Toxicity Reduction of Euphorbia kansui Stir-Fried with Vinegar Based on Conversion of 3-O-(20E,40Z-Decadi-enoyl)-20-O-acetylingenol

Article abstract of DOI:10.3390/molecules24203806

The dried roots of Euphorbia kansui S.L.Liou ex S.B.Ho have long been used to treat edema in China. However, the severe toxicity caused by Euphorbia kansui (EK) has seriously restricted its clinical application. Although EK was processed with vinegar to reduce its toxicity, the detailed mechanisms of attenuation in toxicity of EK stir-fried with vinegar (VEK) have not been well delineated. Diterpenoids are the main toxic ingredients of EK, and changes in these after processing may be the underlying mechanism of toxicity attenuation of VEK. 3-O-(2⁰E,4⁰Z-decadienoyl)-20-O-acetylingenol (3-O-EZ) is one of the diterpenoids derived from EK, and the content of 3-O-EZ was significantly reduced after processing. This study aims to explore the underlying mechanisms of toxicity reduction of VEK based on the change of 3-O-EZ after processing with vinegar. Based on the chemical structure of 3-O-EZ and the method of processing with vinegar, simulation experiments were carried out to confirm the presence of the product both in EK and VEK and to enrich the product. Then, the difference of peak area of 3-O-EZ and its hydrolysate in EK and VEK were detected by ultra-high-performance liquid chromatography (UPLC). Furthermore, the toxicity effect of 3-O-EZ and its hydrolysate, as well as the underlying mechanism, on zebrafish embryos were investigated. The findings showed that the diterpenoids (3-O-EZ) in EK can convert into less toxic ingenol in VEK after processing with vinegar; meanwhile, the content of ingenol in VEK was higher than that of EK. More interestingly, the ingenol exhibited less toxicity (acute toxicity, developmental toxicity and organic toxicity) than that of 3-O-EZ, and 3-O-EZ could increase malondialdehyde (MDA) content and reduce glutathione (GSH) content; cause embryo oxidative damage by inhibition of the succinate dehydrogenase (SDH) and superoxide dismutase (SOD) activity; and induce inflammation and apoptosis by elevation of IL-2 and IL-8 contents and activation of the caspase-3 and caspase-9 activity. Thus, this study contributes to our understanding of the mechanism of attenuation in toxicity of VEK, and provides the possibility of safe and rational use of EK in clinics.

Full text of DOI:10.3390/molecules24203806

molecules
Article
Toxicity Reduction of Euphorbia kansui Stir-Fried
with Vinegar Based on Conversion of
3-O-(20E,40Z-Decadi-enoyl)-20-O-acetylingenol
Qiao Zhang 1, Yi Zhang 1, Shi-Kang Zhou 1, Kan Wang 1, Min Zhang 1, Pei-Dong Chen 1,
Wei-Feng Yao 1, Yu-Ping Tang 2, Jian-Hua Wu 2 and Li Zhang 1,
*
1
Jiangsu Key Laboratory for High Technology Research of TCM Formulae, National and Local Collaborative
Engineering Center of Chinese Medicinal Resources Industrialization and Formulae Innovative Medicine
and Jiangsu Collaborative Innovation Center of Chinese Medicinal Resources Industrialization,
Nanjing University of Chinese Medicine, Nanjing 210023, China; zhangqiao@njucm.edu.cn (Q.Z.);
zhangyi@njucm.edu.cn (Y.Z.); zsk6428249@163.com (S.-K.Z.); wangkan86@hotmail.com (K.W.);
nzyzhm@163.com (M.Z.); chenpeidong1970@163.com (P.-D.C.); yaowf@njucm.edu.cn (W.-F.Y.)
Key Laboratory of Shaanxi Administration of Traditional Chinese Medicine for TCM Compatibility,
Shaanxi University of Chinese Medicine, Xi’an 712046, China; yupingtang@sntcm.edu.cn (Y.-P.T.);
wu0700@126.com (J.-H.W.)
2
*
Correspondence: zhangli@njucm.edu.cn; Tel.: +86-025-85811524 or +86-025-85811519
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 30 September 2019; Accepted: 20 October 2019; Published: 22 October 2019
Abstract: The dried roots of Euphorbia kansui S.L.Liou ex S.B.Ho have long been used to treat edema
in China. However, the severe toxicity caused by Euphorbia kansui (EK) has seriously restricted its
clinical application. Although EK was processed with vinegar to reduce its toxicity, the detailed
mechanisms of attenuation in toxicity of EK stir-fried with vinegar (VEK) have not been well delineated.
Diterpenoids are the main toxic ingredients of EK, and changes in these after processing may be the
underlying mechanism of toxicity attenuation of VEK. 3-O-(20E,40Z-decadienoyl)-20-O-acetylingenol
(3-O-EZ) is one of the diterpenoids derived from EK, and the content of 3-O-EZ was significantly
reduced after processing. This study aims to explore the underlying mechanisms of toxicity reduction
of VEK based on the change of 3-O-EZ after processing with vinegar. Based on the chemical structure
of 3-O-EZ and the method of processing with vinegar, simulation experiments were carried out to
confirm the presence of the product both in EK and VEK and to enrich the product. Then, the difference
of peak area of 3-O-EZ and its hydrolysate in EK and VEK were detected by ultra-high-performance
liquid chromatography (UPLC). Furthermore, the toxicity effect of 3-O-EZ and its hydrolysate, as well
as the underlying mechanism, on zebrafish embryos were investigated. The findings showed that the
diterpenoids (3-O-EZ) in EK can convert into less toxic ingenol in VEK after processing with vinegar;
meanwhile, the content of ingenol in VEK was higher than that of EK. More interestingly, the ingenol
exhibited less toxicity (acute toxicity, developmental toxicity and organic toxicity) than that of 3-O-EZ,
and 3-O-EZ could increase malondialdehyde (MDA) content and reduce glutathione (GSH) content;
cause embryo oxidative damage by inhibition of the succinate dehydrogenase (SDH) and superoxide
dismutase (SOD) activity; and induce inflammation and apoptosis by elevation of IL-2 and IL-8
contents and activation of the caspase-3 and caspase-9 activity. Thus, this study contributes to our
understanding of the mechanism of attenuation in toxicity of VEK, and provides the possibility of
safe and rational use of EK in clinics.
Keywords: Euphorbia kansui; diterpenoids; vinegar processing; zebrafish toxicity
Molecules 2019, 24, 3806; doi:10.3390/molecules24203806
www.mdpi.com/journal/molecules

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1. Introduction
Processing of traditional Chinese medicines (TCM) is a traditional technique of “PaoZhi (Chinese:
炮制)”, which has been applied in China for thousands of years. The processing of TCM has an
important influence on clinical safe applications of toxic TCM, as different changes can be effected after
processing, such as reduced toxicity and improved efficacy. Chemical compositions of some drugs can
change after processing, which leads to changes in efficacy and toxicity. For example, with Aconitum
tubers and Strychnos nux-vomica L., studies have shown that the toxicity of Aconitum tubers was reduced
after processing, which was due to that the diester-diterpenoid alkaloids (aconitine and hypaconitine)
being hydrolyzed into monoesters-diterpenoid and amines-diterpenoid alkaloids [
of Strychnos nux-vomica L. was reduced after processing, which was caused by the indole alkaloids
(strychnine and brucine) converted to less toxic nitrogen oxides [ ]. Meanwhile, the contents of
1,2], and the toxicity
3,4
aconitine, hypaconitine, strychnine and brucine all decreased after processing. Therefore, confirming
the compositional changes of the drug and comparing the differences in efficacy and toxicity before
and after being processed could provide a basis for revealing the processing mechanism of TCM.
Kansui (Chinese: 甘遂), the dried roots of Euphorbia kansui S.L.Liou ex S.B.Ho (EK), recorded in
Shennong-Bencao, is widely used in TCM for clinical treatment of edema, ascites, cancer, HIV,
intestinal obstruction and asthma [
caused by EK has restricted its clinical applications [
5
–
7
]. However, the severe hepatotoxicity and gastrointestinal toxicity
]. Previous studies showed that the structure type
8
of the compounds in EK including diterpenes, triterpenes, flavonoids, phenolic and acids. Among them,
diterpenoids (ingenane and jatrophane type) and triterpenoids are the major bioactive components
from EK, and are responsible for the toxicity effect of EK [9,10]. In contrast to the triterpenoids,
the diterpenoids of EK exhibit stronger cytotoxic activity, indicating that EK may exert systemic toxicity
effect mainly though the actions of diterpenoids. Notably, 3-O-(20E,40Z-decadienoyl)-20-O-acetylingenol
(3-O-EZ), a major diterpenoid of EK, plays a significant role in the process of toxicity effect of EK [11
Recent studies have provided compelling clues that EK was processed with vinegar before being
used in the clinic to minimize the toxicity of EK [ ]. As we know, the chemical compositions determine
,12].
5,8
the efficacy and toxicity of TCM to some extent, so the reduced toxicity of EK after processing is bound
to cause changes in composition. Research has shown that the LC50 value of ethyl acetate extracted
from VEK (6.62
content of 3-O-EZ decreased by 12.79% in ethyl acetate of VEK compared with EK, suggesting that
the change of diterpenoids is correlated with the attenuated toxicity of VEK [ ]. Moreover, numerous
studies have demonstrated that the content of diterpenoids in EK was mostly reduced after being
processed with vinegar [13 14]. Nevertheless, the detail mechanisms of attenuation in toxicity of VEK
±
1.24
µg/mL) was greater than EK (2.78
±
0.86 µg/mL) in embryos of zebrafish, and the
7
,
mediated by processing with vinegar have not been sufficiently explored as yet.
At present, it is generally known that the zebrafish is a valuable biological model for toxicity
research [15
,
16]. Compared to other animal experimental models, the zebrafish model is much less
]. In particular, zebrafish embryos have been well
time consuming and comes with a lower dosage [
7
recognized as an alternative to traditional experimental animals [17].
Based on the above reports, it would be postulated that the ester bond of diterpenoids in EK may
be broken and converted into less toxic compounds after processing, and changes in structure and
content of diterpenoids may contribute to the toxicity reduction of VEK. Thus, in the present study,
the conversion reaction of 3-O-EZ was conducted by simulating vinegar processing of EK for validating
the changes in structure diterpenoids. Meanwhile, determination of the peak areas of 3-O-EZ and its
hydrolysate in EK and VEK was conducted. Finally, acute toxicity, developmental toxicity and organic
toxicity of 3-O-EZ and its hydrolysate on zebrafish embryos were compared involved in oxidative
damage, immune response and cell apoptosis. This study could provide evidence for the material
basis and mechanism of attenuation in toxicity of VEK.

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2. Results
2.1. Identification of Hydrolysate
The positive and negative ion mass spectrum of hydrolysate are shown in Figure S1. The positive
ion mass spectrometry cracking peak of hydrolysate was provided as follows: m/z 371 [M + Na]+, 349
[M + H]+, 331 [M + H
−
H2O]+, 313 [M + H
−
2H2O]+, 295 [M + H
−
3H2O]+, 267 [M + H
−
3H2O
CO]+. The negative ion mass spectrometry cracking peak of hydrolysate was provided as follows:
m/z 347 [M H2O
H]−, 329 [M H2O]−, 301 [M CO]−. The possible process of cracking
is shown in Figure S2. The 1H-NMR and 13C-NMR data of hydrolysate are shown in Table S1 and
Figure S3. Compared to the literature [18 19], the hydrolysate was determined to be ingenol and the
−
−
−
H
−
−
H
−
−
,
conversion process between compounds 3-O-EZ and ingenol is shown in Figure 1.
Figure 1. The conversion process between compounds 3-O-EZ and ingenol.
2.2. Peak Areas Comparison of 3-O-EZ and Ingenol in EK and VEK
As shown in Figure 2, the peak area of the 3-O-EZ in EK was 113577 at 254 nm, which was higher
than that of VEK (81855), whereas the peak area of the ingenol in VEK was 33962 at 210 nm, which was
greater than that of EK (12835).
Figure 2. UPLC of the 3-O-EZ and ingenol in Euphorbia kansui (EK) and EK stir-fried with vinegar (VEK).

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2.3. Comparison of Toxicity of 3-O-EZ and Ingenol
2.3.1. Acute Toxicity
The acute toxicity curves of 3-O-EZ are shown in Figure S4. 3-O-EZ presented definite toxicity
with LC100 value of 0.990 g/mL and LC0 value of 0.166 g/mL, and the LC50 value of 3-O-EZ was
0.412 0.01 g/mL. The pre-experimental result showed that the LC100 and LC0 of the ingenol group
on zebrafish embryos was greater than 100 g/mL when compared with those in 3-O-EZ group,
µ
µ
±
µ
µ
suggesting that ingenol was less toxic to embryos than 3-O-EZ. Thus, the LC50 of ingenol was no
longer performed.
2.3.2. Developmental Toxicity
Compared to the control group, 3-O-EZ group can reduce the heart rate of zebrafish embryos with
dose-dependent relationship, while the ingenol group was not changed (Table 1). As shown in Figure 3,
compared to the control group, three dose groups of 3-O-EZ appeared pericardial edema, and the high
and low dose groups of 3-O-EZ exhibited scoliosis. However, all ingenol treatment groups showed no
obvious pericardial edema and scoliosis compared with the control group.
Table 1. The result of heart rate and SV-BA value in zebrafish embryos (mean ± SD, n = 10).
Group
Concentration (µg/mL)
Heart Rate
SV-BA
Control
0.1%(DMSO)
52.90 ± 1.52
171.09 ± 4.08
0.412
0.206
0.103
26.20 ± 1.99 **
38.80 ± 1.93 **
50.60 ± 1.83
51.60 ± 1.35
51.80 ± 1.99
52.50 ± 2.27
286.32 ± 3.04 **
263.79 ± 2.23 **
193.24 ± 2.68 *
194.48 ± 1.48
171.62 ± 3.44
174.34 ± 2.38
3-O-EZ
ingenol
0.412
0.206
0.103
* p < 0.05, ** p < 0.01 compared to control group.
Figure 3. Abnormal embryos exposed to control, 3-O-EZ and ingenol groups. The control group was
treated with the embryo medium, while the 3-O-EZ and ingenol groups were dissolved in the embryo
medium with high (H, 0.412 ug/mL), moderate (M, 0.206 ug/mL) and low (L, 0.103 ug/mL) dose groups.
Each treatment group showed varying degrees of pericardial edema (PE) and scoliosis (S).

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2.3.3. Organic Toxicity
The distance between the Sinus venosus (SV) and Bulbus arteriosus (BA) of the heart was
determined (Table 1). Compared to the control group, 3-O-EZ group can increase the distance
of SV-BA with dose-dependent changes, while the changes of three ingenol groups were not
observed. Acridine orange (AO) staining was used to estimate whether 3-O-EZ and ingenol
groups can cause apoptosis in zebrafish embryos (Figure 4). Compared to the control group,
increased fluorescence intensity and areas were observed in the embryos under 3-O-EZ treatment groups
with dose-dependence, while no significant change was observed in all ingenol treatment groups.
Figure 4. The results of AO staining in zebrafish embryos (heart, H).
The gastrointestinal motility rates of zebrafish embryos in different groups were determined
(Table 2). Compared to the control group, 3-O-EZ groups can retard the gastrointestinal motility rate
with dose-dependence, while no significant change was observed in all ingenol treatment groups.
The relative area of zebrafish gastrointestinal (GI) in each group was measured. As shown in Figure 5,
compared to the control group (defined as 100%), the mean relative GI area of zebrafish treated
with 3-O-EZ at concentrations of high, moderate and low were 62%, 87% and 95%, respectively
(Table 2), while no significant change was observed in all ingenol treatment groups (104%, 104% and
103%, respectively).
Table 2. The result of gastrointestinal motility rate and area in zebrafish embryos (mean
±
SD, n = 10).
Group
Concentration (µg/mL)
Gastrointestinal Motility Rate
Gastrointestinal Area
Control
0.1%(DMSO)
10.50 ± 0.97
97,771 ± 1073
0.412
0.206
0.103
3.90 ± 0.99 **
7.20 ± 1.03 **
9.20 ± 1.40
10.10 ± 1.10
9.80 ± 1.14
10.60 ± 1.35
61,222 ± 2012 **
86,111 ± 1962 **
93,591 ± 1411 **
101,825 ± 1835
102,635 ± 2335
101,366 ± 1618
3-O-EZ
ingenol
0.412
0.206
0.103
** p < 0.01 compared to control group.

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Figure 5. The results of calcein staining in the gastrointestinal (GI) tract of zebrafish embryos.
2.3.4. Measurement of SDH, SOD Activities, GSH and MDA Content
Compared to the control group, the activities of succinate dehydrogenase (SDH), superoxide
dismutase (SOD), and glutathione (GSH) content were significantly decreased in the high (0.412 g/mL)
and moderate (0.206 µg/mL) 3-O-EZ groups. The content of malondialdehyde (MDA) was obviously
increased in the high (0.412 g/mL) and moderate (0.206 g/mL) 3-O-EZ groups compared with the
µ
µ
µ
control group. Compared to the control group, the activities of SDH and SOD, GSH and MDA content
almost exhibited no significant changes in the different ingenol groups, although the MDA content
was increased in the moderate (0.206 µg/mL) ingenol group (Figure 6).
2.3.5. Measurement of Caspase Activity, IL-2 and IL-8 Content
The contents of Interleukin-2 (IL-2) and Interleukin-8 (IL-8) were obviously higher in all 3-O-EZ
treatment groups than in the control group, while all ingenol treatment groups showed no significant
changes compared with the control group. The activities of caspase-3 and caspase-9 were increased in
3-O-EZ treatment groups than the control group, except for the low 3-O-EZ group, while there was
no significant difference between the ingenol group and the control group in the effect of caspase-3
activity. The activity of caspase-9 was increased in the high and moderate ingenol groups compared
with the control group (Figure 7).

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Figure 6. The enzyme activities of SDH and SOD, GSH and MDA content in zebrafish embryos
(* p < 0.05, ** p < 0.01 compared to control group).
Figure 7. Caspase-3, Caspase-9 activities and IL-2, IL-8 content in zebrafish embryos. (* p < 0.05,
** p < 0.01 compared to control group).

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2.3.6. Gene Expression
The expression levels of sdha and GPx were significantly decreased at 0.412 and 0.206
µg/mL of
3-O-EZ, respectively, compared with the control group, while the ingenol groups showed no significant
changes, except in the high (0.412 g/mL) concentration ingenol group (Figure 8). The expression
levels of TNF- and IL-8 were significantly increased at 0.412 and 0.206 g/mL of 3-O-EZ treatment,
respectively, while no obvious enhancement of TNF- and IL-8 was observed in the ingenol groups.
The expression levels of the apoptosis-related genes were significantly up-related at 3-O-EZ group
(0.412 g/mL) compared with the control group, while there was no significant difference between the
µ
α
µ
α
µ
ingenol group and the control group in the levels of the apoptosis-related genes (Figure 8).
Figure 8. Expression of genes related to oxidative stress, immunity and apoptosis in zebrafish embryos
(* p < 0.05, ** p < 0.01 compared to control group).

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3. Discussion
Numerous studies confirmed that the contents of toxic components decreased after EK was
processed with vinegar. However, what happened during the conversion of these components
from VEK and the relationship between structure and toxicity were unknown. As EK had severe
hepatotoxicity, gastrointestinal toxicity, irritation, etc., it needed to be processed in clinical applications
to reduce its toxicity. According to the 2015 edition of the Chinese Pharmacopoeia and clinical practice,
EK’s processing method is to be stir-fried with vinegar. Rice vinegar is a kind of Chinese medicine
excipient and mainly contains acetic acid, lactic acid, tyrosine, etc. [5,20]. Previous studies had
manifested that the content of 3-O-EZ was reduced in VEK, so, we speculated that 3-O-EZ in EK
may convert into ingenol after processing. Thus, the conversion reaction of 3-O-EZ was conducted
for validating the change of 3-O-EZ and enriching ingenol. The present UPLC result showed that
both 3-O-EZ and ingenol were found in EK and VEK; however, the content of 3-O-EZ was reduced in
VEK, and the content of ingenol was increased in VEK, indicating that the 3-O-EZ in EK could change
into ingenol after processing with vinegar. The simulation reaction of 3-O-EZ demonstrated that the
conversion process from 3-O-EZ to ingenol may be due to the fracture of the ester bond; meanwhile,
the ingenol was obtained and enriched for toxicity evaluation on zebrafish embryos.
Zebrafish embryos are small and relatively transparent, and multiple organs can be observed
directly under the microscope. The heart is an important organ in the development of zebrafish
embryos [21], and the gastrointestinal tract plays a major role in mucosal homeostasis, barrier function
and immunity [22]. However, abnormal heart rate, pericardial edema, and gastrointestinal motility
lead to abnormality of embryonic development or even death [23
,24]. In the present study, the LC50
value of ingenol (>100 g/mL) was far greater than 3-O-EZ (0.412 0.01 µ
µ
±
g/mL), and zebrafish embryos
treated with 3-O-EZ showed bradycardia and pericardial edema, increased SV-BA value, and decreased
GI motility rate and mean relative GI area. While the above changes are not found in all ingenol
treatments, these results indicated that 3-O-EZ possessed cardiotoxicity and gastrointestinal toxicity in
zebrafish embryos, which may lead to the abnormal development of zebrafish embryos.
The activity of SDH binding to mitochondrial inner membrane represents the function of
mitochondria [23]. Conversely, mitochondrial dysfunction and decreased SDH activity can lead
to oxidative damage. Previous studies confirmed that the oxidative damage was considered as an
important mechanism of organ damage [
content were related to the antioxidant capacity of cells [25
product produced by free radical attack, and the level of change in MDA reflected the degree of
damage to cells [ 23]. In this experiment, the activity of SDH and related gene expression (sdha) were
significantly decreased at 0.412 and 0.206 g/mL of 3-O-EZ, respectively, that 3-O-EZ may lead to
damage of mitochondrial function. In addition, the activity and expression levels of the antioxidant
8
], and the activity and expression levels of SOD and GSH
,
26]. Besides, MDA was a lipid peroxidation
8,
µ
enzyme were significantly decreased at 0.412 and 0.206
to an abnormality during embryonic development.
µg/mL of 3-O-EZ, respectively, which may lead
Furthermore, there was a close relationship between inflammation and oxidative damage. IL-2 is a
cytokine with an autocrine survival and proliferation signal for T cells. IL-8 is an important chemotactic
and pro-inflammatory cytokine, which has been confirmed to participate in immune regulation and
play an important role in the regulation of inflammation [8,27,28]. In this study, the levels of IL-2, IL-8
and immune-related gene expression were markedly up-regulated in zebrafish embryos treatment
with 3-O-EZ, suggesting that the EK induced inflammatory response was mediated by 3-O-EZ.
Previous studies demonstrated that oxidative damage and inflammation can lead to apoptosis [23].
The inhibition of anti-apoptotic protein Bcl-2 and activation of caspase-3, caspase-9 and Bax can induce
apoptosis of cells, and the ratio of Bcl-2/Bax was a crucial element of apoptotic cell death [
study, the activity of caspase-3 and caspase-9 as well as immune-related gene expression were markedly
up-regulated in zebrafish embryos treatment with 0.412 g/mL 3-O-EZ. These results indicated that
8]. In this
µ
cell apoptosis was induced by 3-O-EZ in zebrafish embryos, and this may be the explanation for
EK-induced abnormal embryonic development and even death.

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Taken together, 3-O-EZ could inhibit the SDH and SOD activity, reduce the GSH content,
and increase MDA content, and ultimately lead to embryo oxidative damage. Besides, 3-O-EZ can
induce inflammation and apoptosis by increasing IL-2 and IL-8 contents and activating caspase-3 and
caspase-9 activity. Moreover, the ingenol showed no obvious toxic effect and changes of zebrafish
embryonic development. The 3-O-EZ exhibited toxic effect with a dose-dependent relationship on
zebrafish embryos, and the toxicity of ingenol was less than 3-O-EZ. These results may be the reason
for zebrafish embryo development abnormality or even death caused by 3-O-EZ.
All results mentioned above validated our previous assumption in the Introduction and may
explain why vinegar-processing can significantly reduce toxicity of EK. However, we only evaluated
the toxicity and did not evaluate the efficacy, and the structure-efficacy of 3-O-EZ and ingenol will be
further studied in an H22 mouse hepatoma ascites model.
4. Materials and Methods
4.1. Chemicals and Reagents
3-O-(20E,40Z-Decadienoyl)-20-O-acetylingenol (3-O-EZ) was extracted from EK and purified in
our laboratory (purity determined by HPLC-UV were more than 98%) [9]. Acetonitrile and formic acid
of HPLC grade were purchased from Merck Co. (Darmstadt, Germany). Acetic acid, tetrahydrofuran
and dichloromethane were of analytical grade. Ultra-pure water was purified by a Milli-Q academic
water purification system (Millipore, Billerica, MA, USA). AO was purchased from Shanghai Macklin
Biochemical Co. Ltd. (Shanghai, China). Calcein was purchased from Aladdin industrial Corporation
(Shanghai, China).
4.2. Zebrafish Embryos
Zebrafish were purchased from Nanjing YSY Biotech Company LTD (Nanjing, China). As the
previous research described [7], the embryos were collected for further analysis.
4.3. Materials
The dried root of EK was collected from Red River valley of Baoji, Shaanxi province of China, in
October 2017 and was identified by Prof. Qinan Wu (Nanjing University of Chinese Medicine, Nanjing,
210023, China). The voucher specimen (20171015) has been deposited in the Herbarium of college
of pharmacy, Nanjing University of Chinese Medicine (Nanjing, Jiangsu, China). A total of 100 g of
cleaned EK was immersed in 30 g vinegar and stir-fried at 260 ◦C until slight scorched spots appeared
to obtain VEK; then the dried EK and VEK were crushed into powder (65-mesh) [7].
4.4. Preparation and Analysis of Hydrolysate
4.4.1. Conversion Reaction
3-O-EZ was accurately weighed (24.5 mg) and put into a 10 mL ampoule. Then, the appropriate
amount of tetrahydrofuran was added to promote dissolving. Lastly, 2 mL of 3 mol/L acetic acid
solution was added and stored in electro-thermostatic blast oven (EBO) (DHG-9023A, Shanghai, China)
at 120 ◦C for 3 h. After the sample was cooled, it was extracted three times with dichloromethane and
concentrated up to dryness to obtain hydrolysate.
4.4.2. Purification and Analysis of Hydrolysate
The hydrolysate was purified by a Waters 1525 with a 2996 Diode Array Detector (DAD) detector
(Waters, Milford, CT, USA). The structure of the hydrolysate was analyzed by ultra-high-performance
liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UPLC-QTOF/MS),
the UPLC-MS analysis was performed on a Waters ACQUITY UPLC system (Waters, Milford, MA,
USA) and a Synaptt Q-TOF (Waters, Manchester, UK) coupled with an electrospray ionization (ESI)

Molecules 2019, 24, 3806
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source. The hydrolysate was obtained for enrichment, and confirmed by Nuclear Magnetic Resonance
(NMR) (Bruker, Karlsruhe, Germany).
4.5. Determination of the Peak Areas of 3-O-EZ and Hydrolysate in EK and VEK
The powder of EK and VEK (about 2 g) were weighed and put into a stoppered conical flask.
Then, 25 mL of ethyl acetate was carefully added, weighed and ultrasonicated (power 250 w, frequency
50 kHz) for 40 min. Furthermore, the loss of solvent was replenished with ethyl acetate, filtered
and concentrated up to dryness. Lastly, the residue was dissolved in methanol and transferred to
2 mL volumetric flask. Hydrolysate was prepared individually at concentrations of 0.512 mg/mL.
All solutions were stored at 4 ◦C until further analysis.
The mobile phase was composed of acetonitrile (A) and 0.1% formic acid in water (B) with a
gradient elution as follows: 0–9 min, 40–59% A; 9–10 min, 59–64% A; 10–14 min, 64–80% A; 14–22 min,
80–99% A; 22–27 min, 99% A; 27–28 min, 99–40% A; 28–30 min, 40% A. The flow rate was set at
0.4 mL/min and the volume of injection was 2 µL.
4.6. Comparison of Toxicity of 3-O-EZ and Hydrolysate
4.6.1. Acute Toxicity
The appropriate amount of 3-O-EZ and hydrolysate were weighed accurately and dissolved with
0.1% DMSO embryo medium (mixture of 15 mM NaCl, 0.5 mM KCl, 1 mM CaCl2, 1 mM MgSO4,
0.15 mM KH2PO4, 0.05 mM NH2PO4, and 0.7mM NaHCO3) [7]. Then, the concentrations of 3-O-EZ
and hydrolysate solution were prepared at 1.41 and 1.16 mg/mL, respectively. The solution was stored
at 4 ◦C until further analysis.
The six different concentration groups of 3-O-EZ (1.763, 1.058, 0.635, 0.381, 0.228, 0.137
µg/mL)
and hydrolysate (116, 92.8, 74.24, 59.39, 47.51, 30.01 g/mL) were set up. After 10 h, zebrafish embryos
µ
were seeded in a 24-well plate with ten zebrafish embryos per well. Zebrafish embryos were divided
into three groups: Control group (0.1% DMSO embryo medium) (n = 20), 3-O-EZ group (n = 20) and
hydrolysate group (n = 20). Then, the three groups were incubated in triplicate at 28 ◦C. The mortality
date of zebrafish embryos at different drug concentrations was detected under a microscope after 1, 2,
3, and 4 days, and the LC100 and LC0 of 3-O-EZ and hydrolysate were obtained.
According to the LC100 and LC0 of 3-O-EZ, six concentration groups of 3-O-EZ (0.990, 0.693, 0.485,
0.339, 0.238, 0.166 µg/mL) were set up. After 10 h incubation, zebrafish embryos were seeded in 24-well
plate with ten zebrafish embryos per well, and incubated in triplicate at 28 ◦C. The 3-O-EZ group was
conducted in the same way as mentioned above. After 96 h incubation, the mortality of zebrafish
embryos in each group was calculated.
4.6.2. Developmental Toxicity
To further evaluate developmental toxicity (Heart rate, Pericardial edema and Scoliosis) of
zebrafish embryos induced by 3-O-EZ and hydrolysate, high (0.412 µg/mL), moderate (0.206 µg/mL)
and low (0.103 µg◦ /mL) concentrations of 3-O-EZ and hydrolysate were given in triplicate for 96 h
incubation at 28 C. After 96 h incubation, zebrafish embryos were anesthetized and mounted on
double concave slides in 3% methylcellulose. Then, the images were visualized under a DMi8 inverted
microscope (Leica, Berlin, Germany), and the heart rate within 20 s were recorded.
4.6.3. Cardiotoxicity
The treatment of 3-O-EZ and hydrolysate on zebrafish embryos was the same as “Developmental
toxicity”, and SV-BA distance was measured using Image-Pro Plus 6.0 (IPP 6.0, Media Cybernetics,
Silver Spring, MD, USA) software on the acquired images.

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Meanwhile, zebrafish embryos were transferred into AO solution (4 mg/L) in darkness for 1.5 h at
room temperature [21]. Then, the embryos were gently rinsed three times with phosphate buffered
saline (PBS) (Gibco, Thermo Fisher, Waltham, MA, USA).
4.6.4. Gastrointestinal Toxicity
The embryos were kept in the embryo medium for 96 h, and high (0.412
µg/mL), moderate
(0.206 g/mL) and low (0.103 g/mL) concentrations of 3-O-EZ and hydrolysate were given in triplicate
µ
µ
for 120 h incubation at 28 ◦C. After 120 h incubation, gastrointestinal peristalsis rates within 1 min were
recorded under a microscope. And embryos were transferred into calcein solution (0.2%) in darkness
for 10 min at room temperature [29]. Then, the embryos were gently rinsed three times with embryo
medium, and gastrointestinal areas were measured using IPP 6.0 software on the acquired images.
4.6.5. SDH and SOD Activity, GSH and MDA Content Analysis
The treatment of 3-O-EZ and hydrolysate on zebrafish embryos was the same as “Developmental
toxicity”. After 96 h incubation, 50 embryos were randomly selected, and subsequently resuspended in
300 uL PBS. Then, the samples were centrifuged at 4000 rpm for 10 min at 4 ◦C (JX-FSTPRP-24, Jingxin,
Shanghai, China) and the supernatant was recovered. The activities of SDH, SOD, and the content of
MDA and GSH were measured using assay kits (Jiancheng, Nanjing, China). The levels of total protein
were determined using a bicinchoninic acid (BCA) protein assay kit (Jiancheng, Nanjing, China).
4.6.6. Caspase Activity as well as IL-2 and IL-8 Content Analysis
After 96 h incubation, 30 embryos were employed to measure the caspase-3 and caspase-9 activities.
The enzyme activities were determined using assay kits (Jiancheng, Nanjing, China). In addition,
the levels of IL-2 and IL-8 of embryos were quantified using enzyme-linked immunosorbent assay
(ELISA) kits (Jiancheng, Nanjing, China).
4.6.7. Gene Expression Analysis
Total RNA was extracted with Trizol from embryos. The transcription levels of genes related to
oxidation, apoptosis, immunity and mitochondria were measured by quantitative real-time polymerase
chain reaction (qRT-PCR). The primers are provided as Table 3. The 2−∆∆Ct method was used to analyze
the real-time PCR data.
4.7. Statistical Analysis
The LC50 was calculated by SPSS Statistics 17.0 software (International Business Machines
Corporation, Armonk, NY, USA). One-way analysis of variance (ANOVA) followed by a Dunn’s
multiple comparison test was used for comparing between control group and treated groups. It was
considered to be statistically significant when the p value was below 0.05.

Molecules 2019, 24, 3806
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Table 3. Sequences of the primer pairs used in the qRT-PCR.
Gene
Primer
References
Forward
Reverse
50-AGAGCTATGAGCTGCCTGACG-30
50-CCGCAAGATTCCATACCCA-30
β-actin
[30]
Forward
Reverse
50-TGGTATGCCGTTCAGCCGTA-30
50-GGCCAAGTCTTTGGCATTGG-30
50-AGATGTCATTCCTGCACACG-30
50-AAGGAGAAGCTTCCTCAGCC-30
50-GCTGGATCTTCAAAGTCGGGTGTA-3
50-TGTGAGTCTCAGCACACTTCCATC-30
50-GTCGCTGCATTGAAACAGAA-30
50-CTTAACCCATGGAGCAGAGG-30
50-TCACTCGTTCAGACCCTCAT-30
50-ACGCTTTCCACGCACAT-30
50-GGCTATTTCAACCAGGGTTCC-30
50-TGCGAATCACCAATGCTGT-30
50-CCGCTGCCCATCACTA-30
50-ATCCTTTCACGACCATCT-30
50-CTGAGGCAAGCCATAATCG-30
50-AGAGGACATGGGAATAGCGT-30
[23]
[23]
[30]
[23]
[30]
[30]
[23]
[30]
sdha
GPx
Forward
Reverse
Forward
Reverse
TNF-α
IL-8
Forward
Reverse
Forward
Reverse
Bcl-2
Forward
Reverse
Bax
Forward
Reverse
Caspase-3
Caspase-9
Forward
Reverse
5. Conclusions
In summary, the results of the current study revealed that the content of 3-O-EZ was significantly
reduced in VEK, while the levels of ingenol in VEK were significantly increased compared with
EK. Moreover, acute toxicity, developmental toxicity and organic toxicity of 3-O-EZ and ingenol all
demonstrated that ingenol exhibited less toxicity than 3-O-EZ. Together with our previous results on
zebrafish embryos [7], this study contributes to our understanding of the mechanism of attenuation in
toxicity of VEK, and provides the possibility of safe and rational use of EK in clinic.
Supplementary Materials: The following are available online, Figure S1: Positive and negative ion mass spectrum
of hydrolysate. A: 1st ESI MS spectrum of positive; B: 2nd ESI MS spectrum of positive; C: 1st ESI MS spectrum of
negative; D: 2nd ESI MS spectrum of negative, Figure S2: Positive and negative ion mode mass spectrometry
fragmentation process of hydrolysate. A: positive; B: negative, Figure S3: The 1H-NMR and 13C-NMR of
hydrolysate. A: 1H-NMR; B: 13C-NMR, Figure S4: The acute toxicity curves of 3-O-EZ in zebrafish embryos,
Table S1: 1H-NMR and 13C-NMR data of ingenol.
Author Contributions: Conceptualization, L.Z. and Y.-P.T.; methodology, Q.Z., M.Z., S.-K.Z. and K.W.; performing
the experiment, Q.Z., S.-K.Z. and Y.Z.; data acquisition, Q.Z. and P.-D.C.; writing—original draft preparation, Q.Z.;
writing—review and editing, P.-D.C., J.-H.W., W.-F.Y. and L.Z.
Funding: The authors gratefully acknowledge the National Natural Science Foundation of China (81673599,
81373972 and 30973940). This research was also supported by a Project Funded by the Priority Academic Program
Development of Jiangsu Higher Education Institutions (PAPD-2014), Six Talent Peaks Project in Jiangsu Province
(2016-YY-026 and 2010-YY-009), Postgraduate Research & Practice Innovation Program of Jiangsu Province
(KYCX19-1300).
Acknowledgments: This work was completed and supported in Class III Laboratory of Chemistry of Chinese
Materia Medica of State Administration of Traditional Chinese Medicine of China.
Conflicts of Interest: The authors declare no conflicts of interest.

Molecules 2019, 24, 3806
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Abbreviations
EK
Euphorbia kansui
VEK
SV
EK stir-fried with vinegar
Sinus venosus
BA
Bulbus arteriosus
GI
gastrointestinal
SDH
SOD
GSH
MDA
IL-2
Succinate Dehydrogenase
Superoxide Dismutase
Glutathione
Malondialdehyde
Interleukin-2
IL-8
Interleukin-8
EBO
DAD
PBS
qRT-PCR
ANOVA
electro-thermostatic blast oven
Diode Array Detector
Phosphate Buffered Saline
quantitative real-time polymerase chain reaction
One-way analysis of variance
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Sample Availability: Samples of the compounds.3-O-(20E,40Z-decadienoyl)-20-O-acetylingenol and ingenol are
available from the authors.
©
2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).