Studies On The Dynamic Changes Of Chemical Components And The Gene Expression Of PAL And F3H In Mulberry Leaves

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 leavesSericulture 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 leavesThere 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.