Article

Volume 3, Issue ..., 2023, ...

https://doi.org/10.33263/Materials000.000

Profile of secondary metabolites and metal in Jatropha

curcas L. And Reutealis trisperma plants grown in the
media contaminated by gold mining tailings using LC-
MS/MS
1 2* 3
Wa Ode Nur Siti Fatimah , Deden Saprudin , Hamim

1 Department of Chemistry, Faculty of Mathematics and Natural Sciences, IPB University, Bogor, Indonesia;
[email protected] (W.N.S.F);
2 Department of Chemistry, Faculty of Mathematics and Natural Sciences, IPB University, Bogor, Indonesia;
[email protected] (D.S.);
3 Department of Biology, Faculty of Mathematics and Natural Sciences, IPB University, Bogor, Indonesia;
[email protected] (H);
* Correspondence: [email protected] (D.S.)

Scopus Author ID 57216752430

Received: date; Accepted: date ; Published: date
ABSTRACT : The increase of industrial development has negative impact to the environment. One of the
efforts to reduce heavy metals contaminant from the environment is through phytoremediation program.
The purpose of this study was to analyze the merphological response, metal content and changes in
metabolites of the compounds based on LC-MS/MS analysis of Jatropha curcas and Reutealis trisperma
treated with gold mine tailings stress. The study was conducted using a completely randomized design
with two factors consisting of two plant species: Jatropha Curcas and Reutealis trisperma and three
g o l d m i n e tailings concentrations (0, 50, and 100 %). the work procedures carried out are:
planting preparation and tailings treatment, growth observation and sampling for analysis, analysis of
secondary metabolites in plant tissues, identification of metal components in leaves with XRF and
identification of metabolite profiles with LC-MS/MS. Gold mine tailings caused a significant decline in
plant growth represented by plant height and leafnumber. The higher the concentration of tailings, the
more disturbed plant growth. We find about 33 metabolitesthat are thought to be composed of: amino
acids, terpenoids, phenols, flavonoids and other organic compounds. Chemometric results revealed
significant differences in the geographical location metabolites profiles, which J. curcas and R. trisperma
varieties had little effect. This study shows that UHPLC-Q-Orbitrap-HRMS-based metabolomics is
efficient for profiling J. curcas and R. trisperma across various treatment.

Keywords : Phytoremediation, LC-MS/MS, XRF Spectrum , R. Trisperm, J. curcas

© 2023 by the authors. T his article is an open-access article distributed under the terms and conditions of the
Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

1. Introduction

Gold mining is one of the human activities that is at great risk of heavy metal
pollution due to tailings that are produced. Tailings are waste residues of mining
activities that are dominated by sand texture, are poor in nutrients, and often contain

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heavy metal elements [1]. Bayu [2] found that Pb is one of the heavy metals found in
tailings in high concentrations.
The presence of heavy metals in the environment is very dangerous for living
things, and since heavy metals cannot be biologically degraded, they be will
accumulated in the tissues. The higher concentration of metals can be a source of
toxic and carcinogenic materials for living beings [3]. The impact of heavy metal
contamination on humans and animals includes the presence of heavy metal content
in the blood and ends in the onset of various diseases.
Countermeasures of heavy metal waste contained in our envitonment, including
tailings can be carried out with the help of plants (that is known as
phytoremediation). Phytoremediation is the use of plants to remove pollutants from
contaminated soil or waters. This method has been recognized as an efficient and
effective method to reduce contaminants from many sites, including areas
contaminated with heavy metals [4]. Plants that have ability to survive in polluted
environments and have ability to accumulate heavy metals are called
hyperacumulator plants [5,6]. The effectiveness of phytoremediation in reducing
heavy metals depends on the capacity of the plant to absorb and accumulate certain
heavy metals to the canopy part [7], while plants are still able to grow well and have
higher biomass. Therefore, it is necessary to constantly develop efforts to obtain
plants with a high ability to absorb heavy metals as accumulator plants.
There are several criteria for plants that are used as phytoremediation agents of
heavy metals such as having fast growth, producing high biomass, a deep root system
[8] and having tolerant properties, and have ability to absorb heavy metals. Plants
such as Jatropha curcas and Reutealis trisperma can be used as phytoremediators on
land polluted with gold mine waste. The two species can in addition be used as
phytoremediators. J. curcas is a potential plant for phytoremediation of soil or
polluted soil with a wide variety of heavy metals [9]. R. trisperma has a good
adaptability to grow on marginal lands such as, very dry soils, and acidic soils [10],
and is able to adapt to the liquid waste of gold mines [11]. Until now, there have
been no publications related to aspects of J. curcas metabolites and R. trisperma as a
response to heavy metal deposits. Therefore, this study aimed to observe the
morphological response, nutrient content response and metabolite changes response
of the compound based on LC-MS/MS analysis of J. curcas and R. trisperma that get
treated with gold mine tailings.

2. Materials and Methods

2.1. Experimental Methods

Pretreatment, castor bean plants Jatropha curcas and Reutealis trisperma are
germinated in the beds. After germination, the plant is planted in small pots
measuring 7 cm to 1 month old. After 1 month the plant is transferred into polybags
that have been prepared for treatment. The growth medium was prepared using a
mixture of soil and compost in a ratio of 1:3 (v/v), the plants were treated with gold
mine tailings with different concentrations. Each polybag is filled with the mixture
until it reaches a weight of 6 kg per polybag, to help the initial growth, each polybag
is given 500 grams of compost. One plant is planted in each polybag. The study was

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conducted using a complete randomized design with two factors and three tests. The
first factor is 2 types of plants, namely Jatropha curcas and Reutealis trisperma. The
second factor is 3 concentrations of tailings in the growth medium, namely a mixture
of ordinary media without the addition of tailings (as a control) gold mine tailings
with a concentration of 50%, and gold mine tailings with a concentration of 100%.

2.2. Growth observation and sampling for analysis

Plant growth is observed for 3 months with a note, height, number of leaves.
After observing the growth of plants Jatropha curcas and Reutealis trisperma is
ready to be harvested at the age of 3 months for testing on the metabolite profile on
the plant.

2.3. Identification of Metal Element Components on Leaves with XRF

The research method uses microXRF, which is a tool to see the distribution of
elements in a sample. The first step is a sample of Jatropha curcas leaves and intact
Reutealis trisperma is inserted into the chamber. Next turn on the vacuum to 2 mBar.
The goal is to increase the accuracy of the readings on the Light Element.
Furthermore, the sample analyzed is the lower leaf part because the whole leaf is
quite large. Next set of power xray, step size and dwell time for analysis of Jatropha
curcas and Reutealis trisperma samples, the analysis time takes about 30 minutes.
The results came out in the form of images of the distribution of elements in the
Jatropha curcas and Reutealis trisperma [12].

2.4. Analysis of Secondary Metabolites of Plant Tissues

The sample is cleaned and then dried after drying it is mashed then in the
extraction of 10 grams of Jatropha curcas leaf powder and Reutealis trisperma by
dissolving on ethanol PA in a beaker in a ratio of 1:10 and extraction temperature (30
and 40 °C). Ultrasonication was performed for 30 min by using 42 Hz ultrasonic
waves, after which a supernatant filtered extract was collected, and the solvent was
evaporated with a rotary evaporator in vacuum at a temperature of 45 °C to obtain an
extract, then collected in a glass bottle and stored at a temperature of 30 °C [13].

2.5. Identification of metabolites with LC-MS/MS

Identification of chemical components by treating as much as 5 mg of

concentrated extract of the sample is weighed, then dissolved in 1 ml of ethanol. The
dissolving of the extracts used an ultrasonicator for 30 min. The sonication results are
put in a 5 ml measuring flask, then add ethanol until the brick mark. Furthermore, the
solution was filtered with a 0.2 μm PTFE filter membrane and as much as 2.50 μl
filtrate was injected into UHPLC-Q-Orbitrap-MS/MS. Separation of Jatropha curcas
and Reutealis trisperma metabolites using Vanquish Flex UHPLCQ-Orbitrap HRMS
with accucore column C18 (100×2.1 mm, 1.5 m). A gradient elution system with a
flow rate of 0.2 mL/min for 50 min was used to separate metabolites. The
composition of the phase of motion used, namely 0.1 % formic acid in water (A) and
0.1% formic acid in acetonitrile (B) with a gradient elution system of 0-3.45 minutes
(8-25% B), 3.45-6.9 minutes (25-54% B), 6.9-7 minutes (54-100% B), 7-9 minutes

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(100% B), 9-15 minutes (8% B), with positive and negative ion modes [15].

3. Results and Discussion

3.1. Heavy metal content of gold mine tailings and soil
Plant growth is a morphological response that can be directly observed and is
highly influenced by environmental conditions. The measurement of heavy metal
levels carried out in this study was the metal lead (Pb). The determination of heavy
metals tested in this study was based on previous research that Pb had relatively high
levels and very low levels of mercury [16].
Based on the results of the analysis in this study, the pH of the tailings tends to
be alkaline (pH 7.23), while the soil is acidic (pH 4.90). Pb levels in tailings and soil
were 63.35 ppm and 13.43 ppm respectively (Table 1). The Pb levels obtained had
hampered the growth processes of the two plants during the study. The characteristics
of the gold-mine tailings are sand which is dominant at 83.45%, dust at 15.77%, and
clay at 0.90%, while the soil tends to be clay textured at 74.70%, low porosity with a
composition of sand at 11.65%, and dust at 13.70%.
[17] Explained that the presence of heavy metals, especially nonessential heavy
metals, in low amounts can affect plant growth and development. In addition, the Pb
content obtained has also passed the quality standard for potential sources of toxicity
to the environment. According to PP No. 85 1999 concerning the TCLP quality
standard for contaminants in waste for the determination of toxic characteristics, that
the quality standard for lead is 5 ppm.

Table 1 Parameters of heavy metals tailings and soil as a growing medium

Heavy metals
Media
pH Pb (ppm) Hg (ppm) Ag (ppm) As (ppm)
Tailing 7.22 63.31 0.03 0.04 0.06
Soil 4.91 13.44 0.03 0.04 0.06

3.2. Effect of tailings soil concentration on plant growth

Observations on the morphology of the two plants showed that, compared to
control plants (0% tailings), the 50% tailings treatment tended to show growth
inhibition, while the 100% tailings treatment showed significant growth inhibition
in crowns and roots (Figure 1). The results of measurements of the morphological
characters of the two plants showed a significant decrease in response to the 100%
tailings treatment compared to the control plants for 8 weeks (Figure 2). The
morphological characters that experience growth inhibition are plant height,
number of leaves. Observations on the increase in height of jatropha curcas and
Reutealis trisperma plants treated with gold-mine tailings during the study within
8 weeks are presented in (Figure 2).

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A B

Figure 1 Morphology of species J. curcas (A), R. trisperm (B) after tailings

treatment 0 % (T0), 50 % (T50), and 100 % (T100) for 8 Weeks. 30 cm

The results showed that growth inhibition occurred in plant height in line with
the increasing concentration of tailings waste used as a planting medium. This
shows that the tailings treatment has an effect on plant height. The results of the
analysis showed that treatment of gold-mine tailings caused a significant reduction
in plant height and number of leaves in J. curcas but not statistically significant in
R. trisperma (Figure 2).
Treatment of 50% and 100% tailings caused a decrease in plant height in J.
curcas by 9.51% and 28.80%, respectively, while in R.trisperma it tended to
decrease by 15.60% and 25.78%. In addition, 50% and 100% tailings treatment also
caused a decrease in the number of leaves in J. curcas by 26.70% and 47.90%
respectively, while in R. trisperma it tended to decrease by 14.70% and 27.80%
respectively. The greater the concentration of tailings in the soil medium, the lower
the average increase in plant height.
This occurs because of the uptake of heavy metals in plants and results in
inhibition of plant growth. According to [18] namely the presence of heavy metals
such as Pb, Al, Mn, Cr, Hg, and other heavy metals can inhibit the growth of height
and number of plant leaves. Apart from the toxic effect of the heavy metals
contained in the tailings, this decline is related to the availability of organic matter
and nutrients in the planting medium, which was only given 500 grams of compost
at the beginning of the treatment.
Many studies have also found that low concentrations of heavy metals in
growth media reduce plant growth. According to [19] the height of privet plants
decreased with increasing concentrations of heavy metals given. [20], heavy metals
are toxic to legumes because they can cause chlorosis, reduced plant growth,
decreased productivity, and limited nutrient uptake. [21] Also found that growth in
height and number of leaves decreased significantly in the four plants used,
including J. curcas and R. trisperma, after treatment with 100% tailings.

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50 100

High Growth of Jatropha

Reutealis trisperma
High Growth of

curcas Plants (cm)

40 80
Plants (cm) 30 60
20 40
10 20
0 0
3 4 5 6 7 3 4 5 6 7
T ime (week) T ime (week)
A B
20 60
trisperma of Leaf

Plan t Jatropha curcas

Plant Reutealis

15 50
40
10 30

of Leaf
5 20
10
0 0
3 4 5 6 7 3 4 5 6 7
T ime (week) D T ime (week)
C

Figure 2 Height gain of Reutealis trisperma plant (A), Jatropha curcas (B), and
Number of Reutealis trisperma leaves (C), Jatropha curcas leaves (D) on
the influence of concentration tailing T0 (blue), T50 (red), T100 (green)

Growth inhibition is the main common response when plants are under abiotic
stress, including heavy metal stress [22]. The presence of high amounts of non-
essential heavy metals affects plant growth and development, damages leaves, and
reduces plant dry weight [23]. Heavy metal toxicity also reduces enzyme activity
due to metal binding to the site of enzyme activation, which ultimately causes
growth inhibition [24]. Nonetheless, both of these plants survived until the end of
the observation, which shows that these plants have good adaptability to gold-mine
tailings.

3.3 Pb Accumulation in Leaves

Heavy metals can be detected using histochemical methods with specific
reagents. The reagent used to detect Pb is dithizone with a positive red color on the
tissue. Histochemical observations of Pb accumulation were also detected in leaf
tissue. In the leaf tissue, to be precise in the leaf veins, the presence of Pb metal is
found in the vascular tissue, especially in the xylem.
These results indicate that J. curcas and R. trisperma were able to absorb and
distribute Pb metal from the leaf media. No toxic symptoms were found from J.
curcas and R. trisperma, this indicates that J. curcas and R. trisperma can tolerate
heavy metals contained in tailings, although there is a different growth response
between J. curcas and R. trisperma.
Lead accumulation, especially in leaves, was also reported by [16] on Reutalis
trisperma treated with 100% gold-mine tailings where the tailings contained a lot of
lead. Lead was detected in all leaf tissues, such as upper and lower epidermis,
parenchyma and stomata. Furthermore, [20] also reported that lead was detected in

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leaf tissue, especially xylem in Jatropha curcas, Ricinus communis, Reutealis

trisperma, and Melia azedarach plants that were treated with gold-mine tailings
with a concentration of 100%.

3.4 Pb Heavy Metal Levels in Shoots and Roots

In this experiment, the heavy metal content analyzed from plant tissue was Pb
because Pb has a very high concentration in tailings (Table 2). Analysis was carried
out on the crowns and roots of both species after being treated with gold-mine
tailings stress for 8 weeks. The results showed that the Pb content in the roots was
not too different compared to the canopy except for J. curcas, in the 100% tailings
treatment which accumulated Pb in the canopy up to more than 9 times. The 50%
tailings treatment did not induce Pb accumulation in the shoots and roots except for
the roots of R. trisperma (Table 2). The data showed a different typology of the two
plants, tailings treatment caused J. curcas to accumulate Pb in the canopy while R.
trisperma accumulated Pb in the roots (Table 2).

Table 2 Levels of Pb metal in leaf and root tissues of J. curcas, R. trisperma, which
were treated with 0%, 50%, and 100% tailings stress for 8 weeks

Pb Content (ppm)
Plant Shoots Roots
T0 T50 T100 T0 T50 T100
J. curcas 0.40b 0.40b 3.75a 0.40c 0.40c 0.40c
R. trisperma 0.40b 0.40b 0.76b 0.40c 2.45bc 4.50b
Note: Numbers followed by the same letter show results that are not significantly different based on
the DMRT (Duncan Multiple Range Test) test

The results of the analysis of this study showed that the 100% tailings
treatment did not cause a significant increase in the BCF value in both J. curcas and
R. trisperma, while the highest TF value was in J. curcas, which was 7.45 (Table
3). In contrast, in R. trisperma the 100% tailings treatment caused the TF value to
decrease (Table 3).
Plants with low TF values can be used as phytostabilizers, these plants keep
metals outside their tissues and are still able to live without introducing metals into
plant cells [25].
The combination of BCF and TF values < 1 has the potential as a
phytostabilizer indicating that R. trisperma has a higher ratio of Pb levels in roots
than in shoots. This indicates that the species is included in the plant
phytostabilization, which is indicated by the low translocation process from roots to
shoots. [17] Explained that phytostabilizing plants are plants that reduce the
mobility of metals in the soil towards the canopy so that they tend to accumulate
them in their roots.

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Table 3 Bioconcentration factor (BCF) and translocation factor (TF) values of Pb

metal from J. curcas, R. trisperma treated with 0%, 50%, and 100%
tailings stress for 8 weeks

Plant BCF TF
T0 T50 T100 T0 T50 T100
J. curcas 0.06b 0.04c 0.06b 1.00b 1.00b 7.45a
R. trisperma 0.06b 0.09b 0.09b 1.00b 0.20b 0.25b
Note: Numbers followed by the same letter show results that are not significantly different based on
the DMRT (Duncan Multiple Range Test) test

3.5 Metal Components on Jatropha curcas and Reutealis Leaves Castor

Observation of the metal components of pecan leaves and fence spacing using
the XRF micro instrument obtained data on the results of the Reutealis trisperma
spectrum on the control treatment and 100% tailings treatment with observable
metal elements, namely Ca, K, Mg, P, Fe, Ti, Mn and Si, with the value of metal
element levels can be presented on Table 4. There is a difference in the comparison
between the control treatment and the tailings treatment 100% judging from the
existing metal element content, where the Ca in the tailings treatment is 100%
greater than the Ca in the control treatment, the K in the control treatment is greater
than the K in the tailings treatment 100% and Fe in the control treatment is greater
than Fe in the tailings treatment 100%. The data also show that the higher the
concentration of tailings in the soil affects the metal elements present in plants.
As for spectrum Jatropha curcas data on control treatment and 100% tailings
treatment at fence distance, metal data were obtained Ca, Mg, Si, P, S, K, Fe, Mn
and Ti with metal element content values presented in Table 4. There is a
comparative difference between the control treatment and the tailings treatment is
100% fencing distance, judging from the existing metal element content data, where
the Si in the control treatment is greater than the 100% tailings treatment Si, the S
in the control treatment is greater than the 100% tailings treatment S and the K in
the control treatment is greater than the 100% tailings treatment K.
A very prominent difference between the two types of plants is that in
Reutealis trisperma no sulfur (S) element in the leaves is measured because the
element sulfur (S) is so small that it is covered with other metal elements so that it
is not detected in the XRF spectral data, whereas in Jatropha curcas there is
elemental sulfur (S) to appear in the XRF spectrum data. The reason why sulfur (S)
in Reutealis trisperma cannot be detected is because the group's LOD (Limit of
detection) is below 50 ppm. The LOD (Limit of detection) of XRF (X-Ray
Fluorescence) is around 50 ppm, so the possibility of sulfur in Reutealis trisperma
below 50 ppm can be even smaller. This shows that the higher the concentration of
tailings in the soil affects the metal elements present in plants.

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Table 4 Womb metal elements of Jatropha and Reutealis leaves

Mineral Metal Content (mg/100 g)
Fence Jatropha Reutealis
T0 T100 T0 T100
Ca 20.96 26.80 45.82 34.65
K 57.56 55.37 32.60 52.73
P 1.65 1.88 1.88 2.27
Mn 0.25 0.42 0.49 0.75
Fe 0.38 0.28 0.32 0.73
Mg 9.39 8.30 15.77 5.70
Si 7.73 4.87 2.10 1.67
S 1.63 1.22 - -
Ti 0.73 1.05 1.03 1.68

3.6 Secondary Metabolite Profile with LC-MS/MS

Based on the results obtained chromatogram data of jatropha extract J. curcas

and R. trisperma of the two types planted in each tailings treatment, different
separation patterns have a separation pattern similar to each other, but only differ in
the area of each detected peak (Figure 3). This suggests that the distribution of
metabolites in all samples is relatively almost the same only at the level of their
concentration. This suggests that the distribution of metabolites in all samples is
relatively almost the same only at the level of their concentration.
Based on the identification results using the Xcalibur application, 33
compounds were obtained. The identified compounds consist of 6 compounds from
the amino acid group, 9 compounds from the flavonoid group, 4 compounds from the
phenolic acid group, 7 compounds from the triterpenoid group, 6 compounds from
the steroid group and 1 compound from the alkaloid group. The data can be presented
in Table 5.
The absorption of Pb metal that occurred in the leaf metabolites of J. curcas
and R. trisperma occurred in the leaf cells. It can be seen that there were not many
changes due to the 100% tailings treatment. The changes were only seen in the shape
and size of the chloroplasts. Chloroplasts are the part of the leaf cells that are affected
the most due to heavy metal stress, the most frequent change is a decrease in the
number and swelling of the chloroplasts, changes in the shape of the membrane
thylakoids reduced number of grant, increased starch grains and plastoglobuli [26]. In
leaf cells that were treated with 100% treatments, there was also a slight
accumulation of heavy metals in the cell wall. This was because the leaf cell walls
also had a strong affinity for binding to heavy metals, causing accumulation in the
cell wall area and preventing heavy metal ions from entering. In leaf cells [27].

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Figure 3 Sample chromatograms on positive ionization (bottom) and negative

ionization (top) modes of D (Jatropha) and B (Reutealis)

3.6.1 Amino acids Groups

Metabolites of the amino acid group identified in Jatropha and Reutealis
extracts are Valine (1), Tryptophan (5), Leucine (2), Phenylalanine (4), 5-
Aminopentanoate (3), Ethyl 3,5-bis[(4-nitrobenzoyl) amino] benzoate (6). In
Jatropha on average there is a decrease in each tailings concentration of 50 and
100%, while Reutealis increases in tailings 100%, but in tailings concentrations 50%
decreases data can be seen in table 3. This shows that the higher the concentration of
tailings in the soil, the more it affects the growth of the plant.
Leucine (3) and phenylalanine (4) release HCOOH with m/z values of 86 and
120 respectively followed by NH 3 release with m/z values of 69 and 103. The valine
compound (1) is fragmented by releasing CH 2O2 molecules at m/z 72 [M+H-46]+ and
molecule NH2 at m/z 56 [M+H-16]+, while tryptophan (2) was identified in the
positive ionization mode with [M+H]+ at m/z 188, 170 and 143 (Table 4). This group
of compounds is indicated by the presence of M-17 and M-18 fragments which
indicate the release of H 2O and NH3 [28]. 5-Aminopentanoate (5) serves as a
branching point for biosynthesis of plant-derived alkaloids [29], positive ion mode,
undergoes fragmentation at m/z 118[M+H]+, 101[M+H-OH]+, 70[M+H-OH-CH2O]+,
Ethyl 3,5-bis[(4-nitrobenzoyl) amino] benzoate (6) identified in the negative ion
mode, fragmented at m/z 477[M-H]-, 205[M-H-C12H6N3O5]- and 163 [M-H-
C12H6N3O5-C2H2O]-.

3.6.2 Flavonoid Groups

Several flavonoid group compounds that have been successfully identified
using positive ion mode in Jatropha and Reutealis extracts Apigenin (13),
Kaempferol (12), Rutin (10), (+)-dihydrokaempferol (9), Palomid 529 (7),
Daidzein (11), Epicatechin gallate (15), 3-Methylplumbagin (14), Ethyl 1,3-
dihydroxy-2-naphthoate (8). In Jatropha the average has decreased, but there is also
an increase in tailings concentration of 50% of routine compounds, while in
Reutealis the average has a dramatic increase in tailings concentration of 100%, but

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there is also a decrease in tailings concentration of 50%, data can be seen in table 3.
This shows that the higher the concentration of tailings in the soil, the more it
affects the growth of the plant.
Flavonoids play a role in giving a yellow color to higher plants, so it is
suspected that this group of compounds plays an important role in the formation of
the yellow color of Jatropha curcas and kemiri sunan. In addition, flavonoids also
act as natural antioxidants, as well as anti-cancer. The benefits of flavonoids for
plants are that they can influence plant defense responses against micro-organisms
so that plants remain durable, due to the production of nematodes in plant roots.
Rutin (9) is fragmented in the positive ion mode m/z 611[M+H]+,
423[M+H-C11H8O3]+, 329[M+H-C11H8O3-C6H6O]+ and [M+H-C11H8O3-C6H6O-
C9H6O3]+. Kaempferol (8) is fragmented in the positive ion mode, m/z 273[M+H] +,
193[M+H-C2H4O]+, 177[M+H-C2H4O-O]+, and 147[M+H-C2H4O-OCH2O]+ and
Epicatechin gallate (13) undergo fragmentation in a negative ion mode, m/z 441
[M-H]-, 289[M-H-C7H4O4]-, 271[M-H-C7H4O4-H2O]- and 169[M-H-C7H4O4-H2O-
C8H6]-. Apigenin (7) was identified as fragmented in the positive ion mode, m/z
271[M+H]+, 253[M+H-H2O]+, 225[M+H-H2O-CO]+, and 211[M+H-H2O-
COCH2]+. (+)-dihydrokaempferol (10) was identified to undergo fragmentation in
the negative ion mode m/z 288[M-H]-, 259[M-H-CO]-, 243[M-H-CO-O]- and
215[M-H-CO2-CO]-. Palomid 529 (11) was identified as fragmented in the negative
ion mode, m/z 405[M-H]-, 390[M-H-CH3]- and 358[M-H-CH3-CO-2H2]- Daidzein
(12) was identified as fragmented in the positive ion mode m/z 255[M+H] +,
214[M+H-OH-C2]+, 185[M+H-OH-C2-COH]+ and 172[M+H-OH-C2-COH-CH]+.
3-Methylplumbagin (14) was identified as experiencing fragmentation in the
positive ion mode, m/z 203[M+H]+, 188[M+H-CH3]+, 160[M+H-CH3-CO]. Ethyl
1,3-dihydroxy-2-naphthoate (15) was identified as experiencing fragmentation in a
positive ion mode, m/z 233[M+H]+, 218[M+H-CH3]+, 190[M+H-CH3-CO-COH]+.

3.6.3 Phenolics acids Groups

Phenolic Acid Compounds identified in Jatropha and Reutealis extracts
Benzaldehyde (19), Gallic acid (16), Ellagic acid (17) and Methyl gallate (18).
In jatropha on average there is an increase in band area in tailings of 50 and 100%,
while in Reutealis there is a decrease in tape area in tailings 100%, but in tailings
50% there is an increase in band area in benzaldehyde compounds, data can be seen
in table 3. This shows that the higher the concentration of tailings in the soil, the
more it affects the growth of the plant.
The phenolic acid detected in the leaf extract is Benzaldehyde (16), detected
in the positive ion mode, fragmented at m/z 107[M+H] +, 79[M+H-CO]+, 77[M+H-
CO-H2]+. Gallic acid (17), has excellent pharmacological activity, as an active anti-
inflammatory ingredient, without toxic effects, and treats inflammation, is very
easily absorbed orally identified by negative ion mode, undergoes fragmentation at
m/ z 169[M-H]-, 125[M-H-CO2]-, 97[M-H-CO2-CO]- and 81[M-H-CO2-CO-O]-.
Ellagic acid (18) is identified with the positive ion mode. Experiencing

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fragmentation at m/z 300[M+H]+, 283[M+H-HO]+, 230[M+H-HO-3(HO)-H2]+.

Methyl gallate (19) identified with negative ion mode, fragmented at m/z 183[M-
H]-168[M-H-CH3]-, 139[M-H-CH3-CHO]- and 123[M-H-CH3-CHO-O]-

3.6.4 Alkaloid Groups

The alkaloids identified on the leaves are 2-phenylethylamine (20). In Jatropha
there is a decrease in band area per tailings concentration of 50 and 100%, but in
Reutealis it has increased the band-area at a tailings concentration of 100%. The
data can be seen in table 3. This shows that the higher the concentration of tailings
in the soil, the more it affects the growth of the plant.
The alkaloid identified in the leaves is 2-phenylethylamine (20), serves to
analyze the ability to form tyramine by bacteria to evaluate the potential risk of
tyramine biosynthesis in food products (Marcobal et al. 2022), helps to overcome
the dopamine deficit that causes Parkinson's disease, identified with the positive ion
mode, experiencing fragmentation at m/z 122[M+H]+, 105[M+H-NH2]+ and
80[M+H-NH2-C2]+.

3.6.5 Triterpenoid Groups

Terpenoid compounds identified in Jatropha and Reutealis extracts
Hemigossypolone (26), Betulinaldehyde (21), Betulinic acid (22),
Marsformosanone (23) Pomolic acid (26), Lupeol (25) and Myristyl sulfate (24). In
Jatropha on average, there is a decrease in band area with every tailings
concentration of 50-100%, there is also an increase in band area. While the average
Reutealis experienced a decrease in band area with every tailings concentration of
50 and 100%, some also experienced an increase. The data can be seen in table 3.
This shows that the higher the concentration of tailings in the soil, the more it
affects the growth of the plant Tabel 5.
Terpenoids are formed from a combination of isoprene-isoprene consisting
of IPP (isopentenyl diphosphate) and DMAPP (dimethylallyl pyrophosphate).
Terpenoid synthesis occurs through the fusion between the head and tail or tail with
isoprene tail. Two IPPs combine to form monoterpenoids, IPP combines with
DMAPP to form sesquiterpenoids, and two DMAPPs combine to form diterpenoids.
Retro processes or terpenoid bond breaking can occur at the head and tail of
isoprene, such as the synthesis process. The pattern of fragmentation or bond
breaking tends to be the same as that of flavonoids, namely through the retro Aldol
process, which is accompanied by condensation or termination of the H 2O group.
Hemigossypolone (21), identified as experiencing fragmentation in the
negative ion mode, m/z 272[M-H]-, 219[M-H-CH-CH2]-, Betulinic acid (23),
identified as experiencing fragmentation with negative ion mode, m/z 455 [M-H]-,
439[M-H-O]- and 421[M-H-O-H2O]-. Betulinaldehyde (22) was identified as
experiencing fragmentation in a positive ion mode, m/z 441[M+H] +, 315[M+H-
C8H14O]+ and [M+H-C8H14O-COH]+, Marsformosanone (24) was identified as
experiencing fragmentation in positive ion mode, m/z 423[M+H]+, 405[M+H-
H2O]+, 269[M+H-H2O-C10H16]+, 243[M+H-H2O-C10H16-C2H2]+, 229[M+H-H2O-
C10H16-C2H2-CH2]+. Pomolic acid (25) was identified as experiencing
fragmentation in the positive ion mode, m/z 473[M+H] +, 437[M+H-O2-2H2]+ and
409[M+H-O2-2H2-CO]+. Lupeol (26) was identified as experiencing fragmentation

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in the positive ion mode m/z 427[M+H]+, 409[M+H-H2O]+, 229[M+H-H2O-

C14H12]+ and 217[M+H- H2O-C14H12-C]+.

3.6.6 Steroid Groups

Steroid compounds were identified in Jatropha and Reutealis extracts of
sunan 3-Dehydroteasterone (31), 4,4Dimethylzymosterol (32), Rhodalin (29),
Kaempferitrin (28) 4α- Hydroxymethyl-4β-methyl-5αcholesta-8-en-3β-ol (33) and
Rhoifolin (30). At Jatropha, the average increase in band area at all tailings
concentrations was 50 and 100%, while in Reutealis the average experienced a
decrease in band area, but there was also an increase in band area at tailings
concentrations of 50 and 100%. The data can be seen in table 3. This shows that the
higher the concentration of tailings in the soil, the more it affects the growth of the
plant.
The steroid identified in the leaves is 4αhydroxymethyl-4β-methyl-
5αcholesta-8-en-3β-ol (33), with a negative ion mode, fragmented at m/z 429[M-H]-
219[M-H-C14H25-OH]-, 191[M-H-C14H25-OH-C2H4]- and 165[M-H-C14H25-OH-
C2H4-C2H2]-. 3-Dehydrotasterone (31) identified in the positive ion mode,
fragmented at m/z [M+H]+447[M+H-C7H12]+ 351[M+H-C7H12-C5H10O]+ 265. 4,4-
Dimethylzymosterol (32) was identified in the positive ion mode, fragmented at m/z
413[M+H]+, 395[M+H-H2O]+, and 241[M+H-H2O-C9H18-C2H4]+.

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Table 5 Changes in area to control area area (%) leaf Jatropha and Reutealis

Golongan Senyawa Jatropha Reutealis

T50 T100 T50 T100
up down Up down up down up Down

Amino acids
- Leucine x -48,98 X -13,78 x -94,56 96,54 X
- Tryptophan x -7,87 X -90,82 x -84,61 58,94 X
- Phenylalanine x -0,77 X -6,66 x -29,05 x -30,16
Flavonoid
- Rutin 30,44 x 32,52 x x -11,27 x -4,22
- (+)- x -99,95 X -39,81 22,51 X 32,89 X
dihydrokaempferol
- Ethyl 1,3-dihydroxy- x -99,19 X -18,95 x -5,69 94,67 X
2-naphthoate
Phenolics acids
- Gallic acid 4,34 x 8,63 x x -17,37 x -18,02
- Ellagic acid 16,18 x 7,73 x x -12,60 x -4,54
- Methyl gallate 21,09 x 21,19 x 3,13 X x 2,22
- Benzaldehyde 7,70 x 4,23 x 11,91 X x 2,65
Alkaloid
- 2-Phenylethylamine x -38,47 X -42,44 4,75 X 6,67 X
Triterpenoid
- Betulinaldehyde 20,77 x 21,08 x x -10,90 x -10,07
- Lupeol 28,41 x X -24,38 -25,75 x -21,76
- Marsformosanone x -20,47 11,23 x 17,05 X 18,58 X
- Myristyl sulfate x -13,56 X -91,21 10,99 X 16,17 X
Steroid
- Kaempferitrin 70,33 x 86,97 x 16,45 X x -29,34
- Rhodalin 38,33 x 39,31 x x -19,52 x -19,84
- 4,4- x -86,92 47,01 x 15,12 X 14,51 X
Dimethylzymosterol
- 3-Dehydroteasterone 13,27 x 18,44 x 8,88 X 21,62 X
Ket : x = not up/down.

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Table 6 Results of Identification of Secondary Metabolite Compounds of Jatropha and Reutealis leaves
No Metabolite Class RT Formula MW Error MS fragment ion Change in band area to control area (%)
chemical (min) (ppm) (m/z)
Jatropha Reutealis
T50 T100 T50 T100
1 Valine Asam amino 1.02 C5H11NO2 117 -1,9 72, 56 -6,52 -4,44 -28,92 -28,35
2 Leucine Asam amino 1.10 C6H13NO2 131 -1,22 86, 120, 69, 103 -46,98 -13,78 -94,56 96,54
3 5-Aminopentanoate Asam amino 1.23 C15H11N0 2 117 -0,85 118, 101, 70 -6,15 -2,61 -2,23 35,06
4 Phenylalanine Asam amino 1.56 C9H11NO2 165 -2,48 86, 120, 69, 103 -0,77 -6,66 -29,05 -30,16
5 Tryptophan Asam amino 3.04 C11H12N2O2 204 -2,56 86, 120, 69, 103 -7,87 -90,82 -84,61 58,94
6 Ethyl 3,5-bis[(4- Asam amino 14.3 C23H18N4O8 478 -3,76 477, 205, 163 -9,05 -4,68 -85,83 48,11
nitrobenzoyl) amino]
benzoate
7 Palomid 529 Flavonoid 1.13 C24H22O6 406 -0,74 405, 390, 358, 357 -92,39 -41,12 -5,34 -4,31
8 Ethyl 1,3-dihydroxy-2- Flavonoid 4.54 C13H12O4 232 -0,86 233, 218, 190,161 -99,19 -18,95 -5,69 94,67
naphthoate
9 (+)-dihydrokaempferol Flavonoid 4.75 C15H12O6 288 -1,39 288, 259, 243 -99,95 -39,81 22,51 32,89
10 Rutin Flavonoid 7.22 C27H30O16 610 -1,98 611, 423, 329, 167 30,44 32,52 -11,27 -4,22
11 Daidzein Flavonoid 7.23 C15H10O4 254 -2,76 255, 214, 185, 172 -91,18 -16,93 -10,38 -5,26
12 kaempferol Flavonoid 7.62 C15H10O6 286 -1,59 287, 253, 201 19,47 -19,37 -11,12 17,61
13 Apigenin Flavonoid 8.25 C15H10O5 270 -1,22 271, 253, 225, 211 9,39 13,00 10,46 32,94
14 3-Methylplumbagin Flavonoid 9.74 C12H10O2 202 -1,48 203, 188, 160 -8,04 -11,49 16,99 36,89
15 Epicatechin gallate Flavonoid 14.9 C22H18O10 442 -2,04 441, 289, 271, 169 4,51 34,91 -6,93 -35,30
16 Gallic acid Asam fenolat 1.51 C7H6O5 170 -5,52 169, 125, 97, 81 4,34 8,63 -17,37 -18,02
17 Ellagic acid Asam fenolat 5.82 C14H6O8 302 -0,6 300, 283, 230 16,18 7,73 -12,60 -4,54
18 Methyl gallate Asam fenolat 8.03 C8H8O5 184 -5,43 168, 139, 123 21,09 21,19 3,13 2,22
19 Benzaldehyde Asam fenolat 16.9 C7H6O 106 0 107, 79, 77 7,70 4,23 11,91 2,65
20 2-Phenylethylamine Alkaloid 5.36 C8H11N 121 0 122, 105, 80 -38,47 -42,44 4,75 6,67
21 Betulinaldehyde T riterpenoid 5.71 C30H48O2 440 -2,27 441, 315, 286 20,77 21,08 -10,90 -10,07
22 Betulinic acid T riterpenoid 7.91 C30H48O3 456 3,07 455, 439, 421 -25,26 -25,39 -9,79 -10,27
23 Marsformosanone T riterpenoid 8.43 C30H46O 422 -3,32 423, 269, 243, 229 -20,47 11,23 17,05 18,58
24 Myristyl sulfate T riterpenoid 9.64 C14H30O4S 294 -1,3 - -13,56 -91,21 10,99 16,17

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No Metabolite Class RT Formula MW Error MS fragment ion Change in band area to control area (%)
chemical (min) (ppm) (m/z)
Jatropha Reutealis
T50 T100 T50 T100
25 Lupeol T riterpenoid 10,0 C30H50O 426 -3,52 427, 409, 229, 217 28,41 -24,38 -25,75 -21,76
26 Pomolic acid T riterpenoid 10.1 C30H48O4 472 -2,54 473, 437, 409, 313 -88,82 13,38 8,40 3,53
27 Hemigossypolone T riterpenoid 16,2 C15H14O5 274 -2,55 272, 151, 125 -7,14 -90,72 -3,92 86,14
28 Kaempferitrin Steroid 5.59 C27H30O14 578 -2,18 - 70,33 86,97 16,45 -29,34
29 Rhodalin Steroid 6.19 C20H18O11 456 5,06 - 38,33 39,31 -19,52 -19,84
30 Rhoifolin Steroid 6.30 C27H30O14 362 -3,97 - 1,46 18,88 -16,20 17,98
31 3-Dehydroteasterone Steroid 9.04 C28H46O4 446 1,51 447, 351, 265 13,27 18,44 8,88 21,62
32 4,4- Steroid 9.71 C29H48O 412 -2,91 413, 395, 241 -86,92 47,01 15,12 14,51
Dimethylzymosterol
33 4α-Hydroxymethyl- Steroid 9.96 C29H50O2 428 -2,56 429, 219, 191, 165 2,01 5,78 -13,48 -13,46
4β-methyl-5αcholesta-
8-en-3β-ol

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4. CONCLUSION

Tailings treatment of J. curcas and R. trisperma at concentrations of 50% and

100% resulted in a decrease in morphology and plant growth. The 100% tailings
treatment had a greater negative impact on both plant species when compared to the
50% tailings treatment. Overall, the negative effect of tailings treatment on R.
trisperma was lower when compared to J. curcas, this indicated that R. trisperma
had a higher adaptability than J. curcas. The content of metal elements contained in
the leaves of both plants, namely Ca, K, Fe, Ti, Si, P, Mn, and Mg, in the leaves of R.
trisperma, while in J. curcas namely, Ca, K, Fe, Ti, Mn, P, Si, Mg, and S There is a
difference between these 2 plants in that R. trisperma does not contain metal sulfur,
but Jatropha contains metal sulfur, because the XRF detection limit is 50 ppm, it is
possible that sulfur in R. trisperma is below 50 ppm or very small, identification of
compounds in different treatments on gold-mine tailings from J. curcas and R.
trisperma using UHPLC-MS/MS resulted in 33 Chemometric results revealed
significant differences in the geographical location metabolites profiles, which J.
curcas and R. trisperma varieties had little effect. This study shows that UHPLC-Q-
Orbitrap-HRMS-based metabolomics is efficient for profiling J. curcas and R.
trisperma across various treatment.

Funding

This research was funded by the Ministry of Research and Technology/ National Agency for
Research and Innovation, Indonesia, in the Penelitian Dasar Unggulan Perguruan Tinggi (PDUPT)
research grant, grant number 1931/IT3.L1/PN/2021.

Acknowledgments

The authors are thankful to Mr. Topik Ridwan, Tropical Biopharmaca Research Center, IPB
University, for his continuous and kind support.

Conflicts of Interest

The authors declare no conflict of interest.

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