Evaluation of nutrients and related environmental factors for wolfberry (LyciumbarbarumL.) fruitsproduced from indifferent areas of China Wang Yajun1, 2，Liang Xiaojie2，Guo Sujuan1*，Li Yuekun2，Zhang Bo2，Yin Yue2，An Wei2，Cao Youlong2， Zhao Jianhua2 (1. Key laboratory for Silviculture and Conservation,Ministry of Education, Beijing Forestry University, Beijing 100083, China; 2. National Wolfberry Engineering Research Center, Yinchuan 750002, China) *Corresponding author: Sujuan Guo,Ph.D.,E-mail address: [email protected] Abstract: Wolfberry fruit can be Chinese herbal medicine or food, but the fruit quality and the levels of functional components in wolfberries grown in different producing areas are different. In this study, we collected wolfberry fruit samples from 67 gardens distributed in seven wolfberry producing areas in China (Yinchuan and Zhongning in Ningxia, Jingyuan in Gansu, Nuomuhong in Qinghai,Hangjinhouqi and Wulateqianqi in Inner Mongolia, and Jinghe in Xinjiang) to investigated their internal nutrition ingredients and related environmental factors. The wolfberry fruits from seven different areas showed greatly difference in nutritional ingredients, and each of them had special characteristics. Correlation analysis revealed that the internal nutrient ingredients were highly related to each other.Based on the analysis ofratio of total sugar to betaine (sugar-alkali ratio), we created five categoriesof wolfberry quality: high betaine low sugar (sugar-alkali ratio≤15.1), high betaine medium sugar (15<sugar-alkali ratio≤60), medium betaine medium sugar(60<sugar-alkali ratio≤121), medium betaine high sugar (121<sugar-alkali ratio≤165), and low betaine high sugar(sugar-alkali ratio>165). Zhongning of Ningxia is the geo-authentic habitat of wolfberry, where produces the best wolfberry in quality, which is richer in main pharmacological ingredients (e.g. LBP) and three main micronutrients (iron, manganese and selenium) and shows a proper sugar-alkali ratio. In addition, correlation analysis showed that altitude and average temperature difference between day and night were both positively and significantly associated with wolfberry flavonoids content, while annual sunshine duration was negativelyrelated with bataine contents of wolfberry fruit. Keywords:Wolfberry(Lyciumbarbarum L.); Nutrition ingredients; Production area; Quality evaluation Introduction Lyciumbarbarum L. (Solanaceae), a perennial, deciduous shrub (Cao & He, 2013), is commonly known as wolfberry or wolfberry fruit (Bondia-Pons et al. 2014) and is the main economic tree in the northwest arid area of China. Wolfberryfruit has many pharmacological effects, such as enhancing immunity, anti-aging, anti-tumorigenesis, and anti-oxidative (Chang & So, 2008; Lin et al., 2008; Zheng & Hu, 2008; Ma et al., 2009). Modern medical studieshave foundthat wolfberry fruit contains several function ingredients,Lyciumbarbarum polysaccharide (LBP), medlar total saccharide, betaine, carotenoids, flavonoids, and pharmacodynamic amino acids (Qian et al., 2004; Liu et al., 2008; Dong et al., 2009; Liu et al., 2014). Wolfberry fruit is also rich in sugar and organic acids (Xie et al., 2001; Zhang et al., 2016). Among these ingredients, LBP is considered as the most important ingredients determining pharmacological effects of 1 wolfberry fruit, followed by betaine and flavonoids. Generally, the nutrients in wolfberry fruitmainly include twosubstances with opposite characteristics: total sugar representsacidic substances and betaine representsalkaline substances. The contents of these two substances and their equilibrium ultimately determines the quality of wolfberry fruit (Gao and Li, 2003; Zhang et al., 2008). And the wolfberry with a balanced ratio of total sugar to betaine (sugar-alkali ratio) is of high quality. In China, the main cultivated wolfberry variety is ‘Ningqi 1’, a species of L.barbarumL., which has the largest planting area through the country and accounts for more than 80% of the total cultivation. As the geo-authentic habitat of wolfberry, Ningxia promotes wolfberry as a local strategic advantage of leading industry. Driven by Ningxia, the planting of wolfberry has expanded into other regions of China,and the planting area is specially enlarged in Xinjiang, Qinghai, Inner Mongolia, and Gansu. Currently, the total planting area of wolfberry in China is over 1.33×105 ha. Development of the wolfberry industry in China, especially in the northwest arid area, not only protects the fragile ecological environmentand effectively increases the economic efficiency for farmers, but also solves the employment problem of surplus labor and maintains social stability, which results insignificant social, economic, and ecological benefits. The production areas of wolfberry are mainly in northwest China. These areas differ in ecology and climatic conditions greatly, such as altitude, drought, salinity, sunshine hours, diurnal temperature difference, as well as soil nutrient status, which affect the development of wolfberry fruit and the formation of functional components inside. Dong et al. (2009) analyzed the total flavonoid content of wolfberryfrom five areas of China and found that the total flavonoid content was ranked as: Zhongning (Ningxia)>Shahai (Inner Mongolia)>Nuomuhong(Qinghai)>Qitai (Xinjiang)>Jinghe (Xinjiang)>Julu (Hebei). Zheng et al. (2010) tested the sugar content of two wolfberryvarietiesfrom Ningxia, Inner Mongolia, Xinjiang, and Hebei altogether four regions, and results showed that the different levels of soil salt, pH, organic matter, and available nitrogen affected sugarcontent. Zhang et al. (2012) analyzed the 100-kernel weight of ‘Ningqi 1’ dried wolfberry fruit producedfrom six regions, and found that they were different and in the following order: Qinghai>Gausu>Xinjiang>Xingxia>Inner Mongolia>Hebei. Linnan et al. (2013) evaluated the wolfberry fruits from Ningxia, Gansu and Qinghai and found that climate had great effects on the development of external phenotype and internal quality of wolfberry fruit. Zhang et al. (2014a, b) investigated external traits and internal nutrient contents of three varieties of wolfberry fruits from three planting regions, indicating that the 100-kernel weight and fruit shape index of wolfberry fruit produced in Nuomuhong (Qinghai) were higher than thoseproduced in Tongxin (Ningxia) and Wusu (Xinjiang), while the active ingredientcontents varied greatly with different planting regions. Even inTongxin of Ningxia,a geo-authentic habitat of wolfberry, the intrinsic qualitiesof wolfberrywerealso affected by soil nutrients and salinity and the intrinsic composition variedsignificantlywith different planting locations (Léchaudel et al., 2005; Niu et al., 2005; Xu et al., 2005; Zhang et al., 2005a, b; Kang et al., 2008; Liu et al., 2015). In previous studies, researchers analyze the intrinsic nutrient composition of wolfberry fruits from differentareas and obtained some valuable results. However, these studies suffer from a small number of sample sites and the sampling method is not clearly explained, therefore, these samples may not accurately represent the characteristics of wolfberryfrom typical ecological areas. In this study, we collected wolfberry samples from various sites in typical ecological regions where 2 wolfberry is grown, and analyzed their main nutrient ingredients and related growing conditions, thereby revealing the key factors influencing the intrinsic quality of wolfberry. This study would provide a theoretical basis for evaluation of wolfberryquality from different producing regions. 2. Sampling area and sampling methods 2.1 General information of sampling area This study is based on the five main wolfberry production regions in China, which include Ningxia, Xinjiang, Qinghai, Inner Mongolia, and Gansu. Samples are specifically collected from seven typical wolfberry areas in the five regions, which are Wulateqianqi and Hangjinhouqiin in Inner Mongolia, Yinchuan City and Zhongning County in Ningxia, Jingyuan County of Gansu, Nuomuhong Farm in Qinghai, and Jinghe County in Xinjiang. Detailed climate conditions of each sampling area are in Table 1. In the seven sampling areas, altitudes range from 290 to 2780 ma.s.l.; the annual mean temperatures range from 5.2 to 10.5°C; annual precipitations range from 42.2 to 243.9 mm; annual sunshine durations range from 2700 to 3220 h, and mean diurnal temperatures range from 11.8 to 15.2°C. Overall, the climates of the seven sampling areas accuratelyreflect the distribution of climates for wolfberry cultivation in China. For example, among these areas, Zhongningin Ningxia has a moderate altitude and sunshine duration, but hasthe highest annual mean temperature, annual rainfall, anda largerdiurnal temperature. While, Nuomuhongin Qinghai hasthe highest altitude and the largest diurnal temperature among the seven areas, butits annual temperature and rainfall is relatively low. 2.2 Wolfberry fruit sample As ‘Ningqi 1’ could represent the wolfberry varieties of northwest of China, it was chosen as study materials. From each of the seven sampling areas, we randomly selected six-twelve wolfberry gardens where 6-8-year old ‘Ningqi 1’ was cultivated. Then we collected a total of 3 kg of wolfberry fruit with peduncle from different parts of the three trees. All the samples were obtained at the best harvest time. In total, 67 wolfberry fruit samples were collected. These samples were numbered, dried in time and vacuum packed for further analysis. 2.3 Nutrient evaluation and analysis