The L. barbarum (one of Solanaceous defoliated shrubbery) mainly grows
in Northwestern of China, Southwestern Europe and the Mediterranean regions
(Zhang et al. 2010; Amagase and Farnsworth 2011). In China, L. barbarum is cultivated in
Ningxia, Qinghai, Xinjiang and Gansu provinces, and Zhongning County in Ningxia
is considered to be the geographical origin of L. barbarum. Various components have been identified in L. barbarum, such as L. barbarum
polysaccharides, zeabroside, β-sitosterol, p-coumaric acid, various
vitamins (Chang and Ko 2008), minerals and antioxidants (Yao et al.
2011). Recent studies have shown that L.
barbarum has multiple pharmacological effects, such as boosts immunity, the
liver protection, circulation promotion (Potterat 2010), and inflammation elimination
(Li et al. 2007). Because of its many benefits to human body, the
consumption of L. barbarum has
increased greatly. There are various products of L. barbarum on the market today, such as, milk, wine, juice,
preserved fruit, and so on. However, it may also contain harmful elements,
pesticide residues and other harmful substances that endanger human health,
which also need to be studied.
Harmful elements pollution has become a global
environmental problem. Heavy metals can not only accumulate in animal products
(Al Bratty et al. 2018; Ali et al. 2020) but also accumulate in
crops and plants and pass to humans through the food chain. Industrial and
agricultural activities have significantly increased the content of heavy
metals in the soil (Bai et al. 2019).
The proceeding of L. barbarum commercialization may introduce harmful
elements. For example, it is easy to absorb moisture and mold due to the high
sugar content, and sulfur fumigation method were used by medicine farmers to
prevent it mildew and extend its shelf life (Wang et al. 2019b).
However, the sulfur used in sulfur fumigation is natural sulfur, which contains
many heavy metals (Liu et al. 2012a), so harmful element contents of
sulfur-fumigated L. barbarum
may increase. These harmful elements can get into the body when L. barbarum were eaten by
consumer. Some studies have shown harmful elements can damage people's health.
Arsenic exposure may lead to cancer and cardiovascular disorder (Yang et al.
2018). Long-term absorption Pb destroys the nervous
system; inhibit the intellectual development of children (Zhao 2018). Chronic
exposure to Cd poses harmful effects like prostatic proliferative lesions and
kidney dysfunction (Żukowska and Biziuk 2008). Too much Cu causes damage
to the respiratory and metabolic systems (Qiao 2018). Excessive consumption of
Zn for a long time leads to chronic poisoning (Wang and Liu 2007). Although Al
was not a heavy metal, it was widely found in food (Liang 2016) and related to
the pathogenesis of Alzheimer's disease (AD) (Miu et al. 2003).
In most studies, ICP-AES was used to detect element
contents in L. barbarum (Yang
2010; Liu et al. 2012). Inductively coupled plasma mass spectrometry
(ICP-MS) was also often used for the determination of various elements in L. barbarum (Wang and Zhang
2012). Furthermore, inductively coupled plasma optical emission spectrometry
(ICP-OES) was used to measure several elements in L. barbarum simultaneously (Sa et al. 2019). Li et
al. (2018) determined As
and Cu in L. barbarum by
atomic fluorescence spectrometry (AFS). ICP-OES possesses wide linear range,
low detection limits, good sensitivity, widespread instrument availability and
reasonable cost (Waheed et al. 2018;
Sa et al. 2019). Therefore, in this research, ICP-OES was used to
determine the contents of Al, As, Cd, Pb, Cu, Ni and Zn in L. barbarum.
At present, the existing
researches mainly focused on the determination of element contents in L. barbarum from farmland. In fact, only a
small percentage of people purchase L. barbarum from farmland, and most
consumers obtained L. barbarum from supermarkets. Therefore, it is
necessary to make a comparative study on the harmful element contents of L. barbarum from these two sources. In the present study, L. barbarum samples were
collected from geographical origin fields in Zhongning country and supermarkets
in Yinchuan city, the contents of Al, As and five heavy metals (Cd, Pb, Cu, Ni
and Zn) were detected. The objectives of this study were: 1) to determine seven
harmful element contents in L.
barbarum from two sources
and know about the present situation of seven harmful element contamination in it; 2) to understand the correlation
of element in L. barbarum; 3)
to analyze the differences of harmful
element contents in L.
barbarum from different sources and give proper advice for customers
to purchase L. barbarum.
Materials and Methods
Sample collection
Zhongning County was located in the middle of Ningxia hui autonomous
region and the southern part of Ningxia plain. It is a transitional belt
between Inner Mongolia plateau and Loess plateau and belongs to the north
temperate continental monsoon climate zone. The annual average temperature is
9.5°C, the annual average sunshine hours are 2979.9 h, and the annual average
precipitation is 202.1 mm.
Sixteen L. barbarum
samples were randomly collected in production gardens from Zhongning in June
2018 (the time when L. barbarum was at maturity). The plant of L. barbarum has been cultivated
for five-ten years in each production garden. The geographic coordinates of
sampling sites (Table 1) were recorded with a global positioning system (Zeng et
al. 2016). At each production gardens, five normal L. barbarum plants which were planted near the middle of the
garden were selected to collect fresh fruits of L. barbarum. The mature tree was 1.5–2.0 meters in height
with several stout main branches, green elliptic leaves and purplish red fruit
with a length of 5–20 mm and a diameter of 3–10 mm (Fig. 1). Five simultaneous
samples of L. barbarum were
collected using the five-point method and then mixed evenly by quartering. The
samples were packed into polyethylene bags, marked, and then immediately
transported to the laboratory. Ten samples were purchased from supermarkets in
Yinchuan. In order to ensure that the source was Zhongning County, the
selection principles were set as follows: when purchasing L. barbarum with bag, Ningxia Zhongning should be list as
producing area on the package; when purchasing the bulk L. barbarum, the sellers were inquired about the
sample's place of origin and only that from Ningxia Zhongning were purchased.
All L. barbarum samples were
identified by Professor Liming Zhang, School of Pharmacy, Ningxia Medical
University.
Element contents were determination
The treatment and determination of elements in L. barbarum were carried out according to the experimental
method optimized by Zhang et al. (2020). Specifically, L. barbarum
was dried in oven (DHG-9030A, Shanghai Yiheng Technology Co., Ltd.,
China) at 60°C to constant weight and crushed by grinder (Tianjin Taisite
Instrument Co., Ltd., China). 0.2 g samples
were weight accurately by electronic analytical balance (METTLER TOLEDO
Instrument Co., Ltd., Shanghai, China) to digestion tube and 10 mL of mixed
acid (nitric acid: perchloric acid= 4:1, V/V, GR, Sinopharm Chemical
Reagent Co., Ltd., Shanghai, China) was added. After pre-digestion overnight, 4
mL of hydrogen peroxide (AR, Sinopharm Chemical Reagent Co., Ltd., Shanghai,
China) was added to each digestion tube and then the solution continue to
digest in electric digester (AED-automatic electric digester, Beijing Institute
of Chemical Metallurgy, Nuclear Industry, China). The temperature of electric
digester was set as follows: initial temperature was 60°C, followed by 120°C
for 2 h and stable at 180°C until all solution was removed, and crystals
appeared in the bottom of the tube. The crystals were dissolved with ultrapure
water (18.25 MΩ cm) prepared with a Ultra-pure water meter (You Pu,
Sichuan, China) and diluted to 10 mL. Element contents were determined by
ICP-OES (Varian 710-ES, USA) and the working parameters were listed in Table 2.
A series of standard solutions with concentrations of 0.2, 0.5, 1.0, 2.0, 5.0, 10.0 µg/mL were
prepared to draw standard curves. One sample was selected and repeated 11 times
to obtain the precision of the instrument. An appropriate amount of standard
solution (Multi-element standard Solution, SGB-YYA230011, China) was added to
the sample to calculate the recovery after the same treatment. Blank reagents
were used as control. All glass instruments used in this experiment were washed
by tap water and ultrapure water in sequence.
Table 1: The geographic location of 16 samples
collected from geographical origin fields
|
Site No. |
Site name |
Longitude (E) |
Latitude (N) |
Altitude (m) |
|
1 |
Nantan |
105°2783838 |
37°4822849 |
1176 |
|
2 |
Aiwan |
105°3331736 |
37°4766686 |
1170 |
|
3 |
Yongjiayingzi |
105°4388490 |
37°4672515 |
1165 |
|
4 |
Xigoucunerdui |
105°4280175 |
37°4091588 |
1267 |
|
5 |
Damaoshangdian |
105°2971930 |
37°4411168 |
1285 |
|
6 |
Dazhanchang |
105°5653789 |
37°3994330 |
1229 |
|
7 |
Caotai |
105°4904596 |
37°4298687 |
1230 |
|
8 |
Xishawo* |
105°5300730 |
37°4660187 |
1168 |
|
9 |
Xishawo* |
105°5300728 |
37°4659968 |
1172 |
|
10 |
Jiaozishan |
105°6157770 |
37°4623103 |
1156 |
|
11 |
Sjilaba |
105°6345072 |
37°4033717 |
1195 |
|
12 |
Yezhugou |
105°6637701 |
37°3754277 |
1192 |
|
13 |
Baituliang |
105°6724752 |
37°4348742 |
1153 |
|
14 |
Zhaozhuang |
105°7032677 |
37°4847923 |
1143 |
|
15 |
Haizhuang |
105°7018849 |
37°5121626 |
1141 |
|
16 |
Wenzhuang |
105°6547834 |
37°4754650 |
1141 |
*The two samples were from the same village, while they
got from different sampling site
%201565-1572_files/image002.jpg)
Fig. 1: Lycium barbarum L.
Statistical analysis
The means and standard deviation (SD) (Wan et al. 2014) were
calculated using Microsoft Excel 2010. The Mann-Whitney U test (Fay and Yaakov
2018) and Pearson correlation analysis (Chee 2013) were performed in S.P.S.S.
17.0. P-values less than 0.05 in
Mann-Whitney U test and less than 0.01 in Pearson correlation were considered
statistically significant.
Results
The results of linearity, accuracy, precision and correlation
coefficients were listed in Table 3. Recoveries of elements were ranged from
75.2 to 124.3%, and RSD values of all elements were less than 5%.
Methodological verification results showed that the method can be used to
determine the contents of elements in L. barbarum.
Harmful element levels in L. barbarum
The general information of element concentrations in L. barbarum
samples were showed in Table 4 and the contents of Al, Zn, Ni and Cu were presented
in Fig. 2. In sixteen samples that obtained from geographical origin fields,
As, Cd and Pb were not found; Ni was found in thirteen samples; Al, Zn and Cu
were detected in all samples. The average (range) contents (mg·kg-1)
of Al, Zn, Cu and Ni were listed
in Table 4. The variation coefficients (CV, %) result indicated that the
contents of four metals in different sampling points varied greatly and the sequence was Ni > Al > Cu > Zn.
In ten samples from supermarkets, As and Pb were also
not detected; Cd was found in two samples; Ni were found in nine samples and
Al, Zn and Cu were detected in all samples. The average (range) contents (mg·kg-1)
of four elements were listed in Table 4. The variation coefficients result also
stated that the contents of four elements in supermarkets samples changed
greatly and the sequence of variation coefficients (%) of them was Ni > Al
> Zn > Cu.
Table 2: ICP-OES working parameters
|
Parameters |
Numerical
value |
|
Power
(KW) |
1.00 |
|
Plasma
gas flow (L·min-1) |
15.0 |
|
Auxiliary
gas flow (L·min-1) |
1.50 |
|
Nebulizer
pressure (kPa) |
200 |
|
One
reading time (s) |
5 |
|
Pump
speed (rpm) |
15 |
|
Cleaning
time (s) |
10 |
|
Condition |
All
spectral lines |
Table 3: The results of methodological verification
|
Element |
Linear regression equations |
Correlation coefficients |
Recovery (%) |
RSD (%) |
|
Al |
y=0.8781x + 0.1124 |
0.9992 |
91.4 |
1.77 |
|
Ni |
y=1.0310x
- 0.0277 |
0.9993 |
111.7 |
4.08 |
|
Zn |
y=0.9433x + 0.0497 |
0.9999 |
113.3 |
2.07 |
|
As |
y=1.2095x - 0.1239 |
0.9985 |
97.5 |
3.90 |
|
Pb |
y=0.9584x + 0.0774 |
0.9990 |
92.0 |
1.49 |
|
Cd |
y=0.9294x + 0.0663 |
0.9997 |
124.3 |
1.13 |
|
Cu |
y=0.9124x + 0.0766 |
0.9997 |
75.2 |
3.27 |
Over-standard rate
For Cu, International Standardization Organization (ISO) and the United
States Pharmacopoeia (USP) have not stipulated its limit value in Chinese
herbal medicine. According to the limit value (20 mg·kg-1)
stipulated in Chinese Pharmacopoeia (CHP 2015), the Cu content in all samples
not exceeded the standard. Comparing with the standard (10 mg·kg-1)
of Malaysia (ISO 2015), all the supermarket samples were under the limit, while
Cu content in one sample from geographical origin fields exceeded the standard,
with an over-standard rate of 12.50%. In terms of Cd, according to the
standards of ISO (2015), the Cd contents in two detected supermarket samples
were not exceeded, but comparing to the standards of the United States (USPC 2018),
China (CHP 2015) and Malaysia (ISO 2015), there was one sample exceeded the
limit with over-standard rate of 10%.
Only five kinds of harmful elemental limits (As, Cd, Pb,
Cu and Hg) were stipulated in Chinese herbal medicine in most countries and
regions and no available standards for Al, Zn and Ni. Considering L. barbarum was one of medicinal
and edible plants, the contents of Al, Zn and Ni were compared with the
relevant standard in foods stipulated in China. Al is only for flour and Ni is
only for oil and its products and we thought these limits were not suitable to
compare the contents of Al and Ni in L.
barbarum. For Zn, different kinds of food have different limits.
Considering the amount, frequency and way of intake of L. barbarum, the over-standard rate of Zn in this study were
calculated by comparing to the limit value of Zn for fruits (5 mg·kg-1)
and vegetables (20 mg·kg-1). All samples were over the limit and the
over-standard rate was 100% when compared with fruit limits, while the
over-standard rate was 6.25% (geographical origin field samples) and 10%
(supermarkets samples) when compared with vegetables limit.
Correlation analysis
Since, As and Pb were not detected and Cd was only detected in two
samples, the correlation analysis of Al, Zn, Cu and Ni was only conducted in
this study. The result was shown in Table 5. Most elements were positively
correlated except for the negative correlation between Zn and Al. There was a
significant positive correlation between Cu and Zn (P < 0.01). The result showed that Al and Zn had antagonistic
effects, while other elements had synergistic effects in L. barbarum.
Mann-whitney U test
Due to the small sample size and independent samples, Mann-Whitney U
test in Non-parametric test was used in this study to analyze the differences
of metal contents in L. barbarum from different sources. Since, As and
Pb were not detected, only Al, Zn, Cu, Ni and Cd were analyzed. The
Mann-Whitney U test result showed that there was a significant difference (P
< 0.05) in Al content while there were no significant differences in
contents of Cu (P=0.078), Ni (P=0.692), and Zn (P=0.833).
Discussion
For seven elements analyzed in this experiment, Pb and As were not found neither in sixteen geographical origin
fields samples nor in ten supermarkets samples. Cd and Ni had different degrees
of detection and their detective rate in samples from supermarkets (20 and 90%)
was higher than that in samples from geographical origin field (0 and 81.25%).
Al, Cu and Zn were detected in all samples. The average contents of Al, Ni, Zn
and Cu in L. barbarum from geographical origin fields were higher than that in samples
from supermarkets. The Al content in the supermarkets samples was almost one
third of that in the samples from geographical origin fields. The contents of
Cu, Ni and Zn were both lower than that of the samples from geographical origin
fields. It was speculated that the contents of Al, Ni, Zn and Cu were reduced,
while Cd was introduced in some samples during the post processing,
transportation and storage of L.
barbarum.
The element contents in L. barbarum from Ningxia were also determined in other
studies. Kai et al. (2020a)
determined the contents of 49 inorganic elements in red L. barbarum,
black L. barbarum and yellow L. barbarum collected in the same
planting base of Zhongning, Ningxia. They found that contents of these elements
varied greatly in three kinds of L. barbarum.
Since red L. barbarum were analyzed
in this study, the element contents in red L. barbarum were compared with our result. The contents of
Cu, Ni and Zn were consistent with that of this research, while Al content
(217.6 mg·kg-1) was obviously higher than that of the present study
(80.75 mg·kg-1). Besides, Kai et al. (2020b) also discussed
26 elements contents in four places from Ningxia and four varieties of L. barbarum and concluded that
there was no significant difference in elements contents of different varieties
of L. barbarum, while
significant difference was found in the contents of elements in L. barbarum
from different places (Wang and Zhang 2012) measured the contents of twelve
heavy metals in L. barbarum
obtained from farmers in Yinchuan, Ningxia. Ni content (1.405 mg·kg-1)
was consistent with our result (1.78 mg·kg-1), while As, Pb, and Cd
were also detected in their report which were not in agreement with our study.
It was speculated that the difference of element contents may be caused by
different cultivated environment (Kai et al. 2020b). However, the
specific reasons need further study.
Other previous researchers also analyzed the element
contents in L. barbarum from
supermarkets or markets. However, in recent years, the research on L. barbarum from Ningxia mainly
focused on the samples from geographical origin fields and lacked the research
on commercial L. barbarum
(Yang and Li 2006) determined trace elements contents in commercial L. barbarum
and the contents of Cu (10.643 mg·kg-1) and Zn (58.056 mg·kg-1)
in their research were both higher than our result (6.13 mg·kg-1,
13.82 mg·kg-1) (Zhao et al. 2007) analyzed the contents of
trace elements in commercial L. barbarum,
and Cu content (9.471 mg·kg-1) and Zn content (56.056 mg·kg-1)
were also higher than that in this study. Although the contents of Cu and Zn in
above two studies were both higher than that in this study, their results were
basically consistent with each other. The possible reasons for this discrepancy
were as follows: for one thing, the cultivated environments in Zhongning have
changed in these ten years. For another, the monitoring of Chinese herbal
medicine processing were strengthened since 2008 in China (Duan et al.
2011), which would reduce the introduction of harmful elements.
Table 4: The contents of detected toxic elements in two sources
of samples
|
Element |
Mean ± SD |
Median |
Range |
CV (%) |
||||
|
|
aG |
bS |
G |
S |
G |
S |
G |
S |
|
Al |
80.75 ± 29.26 |
28.71 ± 10.65 |
71.29 |
31.11 |
49.43 - 139.78 |
6.44 - 43.65 |
36.23 |
37.10 |
|
Cu |
7.61 ± 2.27 |
6.13 ± 0.82 |
9.88 |
6.38 |
4.73 - 12.16 |
5.01 - 7.33 |
29.83 |
13.38 |
|
Ni |
1.78 ± 1.55 |
1.00 ± 0.90 |
0.66 |
0.34 |
cND - 5.28 |
ND - 2.70 |
87.08 |
90.00 |
|
Zn |
14.03 ± 3.77 |
13.82 ± 4.23 |
14.62 |
16.86 |
8.54 - 20.95 |
8.29 - 22.68 |
26.87 |
30.61 |
aG: Samples from geographical origin fields; bS: Samples from supermarkets; cND means not detected
Table 5: Correlation analysis of elements in L. barbarum
|
Element |
Al |
Ni |
Zn |
Cu |
|
Al |
1.000 |
|
|
|
|
Ni |
0.090 |
1.000 |
|
|
|
Zn |
-0.219 |
0.184 |
1.000 |
|
|
Cu |
0.063 |
0.005 |
**0.667 |
1.000 |
**: P ＜ 0.01
%201565-1572_files/image004.png)
Fig. 2: The concentration of Al, Ni, Zn and Cu in samples. Note:
1-16 samples from geographical origin fields, 17-26 samples from supermarkets
As showed in Table 1, the sixteen sampling points in
this study possessed similar background environment. So it was speculated the
element content difference in L. barbarum
from geographical origin fields may be caused by anthropogenic
factor. However, for the
supermarkets L. barbarum,
although the growing environment was similar to that of the geographical origin
fields samples, the harmful elements may not only come from agricultural
chemicals used in the growing process, but may also be affected by the
commercialization procedure of L. barbarum.
Since there were many possible sources of harmful elements in L. barbarum, it was necessary to
identify the source of elements and analyze the contribution of possible
influencing factors in the future.
Kai et al. (2020b)
also analyzed element correlation in L.
barbarum and they found Zn and Cu was significantly positive correlated
(P < 0.01) which was consistent with our result. However, Ni and Al,
Ni and Cu were significant positive correlation (P < 0.05) in their
research, while Ni and Al, Ni and Cu were positive correlated in the present
study. In addition, in other studies on the correlation of Chinese herbal
medicine elements, although the correlation of other elements was different
from that of L. barbarum
in this study, the correlation of Zn and Cu was consistent. For example, in Radix
A stragali (Lei et al. 2008), Alisma plantago-aquqtica
(Zhang 2010), Bletilla atriata (Zhang et al. 2020), Zn and Cu
were also found significantly positive correlated (P < 0.05). In Portulaca
oleracea L. (Ye et al. 2019) and Conyza blinii H. Lév (Zheng et
al. 2016), Zn and Cu were found positively correlated. It was speculated
that this may be related to the interaction between Zn and Cu, while the
specific reasons were yet to be studied.
Some scholars have compared the correlation between
element content and soil element content in Chinese herbal medicines (Qi et
al. 2014) discussed the contents and correlations of rare earth elements in
soil and fruit of L. barbarum
and the results showed that the soil and fruit rare earth elements were
negatively correlated (Wang et al. 2019a) studied the content of Cu in
different parts of L. barbarum
and soil, and their results indicated that Cu content in fruit was positively
correlated with that in soil. (Zhou et al. 2018) analyzed the
correlation between element content and soil element content in Paeoniae Radix
Alba in Bozhou, and found that the contents of Cr, Cd, Hg and Cu in the soil
were significantly positively correlated with those in the herbs (P <
0.05) (Zhang et al. 2018) conducted a comparative study on the content
of heavy metals in Chrysanthemum indicum and the soil, and found that Pb
and Cd were positively correlated with the corresponding elements in soil (P
< 0.05).
In addition to soil, Chinese herbal medicine would be
exposed to water, air, possible fertilizers and pesticides during the growth
and more harmful elements may be introduced during transportation and storage
procedure. However, the current research mainly focused on the correlation of
element content between plants and soil, and there was still a lack of
correlation of element content between plants and other factors, which should
be analyzed systematically in further study.
Moreover, only five kinds of harmful elements limits
were stipulated in Chinese herbal medicine. In fact, the excessive contents of
other elements were also harmful to the human body. So it is necessary to
establish limits of other elements for Chinese herbal medicine. Furthermore, there
are many kinds of Chinese herbal medicine and the consumption of them varied
greatly. This can lead diverse amount of element enter human body and would
posed different health effects. So the universal standard was not that
applicable to all Chinese herbal medicine. Therefore, it is also necessary to
build the limits of harmful metals for a specific Chinese herbal medicine. For
example, (Zhang et al. 2019a, b) established the limits of As, Cd, Cr,
Pb, Cu, Ni and Zn for Scutellaria baicalensis Georgi (Zhu et
al. 2018) built the limits of As, Cd, Cr, Pb, Cu and Hg in Glycyrrhizae Radix et Rhizoma
(Zhu et al. 2016) also built the limits of As in three kinds of
medicinal and edible Chinese herbs (Dioscorea opposita Thunb, Crataegus
pinnatifida Bge and Ziziphus jujuba Mill).
Conclusion
The contents of seven harmful metals in L. barbarum obtained from geographical
origin fields of Zhongning County and supermarkets in Yinchuan City were
analyzed. The experimental method can be used to detect harmful element
contents in L. barbarum. The
average contents of Al, Zn, Ni and Cu in L. barbarum
from geographical origin fields were higher than that from supermarkets.
Significant difference exist in Al content of L. barbarum from two sources (P <
0.05), while no differences were found in concentrations of Cu, Ni, Zn and Cd
between two sources of L. barbarum.
Cu in two samples from geographical origin fields exceeded the limit of
Malaysia with an over-standard rate of 12.50%. Cd in one sample from
supermarkets exceeded the limits of the US, Malaysia and China with an
over-standard rate of 10%. Al, Zn and Ni didn’t have available standards. Al
and Zn were negatively correlated, while other elements were positively
correlated with significance in Zn and Cu (P < 0.01). When
considering harmful element contamination, it was safer for consumers to
purchase L. barbarum from
supermarkets.
Acknowledgements
The present study was financially supported by the National Natural
Science Foundation of China (21966025 and 21667023) and the Ministry of
Education ‘ChunHui’ plan project (grant nos. Z2016068).
Author Contributions
Yahong Zhang and Meilin Zhu collected Lycium barbarum samples. Ningchuan Feng and Meilin Zhu
conceived and designed the experiments. Le Jian and Yuanyuan Gao assessed the
quality of studies, contributed to the analysis and interpretation of the data.
Yahong Zhang performed the experiments and wrote the initial draft. Yuanyuan
Gao and Meilin Zhu critically revised the manuscript. All authors read and
approved the final manuscript.
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