红黑二丸中甾醇类物质提取、鉴定及生理活性的研究
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摘要
红黑二丸,是秋海棠科植物中中华秋海棠的根茎。本文以红黑二丸为研究对象,通过比较不同提取方法提取海棠根茎中甾醇的效果,确定了最佳提取方法,并采用单因素试验和正交试验对影响提取方法的各个因素进行了条件优化,确定了红黑二丸中甾醇类物质的最佳提取工艺；采用硅胶吸附法对红黑二丸中的甾醇类物质进行了分离纯化,对影响硅胶吸附的温度、时间、吸附液浓度、吸附剂种类及浓度等因素进行了条件优化；并采用紫外-可见吸收光谱法(UV-Vis)、红外光谱法(FT-IR)、高效液相色谱法(HPLC)及气质联用分析法(GC-MS)对分离纯化后的甾醇类物质进行了定性分析；应用β-环糊精分子包埋法将植物甾醇制成一种抗氧化缓释微胶囊,优化了微胶囊的制作工艺；探讨了红黑二丸中甾醇类物质对与日常生活相关的大肠杆菌、金黄色葡萄球菌、铜绿假单胞菌,沙门氏菌和志贺氏菌、空气中常见的枯草芽孢杆菌、冷藏食品腐败菌荧光假单胞菌、致霉变菌黑曲霉和桔青霉等微生物的抑菌活性；并通过动物试验研究了红黑二丸中的甾醇类物质的降血脂作用效果。
结论如下：
(1)采用溶剂结晶法、超声波法及超临界CO2萃取法等三种方法比较了不同方法提取红黑二丸中甾醇类物质的效果。结果显示,超声波法提取的甾醇含量最高,达到0.76 mg/g,提取率也最高,达到84.4 mg/g；溶剂结晶法提取的红黑二丸中的甾醇含量最低,达0.69 mg/g,得率为75.8%；超临界C02萃取法提取得到的甾醇含量和超声波法提取的含量相差不大。因此,本文选择超声波法作为后续提取红黑二丸中甾醇类物质的实验方法。
(2)采用单因素试验和正交试验优化了红黑二丸中的甾醇类物质的提取工艺。结果显示,最佳提取条件为料液比1：24,提取温度为55℃,提取时间为52 min,在此条件下提取得到的红黑二丸中甾醇含量为1.78 mg/g；并采用HPLC对红黑二丸中的甾醇类物质进行了分析。
(3)以吸附率作为评价指标,采用单因素试验优化了硅胶吸附红黑二丸中甾醇类物质的最佳工艺。结果显示,最佳吸附条件为吸附时间2.5 h,吸附温度为35℃,吸附液浓度为0.4 mg/mL,洗脱剂为石油醚：乙酸乙酯=10：1,在该条件下,红黑二丸中甾醇类物质的吸附率为87.54%。
(4)采用紫外、红外、高效液相及气质-联用对提取得到的红黑二丸中的甾醇类物质进行了结构表征。结果显示,分离纯化后的植物甾醇都表现出了甾醇的特征峰,而且干扰较少,分离纯化效果较好；GC-MS结果显示,红黑二丸中的甾醇类物质有四种,其中β-谷甾醇含量最高,达43.42%；其次是豆甾醇,含量达11.68%；然后是豆甾-5,24(28)-双烯-3p-醇,含量11.23%；麦角甾-5-烯-3β-醇,含量9.13%。
(5)以包埋率为评价指标,采用单因素试验和正交试验对影响微胶囊作用的四个条件(温度、时间、加水量及β-环糊精用量)进行了条件优化,最佳工艺条件为：加水量60 mL,搅拌温度50℃,搅拌时间50 min,甾醇(mL)：p-环糊精(g)=1：10。在该条件下所制作的微胶囊产品为乳白色,粉末状,颗粒流动性较好,含水量为3.36%,溶解度为72.27%；密度为0.50 g/mL。采用测定POV值的方法研究了β-谷甾醇、微胶囊化p-谷甾醇及VE在猪油中的抗氧化效果,结果显示微胶囊化p-谷甾醇具有较强的抗猪油油脂氧化能力,其抗氧化效果优于p-谷甾醇和VE；甾醇抗氧化效果呈剂量效应,剂量越大,其抗氧化效果越明显。
(6)红黑二丸甾醇类物质对供试的金黄色葡萄球菌、枯草芽孢杆菌、荧光假单胞菌、铜绿假单胞菌、大肠杆菌、沙门氏菌及志贺氏菌均有一定的抑制效果,且都随着甾醇浓度的提高,抑菌作用越明显。红黑二丸中甾醇类物质对霉菌具有一定的抑制效果,且对黑曲霉和橘青霉抑制效果相当。
(7)红黑二丸甾醇类物质对实验性小鼠高脂血症和动脉硬化具有较好的预防和治疗作用。
This study investigated the best extraction method of sterols from the rhizome of Chinese Begonia (Begonia grandis var. sinensis). Single factor and orthogonal experiments were used to determine the optimal extraction conditions of sterols. The extracted sterols were separated and purified by silica gel adsorption method. The effects of temperature, time, concentrations and kinds of adsorbents on the separation of sterols were also studied. Then the purified sterols were characterized by UV-vis absorption spectra (UV-Vis), infrared spectroscopy (IR), High Performance Liquid Chromatography (HPLC) and Gas Chromatography-Mass Spectrometry (GC-MS).β-Cyclodextrin was used as a wall material to prepare sterols micro-capsules, and the technology was optimized. The antimicrobial activity of sterol extracts against bacteria and fungi were also investigated. In addition, the effect of sterols on the blood-lipid lowering was performed using animal experiments.
The main research subjects and results are listed as follows:
1, Three methods were used to extract the sterol from rhizome of Chinese Begonia. The results showed that ultrasonic technology was the best methods for extracting sterol. The yield using this method was 0.76 mg/g of sterol. The extraction rate was also the highest, which achieved 84.4 mg/g. While the yied and the extraction rate of the other two methods were lower than ultrasonic technology.
2, The optimal conditions of ultrasonic technology were investigated using single factor experiment and orthogonal experiment. The optimal conditions of this methods were listed as follows:material to liquid ratio at 1'.24, extraction temperature 55℃, extraction time 52 min. The content of sterols extracted at this condition was 1.78 mg/g.
3, Sterols were isolated and purified for the purpose of evaluating the adsorption rate using silica gel. The orthogonal test results showed that the optimum conditions of silica static adsorption were as follows:adsorption time 2.5 h, adsorption temperature 35℃, adsorption concentration 0.4mg/mL, elution agent for the petroleum ether/ethyl acetate was 10:1. Under this optimum conditions, the adsorption rate was found to be 87.54%.
4, The structure characterization of the sterols were performed by UV-Vis absorption (UV-Vis), infrared spectroscopy (IR), High-performance Liquid Chromatography (HPLC) and Gas Chromatography-Mass Spectroscopy (GC-MS).β-Sitosterol, stigmasterol, methylenecholest-5,24(28)-diene-3β-ol and ergosta-5-en-3β-ol were detected in rhizome of Chinese Begonia. Among these sterols,β-sitosterol was the most abundant, accounted for 43.42% of the total concentration, followed by stigmasterol.
5, P-Cyclodextrin was used as wall material. The orthogonal test results showed that the optimum conditions for the produce of the sterol microencapsulation were:water addition 60mL, mixing temperature 50℃, stirring time 50 min, sterol (mL):β-cyclodextrin (g) 1:10 (V:m). The finished sterol microencapsulation had a strong antioxidant capacity in comparison with vitamin E,β-sitosterol. The antioxidant capacity enhanced with the concentration increased.
6, Antimicrobial activity of sterol extracts against bacteria and fungi by Oxford plate assay and by growth rate method, respectively. The minimum inhibitory concentrations (MIC) were tested by two-fold serial dilution method. The results showed that sterol extracts had strong inhibition on microorganism with broad-spectrum. The similar effect of antimicrobial activity on gram positive bacterias and gram negative bacterias were observed. It could also inhibit the growth of fungi.
7, The results of the animal experiment showed that great effect of sterols on the blood-lipid lowering was obtained.
结论如下：
(1)采用溶剂结晶法、超声波法及超临界CO2萃取法等三种方法比较了不同方法提取红黑二丸中甾醇类物质的效果。结果显示,超声波法提取的甾醇含量最高,达到0.76 mg/g,提取率也最高,达到84.4 mg/g；溶剂结晶法提取的红黑二丸中的甾醇含量最低,达0.69 mg/g,得率为75.8%；超临界C02萃取法提取得到的甾醇含量和超声波法提取的含量相差不大。因此,本文选择超声波法作为后续提取红黑二丸中甾醇类物质的实验方法。
(2)采用单因素试验和正交试验优化了红黑二丸中的甾醇类物质的提取工艺。结果显示,最佳提取条件为料液比1：24,提取温度为55℃,提取时间为52 min,在此条件下提取得到的红黑二丸中甾醇含量为1.78 mg/g；并采用HPLC对红黑二丸中的甾醇类物质进行了分析。
(3)以吸附率作为评价指标,采用单因素试验优化了硅胶吸附红黑二丸中甾醇类物质的最佳工艺。结果显示,最佳吸附条件为吸附时间2.5 h,吸附温度为35℃,吸附液浓度为0.4 mg/mL,洗脱剂为石油醚：乙酸乙酯=10：1,在该条件下,红黑二丸中甾醇类物质的吸附率为87.54%。
(4)采用紫外、红外、高效液相及气质-联用对提取得到的红黑二丸中的甾醇类物质进行了结构表征。结果显示,分离纯化后的植物甾醇都表现出了甾醇的特征峰,而且干扰较少,分离纯化效果较好；GC-MS结果显示,红黑二丸中的甾醇类物质有四种,其中β-谷甾醇含量最高,达43.42%；其次是豆甾醇,含量达11.68%；然后是豆甾-5,24(28)-双烯-3p-醇,含量11.23%；麦角甾-5-烯-3β-醇,含量9.13%。
(5)以包埋率为评价指标,采用单因素试验和正交试验对影响微胶囊作用的四个条件(温度、时间、加水量及β-环糊精用量)进行了条件优化,最佳工艺条件为：加水量60 mL,搅拌温度50℃,搅拌时间50 min,甾醇(mL)：p-环糊精(g)=1：10。在该条件下所制作的微胶囊产品为乳白色,粉末状,颗粒流动性较好,含水量为3.36%,溶解度为72.27%；密度为0.50 g/mL。采用测定POV值的方法研究了β-谷甾醇、微胶囊化p-谷甾醇及VE在猪油中的抗氧化效果,结果显示微胶囊化p-谷甾醇具有较强的抗猪油油脂氧化能力,其抗氧化效果优于p-谷甾醇和VE；甾醇抗氧化效果呈剂量效应,剂量越大,其抗氧化效果越明显。
(6)红黑二丸甾醇类物质对供试的金黄色葡萄球菌、枯草芽孢杆菌、荧光假单胞菌、铜绿假单胞菌、大肠杆菌、沙门氏菌及志贺氏菌均有一定的抑制效果,且都随着甾醇浓度的提高,抑菌作用越明显。红黑二丸中甾醇类物质对霉菌具有一定的抑制效果,且对黑曲霉和橘青霉抑制效果相当。
(7)红黑二丸甾醇类物质对实验性小鼠高脂血症和动脉硬化具有较好的预防和治疗作用。
This study investigated the best extraction method of sterols from the rhizome of Chinese Begonia (Begonia grandis var. sinensis). Single factor and orthogonal experiments were used to determine the optimal extraction conditions of sterols. The extracted sterols were separated and purified by silica gel adsorption method. The effects of temperature, time, concentrations and kinds of adsorbents on the separation of sterols were also studied. Then the purified sterols were characterized by UV-vis absorption spectra (UV-Vis), infrared spectroscopy (IR), High Performance Liquid Chromatography (HPLC) and Gas Chromatography-Mass Spectrometry (GC-MS).β-Cyclodextrin was used as a wall material to prepare sterols micro-capsules, and the technology was optimized. The antimicrobial activity of sterol extracts against bacteria and fungi were also investigated. In addition, the effect of sterols on the blood-lipid lowering was performed using animal experiments.
The main research subjects and results are listed as follows:
1, Three methods were used to extract the sterol from rhizome of Chinese Begonia. The results showed that ultrasonic technology was the best methods for extracting sterol. The yield using this method was 0.76 mg/g of sterol. The extraction rate was also the highest, which achieved 84.4 mg/g. While the yied and the extraction rate of the other two methods were lower than ultrasonic technology.
2, The optimal conditions of ultrasonic technology were investigated using single factor experiment and orthogonal experiment. The optimal conditions of this methods were listed as follows:material to liquid ratio at 1'.24, extraction temperature 55℃, extraction time 52 min. The content of sterols extracted at this condition was 1.78 mg/g.
3, Sterols were isolated and purified for the purpose of evaluating the adsorption rate using silica gel. The orthogonal test results showed that the optimum conditions of silica static adsorption were as follows:adsorption time 2.5 h, adsorption temperature 35℃, adsorption concentration 0.4mg/mL, elution agent for the petroleum ether/ethyl acetate was 10:1. Under this optimum conditions, the adsorption rate was found to be 87.54%.
4, The structure characterization of the sterols were performed by UV-Vis absorption (UV-Vis), infrared spectroscopy (IR), High-performance Liquid Chromatography (HPLC) and Gas Chromatography-Mass Spectroscopy (GC-MS).β-Sitosterol, stigmasterol, methylenecholest-5,24(28)-diene-3β-ol and ergosta-5-en-3β-ol were detected in rhizome of Chinese Begonia. Among these sterols,β-sitosterol was the most abundant, accounted for 43.42% of the total concentration, followed by stigmasterol.
5, P-Cyclodextrin was used as wall material. The orthogonal test results showed that the optimum conditions for the produce of the sterol microencapsulation were:water addition 60mL, mixing temperature 50℃, stirring time 50 min, sterol (mL):β-cyclodextrin (g) 1:10 (V:m). The finished sterol microencapsulation had a strong antioxidant capacity in comparison with vitamin E,β-sitosterol. The antioxidant capacity enhanced with the concentration increased.
6, Antimicrobial activity of sterol extracts against bacteria and fungi by Oxford plate assay and by growth rate method, respectively. The minimum inhibitory concentrations (MIC) were tested by two-fold serial dilution method. The results showed that sterol extracts had strong inhibition on microorganism with broad-spectrum. The similar effect of antimicrobial activity on gram positive bacterias and gram negative bacterias were observed. It could also inhibit the growth of fungi.
7, The results of the animal experiment showed that great effect of sterols on the blood-lipid lowering was obtained.
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