桑叶化学成分的动态变化与PAL、F3H基因的表达水平研究
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摘要
桑叶的药用始载于《神农本草经》桑白皮项下。中国是桑的资源大国,桑树的自然分布和人工栽培几乎遍及全国。《中华人民共和国药典》2010年版规定,桑叶来源于桑科植物桑Morus alba L.的干燥叶。味甘、苦,性寒,归肺、肝经。具有疏散风热,清肺润燥,清肝明目之功。多酚类成分如绿原酸：、黄酮类化合物等为桑叶中主要的活性成分。桑叶多酚类成分合成途径中关键酶基因有PAL、F3H基因等。不同种质的桑叶,其品质存在一定的差异,为从分子机制方面阐明影响桑叶药材品质的因素,本研究对桑叶的化学成分动态变化及其PAL、F3H基因的表达水平进行了研究,结果如下。
1.桑叶的化学成分动态变化研究
本研究选取了在四川省大面积栽种的桑Morus alba L.的两个栽培品种,即红果2号和保坎61号,并分别在每个居群定点选择8株树,应用高效液相色谱法,对桑叶的化学成分进行动态变化追踪,建立了桑叶的HPLC指纹图谱,探寻成分变化规律。由该方法得到两个栽培品种4～11月的128批次样品的指纹图谱,并标定出11个特征峰。得出桑叶化学成分的动态累积结果：
(1)相同栽培品种、同一居群,在相同采摘时间下的样本,化学成分的组成及含量差异较小(相似度均在0.9左右,样本间的总峰面积无显著差异)；而在不同的采摘时间下的样本,化学成分的组成相近(相似度均在0.9左右),但含量有较大差异,如保坎61号共有峰最低总峰面积(7月)为最高总峰面积(11月)的31%,红果2号最低总峰面积(7月)为最高总峰面积(4月)的28%。
(2)保坎61号和红果2号的HPLC指纹图谱相似,其化学成分的组成相近(两个栽培品种间的相似度在0.9左右)。相同采摘时间的两个栽培品种,化学成分含量有较大差异,如10月时红果2号的绿原酸含量仅为保坎61号的50%；不同采摘时间的两个栽培品种共有峰总峰面积动态变化趋势基本一致,从4-7月整体呈下降趋势,7-11月整体呈上升趋势。
(3)绿原酸、芦丁和紫云英苷为桑叶主要的多酚类成分。本实验测得的3种成分的含量数据显示,绿原酸、芦丁和紫云英苷的含量及三者的总含量均同11个共有峰总峰面积的变化趋势一致,即从4-7月整体呈下降趋势,7-11月整体呈上升趋势。且3种成分的总含量及共有峰总含量均在11月较高,此结果支持了传统冬桑叶入药的要求。
2.PAL、F3H基因在桑叶中的表达水平研究
多酚类成分的合成途径是一个庞大的系统,有多个关键酶基因参与其中,PAL基因是其合成途径中第一个关键酶,F3H也是其中的一个关键酶,在整个合成途径中两者都具有一定的代表性。本实验对桑PAL、F3H基因在桑叶中的表达水平的进行了研究。所选定了两株桑(Morus alba L.)MA-1、MA-2,于每月中旬采摘,同时对其桑叶及桑枝进行化学成分的动态变化研究。最后结合16批次样品的PAL、F3H基因在桑叶中的表达量和桑叶及桑枝的多酚类成分动态变化分析,得到以下结果：
(1)通过克隆桑PAL、F3H基因,经测序后得到两个基因的片段,在Genbank进行同源性分析,验证了PAL基因在桑叶中的表达,同时首次发现了F3H基因在桑叶中的存在,证明了F3H基因在桑叶中有表达。
(2)首次对PAL、F3H基因在桑叶中的表达水平进行了研究,发现了两个基因在植物不同生长发育时期的桑叶中存在表达差异。如MA-1的PAL基因的最低表达量(10月)为最高表达量(9月)的13%,MA-2的PAL基因的最低表达量(11月)为最高表达量(9月)12%；MA-1的F3H基因的最低表达量(8月)为最高表达量(9月)的21%,MA-2的F3H基因的最低表达量(10月)为最高表达量(4月)的20%。
(3)MA-1和MA-2桑叶中的PAL、F3H基因表达的变化趋势基本一致。PAL基因4月～6月整体呈上升趋势,6月～8月整体呈下降趋势,8月～9月再上升,然后到11月整体呈下降趋势。F3H基因4月～8月整体呈下降趋势,9月～11月的趋势与PAL基因基本相同。
(4)首次将PAL、F3H基因的在桑叶中的表达量与桑叶中的多酚类成分绿原酸、芦丁、紫云英苷以及这3种成分的总含量进行相关性分析,得到数据,如MA-1的的相关系数r在0.20～0.70之间,MA-2的相关系数在0.40～0.75之间,发现了两个基因均分别与桑叶中绿原酸、芦丁、紫云英苷含量以及3种成分总含量存在不同程度的正相关。其中,PAL基因与3种成分的相关性较大,而F3H基因与3种成分的相关系性较小；PAL基因的相关系数为F3H基因的1.4-2.2倍。以上结果说明PAL、F3H基因对多酚类成分有调控作用,其中PAL基因的调控作用相对较大。
(5)首次将PAL、F3H基因的在桑叶中的表达量与桑枝中的多酚类成分绿原酸、芦丁、紫云英苷以及这3种成分的总含量进行相关性分析。发现仅MA-1的PAL、F3H基因与芦丁含量存在低度正相关,相关系数r为0.313、0.115； MA-2的PAL、F3H基因与芦丁含量存在低度正相关,相关系数r为0.426、0.090；其余均无相关性。说明PAL、F3H基因在桑叶中的表达量同桑枝中化学成分的动态变化的相关性不大。
(6)比较分析了两个产地桑叶化学成分动态变化的相关性,由HPLC图谱可看出,两个产地桑叶在化学成分组分上是一致的,但含量有很大差异。
本研究揭示了影响中药品质的重要因素,认为遗传、环境、栽培管理技术等对桑PAL、F3H基因的表达水平及多酚类成分积累有较大影响。阐明了桑叶品质形成的分子机制。认为药用植物的不同代谢途径与其种类、药用部位、不同生长发育时期的状况及环境条件有关。多酚类成分或其他化学成分的生物合成存在多个关键制约因素,只要我们通过类似的研究,掌握控制生物合成的关键因素,再优化条件,就能从内因和外因两方面来提升中药品质。
The medicinal uses of the mulberry leaf were first recorded in the article of White Mulberry Root-bark in Shengnong's herbal. Mulberry trees are indigenous to China, where they are widely cultivated almost all over the country. The Pharmacopoeia of the People's Republic of China (2010edition) has named the source of the mulberry leaf as from the dried leaf of Morus alba L., family Moraceae. It is bittersweet in flavor, cold in nature and applicative to the lungs and liver meridians. It's effective for expelling wind and heat, clearing away lung-heat and moisturizing dryness, and clearing away liver-fire to treat eye disease. Its main active components are polyphenols such as chlorogenic acid and flavonoids. To identify the factors which lead to the differing in molecular mechanisms of different mulberry leaves, studies were carried out to research the dynamic changes of chemical components and the gene presence of PAL and F3H in mulberry leaves. The results are as follows:
1. Study on the dynamic changes of chemical components in mulberry leaves
Sericulture is a predominant industry in Sichuan. The study selected "Hong-guo No.2" and "Bao-kan No.61",2strains of Morus alba L., widely cultivated in the silkworm farms of Langzhong, in Sichuan. The study included the dynamical tracking of the active component's change in mulberry leaves from8designated trees from each strain, the HPLC fingerprint of mulberry leaves was established to explore its varying characteristics.128batches of samples collected from April to November were tested to get their fingerprints as well as11characteristic peaks calibrated. The following are the outcome:
(1) For the same strain and population, at the same picking time, the difference of the active ingredient's composition and content was small (similarity was around0.9with no significant difference in the total peak area between the samples); However, for those with different picking times, although the composition was also similar (similarity around0.9), the content appeared to be quite different. For instance, the aggregate peaks in "Bao-kan No.61" in July was lowest, which was only31%of the highest its counterpart achieved in November. And for "Hong-guo No.2", the proportion was only28%when its lowest peaks in July was compared to the highest its counterpart obtained in April.
(2) Samples taken from the2strains, regardless of picking time, showed that the composition of the active ingredient was similar (similarity around0.9). But they differed greatly in content, even those with same picking time. For example in October, the content of chlorogenic acid in "Hong-guo No.2" was only50%of its counterpart in "Bao-kan No.61". But the dynamic trend of aggregate common peaks between the2strains was basically the same, which exhibited an overall downward trend from April to July but inclining upward from July to November.
(3) Chlorogenic acid, rutin, and astragalin are the main polyphenols in mulberry leaves. Tests conducted on the3components showed their individual and total content varied with the aggregate of11common peaks, which was an overall decrease from April to July but increased from July to November. The highest content was reached in November. This result validates the traditional preference of using mulberry leaves for medicinal purpose in winter.
2. The gene presence of PAL and F3H in mulberry leaves
There are multiple key enzyme genes in the synthetic route of polyphenols. This study selected the PAL and F3H genes respectively, in the upstream and midstream of the route, to study their presence level. The samples were sourced from the leaves of MA-1MA-2, two mulberry trees(Morus alba L.) cultivated in the campus of Chengdu University of Traditional Chinese Medicine (TCM) and picked in the middle of each month. Concurrently, the dynamic changes of the active ingredient both in their leaves and branches were also studied. The following were major findings after16batches of test:
(1) By the cloning of PAL and F3H, their fragments were obtained after gene sequencing. Supported by homology analysis using Genbank, the study validates PAL gene presence in mulberry leaves. Meanwhile, the presence of F3H gene fragment was also discovered in mulberry leaves in this study.
(2) This is the first study on the presence level of PAL and F3H in mulberry leaves. A varying degree of presence of these two genes was found in the leaf s different growth period. For MA-1, the lowest presence amount of PAL was in October at only13%of the highest its counterpart exhibited in September while the proportion in MA-2was12%when comparing its lowest in November to the highest in September. For F3H, the lowest presence amount in MA-1was in August at21%of the highest its counterpart exhibited in September and the lowest in MA-2was in October at20%of its counterpart's highest in April.
(3) The variation in gene presence of PAL and F3H in MA-1and MA-2were basically the same in their trends. The overall presence of PAL increased from April to June, then decreased from June to August; increased again from August to September, and then decreased until November. Decreasing from April to August, the presence of F3H continued its decrease from September to November which showed the same trend as PAL's.
(4) For the first time, the correlation was analyzed between the presence amount of PAL, F3H and both of the individual and total content of polyphenols in mulberry leaves, including chlorogenic acid, rutin and astragalin. The results showed the correlation coefficients (r) ranged from0.20to0.70in MA-1and0.40to0.75in MA-2. These2genes have varying degrees of positive correlation with the individual and total content of the above3polyphenols. Moreover, the tests showed that PAL has more correlation than F3H.
(5) Also for the first time, the correlation was analyzed between the presence amount of PAL, F3H in mulberry leaves and both of the individual and total content of polyphenols in mulberry branches, including chlorogenic acid, rutin and astragalin. It's found the PAL and F3H in MA-1had a low positive correlation with the content of rutin (r=0.313,0.115respectively). The PAL and F3H in MA-2had a low positive correlation with the content of rutin (r=0.4260.090respectively). The rest didn't show any correlation. The analysis also revealed that the presence amount of PAL and F3H in mulberry leaves didn't seem to have any correlation with the dynamic changes of the active ingredient in mulberry branches.
(6) The difference in the dynamic changes of the active ingredient in mulberry leaves from2production areas was compared by HPLC. The result showed that their constituents were the same but the content was very different.
This study establishes the important factors affecting the quality of mulberry leaves, which include heredity, environment and technique of cultivation and management. These factors all have a great impact on the gene presence of PAL and F3H and also on the accumulation of polyphenols. The mulberry leaf's quality is also linked to molecular mechanism, which provides basis for the formation mechanism of TCM's quality from molecular level. It is believed that the TCM's research should endeavor to discover by what means and how the internal and external causes affect the formation and accumulation of the active ingredient. The gene regulation and regulatory mechanisms of secondary metabolites in medicinal plant are key factors affecting TCM's quality. To identify the internal and external factors impacting the quality of TCM, the key enzyme genes should be identified first by examining the medicinal plant's secondary metabolites path. This should then be followed by a study into their regulatory mechanism while taking into account the external factors such as soil, climate and techniques of cultivation and management.
1.桑叶的化学成分动态变化研究
本研究选取了在四川省大面积栽种的桑Morus alba L.的两个栽培品种,即红果2号和保坎61号,并分别在每个居群定点选择8株树,应用高效液相色谱法,对桑叶的化学成分进行动态变化追踪,建立了桑叶的HPLC指纹图谱,探寻成分变化规律。由该方法得到两个栽培品种4～11月的128批次样品的指纹图谱,并标定出11个特征峰。得出桑叶化学成分的动态累积结果：
(1)相同栽培品种、同一居群,在相同采摘时间下的样本,化学成分的组成及含量差异较小(相似度均在0.9左右,样本间的总峰面积无显著差异)；而在不同的采摘时间下的样本,化学成分的组成相近(相似度均在0.9左右),但含量有较大差异,如保坎61号共有峰最低总峰面积(7月)为最高总峰面积(11月)的31%,红果2号最低总峰面积(7月)为最高总峰面积(4月)的28%。
(2)保坎61号和红果2号的HPLC指纹图谱相似,其化学成分的组成相近(两个栽培品种间的相似度在0.9左右)。相同采摘时间的两个栽培品种,化学成分含量有较大差异,如10月时红果2号的绿原酸含量仅为保坎61号的50%；不同采摘时间的两个栽培品种共有峰总峰面积动态变化趋势基本一致,从4-7月整体呈下降趋势,7-11月整体呈上升趋势。
(3)绿原酸、芦丁和紫云英苷为桑叶主要的多酚类成分。本实验测得的3种成分的含量数据显示,绿原酸、芦丁和紫云英苷的含量及三者的总含量均同11个共有峰总峰面积的变化趋势一致,即从4-7月整体呈下降趋势,7-11月整体呈上升趋势。且3种成分的总含量及共有峰总含量均在11月较高,此结果支持了传统冬桑叶入药的要求。
2.PAL、F3H基因在桑叶中的表达水平研究
多酚类成分的合成途径是一个庞大的系统,有多个关键酶基因参与其中,PAL基因是其合成途径中第一个关键酶,F3H也是其中的一个关键酶,在整个合成途径中两者都具有一定的代表性。本实验对桑PAL、F3H基因在桑叶中的表达水平的进行了研究。所选定了两株桑(Morus alba L.)MA-1、MA-2,于每月中旬采摘,同时对其桑叶及桑枝进行化学成分的动态变化研究。最后结合16批次样品的PAL、F3H基因在桑叶中的表达量和桑叶及桑枝的多酚类成分动态变化分析,得到以下结果：
(1)通过克隆桑PAL、F3H基因,经测序后得到两个基因的片段,在Genbank进行同源性分析,验证了PAL基因在桑叶中的表达,同时首次发现了F3H基因在桑叶中的存在,证明了F3H基因在桑叶中有表达。
(2)首次对PAL、F3H基因在桑叶中的表达水平进行了研究,发现了两个基因在植物不同生长发育时期的桑叶中存在表达差异。如MA-1的PAL基因的最低表达量(10月)为最高表达量(9月)的13%,MA-2的PAL基因的最低表达量(11月)为最高表达量(9月)12%；MA-1的F3H基因的最低表达量(8月)为最高表达量(9月)的21%,MA-2的F3H基因的最低表达量(10月)为最高表达量(4月)的20%。
(3)MA-1和MA-2桑叶中的PAL、F3H基因表达的变化趋势基本一致。PAL基因4月～6月整体呈上升趋势,6月～8月整体呈下降趋势,8月～9月再上升,然后到11月整体呈下降趋势。F3H基因4月～8月整体呈下降趋势,9月～11月的趋势与PAL基因基本相同。
(4)首次将PAL、F3H基因的在桑叶中的表达量与桑叶中的多酚类成分绿原酸、芦丁、紫云英苷以及这3种成分的总含量进行相关性分析,得到数据,如MA-1的的相关系数r在0.20～0.70之间,MA-2的相关系数在0.40～0.75之间,发现了两个基因均分别与桑叶中绿原酸、芦丁、紫云英苷含量以及3种成分总含量存在不同程度的正相关。其中,PAL基因与3种成分的相关性较大,而F3H基因与3种成分的相关系性较小；PAL基因的相关系数为F3H基因的1.4-2.2倍。以上结果说明PAL、F3H基因对多酚类成分有调控作用,其中PAL基因的调控作用相对较大。
(5)首次将PAL、F3H基因的在桑叶中的表达量与桑枝中的多酚类成分绿原酸、芦丁、紫云英苷以及这3种成分的总含量进行相关性分析。发现仅MA-1的PAL、F3H基因与芦丁含量存在低度正相关,相关系数r为0.313、0.115； MA-2的PAL、F3H基因与芦丁含量存在低度正相关,相关系数r为0.426、0.090；其余均无相关性。说明PAL、F3H基因在桑叶中的表达量同桑枝中化学成分的动态变化的相关性不大。
(6)比较分析了两个产地桑叶化学成分动态变化的相关性,由HPLC图谱可看出,两个产地桑叶在化学成分组分上是一致的,但含量有很大差异。
本研究揭示了影响中药品质的重要因素,认为遗传、环境、栽培管理技术等对桑PAL、F3H基因的表达水平及多酚类成分积累有较大影响。阐明了桑叶品质形成的分子机制。认为药用植物的不同代谢途径与其种类、药用部位、不同生长发育时期的状况及环境条件有关。多酚类成分或其他化学成分的生物合成存在多个关键制约因素,只要我们通过类似的研究,掌握控制生物合成的关键因素,再优化条件,就能从内因和外因两方面来提升中药品质。
The medicinal uses of the mulberry leaf were first recorded in the article of White Mulberry Root-bark in Shengnong's herbal. Mulberry trees are indigenous to China, where they are widely cultivated almost all over the country. The Pharmacopoeia of the People's Republic of China (2010edition) has named the source of the mulberry leaf as from the dried leaf of Morus alba L., family Moraceae. It is bittersweet in flavor, cold in nature and applicative to the lungs and liver meridians. It's effective for expelling wind and heat, clearing away lung-heat and moisturizing dryness, and clearing away liver-fire to treat eye disease. Its main active components are polyphenols such as chlorogenic acid and flavonoids. To identify the factors which lead to the differing in molecular mechanisms of different mulberry leaves, studies were carried out to research the dynamic changes of chemical components and the gene presence of PAL and F3H in mulberry leaves. The results are as follows:
1. Study on the dynamic changes of chemical components in mulberry leaves
Sericulture is a predominant industry in Sichuan. The study selected "Hong-guo No.2" and "Bao-kan No.61",2strains of Morus alba L., widely cultivated in the silkworm farms of Langzhong, in Sichuan. The study included the dynamical tracking of the active component's change in mulberry leaves from8designated trees from each strain, the HPLC fingerprint of mulberry leaves was established to explore its varying characteristics.128batches of samples collected from April to November were tested to get their fingerprints as well as11characteristic peaks calibrated. The following are the outcome:
(1) For the same strain and population, at the same picking time, the difference of the active ingredient's composition and content was small (similarity was around0.9with no significant difference in the total peak area between the samples); However, for those with different picking times, although the composition was also similar (similarity around0.9), the content appeared to be quite different. For instance, the aggregate peaks in "Bao-kan No.61" in July was lowest, which was only31%of the highest its counterpart achieved in November. And for "Hong-guo No.2", the proportion was only28%when its lowest peaks in July was compared to the highest its counterpart obtained in April.
(2) Samples taken from the2strains, regardless of picking time, showed that the composition of the active ingredient was similar (similarity around0.9). But they differed greatly in content, even those with same picking time. For example in October, the content of chlorogenic acid in "Hong-guo No.2" was only50%of its counterpart in "Bao-kan No.61". But the dynamic trend of aggregate common peaks between the2strains was basically the same, which exhibited an overall downward trend from April to July but inclining upward from July to November.
(3) Chlorogenic acid, rutin, and astragalin are the main polyphenols in mulberry leaves. Tests conducted on the3components showed their individual and total content varied with the aggregate of11common peaks, which was an overall decrease from April to July but increased from July to November. The highest content was reached in November. This result validates the traditional preference of using mulberry leaves for medicinal purpose in winter.
2. The gene presence of PAL and F3H in mulberry leaves
There are multiple key enzyme genes in the synthetic route of polyphenols. This study selected the PAL and F3H genes respectively, in the upstream and midstream of the route, to study their presence level. The samples were sourced from the leaves of MA-1MA-2, two mulberry trees(Morus alba L.) cultivated in the campus of Chengdu University of Traditional Chinese Medicine (TCM) and picked in the middle of each month. Concurrently, the dynamic changes of the active ingredient both in their leaves and branches were also studied. The following were major findings after16batches of test:
(1) By the cloning of PAL and F3H, their fragments were obtained after gene sequencing. Supported by homology analysis using Genbank, the study validates PAL gene presence in mulberry leaves. Meanwhile, the presence of F3H gene fragment was also discovered in mulberry leaves in this study.
(2) This is the first study on the presence level of PAL and F3H in mulberry leaves. A varying degree of presence of these two genes was found in the leaf s different growth period. For MA-1, the lowest presence amount of PAL was in October at only13%of the highest its counterpart exhibited in September while the proportion in MA-2was12%when comparing its lowest in November to the highest in September. For F3H, the lowest presence amount in MA-1was in August at21%of the highest its counterpart exhibited in September and the lowest in MA-2was in October at20%of its counterpart's highest in April.
(3) The variation in gene presence of PAL and F3H in MA-1and MA-2were basically the same in their trends. The overall presence of PAL increased from April to June, then decreased from June to August; increased again from August to September, and then decreased until November. Decreasing from April to August, the presence of F3H continued its decrease from September to November which showed the same trend as PAL's.
(4) For the first time, the correlation was analyzed between the presence amount of PAL, F3H and both of the individual and total content of polyphenols in mulberry leaves, including chlorogenic acid, rutin and astragalin. The results showed the correlation coefficients (r) ranged from0.20to0.70in MA-1and0.40to0.75in MA-2. These2genes have varying degrees of positive correlation with the individual and total content of the above3polyphenols. Moreover, the tests showed that PAL has more correlation than F3H.
(5) Also for the first time, the correlation was analyzed between the presence amount of PAL, F3H in mulberry leaves and both of the individual and total content of polyphenols in mulberry branches, including chlorogenic acid, rutin and astragalin. It's found the PAL and F3H in MA-1had a low positive correlation with the content of rutin (r=0.313,0.115respectively). The PAL and F3H in MA-2had a low positive correlation with the content of rutin (r=0.4260.090respectively). The rest didn't show any correlation. The analysis also revealed that the presence amount of PAL and F3H in mulberry leaves didn't seem to have any correlation with the dynamic changes of the active ingredient in mulberry branches.
(6) The difference in the dynamic changes of the active ingredient in mulberry leaves from2production areas was compared by HPLC. The result showed that their constituents were the same but the content was very different.
This study establishes the important factors affecting the quality of mulberry leaves, which include heredity, environment and technique of cultivation and management. These factors all have a great impact on the gene presence of PAL and F3H and also on the accumulation of polyphenols. The mulberry leaf's quality is also linked to molecular mechanism, which provides basis for the formation mechanism of TCM's quality from molecular level. It is believed that the TCM's research should endeavor to discover by what means and how the internal and external causes affect the formation and accumulation of the active ingredient. The gene regulation and regulatory mechanisms of secondary metabolites in medicinal plant are key factors affecting TCM's quality. To identify the internal and external factors impacting the quality of TCM, the key enzyme genes should be identified first by examining the medicinal plant's secondary metabolites path. This should then be followed by a study into their regulatory mechanism while taking into account the external factors such as soil, climate and techniques of cultivation and management.
引文
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