杨梅叶原花色素的研究
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摘要
原花色素(Proanthocyanidins)是广泛存在于植物中的具有C6·C3·C6基本构架的多酚类物质,其中原花青素型原花色素分布最为广泛。大量研究结果表明原花色素具有抗氧化、抗菌、抗癌以及预防和治疗心血管疾病等活性功能。杨梅(Myrica rubra Sieb. et Zucc.)是我国南方特产水果,目前有关杨梅的研究利用主要以杨梅果实为主。杨梅树枝繁叶茂,四季常青,每年修剪下来的杨梅叶常被当做废弃物而丢弃。已有研究表明杨梅叶中富含酚类物质,具有抗氧化、抗菌抗病毒等生物活性,是一种具有开发潜力的植物资源。
本文主要以杨梅叶为研究对象,从中提取分离原花色素,进而对其结构组成进行了鉴定,在此基础之上分析了它的抗氧化和抗癌活性及机理,评价了它的食用安全性,主要的研究结果如下：
以荸荠种杨梅叶为原料,采用Sephadex LH-20等分离方法分离纯化杨梅叶原花色素,采用光谱分析、酸催化降解、HPLC-MS及NMR等方法分析了原花色素的结构组成。结果显示杨梅叶可提取(EPAs)及不可提取原花色素(UEPAs)均为原飞燕草素,可提取原花色素的末端单元只含有表桔儿茶素桔酸酯,延伸单元主要含有表桔儿茶素桔酸酯,此外还含有少量表桔儿茶素。所有组成单元通过B型键连接而成,即杨梅叶原花色素为B型原花色素,未检测到A型原花色素。杨梅叶原花色素平均聚合度为6.5。不可提取原花色素主要由表桔儿茶素桔酸酯组成,还含有少量表桔儿茶素。杨梅叶原花色素的以上结构组成特点显示其在已有研究材料中具有独特性。
研究了不同杨梅叶中原飞燕草素的组成(NP-HPLC-DAD法和酸催化降解法),结果显示所有杨梅叶原花色素都包含有EPAs和UEPAs,不同品种不同嫩度杨梅叶中的原飞燕草素的组成是不同的。对于荸荠种杨梅叶,无论是EGCG含量,还是原飞燕草素低聚体和高聚体含量,嫩叶中都高于次嫩叶和老叶,同样地,UEPAs含量也是嫩叶高于其他叶。而对于东魁种杨梅叶情况则有所变化,除UEPAs含量是嫩叶中最高外,其他如EGCG、原飞燕草素低聚体和高聚体的含量都是老叶中含量最高,另外两种嫩度杨梅叶差别不大。在本文中,用NP-HPLC法分析原花色素组成,结果说明无论是荸荠种还是东魁种杨梅叶,其二聚体、三聚体和四聚体的含量总合大于聚合度大于4的EPAs。
利用HPLC-DAD-ESIMS等方法分析了杨梅叶中原花色素低聚体及其他小分量多酚类物质。鉴定出了四种黄烷-3-醇单体、三种原飞燕草素二聚体以及两种原飞燕草素三聚体。鉴定出的其他酚类物质有没食子酸和六种黄酮糖苷,其中杨梅黄酮糖苷为主要组成部分。此外还分析了荸荠和东魁两种杨梅叶不同嫩度叶子中原飞燕草素等多酚类物质的含量,结果显示两栽培品种嫩叶中总酚及原飞燕草素含量均最高。
采用DPPH法和FRAP法测定了杨梅叶提取物抗氧化能力,分析了抗氧化成分,并采用响应面分析法优化了主要抗氧化成分原花色素的提取工艺。结果显示杨梅嫩叶提取物抗氧化能力最强,如在DPPH体系中,其EC50值为4.15μg/mL(荸荠)、6.70μg/mL(东魁),荸荠嫩叶抗氧化能力强于葡萄籽原花色素：提取物抗氧化能力与原花色素含量呈线性正相关,相关系数分别为0.963(DPPH)、0.97(FRAP)；丙酮浓度、时间、料液比和温度对原花色素提取量影响显著,优化后的工艺参数为丙酮浓度56.93%、时间31.98min、料液比1：44.52、温度50.00℃,在此条件下,预计原花色素提取量为121.2mg/g,验证实验结果为117.3±5.1 mg/g,两者差异不显著。
以荸荠种和东魁种嫩叶为原料,研究热水提取温度和提取时间对提取物中总酚含量、原花色素含量、原花色素组成及提取物抗氧化活性的影响。总酚和原花色素含量一般均随提取温度的升高和提取时间延长而增加,但是超过一定温度或时间时其含量略有下降。当提取条件一定时,荸荠种总酚和原花色素含量低于东魁种。提取条件对不同聚合度原花色素含量也有影响,一般在较低温度时(不超过75℃)低聚体部分的含量会随提取温度的升高而增加,而在较高温度时含量减少,高聚体部分的含量随提取温度的升高和提取时间的延长而增加。
不同体外抗氧化试验体系(DPPH、ABTS、铁氰化钾、FRAP、螫合金属离子)分析结果表明杨梅叶原飞燕草素具有很好的抗氧化活性,与抗坏血酸、葡萄籽原花青素和杨梅叶中的其他黄酮醇和酚酸比较,杨梅叶原花色素清除自由基的能力仅次于没食子酸,显著强于抗坏血酸、葡萄籽原花青素以及杨梅叶黄酮醇,还原能力强于葡萄籽原花青素,螯合金属离子的能力较弱,与EDTA无可比性。将杨梅叶多酚及原花色素添加到油脂中去,它们都能够有效缓解油脂的氧化腐败,杨梅叶原花色素缓解油脂氧化的能力显著强于杨梅叶多酚及BHT。杨梅叶原花色素平均聚合度对其清除自由基能力影响显著,其清除自由基能力随平均聚合度的增加而增强。相对而言,平均聚合度对杨梅叶原花色素的还原力和螯合金属的能力影响较小,随着平均聚合度的增加,其还原力逐渐减小,金属螯合能力逐渐增强。
选用卵巢癌(OVCAR-3和CP-70)和前列腺癌(DU-145和PC-3)各两种细胞株系分析了以原花色素为主要成分的杨梅叶多酚(BLP)的抗癌活性。BLP能够显著抑制卵巢癌细胞的增殖,而对前列腺癌细胞的增殖无抑制作用。BLP能够抑制血管生成主要因子VEGF的表达,并能够抑制调节VEGF表达的HIF-1α和p-AKT基因的表达,且对p-AKT的抑制作用强于对HIF-1α。BLP也能够显著抑制所选癌细胞中cMyc的表达。BLP对所选癌细胞中NF-κB表达的影响因细胞而异。BLP对所选癌细胞中保护性蛋白PTEN的表达有不同程度的抑制作用,综合研究结果可以推测BLP抗卵巢癌和前列腺癌的作用是对以上几种基因表达综合调节的结果。
通过急性毒性试验及30天大鼠喂养试验评价了杨梅叶酚类提取物的安全性,结果表明受试动物各项指标正常,初步说明杨梅叶提取物具有食用安全性。
综上所述,杨梅叶原花色素是一种结构独特、具有强抗氧化活性的酚类物质,同时杨梅叶原花色素还具有抗癌的活性功能,因此杨梅叶是一种具有广阔开发前景的优良资源。
Proanthocyanidins which have a C6·C3·C6 skeleton in their chemical structures blong to a class of natural phenolic compounds found abundantly in the plant kingdom. The most widely distributed proanthocyanidins in plants are of procyanidin type. Proanthocyanidins are known for their notable pharmacological effects, which include anti-oxidative, antimicrobial, and antitumor properties, and confer cardioprotective activities. Bayberry (Myrica rubra Sieb. et Zucc.), cultivated mainly in southern China, is a special fruit in our country. At present, bayberry fruits are the main materials of research and production. Foliar growth of bayberry tree is luxuriant, and leaves remain green throughout the year. Bayberry trees are pruned every year more than once. Generally, the leaves are discarded generally and remain underutilized. Bayberry leaves are rich in phenolics. Extracts of bayberry leaves are reported to have anti-oxidative, antimicrobial, and antiviral properties. Bayberry leaves are natural source with potential economic and medicinal values.
In this study, structures of proanthocyanidins extracted from bayberry leaves were characterized. Antioxidant and anticancer activities were also determined and the mechanism was analyzed. Then the safety of bayberry leaf extract was evaluated. Results were summarized as follows:
The structural features of bayberry leaf proanthocyanidins of Biqi were investigated by using fractionation on a Sephadex LH-20 column, acid-catalysis in the presence of excess phloroglucinol, HPLC coupled to photodiode array detection or UV detection and electrospray ionization MS (HPLC-DAD-UV/ESI-MS) and NMR analysis. The extractable and unextractable proanthocyanidins (EPAs and UEPAs) were of prodelphinidin type. In fact, epigallocatechin-3-O-gallate (EGCG) and traces of epigallocatechin (EGC) were detected as the extension units, but only EGCG was present in the terminal units. The EPAs were of B-type proanthocyanidins. The mean degree of polymerization (mDP) of bayberry leaf EPAs was 6.5. The UEPAs were highly polymerized prodelphinidins consisting of EGCG and traces of EGC. The EPAs and UEPAs consist mostly of EGCG, which is unusual in the plant kingdom.
EPAs and UEPAs of different bayberry leaves were investigated by using NP-HPLC-DAD analysis and acid-catalysis. Composition of prodelphinidins in bayberry leaves with different cultivar and different leaf age was different from each other. In bayberry leaves of Biqi, the contents of EGCG, oligomer, polymer, and UEPAs of immature leaves were higher than those of intermediate and mature leaves. In bayberry leaves of Dongkui, content of UEPAs of immature leaves was highest, and contents of EGCG, oligomer, and polymer of mature leaves were highest. Sum contents of dimer, trimer, and tetramer determined by NP-HPLC-DAD were higher than those of polymers with DP>4 in both Biqi and Dongkui bayberry leaves.
Individual prodelphinidin oligomers and other low-molecular-weight phenolic compounds in bayberry leaves were investigated by HPLC-DAD-ESIMS. Four flavan-3-ol monomers, three prodelphinidin dimmers, and two trimers were identified. Only one phenolic acid was identified as gallic acid. Six flavonol glycosides were also identified and myricetin deoxyhexoside (535.4-853.0 mg/100 g) was the major flavonol glycoside. Among the three categories, immature leaves of both cultivars contained the highest level of total phenolics and prodelphinidins.
The antioxidant activity of the bayberry leaf extract determined by DPPH·and FRAP assay was comparable with ascorbic acid. Prodelphinidins in the extract had a significant positive correlation with the antioxidant activities of bayberry leaf extract. The correlation coefficients (R) were 0.963 and 0.970 for DPPH·and FRAP assay, respectively. In order to develop a new natural antioxidant, a central composite design was employed when the yield of prodelphinidins was selected as the response value to investigate the extraction. The best extraction condition involving acetone concentration, time, solid-liquid ratio and temperature was obtained for the maximum extraction of prodelphinidins by using response surface methodology (RSM). The extraction yield of prodelphinidins was affected significantly by the extraction variables. The optimal conditions obtained by RSM include 56.93% acetone,31.98 min time,1:44.52 solid-liquid ratio, and 50.00℃temperature. Under the optimal condition, the experimental yield of prodelphinidins was 117.3±5.1 mg/g, which was not different from the predicted value significantly.
Immature bayberry leaves of two cultivars, Bigi and Dongkui, were selected to investigate the effect of water temperature and extraction time on contents of total phenolics and proanthocyanidins, composition of proanthocyanidins, and the antioxidant activity of the hot water extract. The results indicated that the contents of total phenolics and proanthocyanidins were higher at higher water temperature and on the longer extraction time, then the contents dropped if the temperature was too high or the extraction time was too long. The contents of total phenolics and proanthocyanidins of Biqi cultivar were lower than that of Dongkui cultivar under the same extraction condition. Generally, higher contents of oligomers can be obtained at higher temperature. However, the content finally dropped when temperature exceeded 75℃. Higher contents of polymers can be obtained at higher temperature and over longer duration.
The antioxidant activity of bayberry leaf proanthocyanidins (BLPAs) was evaluated by several antioxidant assays with different antioxidant mechanisms (e.g. DPPH, ABTS, PF, PFAP, Metal chelation). In all tested assays, BLPAs exhibited strong antioxidant activities. The DPPH radical scavenging capacity of BLPAs was weaker than that of gallic acid but stronger than that of grape seed procyanidins and bayberry leaf flavonols. Reducing activity of BLPAs was also stronger than that of grape seed procyanidins and bayberry leaf flavonols. The metal chelating activity of BLPAs was very weak compared to EDTA. Bayberry leaf phenolic (BLP) and BLPAs can inhibit lipid oxidation in oils. The inhibition activity of BLPAs was stronger than BLP and BHT. The mDP of BLPAs had significant effect on their radical scavenging activity while no significant effect on reducing and metal chelating activity.
Ovarian (OVCAR-3 and CP-70) and prostate (DU-145 and PC-3) cancer cell lines were selected to estimate anticancer activity of bayberry leaf polyphenols (BLP). The main compounds in BLP were proanthocyanidins. BLP significantly inhibited cell viability in ovarian cancer cell lines while can not inhibit cell viability in prostate cancer cell lines. BLP can inhibit VEGF expression in all cancer cell lines selected. HIF-la and p-AKT levels which were implicated in VEGF expression were also inhibited by BLP. cMyc expression was down regulated by BLP in selected cancer cell lines. Inhabitation of NF-κB expression in cancer cell lines was different from each other. PTEN which play a role in suppressing tumor growth was also be inhibited by BLP. Then it was indicated that the anticancer activity of BLP was a comprehensive action of these factors studied.
Through acute toxity test and 30 days feeding study methods, it can be concluded that bayberry leaf extract was safe.
In conclusion, bayberry proanthocyanidins were phenolic compounds with unusual structures and strong antioxidant activity. They also had anticancer capacity. Bayberry leaf can be a good source of novel functional food.
本文主要以杨梅叶为研究对象,从中提取分离原花色素,进而对其结构组成进行了鉴定,在此基础之上分析了它的抗氧化和抗癌活性及机理,评价了它的食用安全性,主要的研究结果如下：
以荸荠种杨梅叶为原料,采用Sephadex LH-20等分离方法分离纯化杨梅叶原花色素,采用光谱分析、酸催化降解、HPLC-MS及NMR等方法分析了原花色素的结构组成。结果显示杨梅叶可提取(EPAs)及不可提取原花色素(UEPAs)均为原飞燕草素,可提取原花色素的末端单元只含有表桔儿茶素桔酸酯,延伸单元主要含有表桔儿茶素桔酸酯,此外还含有少量表桔儿茶素。所有组成单元通过B型键连接而成,即杨梅叶原花色素为B型原花色素,未检测到A型原花色素。杨梅叶原花色素平均聚合度为6.5。不可提取原花色素主要由表桔儿茶素桔酸酯组成,还含有少量表桔儿茶素。杨梅叶原花色素的以上结构组成特点显示其在已有研究材料中具有独特性。
研究了不同杨梅叶中原飞燕草素的组成(NP-HPLC-DAD法和酸催化降解法),结果显示所有杨梅叶原花色素都包含有EPAs和UEPAs,不同品种不同嫩度杨梅叶中的原飞燕草素的组成是不同的。对于荸荠种杨梅叶,无论是EGCG含量,还是原飞燕草素低聚体和高聚体含量,嫩叶中都高于次嫩叶和老叶,同样地,UEPAs含量也是嫩叶高于其他叶。而对于东魁种杨梅叶情况则有所变化,除UEPAs含量是嫩叶中最高外,其他如EGCG、原飞燕草素低聚体和高聚体的含量都是老叶中含量最高,另外两种嫩度杨梅叶差别不大。在本文中,用NP-HPLC法分析原花色素组成,结果说明无论是荸荠种还是东魁种杨梅叶,其二聚体、三聚体和四聚体的含量总合大于聚合度大于4的EPAs。
利用HPLC-DAD-ESIMS等方法分析了杨梅叶中原花色素低聚体及其他小分量多酚类物质。鉴定出了四种黄烷-3-醇单体、三种原飞燕草素二聚体以及两种原飞燕草素三聚体。鉴定出的其他酚类物质有没食子酸和六种黄酮糖苷,其中杨梅黄酮糖苷为主要组成部分。此外还分析了荸荠和东魁两种杨梅叶不同嫩度叶子中原飞燕草素等多酚类物质的含量,结果显示两栽培品种嫩叶中总酚及原飞燕草素含量均最高。
采用DPPH法和FRAP法测定了杨梅叶提取物抗氧化能力,分析了抗氧化成分,并采用响应面分析法优化了主要抗氧化成分原花色素的提取工艺。结果显示杨梅嫩叶提取物抗氧化能力最强,如在DPPH体系中,其EC50值为4.15μg/mL(荸荠)、6.70μg/mL(东魁),荸荠嫩叶抗氧化能力强于葡萄籽原花色素：提取物抗氧化能力与原花色素含量呈线性正相关,相关系数分别为0.963(DPPH)、0.97(FRAP)；丙酮浓度、时间、料液比和温度对原花色素提取量影响显著,优化后的工艺参数为丙酮浓度56.93%、时间31.98min、料液比1：44.52、温度50.00℃,在此条件下,预计原花色素提取量为121.2mg/g,验证实验结果为117.3±5.1 mg/g,两者差异不显著。
以荸荠种和东魁种嫩叶为原料,研究热水提取温度和提取时间对提取物中总酚含量、原花色素含量、原花色素组成及提取物抗氧化活性的影响。总酚和原花色素含量一般均随提取温度的升高和提取时间延长而增加,但是超过一定温度或时间时其含量略有下降。当提取条件一定时,荸荠种总酚和原花色素含量低于东魁种。提取条件对不同聚合度原花色素含量也有影响,一般在较低温度时(不超过75℃)低聚体部分的含量会随提取温度的升高而增加,而在较高温度时含量减少,高聚体部分的含量随提取温度的升高和提取时间的延长而增加。
不同体外抗氧化试验体系(DPPH、ABTS、铁氰化钾、FRAP、螫合金属离子)分析结果表明杨梅叶原飞燕草素具有很好的抗氧化活性,与抗坏血酸、葡萄籽原花青素和杨梅叶中的其他黄酮醇和酚酸比较,杨梅叶原花色素清除自由基的能力仅次于没食子酸,显著强于抗坏血酸、葡萄籽原花青素以及杨梅叶黄酮醇,还原能力强于葡萄籽原花青素,螯合金属离子的能力较弱,与EDTA无可比性。将杨梅叶多酚及原花色素添加到油脂中去,它们都能够有效缓解油脂的氧化腐败,杨梅叶原花色素缓解油脂氧化的能力显著强于杨梅叶多酚及BHT。杨梅叶原花色素平均聚合度对其清除自由基能力影响显著,其清除自由基能力随平均聚合度的增加而增强。相对而言,平均聚合度对杨梅叶原花色素的还原力和螯合金属的能力影响较小,随着平均聚合度的增加,其还原力逐渐减小,金属螯合能力逐渐增强。
选用卵巢癌(OVCAR-3和CP-70)和前列腺癌(DU-145和PC-3)各两种细胞株系分析了以原花色素为主要成分的杨梅叶多酚(BLP)的抗癌活性。BLP能够显著抑制卵巢癌细胞的增殖,而对前列腺癌细胞的增殖无抑制作用。BLP能够抑制血管生成主要因子VEGF的表达,并能够抑制调节VEGF表达的HIF-1α和p-AKT基因的表达,且对p-AKT的抑制作用强于对HIF-1α。BLP也能够显著抑制所选癌细胞中cMyc的表达。BLP对所选癌细胞中NF-κB表达的影响因细胞而异。BLP对所选癌细胞中保护性蛋白PTEN的表达有不同程度的抑制作用,综合研究结果可以推测BLP抗卵巢癌和前列腺癌的作用是对以上几种基因表达综合调节的结果。
通过急性毒性试验及30天大鼠喂养试验评价了杨梅叶酚类提取物的安全性,结果表明受试动物各项指标正常,初步说明杨梅叶提取物具有食用安全性。
综上所述,杨梅叶原花色素是一种结构独特、具有强抗氧化活性的酚类物质,同时杨梅叶原花色素还具有抗癌的活性功能,因此杨梅叶是一种具有广阔开发前景的优良资源。
Proanthocyanidins which have a C6·C3·C6 skeleton in their chemical structures blong to a class of natural phenolic compounds found abundantly in the plant kingdom. The most widely distributed proanthocyanidins in plants are of procyanidin type. Proanthocyanidins are known for their notable pharmacological effects, which include anti-oxidative, antimicrobial, and antitumor properties, and confer cardioprotective activities. Bayberry (Myrica rubra Sieb. et Zucc.), cultivated mainly in southern China, is a special fruit in our country. At present, bayberry fruits are the main materials of research and production. Foliar growth of bayberry tree is luxuriant, and leaves remain green throughout the year. Bayberry trees are pruned every year more than once. Generally, the leaves are discarded generally and remain underutilized. Bayberry leaves are rich in phenolics. Extracts of bayberry leaves are reported to have anti-oxidative, antimicrobial, and antiviral properties. Bayberry leaves are natural source with potential economic and medicinal values.
In this study, structures of proanthocyanidins extracted from bayberry leaves were characterized. Antioxidant and anticancer activities were also determined and the mechanism was analyzed. Then the safety of bayberry leaf extract was evaluated. Results were summarized as follows:
The structural features of bayberry leaf proanthocyanidins of Biqi were investigated by using fractionation on a Sephadex LH-20 column, acid-catalysis in the presence of excess phloroglucinol, HPLC coupled to photodiode array detection or UV detection and electrospray ionization MS (HPLC-DAD-UV/ESI-MS) and NMR analysis. The extractable and unextractable proanthocyanidins (EPAs and UEPAs) were of prodelphinidin type. In fact, epigallocatechin-3-O-gallate (EGCG) and traces of epigallocatechin (EGC) were detected as the extension units, but only EGCG was present in the terminal units. The EPAs were of B-type proanthocyanidins. The mean degree of polymerization (mDP) of bayberry leaf EPAs was 6.5. The UEPAs were highly polymerized prodelphinidins consisting of EGCG and traces of EGC. The EPAs and UEPAs consist mostly of EGCG, which is unusual in the plant kingdom.
EPAs and UEPAs of different bayberry leaves were investigated by using NP-HPLC-DAD analysis and acid-catalysis. Composition of prodelphinidins in bayberry leaves with different cultivar and different leaf age was different from each other. In bayberry leaves of Biqi, the contents of EGCG, oligomer, polymer, and UEPAs of immature leaves were higher than those of intermediate and mature leaves. In bayberry leaves of Dongkui, content of UEPAs of immature leaves was highest, and contents of EGCG, oligomer, and polymer of mature leaves were highest. Sum contents of dimer, trimer, and tetramer determined by NP-HPLC-DAD were higher than those of polymers with DP>4 in both Biqi and Dongkui bayberry leaves.
Individual prodelphinidin oligomers and other low-molecular-weight phenolic compounds in bayberry leaves were investigated by HPLC-DAD-ESIMS. Four flavan-3-ol monomers, three prodelphinidin dimmers, and two trimers were identified. Only one phenolic acid was identified as gallic acid. Six flavonol glycosides were also identified and myricetin deoxyhexoside (535.4-853.0 mg/100 g) was the major flavonol glycoside. Among the three categories, immature leaves of both cultivars contained the highest level of total phenolics and prodelphinidins.
The antioxidant activity of the bayberry leaf extract determined by DPPH·and FRAP assay was comparable with ascorbic acid. Prodelphinidins in the extract had a significant positive correlation with the antioxidant activities of bayberry leaf extract. The correlation coefficients (R) were 0.963 and 0.970 for DPPH·and FRAP assay, respectively. In order to develop a new natural antioxidant, a central composite design was employed when the yield of prodelphinidins was selected as the response value to investigate the extraction. The best extraction condition involving acetone concentration, time, solid-liquid ratio and temperature was obtained for the maximum extraction of prodelphinidins by using response surface methodology (RSM). The extraction yield of prodelphinidins was affected significantly by the extraction variables. The optimal conditions obtained by RSM include 56.93% acetone,31.98 min time,1:44.52 solid-liquid ratio, and 50.00℃temperature. Under the optimal condition, the experimental yield of prodelphinidins was 117.3±5.1 mg/g, which was not different from the predicted value significantly.
Immature bayberry leaves of two cultivars, Bigi and Dongkui, were selected to investigate the effect of water temperature and extraction time on contents of total phenolics and proanthocyanidins, composition of proanthocyanidins, and the antioxidant activity of the hot water extract. The results indicated that the contents of total phenolics and proanthocyanidins were higher at higher water temperature and on the longer extraction time, then the contents dropped if the temperature was too high or the extraction time was too long. The contents of total phenolics and proanthocyanidins of Biqi cultivar were lower than that of Dongkui cultivar under the same extraction condition. Generally, higher contents of oligomers can be obtained at higher temperature. However, the content finally dropped when temperature exceeded 75℃. Higher contents of polymers can be obtained at higher temperature and over longer duration.
The antioxidant activity of bayberry leaf proanthocyanidins (BLPAs) was evaluated by several antioxidant assays with different antioxidant mechanisms (e.g. DPPH, ABTS, PF, PFAP, Metal chelation). In all tested assays, BLPAs exhibited strong antioxidant activities. The DPPH radical scavenging capacity of BLPAs was weaker than that of gallic acid but stronger than that of grape seed procyanidins and bayberry leaf flavonols. Reducing activity of BLPAs was also stronger than that of grape seed procyanidins and bayberry leaf flavonols. The metal chelating activity of BLPAs was very weak compared to EDTA. Bayberry leaf phenolic (BLP) and BLPAs can inhibit lipid oxidation in oils. The inhibition activity of BLPAs was stronger than BLP and BHT. The mDP of BLPAs had significant effect on their radical scavenging activity while no significant effect on reducing and metal chelating activity.
Ovarian (OVCAR-3 and CP-70) and prostate (DU-145 and PC-3) cancer cell lines were selected to estimate anticancer activity of bayberry leaf polyphenols (BLP). The main compounds in BLP were proanthocyanidins. BLP significantly inhibited cell viability in ovarian cancer cell lines while can not inhibit cell viability in prostate cancer cell lines. BLP can inhibit VEGF expression in all cancer cell lines selected. HIF-la and p-AKT levels which were implicated in VEGF expression were also inhibited by BLP. cMyc expression was down regulated by BLP in selected cancer cell lines. Inhabitation of NF-κB expression in cancer cell lines was different from each other. PTEN which play a role in suppressing tumor growth was also be inhibited by BLP. Then it was indicated that the anticancer activity of BLP was a comprehensive action of these factors studied.
Through acute toxity test and 30 days feeding study methods, it can be concluded that bayberry leaf extract was safe.
In conclusion, bayberry proanthocyanidins were phenolic compounds with unusual structures and strong antioxidant activity. They also had anticancer capacity. Bayberry leaf can be a good source of novel functional food.
引文
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