CN106074636B - 人面果叶提取物的应用及制备方法 - Google Patents
人面果叶提取物的应用及制备方法 Download PDFInfo
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- CN106074636B CN106074636B CN201610570472.XA CN201610570472A CN106074636B CN 106074636 B CN106074636 B CN 106074636B CN 201610570472 A CN201610570472 A CN 201610570472A CN 106074636 B CN106074636 B CN 106074636B
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Abstract
本发明公开了一种人面果叶提取物的应用,所述人面果叶提取物含有人面果叶的总黄酮提取物,所述人面果叶提取物用于制备降尿酸和抗痛风的药物。本发明经研究发现,人面果叶的醇提物、总黄酮部位及其化合物福木甙(Fukugiside)、福树黄素(Fukugetin)和白果素(Bilobetin)具有极显著的降尿酸效果,比所有已知的降尿酸植物提取物均要明显,甚至远超阳性药别嘌醇的效果,且还有较好的抗痛风的作用,真正达到了安全有效的目的,极具开发应用前景。
Description
技术领域
本发明属于中药领域,具体涉及一种人面果叶提取物及其制备方法和应用。
背景技术
高尿酸血症最有代表性的特点是循环血液中的尿酸高出正常水平(大于7.0mg/dl)并长时间维持。随之社会经济与科技水平不断发展,人类寿命平均水平也比过去有所提高,随之而来的是社会老龄化的问题。经济发展,人们饮食状况的提高已经到了尤过不及的水平,营养摄入过剩,造就了诸多老年性疾病,如高血压、动肪粥样硬化、糖尿病、高脂血症等。这些疾病的高度流行无一不对人们的生活质量和社会的发展带来负担。大量的流行病学分析报道指出,高尿酸血症并不是孤立单一的疾病,除了进一步诱发成痛风,它还与前面所提及的疾病有不可分割的联系。高尿酸血症是常见的慢性疾病,它在发病初期可以长期保持无症状状况,因而不易被患者所注意,直到发展到痛风发作,疼痛剧烈才被觉察。因此,长期的体内高尿酸状况可能导致身体出现上述的诸多老年性的疾病,疾病发展下去,有可能演变为肾功能不全等病危症状,危害生命。
高尿酸血症与痛风的治疗包括饮食疗法与药物治疗两部分。对于无症状的高尿酸血症患者,多数学者主张采用饮食疗法,即限制嘌呤摄入。而痛风则应严格在饮食疗法的基础上,加用有针对性的药物治疗。
目前西药治疗高尿酸与痛风的靶点明确,机理清楚,但毒副作用明显,若大量或长期服用,极易对人体造成较为严重的毒副作用;中药有明确疗效,毒副作用小,但有效成分与作用机理急需阐明。
人面果叶藤黄科藤黄属植物人面果(Garcinia xanthochymus)的干燥树叶。我国主要分布在云南、广东、广西、台湾等省。树高20-30米,因聚合浆果呈球形或似糯米饭团,故称人面果或大冷饭团。人面果原生于原始森林,属野生植物水果。化学成分研究表明,人面果叶主要含有双黄酮、黄酮及酚酸等成分。现代药理研究表明,人面果叶具有抗炎、抗氧化、改善糖尿病等功效。
发明内容
现有技术中,尚未见有文献报道人面果叶具有降尿酸与抗痛风的药理作用。本发明在前期的药物筛选过程中,发现人面果叶的醇提物及其总黄酮部位具有极显著的降尿酸效果,比所有已知的降尿酸植物提取物均要明显,甚至远超阳性药别嘌醇的效果,且还有较好的保护肾功和抗痛风的作用,真正达到了安全有效的目的,极具开发应用前景。
本发明的目的在于提供人面果叶提取物在制备防治高尿酸与痛风中的应用。
本发明提供了一种人面果叶提取物的应用,所述人面果叶提取物含有人面果叶的总黄酮提取物,所述人面果叶提取物用于制备降尿酸和抗痛风的药物。
优选地,所述人面果叶的总黄酮提取物含有如下化合物:福木甙(Fukugiside)、福树黄素(Fukugetin)和白果素(Bilobetin)。
优选地,福木甙、福树黄素和白果素的质量之和在人面果叶的总黄酮提取物中占50%以上。
本发明还提供一种人面果叶提取物的应用,所述人面果叶提取物为福木甙、福树黄素或白果素的其中之一或其组合,所述人面果叶提取物用于制备降尿酸和抗痛风的药物。
本发明还提供一种人面果叶提取物的应用,所述人面果叶提取物为人面果叶的醇提物,所述人面果叶提取物用于制备降尿酸和抗痛风的药物,其中人面果叶的醇提物的制备方法为:将人面果叶药材以体积百分比浓度为40~80%的乙醇水溶液提取,提取液过滤并回收乙醇后,得人面果叶的醇提物。
本发明还提供一种降尿酸和抗痛风的药物,含有人面果叶的总黄酮提取物、人面果叶的醇提物、福木甙、福树黄素或白果素中的一种或其任意组合。
本发明提供一种人面果叶的总黄酮提取物的制备方法,包括如下步骤:
(1)干燥人面果叶药材粉碎后,用体积百分比浓度为40%-80%的乙醇水溶液提取,提取液过滤并回收乙醇后,得人面果叶的醇提物;
(2)人面果叶的醇提物加水调节浓度,调节pH至7~8,上HPD100大孔吸附树脂柱吸附后,以水洗脱,然后以体积百分比浓度为20~30%的乙醇水溶液洗脱,再以体积百分比浓度为50~70%的乙醇水溶液洗脱,收集50-70%的乙醇水溶液洗脱液,减压回收至无醇味,得到HPD100树脂洗脱液,干燥,即得人面果叶的总黄酮提取物。
优选地,步骤(2)中以NaHCO3调节pH。
本发明提供一种人面果叶提取物的制备方法,包括如下步骤:
(1)干燥人面果叶药材粉碎后,用体积百分比浓度为40%-80%的乙醇水溶液提取,提取液过滤并回收乙醇后,得人面果叶的醇提物;
(2)人面果叶的醇提物加水调节浓度,调节pH至7~8,上HPD100大孔吸附树脂柱吸附后,以水洗脱,然后以体积百分比浓度为20~30%的乙醇水溶液洗脱,再以体积百分比浓度为50~70%的乙醇水溶液洗脱,收集50-70%的乙醇水溶液洗脱液,减压回收至无醇味,得到HPD100树脂洗脱液,干燥,即得人面果叶的总黄酮提取物;
(3)取人面果叶的总黄酮提取物以50~60℃的水溶解,调节浓度后,上30-60目聚酰胺柱,先用水洗脱,再依次用体积百分比浓度20%、35%、60%和75%乙醇洗脱,分别收集35%、60%和75%乙醇洗脱液,减压回收至少量体积后,再冷冻干燥,分别得到化合物福木甙、福树黄素和白果素。
优选地,步骤(2)中以NaHCO3调节pH。
本发明的有益效果:
本发明经研究发现,人面果叶的醇提物、总黄酮部位及其化合物福木甙、福树黄素和白果素具有极显著的降尿酸效果,比所有已知的降尿酸植物提取物均要明显,甚至远超阳性药别嘌醇的效果,且还有较好的抗痛风的作用,真正达到了安全有效的目的,极具开发应用前景。
具体实施方式
下面结合具体实施例对本发明作进一步说明,以使本领域的技术人员可以更好的理解本发明并能予以实施,但所举实施例不作为对本发明的限定。
实施例1人面果叶醇提物的制备
人面果叶购于云南西双版纳,经鉴定为藤黄科藤黄属植物人面果(Garciniaxanthochymus)的干燥树叶。药材干燥后,粉碎机粉碎,过20目筛,用体积百分浓度为60%的乙醇溶液提取2次,所用乙醇溶液的质量依次为人面果叶药材质量的12倍,10倍,每次提取的回流时间一次为2h,1.5h,将2次提取液过滤后合并,回收乙醇,得人面果叶醇提物。
实施例2人面果叶的总黄酮提取物的制备
实施例1中的人面果叶醇提物经HPD100大孔吸附树脂富集,精制获得人面果叶的总黄酮部位;HPD100大孔吸附树脂纯化流程为:将人面果叶醇提物加水调节浓度为0.01g药材/ml,同时以浓度为10%的NaHCO3调节pH为8,上HPD100树脂柱吸附,先用3倍柱体积的水洗脱,再用2倍量柱体积的30%乙醇洗脱,再用3倍量柱体积的60%乙醇洗脱。收集60%的乙醇洗脱液,减压回收至至少量体积后,再冷冻干燥,得人面果叶总黄酮部位;
实施例3人面果叶总黄酮部位中各类成分含量的测定
实施例2中的人面果叶总黄酮提取物,其特征在于应含有3种黄酮类化合物,所述的3种黄酮类化合物为:福木甙、Fukugetin,福树黄素Fukugiside和白果素。且所述的3种化合物的质量之和应不低于总黄酮部位质量的50%。这3种化合物经高效液相色谱法测定,其总质量之和占人面果叶总黄酮部位的55%。
实施例4福木甙、福树黄素和白果素的制备
将实施例2中的人面果叶的总黄酮部位经50-60℃的热水溶解,调节至浓度为0.02g药材/ml,上60目聚酰胺柱,先用水洗脱,再用20%、35%、60%和75%乙醇洗脱。收集35%、60%和75%乙醇洗脱液,减压回收至少量体积后,再冷冻干燥,分别得到化合物福木甙、福树黄素和白果素。且上述3个化合物的纯度经高效液相色谱法测定不低于90%。
实施例5人面果叶醇提物、人面果叶总黄酮部位和福木甙、福树黄素、白果素降尿酸研究
1仪器、材料与试药
1.1仪器和材料
Tissue-Tearor型手持高速组织匀浆机(北京市世安科兴科技开发有限公司),TDL80-2B型台式离心机(上海安亭科技仪器厂),BS124S型电子天平(SartoriusCorporation,Germany),KD-160型鼠称(TANITA公司),Mni shaker培养板振荡器(Kylin-Bell Lab Instruments,海门市其林贝尔仪器制造有限公司),WH-2型漩涡混合仪(上海沪西分析仪器厂),DZF-6020型真空干燥箱(上海一恒科技术有限公司),DG5033A型酶标仪(华东电子),96孔细胞培养板(Corporation,USA),50μL、200μL、1000μL移液器(ThermoElectron Corporation,USA),HH-4型恒温水浴锅(常州国华电器有限公司),MILLI-QBiocel型超纯水装置(Millipore,USA),其它常用器具(手术器械、温度计、离心管、离心管架、烧杯、移液枪头、注射剂、小鼠灌胃针和量筒等)。
1.2试药
别嘌醇片(批号:20150908,江苏方强制药厂有限责任公司),氧嗪酸钾(AR,批号:120202,纯度≥99%,Amresco公司),羧甲基纤维素钠(CMC-Na,分子量300-800,中国医药集团上海化学试剂公司),食用级乙醇(南京鼎新化工轻工有限公司),氯化钠(AR,南京化学试剂有限公司),尿酸测定试剂盒(南京建成生物科技有限公司,批号:20160108)。
1.3实验动物
昆明种雄性小鼠,体重18-22g(华中科技大学实验动物中心提供,动物生产许可证SCXK(鄂)2010-0009)。
2.实验溶液配制
2.1 0.5%的CMC-Na溶液配制
称取CMC-Na 2.5g加入500mL纯净水中,加热,超声使其溶解,静置过夜。
2.2生理盐水的配制
称取NaCl 4.5041g,溶于500mL纯净水中即得。
2.3人面果叶醇提物
称取实施例1中的人面果叶醇提物适量,用CMC-Na溶液稀释至浓度分别为0.4g生药材/mL和0.2g生药材/mL的高、低浓度样品液。人面果叶醇提物的得率为15%(与生药材比较)。
2.4人面果叶总黄酮部位
称取实施例2中的人面果叶总黄酮部位适量,用CMC-Na溶液稀释至浓度分别为0.4g生药材/mL和0.2g生药材/mL的高、低浓度样品液。人面果叶总黄酮部位的得率为8%(与生药材比较)。
2.5福木甙、福树黄素、白果素
称取实施例4中的福木甙、福树黄素、白果素适量,用CMC-Na溶液稀释至浓度分别为2mg/mL和1mg/mL的高、低浓度样品液。
2.6别嘌醇溶液的制备(规格为1片100mg)
取别嘌醇片1片,研碎后加入100mL 0.5%的CMC-Na混悬,配制成浓度为1mg/mL的别嘌醇溶液。
2.7氧嗪酸钾溶液的配制
称取适量,以0.5%CMC-Na混悬,配制成12.5mg/mL的造模液。
3实验方法
3.1动物饲养
昆明种小鼠,分笼饲养于室内,室温(20~22℃),相对湿度35%~50%,自由饮食、饮水。适应性饲养三天后,开始给药。
3.2动物分组及造模
90只昆明种小鼠,按体重随机分为13组,每组10只,分别设为空白组,模型组,别嘌醇组,人面果叶醇提物高剂量组(RMH),人面果叶醇提物低剂量组(RML),人面果叶总黄酮部位高剂量组(RMTH),人面果叶总黄酮提取物高剂量组(RMTL),福木甙高剂量组(FDH),福木甙低剂量组(FDL),福树黄素高剂量组(FTH),福树黄素低剂量组(FTL),白果素高剂量组(BGH)、白果素低剂量组(BGL)。上述13组动物,自由饮食、饮水,空白及模型组灌胃给予0.5%CMC-Na,其它各组每日灌胃给予相应的药物,给药剂量均为为0.2mL/10g,连续给药4天。末次给药前1h,空白组腹腔注射0.2mL/10g的0.5%CMC-Na溶液,其它各组腹腔注射氧嗪酸钾溶液0.2mL/10g,1h后,眼内眦取血0.1mL左右。
3.3样品处理及测定
按照尿酸测定试剂盒进行操作
4实验结果
所得数据用SPSS11.5统计分析软件进行统计分析,分析结果以平均值±SD表示。
表一、不同浓度的大高纯化物对高尿酸小鼠血清尿酸的影响
###p<0.001与空白组比较;*p<0.05;**p<0.01and***p<0.001与模型组比较。
5结论
本实验结果表明,人面果叶醇提物、人面果叶总黄酮提取物和化合物福木甙、福树黄素、白果素均有显著的降低高尿酸小鼠的血清尿酸值,且福木甙的高剂量效果要优于一线降尿酸药物别嘌醇,这样的实验结果是非常鼓舞人的,具有极大的开发前景。
实施例6人面果叶醇提物、人面果叶总黄酮部位和福木甙、福树黄素、白果素抗痛风研究
1仪器、材料与试药
1.1仪器和材料
Tissue-Tearor型手持高速组织匀浆机(北京市世安科兴科技开发有限公司),TDL80-2B型台式离心机(上海安亭科技仪器厂),BS124S型电子天平(SartoriusCorporation,Germany),KD-160型鼠称(TANITA公司),Mni shaker培养板振荡器(Kylin-Bell Lab Instruments,海门市其林贝尔仪器制造有限公司),WH-2型漩涡混合仪(上海沪西分析仪器厂),DZF-6020型真空干燥箱(上海一恒科技术有限公司),DG5033A型酶标仪(华东电子),96孔细胞培养板(Corporation,USA),50μL、200μL、1000μL移液器(ThermoElectron Corporation,USA),HH-4型恒温水浴锅(常州国华电器有限公司),MILLI-QBiocel型超纯水装置(Millipore,USA),其它常用器具(手术器械、游标卡尺、温度计、离心管、离心管架、烧杯、移液枪头、注射剂、小鼠灌胃针和量筒等)。
1.2试药
吲哚美辛(批号:20151001,昆明振华制药有限公司),尿酸钠结晶(AR,批号:150912,纯度≥99%,Sigma公司),羧甲基纤维素钠(CMC-Na,分子量300-800,中国医药集团上海化学试剂公司),食用级乙醇(南京鼎新化工轻工有限公司),氯化钠(AR,南京化学试剂有限公司),IL-1βELISA试剂盒(R&D,US,批号:20160201)。
1.3实验动物
SD雄性大鼠,体重180~220g(华中科技大学实验动物中心提供,动物生产许可证SCXK(鄂)20050008)。
2.实验溶液配制
2.1 0.5%的CMC-Na溶液配制
称取CMC-Na2.5g加入500mL纯净水中,加热,超声使其溶解,静置过夜。
2.2生理盐水的配制
称取NaCl 4.5041g,溶于500mL纯净水中即得。
2.3人面果叶醇提物
称取实施例1中的人面果叶醇提物适量,用CMC-Na溶液稀释至浓度分别为0.4g生药材/mL和0.2g生药材/mL的高、低浓度样品液。人面果叶醇提物的得率为15%(与生药材比较)。
2.4人面果叶总黄酮提取物
称取实施例2中的人面果叶总黄酮部位适量,用CMC-Na溶液稀释至浓度分别为0.4g生药材/mL和0.2g生药材/mL的高、低浓度样品液。人面果叶总黄酮提取物的得率为8%(与生药材比较)。
2.5福木甙、福树黄素和白果素
称取实施例4中的福木甙、福树黄素、白果素适量,用CMC-Na溶液稀释至浓度分别为2mg/mL和1mg/mL的高、低浓度样品液。
2.6吲哚美辛的制备(规格为1片100mg)
取别吲哚美辛片1片,研碎后加入100mL 0.5%的CMC-Na混悬,配制成浓度为1mg/mL的吲哚美辛溶液。
2.7尿酸钠结晶溶液的配制
称取适量尿酸钠结晶,以结晶生理盐水混悬,配制成1mg/mL的造模液。
3实验方法
3.1动物饲养
SD大鼠,分笼饲养于室内,室温(20~22℃),相对湿度35%~50%,自由饮食、饮水。适应性饲养三天后,开始给药。
3.2动物分组及造模
90只SD大鼠,按体重随机分为13组,每组10只,分别设为空白组,模型组,吲哚美辛组,人面果叶醇提物高剂量组(RMH),人面果叶醇提物低剂量组(RML),人面果叶总黄酮部位高剂量组(RMTH),人面果叶总黄酮部位高剂量组(RMTL),福木甙高剂量组(FDH),福木甙低剂量组(FDL),福树黄素高剂量组(FTH),福树黄素低剂量组(FTL),白果素高剂量组(BGH)、白果素低剂量组(BGL)。上述13组动物,自由饮食、饮水,空白及模型组灌胃给予0.5%CMC-Na,其它各组每日灌胃给予相应的药物,给药剂量均为为0.2mL/10g,连续给药4天。末次给药后1h,固定大鼠,将右踝关节弯曲,局部消毒后,用6号注射针自两骨突之间进针,除空白组大鼠注射生理盐水0.05mL外,其余各组大鼠在右关节腔内注射尿酸钠生理盐水混悬液0.05mL,以关节囊对侧鼓起为注入标准,诱导痛风性关节炎的发生。
3.3样品处理及测定
检测指标:(1)关节肿胀度:分别用游标卡尺测量致炎前及致炎后4、12、24、48h大鼠右踝关节的厚度,以致炎前后的差值除以致炎前的厚度×100%作为痛风性关节炎的肿胀度。(2)对大鼠关节及周围软组织中IL-1β的影响:致炎48h取血后,处死动物,取关节滑膜及周围软组织,称质量,在冰浴中制成匀浆,按1∶20比例加生理盐水稀释,4000r/min离心5min,取上清液,分装于Ep管中,于-80℃冰箱保存,按双抗体夹心ELISA法试剂盒说明书进行IL-1β定量检测。
4实验结果
所得数据用SPSS11.5统计分析软件进行统计分析,分析结果以平均值±SD表示。
表二、不同浓度的人面果提取物及化合物对痛风大鼠关节肿胀的影响
###p<0.001与空白组比较;*p<0.05;**p<0.01and***p<0.001与模型组比较。
表三、不同浓度的人面果叶及化合物对痛风大鼠关节组织中IL-1β水平的影响
###p<0.001与空白组比较;*p<0.05;**p<0.01and***p<0.001与模型组比较。
5结论
本实验结果表明,人面果叶醇提物、人面果叶总黄酮提取物位和化合物福木甙、福树黄素、白果素均有显著的改善大鼠痛风性关节肿胀及降低组织中IL-1β水平的效果,且呈现出一定的剂效关系,因此认为人面果叶2种提取物和福木甙、福树黄素、白果素均有较明显的抗痛风性关节炎的作用,且福木甙的效果更为明显。
综合实施例5与实施例6,我们发现,本案例中的人面果叶醇提物、人面果叶总黄酮部位和福木甙、福树黄素、白果素有确切的降尿酸与抗痛风的作用,且其降尿酸效果尤其明显。另外,人面果叶作为一种传统的食品,基本是无毒,如果能够开发成抗痛风与降尿酸的药物,将具有很好的应用前景。
实施例7颗粒剂制备
称取实施例1、2或4中的最佳工艺制备的人面果叶醇提物、人面果叶总黄酮部位或福木甙、福树黄素、白果素,加入适量的辅料糊精,混合均匀,过60目筛,加入适量的无水乙醇,过20目筛挤压制粒,60℃干燥后,得颗粒剂。
实施例8片剂制备
称取实施例1、2或4中的最佳工艺制备的人面果叶醇提物、人面果叶总黄酮部位或福木甙、福树黄素、白果素,加入适量的辅料淀粉和硬脂酸镁,混匀,打片,即得片剂。
以上所述实施例仅是为充分说明本发明而所举的较佳的实施例,本发明的保护范围不限于此。本技术领域的技术人员在本发明基础上所作的等同替代或变换,均在本发明的保护范围之内。本发明的保护范围以权利要求书为准。
Claims (7)
1.一种人面果叶提取物的应用,其特征在于,所述人面果叶提取物含有人面果叶的总黄酮提取物,所述人面果叶的总黄酮提取物含有福木甙(Fukugiside)、福树黄素(Fukugetin)和白果素(Bilobetin),所述人面果叶提取物用于制备降尿酸和抗痛风的药物;
其中,人面果叶的总黄酮提取物的制备方法:将人面果叶药材以体积百分比浓度为40~80%的乙醇水溶液提取,提取液过滤并回收乙醇后,得人面果叶醇提物,再将人面果叶醇提物经HPD100大孔吸附树脂富集,精制即得人面果叶的总黄酮提取物。
2.根据权利要求1所述的应用,其特征在于,福木甙、福树黄素和白果素的质量之和在人面果叶的总黄酮提取物中占50%以上。
3.一种降尿酸和抗痛风的药物,其特征在于,含有人面果叶的总黄酮提取物、人面果叶的醇提物,所述人面果叶的总黄酮提取物含有福木甙、福树黄素和白果素。
4.一种人面果叶的总黄酮提取物的制备方法,其特征在于,包括如下步骤:
(1)干燥人面果叶药材粉碎后,用体积百分比浓度为40~80%的乙醇水溶液提取,提取液过滤并回收乙醇后,得人面果叶的醇提物;
(2)人面果叶的醇提物加水调节浓度,调节pH至7~8,上HPD100大孔吸附树脂柱吸附后,以水洗脱,然后以体积百分比浓度为20~30%的乙醇水溶液洗脱,再以体积百分比浓度为50~70%的乙醇水溶液洗脱,收集50~70%的乙醇水溶液洗脱液,减压回收至无醇味,得到HPD100树脂洗脱液,干燥,即得人面果叶的总黄酮提取物,该人面果叶的总黄酮提取物含有福木甙、福树黄素和白果素。
5.根据权利要求4所述的制备方法,其特征在于,步骤(2)中以NaHCO3调节pH。
6.一种人面果叶提取物的制备方法,其特征在于,包括如下步骤:
(1)干燥人面果叶药材粉碎后,用体积百分比浓度为40~80%的乙醇水溶液提取,提取液过滤并回收乙醇后,得人面果叶的醇提物;
(2)人面果叶的醇提物加水调节浓度,调节pH至7~8,上HPD100大孔吸附树脂柱吸附后,以水洗脱,然后以体积百分比浓度为20~30%的乙醇水溶液洗脱,再以体积百分比浓度为50~70%的乙醇水溶液洗脱,收集50~70%的乙醇水溶液洗脱液,减压回收至无醇味,得到HPD100树脂洗脱液,干燥,即得人面果叶的总黄酮提取物,该人面果叶的总黄酮提取物含有福木甙、福树黄素和白果素;
(3)取人面果叶的总黄酮提取物以50~60℃的水溶解,调节浓度后,上30~60目聚酰胺柱,先用水洗脱,再依次用体积百分比浓度20%、35%、60%和75%乙醇洗脱,分别收集35%、60%和75%乙醇洗脱液,减压回收至少量体积后,再冷冻干燥,分别得到化合物福木甙、福树黄素和白果素。
7.根据权利要求6所述的制备方法,其特征在于,步骤(2)中以NaHCO3调节pH。
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