1

1 The effects of ecological factors on the chemical compounds in

2 Lycium barbarum L.
3
4 Jia Mi1 · Yamei Yan1 · Yuekun Li1 · Yue Yin1 · Lu Lu1 · Qing Luo1 · Xiaoying Li1 · Xuan

5 Zhou1 · Yan Niu2 ·Youlong Cao1

6
7Abstract Lycium barbarum L. is a traditional medicinal and edible plant cultivated in different
8ecological areas in China. In this study, “Ningqi” No.1, one of the main cultivars of L. barbarum
9L., was collected from 10 different important cultivation areas in 5 autonomous regions or
10provinces of China. Partial least squares regression and correlation analysis were used to analyze
11the relationship between 16 ecological factors and 9 phytochemicals of “Ningqi” No.1. There was
12no significant difference in carotenoids or ash in plants from the 5 regions or provinces (p > 0.05),
13but total sugar content and total reducing sugars were lowest in Ningxia (p < 0.05), and total
14polysaccharides (3.61%), flavonoids (0.56%), and Se (0.62 mg/kg) were highest in Ningxia.
15Different chemical compounds were affected by different ecological factors. However, when taken
16as a whole, the most influential ecological factors were elevation, relative humidity, accumulated
17temperature of ≥10℃, annual mean temperature, available potassium, and annual precipitation.
18Except for the available potassium, meteorological, and topographical factors had greatest effects
19on the accumulation of the main chemical compounds rather than soil factors.Different chemical
20compounds of Lycium barbarum L.were affected by different ecological factors. Aside from the
21available potassium, meteorological, and topographical factors had greatest effects on the
22accumulation of the main chemical compounds rather than soil factors.
23Keywords Lycium barbarum L. · Chemical compounds · Ecological factors · Partial least squares
24regression (PLSR)
25
26Introduction
27
28Herbaceous plants (herbs) have been subject to a long process of evolution and continuous

21 Ya-mei Yan, yanyamei@163.com

3 Youlong Cao, youlongchk@163.com
41 Institute of Wolfberry Engineering Technology, Ningxia Academy of Agriculture and Forestry Sciences,
5 Ningxia 750002, China
62 Supervision and Testing Center for Lycium Quality, Ministry of Agriculture, Ningxia 750002, China
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29adaptation to their environment leading to the formation of stable strains. Due to climate, soil,
30terrain, and other factors, some areas have supported the evolution of certain herbs used in geo-
31herbalism (traditional Chinese medicine) (Yang et al. 2017; Gurbuz et al. 2016). Some of the
32phytochemicals found in geoherbs are the material basis for their medicinal effects, and these
33compounds and their levels are the main basis for evaluating their quality. Most of the functional
34ingredients are the plant's primary metabolic compounds (e.g., polysaccharides) or secondary
35metabolic compounds (e.g., flavonoids). They are the product of the interaction of the plant with
36its environment (biological and non-biological) during its evolution, and have played an important
37role in improving the ability of plants to protect themselves and survive, and to coordinate their
38relationship with the environment (Yang 2011). At the same time, the production and modification
39of these compounds is strongly influenced by the environment (Huang et al. 2013; Raúl et al.2012;
40Wang and Zheng 2001; Deng et al.2016; Kaur et al. 2016).
41 Lycium barbarum L. (L. barbarum L.) is a traditional Chinese medicinal geo-herb and edible
42herb (Xie et al. 2016), which can improve eyesight and which exhibits protective effects for liver
43function, as recorded in the Compendium of Materia Medica. It has been shown that L. barbarum
44L. is rich in polysaccharides (Xie et al. 2016), polyphenols (Magiera and Zaręba 2015),
45carotenoids (Hempel et al. 2017), betaines, amino acids, and many functional compounds
46(Amagase and Famsworth 2011; Zhang et al. 2016). L. barbarum L. can encourage development
47of immunity against diseases (Zhao et al. 2015), and has antioxidant effects (Zhang et al. 2016),
48impairing the reproduction system (Yu and Qian 2016), protecting photoreceptor cells and treating
49cardiovascular diseases (Zhu et al. 2016; Tang et al. 2015).
50 Currently, the planted area of L. barbarum L. in China exceeds 134,000 hectares (Lam et al.
512016), and more than 80% of this is for the variety of“Ningqi” No.1. Although L. barbarum is
52found in other countries (Vulić et al. 2016), in China, it is mainly planted in Xinjiang Uygur
53Autonomous Region, Qinghai Province, Ningxia Hui Autonomous Region, Inner Mongolia
54Autonomous Region, and Gansu Province. With continuous expansion in production areas, many
55studies have been conducted on compounds in L.barbarum L. in different areas. However, it is
56rarely systematically and comprehensively reported that the quality difference and ecological
57factors effected on the main chemical compounds under the condition of unified variety and
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58cultivation management mode. sampling has been concentrated on only some of the producing
59regions (Wang 2003), with different varieties of L.barbarum L. or different cultivation conditions
60or management measures (Gao and Li 2003; Zhang et al. 2003). The evaluation criteria have
61usually included fruit morphology or other morphological characteristics, total sugar content, and
62amino acid content (Zhang et al. 2004; Liu et al. 2004). Other functional compounds, such as
63flavonoids and carotenoids, were not analyzed (Zhang et al. 2004). The statistical methods used
64were relatively simple, and the quantitative studies involved mainly single correlations and
65regression analyses (Zhang et al. 2004). The effect of 10 ecological factors on the accumulation of
66functional compounds of L.barbarum L. was reported, but the effect of soil factors was not
67analyzed(Zeng et al. 2015).
68 For this study 10 samples were collected in different ecological environments in China and used
69to examine the correlations between 9 chemical compounds and ecological factors by means of
70bivariate correlation analysis and partial least squares regression (PLSR). Moreover, this study
71systematically investigated the main ecological factors influencing the main chemical compounds
72of L. barbarum L. to provide a reliable basis for testing the quality of L. barbarum L, and a
73theoretical basis for the cultivation of high-quality L. barbarum L. alongside rational utilization of
74ecological resources.
75
76Materials and chemicals
77
78Plant materials and Soil materials
79
80Sampling points for “Ningqi” No. 1 are shown in Fig. 1 and Table 1. Plots bearing plants of the
81same age were selected from the main production regions in Xinjiang, Qinghai, Ningxia, Gansu
82and other provinces or autonomous regions to establish different sampling points in these
83ecological regions. The fruits were harvested at the same stage of maturity. The sampling was
84done at the first maturation of fruits at each testing point. High-quality fruits at 90% maturation,
85without decay and insect damage, were randomly selected. Approximately 10 kg of fruits were
86sampled each time, dried by hot air at 50 °C, and kept at 0–4 °C until testing. Tests were done on 3
87randomly selected samples. Soil sampling time is shown in Table 1. The sampling method was as
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88follows: samples were collected at the beginning of the first flowering; a line of spray-painted
89sampling trees was selected to collect root soil 25 cm away from the main stem; samples were
90collected at a depth of 0-20 and 20-40 cm, totaling 500 g each. The collected soil samples
91corresponded to the fruit samples.
92
93Determination of chemical compounds of L.barbarum L.
94
95Total sugar content, reducing sugar content, total polysaccharide content, total flavonoid content,
96total carotenoid content, amino acid content, betaine content, selenium content, and ash content
97were measured by Ningxia Analysis Test Center in accordance with GB/T15672-2009, GB/T
985009.7-2008, NY/T 1676-2008, NY/T 1295-2007, GB/T12389-1990, GB/T 5009.124-2003, NY/T
991746-2009, GB5009.93-2010 and GB 5009.4-2010, respectively.
100
101Determination of Soil Factors
102
103Measurements of pH, total nitrogen, total phosphorus, total potassium, total salt, available
104phosphorus, available nitrogen, available potassium, organic matter and selenium in the soil were
105made by the Supervision and Inspection Center of Chinese Wolfberry Product Quality of Ministry
106of Agriculture in accordance with LY/T 1239-1999, LY/T 1228-1999, LY/T 1232-1999, LY/T
1071234-1999, LY/T 1251-1999, LY/T 1233-1999, LY/T 1229-1999, LY/T 1236-1999, NY/T 1221.6-
1082006, and NY/T 1104-2006, respectively. The results are shown in Table 2.
109
110Collection of climate factors
111
112The climate data were from China Meteorological Science Data Sharing Service Platform (Table
1131).
114
115Statistical analysis
116
117Bivariate correlation analysis method and partial least squares method (PLSR) were analyzed by
118SPSS21.0 and SIMCA-P software, respectively.
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119
120Results
121
122The Chemical Compounds of “Ningqi” No. 1 in Different Production Areas
123
124The main chemical compounds of “Ningqi” No. 1 in different production areas in 5 different
125regions or provinces is shown in Tables 3 and Tables 4. The amounts of the main chemical
126compounds in different production areas were different, with the levels of carotenoids, flavonoids,
127amino acids, and selenium in sample No.2 being significantly higher than those in other areas.
128There was no significant difference in the level of carotenoids and ash in the 5 regions or
129provinces mentioned above (p > 0.05), but total sugar content and total reducing sugars were
130significantly lower in Ningxia (p < 0.05), and total content of polysaccharides (3.61%), flavonoids
131(0.56%) and Se (0.62 mg/kg) were highest in Ningxia.
132 The level of polysaccharides in sample No.3 was the highest (Table 3), and significantly higher
133(p < 0.05) than that of samples No.6, No.7, No.8, and No.10, but there were no significant
134difference between the levels in No.3 and those in samples No.1, No.2, No.4, No.5, and No.9.
135 The level of selenium in sample No.2 was the highest (Table 3), followed by No.4, with the
136levels >1 mg/kg, both significantly higher than that in other production areas (p < 0.05). Levels of
137Se in samples No. 5 and No. 1 were moderate, and lower in the other 6 production areas.Selenium,
138especially organic selenium, has a variety of physiological functions and uses, such as prevention
139of cardiovascular disease, anti-cancer, improvement in immunity, promotion of fertility and so
140on(Yang et al. 2011). L. barbarum L. from Ningxia contains a higher level of selenium (Table 4).
141
142Pearson Correlations between Ecological Factors and Chemical Compounds in L. Barbarum
143L.
144
145The results of Pearson correlations between ecological factors and chemical compounds in L.
146barbarum L. are shown in Table 5. Among the 10 soil factors tested, there was a significant
147positive correlation between total phosphorus and the accumulation of polysaccharides; available
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148potassium was positively correlated with the accumulation of ash and selenium level; and
149selenium in soil was negatively correlated with total sugar content.
150 The six climatic factors tested were significantly correlated with the chemical compounds in
151L.barbarum L. Sunshine duration was positively correlated with total sugar content, reducing
152sugars and betaine. Annual precipitation was negatively correlated with total sugar content and
153reducing sugars. Annual mean temperature was negatively correlated with the level of betaine, and
154there was a significant positive correlation between annual mean temperature and selenium level.
155Relative humidity was positively correlated with levels of flavonoids and selenium. Accumulated
156temperature of ≥10 ℃ was negatively correlated with the enrichment of betaine and amino acids.
157Accumulation of betaine and amino acids was significantly related to elevation. In general,
158climatic and topography factors had more significant effects on chemical compounds than soil
159factors.
160
161Correlation between the Main Chemical Compounds and Ecological Factors in Different
162Production Areas by PLSR Analysis
163
164Correlation between total sugar and ecological factors
165
166In relation the annual precipitation, total sugar content in L. barbarum L. exhibited the most
167negative regression coefficient, followed by selenium content in soil, and available potassium
168(Fig. 2A). Sunshine hours and accumulated temperature of ≥10℃ showed a higher positive
169correlation with the total sugar. Fig. 2 (B) shows the Variable important values (VIP) for annual
170precipitation (1.972), soil selenium content (1.370), sunshine hours (1.310), accumulated
171temperature of ≥10℃(1.301) and available potassium (1.222) all higher than 1.000. Thus, annual
172precipitation, soil selenium, sunshine hours, accumulated temperature of ≥10℃ and available
173potassium were the main factors affecting the accumulation of total sugar for L. barbarum L..
174
175Correlation between reducing sugars and ecological factors
176
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177In relation to annual precipitation, reducing sugar content in L. barbarum L. exhibited the most
178negative regression coefficient followed by soil selenium content and available potassium (Fig. 3
179A). An accumulated temperature of ≥10℃ showed the largest positive correlation with reducing
180sugars, followed by sunshine hours. Fig. 3B shows the VIP level for annual precipitation (2.000),
181accumulated temperature of ≥10℃ (1.679), sunshine hours (1.176), available potassium (1.190)
182and soil selenium content (1.108) all being higher than 1.000. Thus, the main factors affecting
183reduction in sugar content were annual precipitation, accumulated temperature of ≥10℃, sunshine
184hours, rapidly available potassium and soil selenium content. The dominant factors affecting the
185accumulation of the reducing sugars were the same as those for total sugar.
186
187Correlation between flavonoids and ecological factors
188
189Among the ecological factors, annual mean temperature showed the most negative regression
190coefficient in relation to flavonoid content followed by relative humidity, available potassium and
191total salt (Fig. 4A). Among these, annual mean temperature and annual precipitation were
192positively correlated with the flavonoid content. VIP for annual mean temperature (1.943) was the
193highest compared with other variables, followed by relative humidity (1.717), available potassium
194(1.272) and total salt (1.130) (Fig. 4B). Annual mean temperature, relative humidity, available
195potassium and total salt were the main ecological factors affecting flavonoids content.
196
197Correlation between amino acids and ecological factors
198
199In relation to amino acid content, elevation showed the highest absolute value of the regression
200coefficient followed by accumulated temperature ≥ 10 ℃, total potassium and available
201phosphorus (Fig. 5A). Among the factors, elevation and available phosphorus were positively
202correlated with amino acid content while an accumulated temperature ≥ 10 ℃ and total potassium
203were negatively correlated. VIP for elevation (1.949) was the highest compared with other
204variables, followed by accumulated temperature ≥ 10 ℃(1.758), organic matter (1.065) and total
205potassium (1.038) (Fig. 5B), respectively. Elevation, accumulated temperature≥10 ℃ and total
206potassium were the main ecological factors affecting amino acid content.
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207
208Correlation between betaine and ecological factors
209
210In relation to betaine content, elevation showed the highest absolute value of the regression
211coefficient followed by annual precipitation, rapidly available potassium, available nitrogen and
212annual mean temperature (Fig. 6A). Among these, elevation and available nitrogen were positively
213correlated, while annual precipitation, available potassium and annual mean temperature were
214negatively correlated. VIP for elevation was the highest compared with other variables (1.878),
215followed by annual mean temperature (1.249), available potassium (1.218), accumulated
216temperature ≥10 ℃ (1.144), annual precipitation (1.092), and soil organic matter (1.026) ( Fig. 6
217B). Elevation, annual precipitation, available potassium and annual mean temperature were the
218main ecological factors affecting betaine content.
219
220Correlation between selenium and ecological factors
221
222In relation to selenium content, annual mean temperature showed the highest absolute value of the
223regression coefficient followed by available potassium, relative humidity, total phosphorus, and
224soil pH (Fig. 7A). Among these, annual mean temperature, available potassium and relative
225humidity were positively correlated, while total phosphorus and soil pH was negatively correlated.
226VIP for annual mean temperature (1.740) was the highest compared with that of the other
227variables, followed by available potassium (1.634), relative humidity (1.526) and total salt (1.087)
228( Fig. 7B). Annual mean temperature, relative humidity and available potassium were the main
229factors affecting selenium content.
230Correlation between all the main chemical compounds and ecological factors
231
232Correlations between ecological factors and all the main chemical compounds of L. barbarum
233were analyzed by PLSR. VIP values for elevation (1.509), accumulated temperature of ≥10 ℃
234(1.497), relative humidity (1.345), annual average temperature (1.318), sunshine duration (1.291),
235available potassium (1.250), and annual precipitation (1.124) were all greater than 1.0 (histogram,
236Fig. 8B). Among all the important ecological factors which VIP values were greater than 1.0,
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237sunshine duration and accumulated temperature of ≥10 ℃ were positively correlated with the
238accumulation of compounds in L. barbarum. (Fig. 8A). The results showed that meteorological
239factors had a greater affect than soil factors on accumulation of the main chemical compounds in
240L. barbarum L., except for available potassium. It was apparent that the effects of artificial
241cultivation conditions were less than those of natural ecological conditions. The results analyzed
242by PLSR were similar to those analyzed by bivariate correlation analysis method.
243 It was shown that annual precipitation was apparently and negatively correlated with the
244accumulation of total sugars and reducing sugars, accumulated temperature ≥10 ℃ was apparently
245and positively correlated with the accumulation of total sugar and reducing sugars, The main
246production areas of L. barbarum L. in their studies were in Northwest China, where there is
247generally less annual precipitation. However, compared with the annual precipitation of 160–700
248mm in Ningxia, it was significantly higher than that of Inner Mongolia, Xinjiang, Gansu and
249Qinghai, and had higher accumulated temperature of ≥10 ℃ (2700–3952 ℃), which was
250beneficial to the production of L. barbarum L. having lower total sugar content and lower
251reducing sugar content.
252 Annual mean temperature had a significant positive effect on the accumulation of flavonoids
253and selenium in L. barbarum L. (Table 5, Fig. 4 to Fig. 7). It was suggested that climatic factors
254were more likely to affect the accumulation of flavonoids in plants than were soil factors, and
255among the former, temperature had greatest positive effect on the content of flavonoids in
256plants(Wang and Zheng 2015). It was showed that higher temperature could induce the expression
257of the genes PAL, C4H, 4CL and STS in the secondary metabolic pathway of the medicinal herbs
258and promote the accumulation of flavonoids(Deng et al. 2016). The annual mean temperature in
259Ningxia was 8.0–16 ℃, which was significantly higher than that in other provinces /regions in the
260study. It provided favorable conditions for the production of higher levels of flavonoids and
261selenium in L. barbarum L., but the mechanism involved needs to be further explored.
262Disscussion
263 The analysis of The chemical compounds of “Ningqi” No. 1 in different production areas
264The results of The main chemical compounds of “Ningqi” No. 1 in different production areas
265(Tables 3 and Tables 4) were consistent with the reports of Zeng Fanlin et al (Zeng et al. 2015)
266and Zheng Guo-Qi, et al (Zheng et al.2010). As the main processing method for L. barbarum L. is
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267drying, the higher the total sugar content (especially the content of reducing sugars) the higher the
268drying cost. At the same time, the high sugar content of L. barbarum L. encourages moisture
269absorption and this is not conducive to satisfactory storage(Wu et al. 2015). Therefore, for
270processing and storage, the quality of L. barbarum L. in Ningxia was better than that of the other
271four provinces or regions.
272 Ningxia Region is the main geo-herbalism production area for L. barbarum as recorded in 2015
273edition of "Chinese Pharmacopoeia”, and Zhongning County in Ningxia was the birthplace of
274L.barbarum. L. Our results for polysaccharide content, both in the 10 different producing areas
275and in the five provinces or regions (Table 3), are consistent with the pharmacology of the geo-
276herbalism and the origin of L. barbarum L. (Dong et al. 2008).
277 L. barbarum L. from Ningxia contains a higher level of selenium (Table 4), which is consistent
278with the conclusions of Gao Yu and reports from others for the selenium-rich areas of
279Ningxia(Yang et al. 2015), and according to Table 4, six kinds of compounds, total sugar, reducing
280sugar, betaine, flavones, amino acid and Se, were the main chemical compounds in 5 autonomous
281regions or provinces of China (P<0.05).
282 In recent years, there has been large-scale cultivation of L. barbarum L. in Qinghai, Gansu,
283Inner Mongolia, Xinjiang Uygur Autonomous Region and other regions or provinces of China, but
284the main chemical compounds of the product were quite different. Therefore, in order to ensure
285the product is of the right quality, containing the correct ingredients in the correct proportions, it is
286necessary to pay attention to the source of the plant.
287The analysis of correlation between all the main chemical compounds and ecological factors
288 Elevation was the main ecological factor affecting the accumulation of betaine and amino acids
289in L. barbarum L. (Table 5, Fig. 7, Fig. 5 and Fig. 6). Many studies have shown that higher
290elevation was beneficial for the accumulation of betaine (Kumar et al. 2016) and amino
291acids(Silvanini et al. 2014; Kaur et al. 2016). The elevation of the production areas of L.
292barbarum L. in Qinghai province was between 2800–2900 m, significantly higher than that in
293other provinces or regions where L. barbarum L. were produced, and sunshine hours were greater
294(3000–3212 hours) in the areas of Qinghai province. Therefore, in those areas, ecological
295conditions were conducive to the accumulation of betaine and amino acids. At Xingren in Ningxia,
296the elevation was higher (1764 m), the sunshine duration was longer (2959.2 h), the accumulated
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297temperature of ≥10 ℃ was higher (3270.4 ℃), and the relative humidity was up to 77%, and the
298content of betaine (1.15%) and amino acid (13.47 mg / 100g) were also higher. It is apparent that
299ecological factors are advantageous and of great significance.
300 In conclusion, The main chemical compounds of L. barbarum L. from different regions were
301significantly different. Plants from the Ningxia Autonomous region exhibited the lowest content of
302total sugar and total reducing sugars, and the highest total content of polysaccharides, flavonoids
303and selenium. The ecological factors affecting different chemical compounds varied. However,
304taking all chemical compounds as an indicator, the most influential ecological factors were
305elevation, accumulated temperature of ≥10℃, relative humidity, annual mean temperature,
306available potassium, sunshine duration and annual precipitation. Except for available potassium,
307meteorological factors had a greater effect on the accumulation of the main chemical compounds
308than soil factors did . The results provide a scientific basis for the introduction and cultivation of
309L. barbarum L. and its sustainable development.
310
311Author contribution statement Yamei Yan and Youlong Cao designed the experiments. Yuekun
312Li, Yue Yin and Yan Niu performed the experiments. Jia Mi, Qing Luo, Xuan Zhou and Xiaoying
313Li. analyzed the data. Jia Mi and Lu Lu composed the paper.
314Acknowledgement This work was supported by Grants-in-Aid for scientific research from the
315National Natural Science Foundation of China (31360076), and Pilot project by Ningxia Academy
316of Agriculture and Forestry Science (NKYJ-15-16, NKYZ-16-05).
317
318Compliance with ethical standards
319
320Conflict of interest The authors declare that they have no competing interests.
321
322