经验验证的生物资源:包含多种活性组分,从中不断提取、分离、纯化并测试它们的生理活性,最终应用于健康产品(食品、保健品及药物)中,是现代食品工业和制药工业发展的基础和主要途径。
杜鹃花科越橘属(Vaccinium)植物约300种,我国有47种,资源十分丰富。该属植物有多种生理活性:本草纲目中记载乌饭树(Vaccinium bracteatum Thunb.)具有“利肠胃,补骨髓,固精驻颜,轻身明目”的功效;国外对欧洲越橘(V.myrtillus L.)的研究发现其花色苷对眼科疾病和心血管疾病具有显著疗效;美国药典记载,蔓越橘(V.oxycoccus L.)是对付膀胱炎、尿道感染有效的辅助品;日本则从欧洲和我国东北林区进口越橘属(Vaccinium)植物的冻干粉用于生产保健食品。国内外均有大量关于该属植物活性成分研究的报道。我们研究发现樟叶越橘苷(PPCG)广泛存在于橘属(Vaccinium)植物中,特别是在云南产樟叶越橘(Vaccinium dunalianum)中含量异常高,且其具有多种生理活性,在健康产品中的应用开发前景巨大。
发明内容
本发明的目的是从云南产樟叶越橘中提取樟叶越橘苷(PPCG),以及其在健康产品(食品、保健品及药物)中的应用。
本发明的越橘苷的提取方法是:
取云南产樟叶越橘(Vaccinium dunalianum),粉碎,加水6~8倍,煎煮2~3小时,过滤,滤液用大孔吸附树脂吸附,先用水洗脱,除去寡糖类成分,再用75%乙醇洗脱,收集洗脱液,浓缩至无乙醇,放置,结晶,过滤,结晶再用50%乙醇加热溶解,放置结晶,过滤,50℃以下干燥,得樟叶越橘苷(PPCG);或煎煮液过滤后用乙酸乙酯或丁醇萃取,萃取液浓缩,残留物加75%乙醇溶解,放置,结晶,过滤,结晶再用50%乙醇加热溶解,放置结晶,过滤,50℃以下干燥,得樟叶越橘苷。
越橘苷的应用:
取樟叶越橘苷加入食品或药品中允许加入的助剂,制成饮料、口服液、片剂、胶囊等现有食品和药品产品形式。
取所制成的产品每日口服3次,每次服用相当于樟叶越橘苷25~500mg。可用于高血脂、高胆固醇及血栓性心脑血管疾病治疗;也可用于各种肿瘤疾病的辅助治疗及缓解和减轻肿瘤疾病因放化疗引起的副作用;也可用于细菌、病毒引起的身体发热及炎症治疗。
樟叶越橘苷功能活性试验如下:
一、对血液流变学的影响
1、试验材料
1.1、供试样品
樟叶越橘苷样品(WF-1)为米黄色粉剂,样品批号050922。水溶性差,体外试验用DMSO助溶,口服样品用0.5%羧甲基纤维素助悬,饮用水稀释至一定容积,小鼠按0.2ml/10g体重ig,大鼠按1ml/100g体重ig。
1.2、其他药品试剂
血脂康胶囊由北京北大维信生物科技有限公司生产,批号20050626,阿司匹林由山东新华制药公司提供,二磷酸腺苷(adenosine diphosphate,ADP)、血小板活化因子(plateletactivating facter,PAF)及花生四烯酸(arachidonic acid,AA)系sigma公司产品,血清总胆固醇(TC)、甘油三酯(TG)及高密度脂蛋白-胆固醇(HDL-C)测定试剂盒购自北京中生北控生物科技股份有限公司。灯盏花素片为云南植物药业有限公司产品,批号20040402。
1.3、主要仪器设备
LBY-NJ2型血液凝聚仪由北京普制生精密仪器研究中心生产,日本岛津公司生产CL-770型临床分光光度仪,北京赛科希德SA-6000型锥板粘度计等。
1.4、实验动物
SD大鼠、ICR小鼠为SPF级,家兔为普通级,实验动物生产许可证号:SCXK(滇)2005-0008,由昆明医学院实验动物中心提供。
2、方法与结果
2.1、统计学方法
实验结果用X±S表示,组间差异用t检验,P<0.05表示组间差异有显著性。
2.2、对血小板聚集功能的影响
2.2.1、富血小板血浆(platelet-rich plasma,PRP)和贫血小板血浆(platelet-poorplasma,PPP)的制备。
自清醒家兔颈动脉取血收集于塑料离心管中,用3.8%枸橼酸钠抗凝(血与抗凝剂体积之比为9∶1),1000rpm离心10min得PRP,吸出PRP后剩余血液再以3000rpm离心10min得PPP,PPP用于调零或调整PRP中血小板的数目,试验中血小板数控制在5×1011/L。
2.2.2、血小板聚集性测定
按Born’s比浊法测定样品不同浓度对ADP,PAF和AA诱导的血小板聚集的影响,即将样品与PRP共同温育10min,再加入不同诱导剂(终浓度分别为ADP3μmol/L,PAF7.2nmol/L,AA0.35mmol/L),测定5min内血小板最大聚集率(%)。结果见表1。
表1 WF-1体外对ADP、PAF或AA诱导血小板聚集功能的影响
n=5 x±S -5溶媒对照(0.5%DMSO)比 ap<0.01 bp<0.05
结果表明,WF-1显著抑制ADP诱导的血小板聚集作用,该作用呈一定的浓度相关性;此外,WF-1还有显著抗PAF作用;对AA诱导的血小板聚集,WF-1只在较高浓度有抑制作用(80mg/L)。
2.2.3对小鼠断头后张口喘气时间的影响
选用ICR小鼠50只,雌雄各半,体重18~22g,按体重随机分为5组,即空白对照组,阳性对照组(灯盏花素片100mg、kg-1、d-1)及SC-1低、中、高剂量组和WF-1低、中、高剂量组(分别按50、100、200mg、kg-1、d-1给药),样品用饮用水稀释至一定容积,按0.2ml/10g体重ig,每天一次,空白对照组ig等体积饮用水。连续给药(或水)6天。末次给药后1h进行断头张口喘息时间测定。
结果显示,除灯盏花素片组(100mg/kg)有显著延长小鼠断头后张口喘气时间外,其他各组喘气时间与空白对照组比均无显著差异。WF-1高剂量组张口喘气时间虽较空白对照组略有延长,但无显著性差异(P>0.05)(表2)。
表2 WF-1对小鼠断头张口喘气时间的影响
n=10 x±S 与空白对照组比bP<0.05
2.2.4对出血时间的影响
选用ICR小鼠50只,雌雄各半,体重18~22g,按体重随机分成5组,分组及给药量同前,阳性对照改用阿司匹林(30mg/kg),连续给药5天,末次给药后1h用断尾法进行出血时间测定[3],将小鼠尾尖8mm处剪断,从出血开始(滤纸上出现血迹)计时,每半分钟用滤纸吸去血滴一次,直到出血自然停止(滤纸上无血迹),所计时间即为出血时间。
结果显示,WF-1 100-200g·kg-1·d-1有显著延长小鼠出血时间作用,与ASP作用一致,该作用呈一定的剂量相关性,见表3
表3 WF-1对小鼠尾尖出血时间的影响
n=10 x±S 与空白对照组比ap<0.01,bp<0.05
2.2.5 对凝血时间的影响-毛细玻璃管法
选用ICR小鼠50只,雌雄各半,体重20~24g,按体重随机分为5组,分组及给药量与观察对出血时间影响相同。按分组连续给药5天,末次给药后1h用毛细玻管法进行凝血时间测定。取内径1mm、长10cm毛细玻管自小鼠一侧眼球内眦插入球后静脉丛,深的4~5mm,至血液流入毛细玻管内开始计时,血液注满后将毛细玻管取出平放于桌上,每隔30秒折断二端毛细管约0.5cm,至血凝丝出现为止,所历时间即为凝血时间。
表4 WF-1对小鼠凝血时间的影响
n=10 x±S 与空白对照组比ap<0.01,bp<0.05
结果表明,WF-1有延长小鼠凝血时间作用。
2.2.6对ADP诱发小鼠肺微循环血栓形成的影响
ICR小鼠50只,雌雄各半,体重18~20g,按体重随机分为5组,分组及给药量同前,连续灌胃给药5天,末次给药后1h按文献方法于小鼠尾静脉iv 400mg/kg的ADP生理盐水溶液(0.1ml/10g体重),注毕,观察并记录15min内动物死亡数,计算死亡抑制率。
结果表明:100和200mg/kg体重WF-1有显著降低静脉注射ADP所导致的肺微血管栓塞动物死亡数,该作用呈剂量依赖趋势(表5)
表5 WF-1对ADP致小鼠肺栓塞死亡的保护作用
与空白对照组比aP<0.01,bP<0.05
2.2.7对小鼠实验性高脂血症血脂水平的影响
雄性ICR小鼠50只,体重18~22g,按体重随机分为5组,全部动物饲以含高脂肪,高胆固醇饲料,自由进食,饮水不限。高脂饲料配方为(W∶W):胆固醇2%,蛋黄10%,猪油7%,丙基硫氧嘧啶0.1%,胆酸钠0.3%,基础饲料80.6%,在进食高脂饲料的同时,各给药组按分组灌胃给予不同剂量的药物,空白对照组给相同体积饮用水。5天后摘眼球取禁食12h空腹血,用酶法按试剂盒说明书操作程序,测血清总胆固醇(TC)、甘油三酯(TG)和高密度脂蛋白-胆固醇(HDL-C),结果见表6。
结果表明,WF-1灌胃给药有降低实验性高脂血症小鼠血清TC和TG作用,该作用呈剂量相关性,200mg/kg体重降低TC和TG作用十分显著,与模型组比P<0.01,随着TC下降,HDL-C也有一定降低,但HDL-C/TC比值略有升高,说明WF-1对脂蛋白分布无有害影响。
表6 WF-1对小鼠实验性高脂血症的影响
n=10 x±saP<0.01与高脂对照组比较
2.2.8对血浆粘度的影响
雄性SD大鼠40只,体重200~240g,按体重随机分为4组,分组及给药量同前,高脂饲料喂养,饲料配方不变,连续给药7天,禁食12h后由股动脉取血,1%肝素抗凝,分离血浆(3000rpm,10min),用北京赛科希德SA-6000型锥板粘度计进行血浆粘度测定。结果显示,WF-1无明显影响大鼠血浆粘度作用(表7)
表7WF-1对大鼠血浆粘度的影响
小结:初步实验结果显示,样品WF-1体外试验有较好的抑制ADP、PAF诱导的血小板聚集作用,体内给药能显著降低静脉注射ADP所致的肺微血管栓塞动物死亡数,显著延长小鼠的出、凝血时间,WF-1在高剂量时(200mg/kg)可降低动物实验性高脂血症血清TC和TG水平,但无降低血粘度作用。以上结果提示,WF-1具有抗血栓栓塞作用,具有良好的心脑血管疾病药物开发前景。
二、抗菌消炎作用
1.材料
樟叶越橘提取物、盐酸环丙沙星、强力银翘片。昆明种小鼠,体重18~22g。Wistar大鼠,体重120~200g。菌种有肺炎双球菌菌液(浓度6×108个/mL)、金黄色葡萄球菌菌液(浓度1.2×109个/mL)。电脑数字式体温计,VIS-7220型分光光度计。
2方法
2.1樟叶越橘提取物体内抗菌试验
2.1.1对肺炎双球菌感染小鼠的保护作用
取小鼠60只,随机分为4组,每组15只。分别ig 18%樟叶越橘提取物、9%樟叶越橘提取物、0.8%环丙沙星、生理盐水,均为0.3mL·10g-1体重。各组小鼠每日给药1次,连续给药4d(感染后各组动物继续按上述方法给药,直至试验结束)。将引起小鼠90%死亡的肺炎链球菌菌液(浓度3.16×108个/mL)给小鼠ip 0.5mL/只,观察一周内各组小鼠死亡数。
2.1.2樟叶越橘提取物对金黄色葡萄球菌感染小鼠的保护作用
取小鼠60只,分组和给药方法同2.1.1。将引起小鼠90%死亡的金黄色葡萄球菌菌液(浓度6.6×108个/mL)给小鼠ip 0.5mL/只,观察一周内各组小鼠死亡数。
2.2樟叶越橘提取物抗炎试验
2.2.1樟叶越橘提取物对蛋清所致大鼠足跖肿胀的影响
取大鼠40只,体重120-150g,随机分为4组,每组10只。分别ig18%樟叶越橘提取物、9%樟叶越橘提取物、4.5%强力银翘片、生理盐水,均为1mL·100g-1体重。各组每天给药1次,连续给药4d。末次给药前测量各组大鼠左后足跖容积作为致炎前正常足跖容积,于给药后30min于左后足跖Sc新鲜蛋清0.05mL/只。于注射蛋清后0.5、1、2、4及6h各测足跖容积1次,记录各组测量数据。
2.2.2樟叶越橘提取物对二甲苯所致小鼠耳肿胀的影响
取小鼠40只,体重18-22g,随机分为4组,每组10只。分别ig18%樟叶越橘提取物、9%樟叶越橘提取物、4.5%强力银翘片、生理盐水,均为0.2mL·10g-1体重。各组每天给药1次,连续给药4d。末次给药后1h在乙醚麻醉下,将二甲苯0.02mL涂在小鼠左耳前后两面致炎,致炎后30min将小鼠处死,用7mm直径打孔器在小鼠左右耳相同部位打下耳片,用电子天平称重。记录左右耳片重量,计算肿胀率。
3.结果
3.1体内抗菌试验结果
对照组、樟叶越橘提取物大剂量组、小剂量组、环丙沙星组各15只小鼠腹腔感染肺炎双球菌,死亡数分别是14、7、9、4只。4组各15只小鼠腹腔感染金黄色葡萄球菌,死亡数分别是13、5、8、1只。结果表明,樟叶越橘提取物大剂量组可使腹腔感染肺炎双球菌小鼠和腹腔感染金黄色葡萄球菌小鼠的死亡数明显减少,与对照组比较,差异分别有显著意义(P<0.05)和极显著意义(P<0.01)。
3.2抗炎试验结果
大鼠足跖肿胀试验结果表明,樟叶越橘提取物对蛋清引起的大鼠足跖肿胀有明显抑制作用,大剂量组在致炎后0.5h、1h、2h和4h作用明显,肿胀率与对照组比较差异有显著性;小剂量组在致炎后0.5h、1h和2h作用较明显,肿胀率与对照组比较差异有显著性。
小鼠耳肿胀试验结果表明,樟叶越橘提取物对二甲苯所致小鼠耳肿胀有一定的抑制作用,大剂量组与对照组比较肿胀率差异有显著性。
4.讨论
实验说明樟叶越橘提取物具有一定抗菌、抗炎的作用。
三、抗肿瘤作用(细胞毒)
取浓度为2×105个/mt人胃癌细胞BGC-823混悬液,置于96孔微量培养板,每孔50μl,同时加入等体积的不同浓度樟叶越橘提取物。使终浓度分别为2、4、6和8μg/ml,以空白培养基作为对照。每一实验浓度设4个平行孔。然后在37℃二氧化碳温箱中培养40~48h,每孔加噻唑蓝的PBS溶液20μl(5mg/ml),37℃作用4h后,吸去上清液,每孔加0.10ml二甲基亚砜,振荡5min后,在酶标仪上测定各孔A370nm,计算杀伤率,结果见图1。试验结果表明,樟叶越橘提取物中确存在对肿瘤细胞直接杀伤的成分。
附图说明
图1是本发明的樟叶越橘提取物对B G C-823的杀伤作用。
具体实施方式
下面结合附图和实施例,对本发明作进一步的详细说明,但不是对本发明的限定。
实施例1:
取云南产樟叶越橘(Vaccinium dunalianum)10公斤,粉碎,加水60公斤,煎煮2小时,过滤,滤液用大孔吸附树脂吸附(或乙酸乙酯、丁醇萃取),先用水洗脱,除去寡糖类成分,再用75%乙醇洗脱,收集洗脱液,浓缩至无乙醇,放置,结晶,过滤,结晶再用50%乙醇加热溶解,放置结晶,过滤,50℃以下干燥,得420g越橘苷。取该结晶400g粉碎,加入淀粉310g、蔗糖粉100g、乳糖100g、山梨醇50g,混匀,制粒,干燥,整粒,与40g硬脂酸镁,混匀,压片,制成4000片。
实施例2:
取云南产樟叶越橘(Vaccinium dunalianum)10公斤,粉碎,加水80公斤,煎煮3小时,过滤,丁醇萃取,萃取液浓缩至干,加75%乙醇水使完全溶解,放置,结晶,过滤,结晶再用50%乙醇加热溶解,放置结晶,过滤,50℃以下干燥,得435g樟叶越橘苷。取该结晶400g粉碎,加入蔗糖粉420g、乳糖100g、山梨醇50g,混匀,制粒,干燥,整粒,加入30g硬脂酸镁,混匀,填装成胶囊8000粒。
Claims (5)
1、一种越橘苷的提取方法,其特征在于按以下进行:取樟叶越橘粉碎,加水6~8倍,煎煮2~3小时,过滤,滤液用大孔吸附树脂吸附先用水洗脱,除去寡糖类成分,再用75%乙醇洗脱,收集洗脱液,浓缩至无乙醇,放置,结晶,过滤,结晶再用50%乙醇加热溶解,放置结晶,过滤,50℃以下干燥,得樟叶越橘苷;或煎煮液过滤后用乙酸乙酯或丁醇萃取,萃取液浓缩,残留物加75%乙醇溶解,放置,结晶,过滤,结晶再用50%乙醇加热溶解,放置结晶,过滤,50℃以下干燥,得樟叶越橘苷。
2、用权利要求1所述的樟叶越橘苷的提取方法提取的樟叶越橘苷制备的食品或药品,其特征在于取樟叶越橘苷,加入食品或药品中允许加入的助剂,制成饮料、口服液、片剂、胶囊。
3、权利要求2所述的越橘苷制备的食品或药品的应用,其特征在于可以用于降血脂、胆固醇和抗血栓。
4、权利要求2中所述的越橘苷制备的食品或药品的应用,其特征在于其可以用于肿瘤疾病的治疗。
5、权利要求2中所述的越橘苷制备的食品或药品的应用,其特征在于其可以用于病原性微生物引起的炎症治疗。
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