Spatial Variations of Phytochemistry in Salvia Miltiorrhiza and Environmental Factors

Xiaoyu Chen1, Chao He1, Jie Cui1, Binbin Yan2, Yeye Geng3, Junling Hou3*, Wenquan Wang1,3,4* and Weixu Chen5 11Institute of Medicinal Plant Development, Chinese Academy of Medicinal Sciences and Peking Union Medical Collage, Beijing, China 2College Pharmacy, Chengdu University of Traditional Chinese Medicine, Chengdu, China 3School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing, China 4Engineering Research Center of Good Agricultural Practice for Chinese Crude Drugs, Ministry of Education, Beijing, China 5Shang yao hua yu (LinYi) Tradtional chineese Medicine Resouces Co.,Ltd, Shandong, China


Introduction
functions of metabolites and their evolution but also critical for quality control of S. miltiorrhiza in good agricultural practice.
It is known that medicinal plants have developed their own defense system by producing secondary metabolites which are important sources for pharmacological active products in response to environmental stresses [13,14]. A variety of environmental factors, such as climate, geographic distribution, radiation, and soil nutrition, have been proven to significantly influence the secondary metabolite profile [15][16][17][18]. There have been also some researches about influence of the ecological factors on the bioactive ingredients of S. miltiorrhiza [19,20], but which mostly focus on single factor effect. For example, the study of WANG Wei et al. [21] on the effect of soil moisture on contents of S. miltiorrhiza which reported an appropriate drought stress during cultivation is beneficial for the raise of phenolic acids. The changes of key enzyme gene expression of rosmarinic acid biosynthesis pathway during heat stress was studied [22]. And the study of YU yange et al. [23] on the relationship between inorganic elements in wild-growing plants of S. miltiorrhiza and its soil. Additional investigations into the metabolic profile of plant populations of one species inhabiting varied environments are therefore necessary to understand the complex relationships between secondary metabolites and the habits in which they are produced [18].
It was proposed that global climate change influenced overall ecosystem functions, including the secondary metabolic profiles of plants. Our previous study also implied that the local environment may play an important role in the quality of cultivated S. miltiorrhiza [24]. This raised our interest to test whether the environmental parameters influence the phytochemical diversity in cultivated S. miltiorrhiza, and which is/are the most contributive one(s). Therefore, we enlarged our sampling to include five main regions of cultivation of S. miltiorrhiza in China with more plants, as well as the corresponding climatic and geographic data and soil samples, aiming to further investigate the secondary metabolic variation of cultivated S. miltiorrhiza among geographical regions and assess the effects of different environmental variables on phytochemical variation in terms of both the overall chromatographic fingerprint and six single major compounds of S. miltiorrhiza. This work will provide a reference for controlling the quality of S. miltiorrhiza during cultivation.

Phytochemical Analyses with HPLC
The plant samples were dried uniformly in drying ovens at 40 ℃ and crushed after 0.355mm mesh sieve. The phytochemical diversity of S. miltiorrhiza was presented in terms of variations of both overall chromatographic fingerprints and major single compounds.
The present methods followed those in Gao et al. [25]

Soil Analyses
The collected soil samples were air-dried before screening through 40 mesh. The following attributes of each sample were analyzed: pH, content of available nitrogen (An), available phosphorus (Ap) and available potassium (Ak). Soil analyses were performed according to the method of Bao [26]. Six microelements, Fe, Mn, Ca, Cu, Zn, B and content of total nitrogen (Tn), total phosphorus (Tp) and total potassium (Tk) were provided by Chinese medicine resources geospatial grid information database at Beijing (http://www.tcm-resources.com/).

Climatic Data
Annual and monthly average temperature and precipitation were respectively acquired from the weather stations nearest the sampling sites, which are available from Chinese medicine resources geospatial grid information database at Beijing (http:// www.tcm-resources.com/).

Statistical Analyses
We examined the environmental correlations with both chromatographic fingerprints and the six single compounds based on comparisons within each set of the parameters. We

Climatic, Geographic, and Soil Characteristics of the Cultivation Regions
S. miltiorrhiza has strong growth suitability and is widely distributed. The regions where the cultivated samples were collected are located from 119.0296° E to 104.536373° E, 38.4852° N to 31.0019° N. The annual rainfall in these regions varied from 423 to 1141mm, and the average yearly temperature ranged from 11.7 to 17.0 ℃. Obvious distinctions in geographic factors exist between the sites. Sichuan (SC) is at the highest elevation in altitude and is at the minimum longitude, whereas Shandong (SD) is at the lowest altitude and the maximum longitude. In latitude, Hebei (HB) is at the maximum latitude, whereas SC is at the minimum (Table 1 & Figure 1). These distinctions result in climatic differences among regions. The sampling sites in SC had the highest annual average temperature and precipitation, whereas those in HB were the opposite. The monthly average temperatures were highest in SC, with January to April and October to December had a significantly higher level, except June, July and August were highest in Henan (HN). The soil was slightly alkaline in most sites, except for HB. None of the regions were deficient in nutrients, and the soil in HB and SC exhibited distinctive high concentration of available nitrogen, and the concentration of available potassium in HB was significantly higher. The concentration of organic matter in HN and SC was significantly lower. PCA of climatic and soil factors showed that the environment of the Shanxi (SX) site was closest to the HN site, these sites were distributed in the center of the plot, whereas the HB site was near to the SD site and the SC site was the most distal site (Figure 2). The determinant bringing about these results was temperature, and the next was precipitation.     Figure   3). The loading values of PCA indicated that the fifth, fourth and fifteenth (tanshinone IIA) peaks were the most contributive variable on the first axis and the second peak (RA) was the most on the second axis.  showed the lowest average content of TSI (0.0492 %). Not significant regional variations in SAB and TSIIA were detected. In the SC and SX population, hydrophilic components were low, while lipophilic components were high, which were opposite in the SD and HN population. Dihydrotanshinone I, C.
Tanshinone I E.
Tanshinone IIA F. Among S. miltiorrhiza from different regions. (Error bars indicate standard deviations. Lowercase letters indicate significance at the level of 95% resulted from post hoc multiple comparison of either LSD or Tamhane T2.).

Discussion
Our present results of PCA of fingerprint ( Figure 3)  Ca produced positive correlation with RA. It is known that N, K and P are important macronutrients for plants. N is the main constituent of proteins, chlorophyll, and enzymes involved in photosynthesis and K is needed for vital functions in metabolism, growth, and stress adaptation [32,33]. A S. miltiorrhiza plot experiment showed that plant growth and all the contents of the bioactive components responded negatively to increasing N availability, suggesting that S. miltiorrhiza is not a nitrophile [34]. And another plot experiment showed that the effects of the applications of P and K fertilizers at different growth stages on the root growth and bioactive compounds were shown to vary greatly in S. miltiorrhiza and K negatively affected the accumulations of tanshinones [35].
Microelements are very important to plants, so some scholars do studies about effect of microelements on S. miltiorrhiza. Wang B through sand culture experiments got that the mechanism of Cu and Zn on the accumulation of tanshinones may be that Cu and Zn improve the activity of peroxidase and polyphenol oxidase, which promote transformation of phenolic compounds to terpenes and therefore to increase contents of danshinones [36,37].
Mn increased the activities of PPO and POD, which promote the accumulation of tanshinones in S. miltiorrhiza [38]. So, if lack of these nutrition elements in the soil, we need to fertilizer these elements to guarantee the quality of S. miltiorrhiza. Precipitation also pronouncedly influenced hydrophilic components. Precipitation parameters displayed negative correlation with chemical variations of S. miltiorrhiza, particularly RA ( Figure 5 & Table 3). A plot experiment showed that the effects of drought stress on physiological characteristics were inhibitory, and drought stress could promote the accumulation of phenolic compounds [39]. A drought stress experiment showed drought stress is more advantageous to accumulate phenolic acid composition of S. miltiorrhiza lamina [40]. Water deficit has been reported to increase the expression of different genes involved in the biosynthesis of phenolic compounds [41][42][43]. Temperature also showed pronounced negative correlation with lipophilic components in S. miltiorrhiza, especially DTSI and TSI. Harpagoside displayed significant positive correlations with monthly and annual average temperature, which was related to heat tolerance [44]. High temperature and strong sunshine limited the content of ginsenosides through collecting Panax ginseng samples [45]. Despite reports on the effect of temperature factors on plant secondary metabolism in natural environments, an array of similar work has been carried out in controlled conditions.
In tomato plants, Anthocyanins showed pronounced increased levels when lowering the growth temperature from 24 °C to 18 °C or 12 °C [46]. Accordingly, the chemical variation of lipophilic components in S. miltiorrhiza might be related to cold tolerance. The pattern of differentiation of lipophilic components also corresponds to the longitudinal and latitudinal directions, which were negative with hydrophilic components. A positive correlation between latitude and chemical diversity was detected in juniper (Juniperus communis) needles [47]. Concentrations of anthocyanidin and delphinidin in Vaccinium myrtillus fruits varied significantly across latitude, with higher values from northern latitudes, whereas another anthocyanidin, cyanidin, was opposite [48]. Therefore, different plants or even different metabolites might differ in their responses to latitudinal variation [49]. Our result indicatied that the SC and SX populations were located at the lower latitude and longitude, and the other three were at higher longitude (Table 1).
The factor of latitude comprises a series of other environmental factors, among which temperature and precipitation tend to fall with the growth of latitude.

Conclusion
Our results revealed how chemical differentiation in