# The Correlation between Chemical Composition, as Determined by UPLC-TOF-MS, and Acute Toxicity ofVeratrum nigrum L. and Radix paeoniae alba

Authors: Xianxie Zhang; Yuguang Wang; Qiande Liang; Zengchun Ma; Chengrong Xiao; Hongling Tan; Yue Gao
Journal: Evidence-Based Complementary and Alternative Medicine
 (2014)

---

## Abstract

The eighteen incompatible medicaments is an important theory in traditional Chinese medicine. The theory suggests that drugs in the eighteen incompatible medicaments can be toxic when used together.Veratrum nigrum L. and Radix paeoniae alba belong to the eighteen incompatible medicaments and have been prohibited for thousands of years. This study offers preliminary insight into the mechanism and chemical constituents responsible for the incompatibility and toxicity of these two agents. Specifically, we performed toxicology studies to identify and quantify the constituent substances of the two agents. Experiments revealed that acute toxicity increases when the dose of V. nigrum L. is higher than, or equal to, RPA. UPLC-TOF-MS analysis showed that, although the volumes of V. nigrum L. were the same, the content of some veratrum alkaloids changed significantly and had a trend toward a highly positive correlation r
≥
0.8 with toxicity. This suggests that the increased toxicity of the V. nigrum L. and RPA combination was due mainly to increased content of the special veratrum alkaloids. The cytotoxicity of veratridine in SH-SY5Y cells was decreased with increasing paeoniflorin concentrations. This study provides insight into the mechanism behind the incompatibility theory of TCM.


---

## Body

## 1. Introduction

Traditional Chinese medicine (TCM) is an important part of Chinese culture and makes significant contributions to the prosperity and health of the Chinese population. TCM has become more popular worldwide because of its efficacy and curative effects. However, it is important to ensure that these treatments are safe. Confirming the compatibility of TCM is currently the major method used to ensure its safety and efficacy [1]. TCM has formed a unique incompatibility theory over thousands of years, and the typical principles of prescriptions include the eighteen incompatible medicaments. This means that these specific agents can be toxic when used in combination [2]. However, some studies have used combinations of drugs in the eighteen incompatible medicaments to treat incurable diseases [2]. Therefore, it is important to determine whether these agents are incompatible when used in combination, and the reasons behind any incompatibility.In this study, we usedVeratrum nigrum L. (V. nigrum L.) andRadix paeoniae alba (RPA), which are two agents belonging to the eighteen incompatible medicaments whose concurrent use has been prohibited for thousands of years. Although there are no cases describing the compatibility ofV. nigrum L. and RPA in modern Chinese medicine, fewer than 20 prescriptions using a combination ofV. nigrum L. and RPA have been described to treat incurable diseases such as tumors, hemorrhoids, carbuncle, and breast carbuncle [3]. Therefore, it is necessary to determine whether the use ofV. nigrum L. and RPA in combination should be prohibited, as well as the reasons for any incompatibility [4].V. nigrum L. is the dried roots and rhizomes ofVeratrum nigrum L. It has been used for medicinal purposes for thousands of years in China and was used in Europe during the Middle Ages [5] despite its well-known poisonous characteristics [4]. It is used to treat hypertension, stroke, excessive phlegm, and epilepsy. However, aqueous extracts are toxic and irritate the digestive tract mucosa, nucleus nervi vagi, and central nervous system [6, 7].RPA, the dried root ofPaeonia lactiflora Pall without bark, has been used as a medicinal herb in traditional Chinese medicine for centuries and exerts a wide range of pharmacological activities. In ancient pharmacology, RPA was used to calm liver wind, relieve pain, nourish blood, regulate menstrual functions, and suppress sweating [8]. In modern pharmacology, RPA decoctions could be used to treat rheumatoid arthritis, systemic lupus erythematosus, hepatitis, dysmenorrhea, muscle cramping and spasms, and long-standing fever [4, 9, 10].In this study, UPLC/TOF MS, multivariate statistical analysis, and typical metabolomics approach were used to identify the key chemical markers that are responsible for the increased toxicity and the combination ofV. nigrum L. and RPA. In addition, cellular and mouse acute toxicity assays were used to identify the toxic effects of this combination. The compatibility ofV. nigrum L. and RPA has not yet been validated by modern science; therefore, it is important to provide preliminary insight into the mechanism of their reported incompatibility, as well as to identify and quantify their toxic chemical constituents.


## 2. Materials and Methods

### 2.1. Chemicals and Materials

RPA from Panan County (Zhejiang, China; lot 100714) andV. nigrum L. from Huajia County (Changchun, China; lot 100701) were purchased from Anhui BBCA Tongling Chinese herbal medicine company. Formic acid (CNW Technologies GmbH) and acetonitrile (Fisher Scientific, Fair Lawn, NJ, USA) were both of chromatographic purity. Deionized water was prepared using a Millipore water purification system. Veratridine (D00114760) was purchased from Merck (Merck KGaA, Germany), and paeoniflorin was purchased from Sigma-Aldrich. The MTS cell proliferation assay kit was purchased from Promega. Roswell Park Memorial Institute 1640 (RPMI-1640) and fetal bovine serum (FBS) were purchased from Gibco BRL (Invitrogen, USA).


### 2.2. Preparation of Decoctions

Based on a previous study, theLD
50 of aqueous extracts ofV. nigrum L. and RPA after intragastric administration were 2.566 g/kg and 160 g/kg, respectively [11]. In this study, the dose ofV. nigrum L. was fixed at 2.566 g/kg, and the RPA dose varied from 0.2566 to 25.66 g/kg.The doses ofV. nigrum L. and RPA in each of 12 groups are shown in Table 1. Each group was extracted using deionized water (700 mL) for 1 h during microboiling under reflux. The extracts were filtered through three layers of gauze, and the drug extraction was then repeated. The filtrates were combined and were then concentrated to 100 mL at 60−70°C under reduced pressure. Samples were then shaken, calibrated, and stored at 4°C.Table 1
Doses ofV. nigrumL. and RPA in the 12 decoction groups.
Group
V. nigrumL. dose (g)
RPA dose (g)
Water (mL)
Final volume (mL)
Drug concentration ratio (V. nigrumL.: RPA)
A
6.415
0.6415
700
100
10 : 1
B
6.415
0.802
700
100
8 : 1
C
6.415
1.069
700
100
6 : 1
D
6.415
1.604
700
100
4 : 1
E
6.415
3.208
700
100
2 : 1
F
6.415
6.415
700
100
1 : 1
G
6.415
12.83
700
100
1 : 2
H
6.415
25.66
700
100
1 : 4
I
6.415
38.49
700
100
1 : 6
J
6.415
51.32
700
100
1 : 8
K
6.415
64.15
700
100
1 : 10
L
6.415
0
700
100
—All decoctions were centrifuged at 13,000 rpm for 10 min using a Heraeus Labofuge 400R refrigerated centrifuge (Thermo Scientific, USA). The supernatants were then filtered through a 0.22μM aqueous microporous membrane and were stored at 4°C for UPLC-TOF-MS analysis. Groups were prepared in triplicate [12, 13].


### 2.3. Animals and Experimental Design

Seven-week-old Kunming (KM) mice (18−22 g) were obtained from the Experimental Animal Center of the Academy of Military Medical Sciences with the certificate of conformity SCXK-(Army) 2007-004. Animal subjects were housed at the SPF animal center of the Academy of Military Medical Sciences (Beijing, China) according to the regulations of the animal care committee. Mice were housed at a constant temperature of25
±
1°C with 50
±
20% humidity, with a 12 h light/dark cycle and 10–15 air changes per hour. Mice were allowed free access to water and food during the experimental period.Mice were acclimatized to the facilities and environment for 3 days before the experiments. Two hundred and forty mice were randomized into 12 groups (A–L), with 10 mice per group. Food was removed from all animals 12 h before the experiments. Mice received decoctions at 0.4 mL/10 g via gavage and were observed for 14 days.


### 2.4. UPLC-MS

#### 2.4.1. Liquid Chromatography

UPLC was performed on a Waters Acquity UPLC system (Waters, Milford, MA, USA) equipped with a binary solvent delivery system, an autosampler, and a photodiode-array detection (PDA) system. Chromatography was performed using a Waters ACQUITYBEHC
18 column (100 mm × 2.1 mm, 1.7 μm) [12] The mobile phase consisted of (A) water containing 0.1% formic acid and (B) acetonitrile containing 0.1% formic acid. The eluting conditions were as follows: isocratic 2% B (0-1 min), linear gradients of 2−5% B (1-2 min), 5–20% B (2–5 min), 20–30% B (5–7 min), 30–33% B (7–10 min), 33–36% B (10–13 min), 36–40% B (13–17 min), 40–100% B (17-18 min), 100–2% B (18-19 min), and 2% B (19-20 min). The flow rate was 0.5 mL/min. The sample chamber was maintained at 4°C, with the column at 45°C. The injection volume was 5 μL [12, 14, 15].


#### 2.4.2. Mass Spectrometry

Mass spectrometry was performed using a Waters SYNAPT mass spectrometer equipped with an electrospray ionization (ESI) source. Samples were injected twice: once in positive ESI mode and once in negative ESI mode. The data acquisition range was 100–1500 Da. The lock spray reference scan frequency was 20 s, with a reference cone voltage of 30 V. The MS source temperature was set at 100°C, and the desolvation temperature was 450°C with a gas flow of 900 L/h. The lock mass compound was leucine enkephalin (200 pg/μL), with an m/z of 556.2771 in the positive ion mode and 554.2615 in negative ion mode. The capillary voltages were set to 2.9 kV for ESI+ and 3 kV for ESI−. The cone voltage was 40 kV, and the collision energies were 6 V (trap) and 4 V (transfer), with 2.00 mL/min trap gas flow [16].


### 2.5. SH-SY5Y Cell Cultures and Cell Proliferation Assay

SH-SY5Y cells (ATCC, Manassas, USA) were cultured in RPMI-1640 supplemented with 10% FBS. Cells were maintained at 37°C in an incubator with a saturated humidity atmosphere of 95% air and 5% CO2. They were cultured in a 96-well plate and treated with different drug combinations. An MTS cell proliferation assay was performed, and the OD at 490 nm was read using a VICTOR X plate reader (Perkin Elmer, USA).


### 2.6. Data Analysis

The UPLC-TOF-MS data of all samples were analyzed using MassLynx4.1 software (Waters, Manchester, UK), and principal component analysis (PCA) was used for data analysis. Pearson correlation coefficients were used to identify relationships between the study parameters and mortality. The chemical markers in each group were identified using theV. nigrum L. and RPA chemical databases [12]. For data analyses, we compared the correlation between chemical composition and the acute toxicity ofV. nigrum L. and RPA between the 12 groups. Experimental values are expressed as means ± standard deviations (SD). Statistical analyses were performed using two-tailed Student’s t-tests. A value of P
<
0.01 was considered statistically significant.


## 2.1. Chemicals and Materials

RPA from Panan County (Zhejiang, China; lot 100714) andV. nigrum L. from Huajia County (Changchun, China; lot 100701) were purchased from Anhui BBCA Tongling Chinese herbal medicine company. Formic acid (CNW Technologies GmbH) and acetonitrile (Fisher Scientific, Fair Lawn, NJ, USA) were both of chromatographic purity. Deionized water was prepared using a Millipore water purification system. Veratridine (D00114760) was purchased from Merck (Merck KGaA, Germany), and paeoniflorin was purchased from Sigma-Aldrich. The MTS cell proliferation assay kit was purchased from Promega. Roswell Park Memorial Institute 1640 (RPMI-1640) and fetal bovine serum (FBS) were purchased from Gibco BRL (Invitrogen, USA).


## 2.2. Preparation of Decoctions

Based on a previous study, theLD
50 of aqueous extracts ofV. nigrum L. and RPA after intragastric administration were 2.566 g/kg and 160 g/kg, respectively [11]. In this study, the dose ofV. nigrum L. was fixed at 2.566 g/kg, and the RPA dose varied from 0.2566 to 25.66 g/kg.The doses ofV. nigrum L. and RPA in each of 12 groups are shown in Table 1. Each group was extracted using deionized water (700 mL) for 1 h during microboiling under reflux. The extracts were filtered through three layers of gauze, and the drug extraction was then repeated. The filtrates were combined and were then concentrated to 100 mL at 60−70°C under reduced pressure. Samples were then shaken, calibrated, and stored at 4°C.Table 1
Doses ofV. nigrumL. and RPA in the 12 decoction groups.
Group
V. nigrumL. dose (g)
RPA dose (g)
Water (mL)
Final volume (mL)
Drug concentration ratio (V. nigrumL.: RPA)
A
6.415
0.6415
700
100
10 : 1
B
6.415
0.802
700
100
8 : 1
C
6.415
1.069
700
100
6 : 1
D
6.415
1.604
700
100
4 : 1
E
6.415
3.208
700
100
2 : 1
F
6.415
6.415
700
100
1 : 1
G
6.415
12.83
700
100
1 : 2
H
6.415
25.66
700
100
1 : 4
I
6.415
38.49
700
100
1 : 6
J
6.415
51.32
700
100
1 : 8
K
6.415
64.15
700
100
1 : 10
L
6.415
0
700
100
—All decoctions were centrifuged at 13,000 rpm for 10 min using a Heraeus Labofuge 400R refrigerated centrifuge (Thermo Scientific, USA). The supernatants were then filtered through a 0.22μM aqueous microporous membrane and were stored at 4°C for UPLC-TOF-MS analysis. Groups were prepared in triplicate [12, 13].


## 2.3. Animals and Experimental Design

Seven-week-old Kunming (KM) mice (18−22 g) were obtained from the Experimental Animal Center of the Academy of Military Medical Sciences with the certificate of conformity SCXK-(Army) 2007-004. Animal subjects were housed at the SPF animal center of the Academy of Military Medical Sciences (Beijing, China) according to the regulations of the animal care committee. Mice were housed at a constant temperature of25
±
1°C with 50
±
20% humidity, with a 12 h light/dark cycle and 10–15 air changes per hour. Mice were allowed free access to water and food during the experimental period.Mice were acclimatized to the facilities and environment for 3 days before the experiments. Two hundred and forty mice were randomized into 12 groups (A–L), with 10 mice per group. Food was removed from all animals 12 h before the experiments. Mice received decoctions at 0.4 mL/10 g via gavage and were observed for 14 days.


## 2.4. UPLC-MS

### 2.4.1. Liquid Chromatography

UPLC was performed on a Waters Acquity UPLC system (Waters, Milford, MA, USA) equipped with a binary solvent delivery system, an autosampler, and a photodiode-array detection (PDA) system. Chromatography was performed using a Waters ACQUITYBEHC
18 column (100 mm × 2.1 mm, 1.7 μm) [12] The mobile phase consisted of (A) water containing 0.1% formic acid and (B) acetonitrile containing 0.1% formic acid. The eluting conditions were as follows: isocratic 2% B (0-1 min), linear gradients of 2−5% B (1-2 min), 5–20% B (2–5 min), 20–30% B (5–7 min), 30–33% B (7–10 min), 33–36% B (10–13 min), 36–40% B (13–17 min), 40–100% B (17-18 min), 100–2% B (18-19 min), and 2% B (19-20 min). The flow rate was 0.5 mL/min. The sample chamber was maintained at 4°C, with the column at 45°C. The injection volume was 5 μL [12, 14, 15].


### 2.4.2. Mass Spectrometry

Mass spectrometry was performed using a Waters SYNAPT mass spectrometer equipped with an electrospray ionization (ESI) source. Samples were injected twice: once in positive ESI mode and once in negative ESI mode. The data acquisition range was 100–1500 Da. The lock spray reference scan frequency was 20 s, with a reference cone voltage of 30 V. The MS source temperature was set at 100°C, and the desolvation temperature was 450°C with a gas flow of 900 L/h. The lock mass compound was leucine enkephalin (200 pg/μL), with an m/z of 556.2771 in the positive ion mode and 554.2615 in negative ion mode. The capillary voltages were set to 2.9 kV for ESI+ and 3 kV for ESI−. The cone voltage was 40 kV, and the collision energies were 6 V (trap) and 4 V (transfer), with 2.00 mL/min trap gas flow [16].


## 2.4.1. Liquid Chromatography

UPLC was performed on a Waters Acquity UPLC system (Waters, Milford, MA, USA) equipped with a binary solvent delivery system, an autosampler, and a photodiode-array detection (PDA) system. Chromatography was performed using a Waters ACQUITYBEHC
18 column (100 mm × 2.1 mm, 1.7 μm) [12] The mobile phase consisted of (A) water containing 0.1% formic acid and (B) acetonitrile containing 0.1% formic acid. The eluting conditions were as follows: isocratic 2% B (0-1 min), linear gradients of 2−5% B (1-2 min), 5–20% B (2–5 min), 20–30% B (5–7 min), 30–33% B (7–10 min), 33–36% B (10–13 min), 36–40% B (13–17 min), 40–100% B (17-18 min), 100–2% B (18-19 min), and 2% B (19-20 min). The flow rate was 0.5 mL/min. The sample chamber was maintained at 4°C, with the column at 45°C. The injection volume was 5 μL [12, 14, 15].


## 2.4.2. Mass Spectrometry

Mass spectrometry was performed using a Waters SYNAPT mass spectrometer equipped with an electrospray ionization (ESI) source. Samples were injected twice: once in positive ESI mode and once in negative ESI mode. The data acquisition range was 100–1500 Da. The lock spray reference scan frequency was 20 s, with a reference cone voltage of 30 V. The MS source temperature was set at 100°C, and the desolvation temperature was 450°C with a gas flow of 900 L/h. The lock mass compound was leucine enkephalin (200 pg/μL), with an m/z of 556.2771 in the positive ion mode and 554.2615 in negative ion mode. The capillary voltages were set to 2.9 kV for ESI+ and 3 kV for ESI−. The cone voltage was 40 kV, and the collision energies were 6 V (trap) and 4 V (transfer), with 2.00 mL/min trap gas flow [16].


## 2.5. SH-SY5Y Cell Cultures and Cell Proliferation Assay

SH-SY5Y cells (ATCC, Manassas, USA) were cultured in RPMI-1640 supplemented with 10% FBS. Cells were maintained at 37°C in an incubator with a saturated humidity atmosphere of 95% air and 5% CO2. They were cultured in a 96-well plate and treated with different drug combinations. An MTS cell proliferation assay was performed, and the OD at 490 nm was read using a VICTOR X plate reader (Perkin Elmer, USA).


## 2.6. Data Analysis

The UPLC-TOF-MS data of all samples were analyzed using MassLynx4.1 software (Waters, Manchester, UK), and principal component analysis (PCA) was used for data analysis. Pearson correlation coefficients were used to identify relationships between the study parameters and mortality. The chemical markers in each group were identified using theV. nigrum L. and RPA chemical databases [12]. For data analyses, we compared the correlation between chemical composition and the acute toxicity ofV. nigrum L. and RPA between the 12 groups. Experimental values are expressed as means ± standard deviations (SD). Statistical analyses were performed using two-tailed Student’s t-tests. A value of P
<
0.01 was considered statistically significant.


## 3. Results and Discussion

### 3.1. Mouse Acute Toxicity

Mice in the 12 groups were administered the decoctions by intragastric administration. Death occurred 5−10 min after administration. Toxicity was manifested predominantly as trembling, convulsions, and spasms. The major organs showed no obvious lesions at necropsy by the naked eye [6, 17, 18]. Mouse mortality is shown in Table 2.Table 2
Mice mortality after the intragastric administration of different proportion decoctions ofV. nigrumL. and RPA.
Group
V. nigrumL. dose (g)
RPA dose (g)
Crude drug concentration ratio
Female mortality
Male mortality
Total mortality
A
6.415
0.6415
10 : 1
100%
90%
95%
B
6.415
0.802
8 : 1
100%
100%
100%
C
6.415
1.069
6 : 1
100%
60%
80%
D
6.415
1.604
4 : 1
100%
60%
80%
E
6.415
3.208
2 : 1
90%
70%
80%
F
6.415
6.415
1 : 1
90%
100%
95%
G
6.415
12.83
1 : 2
50%
70%
60%
H
6.415
25.66
1 : 4
60%
60%
60%
I
6.415
38.49
1 : 6
0
20%
10%
J
6.415
51.32
1 : 8
0
0
0
K
6.415
64.15
1 : 10
0
0
0
L
6.415
0
0
70%
70%
70%Group L (with aV. nigrum L. dose of the LD
50 2.566 g/kg) had a mortality of 70%. Groups A−F were treated with the same dose ofV. nigrum L., but the groups fed a lower ratio of RPA had a higher mortality than group L. Groups G−K had a higher proportion of RPA and lower mortality than group L, suggesting that toxicity was minimized. Groups B and F were the most toxic with mortalities of 100% and 95%, respectively. Overall analyses showed that mortality increased when the dose ofV. nigrum L. was less than the dose of RPA. Conversely, toxicity decreased when the proportion of RPA increased. The trends in mortality are shown in Figure 1.Figure 1
Total mortality of mice after the intragastric administration of decoctions containing different proportions ofV. nigrum L. and RPA. The groups represent the combinations shown in Table 1.Female mouse mortality decreased with increasing proportions of RPA, whereas males mortality exhibited two peaks at groups B and F, which both had 100% (Figure2).Figure 2
Mortality of male and female mice after the intragastric administration of decoctions containing different proportions ofV. nigrum L. and RPA. The groups represent the combinations shown in Table 1.


### 3.2. Identification and Quantification of the Chemical Components ofV. nigrum L. and RPA

To explain the cause of acute toxicity of the combined use ofV. nigrum L. and RPA, we compared the chemical composition of the 12 groups using a supervised orthogonal partial least squared discriminant analysis (OPLS-DA). After Pareto scaling with mean centering, the data from both the positive and negative ion modes were displayed as scores plots (Figure 3). The scores plots clearly revealed that the samples were clustered into 12 groups; replicates of same group were comparable, but the 12 groups could be distinguished easily. This suggests that the changes in chemical compositions of the 12 groups were consistent, and the experiment was reproducible.BBThe OPLS-DA score plot of 36 samples obtained using Pareto scaling with mean centering. (a) Positive ion mode. (b) Negative ion mode. The same score plots represent the same group. Both the positive and negative ion modes showed that the samples were clearly clustered into 12 groups. Replicates of the 12 groups could be distinguished easily.
(a)
(b)UPLC-TOF-MS is a rapid, specific, and sensitive method used to identify and quantify individual components. Representative BPI chromatograms of the 36 samples in the positive and negative mode ESI are shown in Figure4.Representative BPI chromatograms. (a) Positive ion mode. (b) Negative ion mode. Some of identified constituents (1–10) are labeled in the BPI chromatogram, and the numbers correspond to Tables3 and 4.
(a)
(b)The contents of some special alkaloids change significantly in different proportion decoctions. To determine which constituents contributed to the differences in toxicity observed with the 12 decoctions, statistical analyses were performed using Pearson’s correlation, and the correlation coefficient (r) described the degree of linear correlation between two variables: total mouse mortality and the chemical composition of the 12 decoctions. The r values ​​were between −1 and +1; r
>
0 indicates that two variables were positively correlated, whereas r
<
0 indicates a negative correlation. The larger the absolute value, the higher the correlation; therefore we defined r
=
0.90
–
1.00 as extremely related and r
=
0.80
–
0.89 as highly related. We extracted the relevant chemical component data when r
≥
0.8 and identified the individual components using the RPA andV. nigrum L. chemical composition databases.According to Pearson’s correlation coefficient, 131 compositions had an extremely positive relationship (r
≥
0.9) with toxicity. However, only 14 of these could be identified and all came fromV. nigrum L. In addition, 35 components were highly positively correlated (r
≥
0.8) to acute toxicity, and these constituents contributed most of the toxicity identified in the 12 decoctions. Therefore, we hypothesized that these components might play important roles in the acute toxicity of this drug combination. We identified that these compounds were all veratrum alkaloids that were detected in each sample in various ionized forms. The chemical components with r
≥
0.8 are shown in Table 3 [7, 19–24].Table 3
The chemical components that were highly positively correlated with toxicity (r
≥
0.8).
Number
r
tR (min)
Assigned identity
Molecular formula
Mean measured mass (Da)
Theoretical exact mass (Da)
Mass accuracy (ppm)
1
0.9779
12.98
3-Veratroylgermine
C36H53NO11
676.3667
675.8061
0.1805
2
0.9661
12.39
Jervine
C27H39NO3
426.3004
425.6035
2.3624
3
0.9589
12.61
Veretramine
C27H39NO2
410.3042
409.6041
2.9751
4
0.9581
13.43
Germanitrine
C39H59NO11
718.4169
717.8859
2.5015
5
0.9402
12.20
Germidine
C34H53NO10
636.3412
635.7853
0.5755
6
0.9393
13.40
Germerine
C37H59NO11
716.3999
693.8645
0.3408
7
0.9390
12.91
3-Angeloylzygadenine
C32H49NO8
576.3557
575.7334
1.2708
8
0.9311
10.35
11-Deoxojervine
C27H41NO2
412.3189
411.6199
6.4392
9
0.9240
1.57
1β,3α-Dihydroxy-5β-jervanin-12-en-11-one
C27H41NO4
444.3088
443.6187
2.4040
10
0.9237
6.96
Jervinone
C27H37NO3
424.2860
423.5876
2.0140
11
0.9182
11.25
Neogermbudine
C37H59NO12
732.3971
709.8639
4.9169
12
0.9143
4.25
Germine
C27H43NO8
510.3059
509.6322
1.5549
13
0.9131
4.18
Neogermine, Veramanine
C27H43NO5
462.3213
461.6340
1.4522
14
0.9034
11.58
Veraline B
C27H45NO3
432.3465
431.6511
2.9646
15
0.8978
11.16
Verdine
C27H41NO5
460.3056
459.6181
1.5249
16
0.8903
11.63
Stenophylline A
C36H51NO11
674.3474
673.7902
9.8655
17
0.8870
10.35
Germitrine
C39H61NO12
736.4278
735.9011
0.8288
18
0.8805
9.14
Polydatin
C20H22O8
429.0937
390.3839
3.4849
19
0.8738
10.81
Cervdine
C32H49NO9
592.3498
591.7328
2.0608
20
0.8675
12.34
3-Veratrum acyl protoveratrine
C36H51NO12
690.3482
689.7896
1.0609
21
0.8671
12.89
Zygadenitic acidδ-lactone-16-angelate
C32H47NO8
574.3408
573.7175
4.8884
22
0.8652
12.32
Maackinine
C39H59NO11
718.4171
717.8859
0.6797
23
0.8641
12.38
Rubijervine; rubivirine; etioline; epirubijervine; Isorubijervine
C27H43NO2
414.3357
413.6358
3.6827
24
0.8639
10.49
Solanidine
C27H43NO3
430.3308
429.6352
3.0494
25
0.8492
10.57
Pseudojervine
C32H49NO8
588.3525
587.7441
1.9710
26
0.8444
11.81
Neojerminalanine
C39H61NO13
752.4244
751.9005
3.0014
27
0.8379
8.43
Zygacine
C29H45NO8
536.3223
535.6695
0.0000
28
0.8358
13.20
Neoverataline B
C27H42N2O9
556.3260
538.6304
4.079
29
0.8201
12.80
3-Veratroylzygadenine
C36H51NO10
696.3187
657.7908
5.3469
30
0.8176
5.83
Zygadenine
C27H43NO7
494.3110
493.6328
1.5434
31
0.8176
0.54
γ-Aminobutyric acid
C4H9NO2
246.0978
103.1198
1.6121
32
0.8160
12.73
Veratrosine
C33H49NO7
616.3528
571.7447
6.8329
33
0.8128
13.22
Stenophylline A
C37H55NO10
674.3901
673.8333
0.5430
34
0.8119
13.34
7-Acetyl-15-methylbutyryl-3- veratroylgermine
C43H61NO13
838.3791
799.9433
1.3104
35
0.8017
12.13
Vanilloylzygadenine
C35H49NO10
644.3444
643.7643
1.51563-Veratroylgermine, jervine, veratramine, germanitrine, germidine, and germerine were the main chemical components that were associated with acute toxicity, with Pearson’s correlation coefficients of 0.9779, 0.9661, 0.9589, 0.9581, 0.9402, and 0.9393, respectively. These compounds are all veratrum alkaloids, and the changes in their content are shown in Figure5. The other 29 components that were highly positively correlated (r
≥
0.8) with acute toxicity are shown in Figure 6.Figure 5
Ion intensity trend plots for 3-veratroylgermine, jervine, veratramine, germanitrine, germidine, and germerine, which were highly positively correlated (r
≥
0.9393) with toxicity.Ion intensity trend plots for the remaining 29 chemical components that were highly positively correlated (r
≥
0.8) with toxicity.
(a)
(b)Interestingly, Pearson’s correlation coefficient identified 24 RPA components that were extremely negatively correlated (r
≤
-
0.9) with toxicity. The chemical components with r
≤
-
0.9 are shown in Table 4 [25–33].Table 4
The chemical components that were highly negatively correlated with toxicity (r
≤
-
0.9).
Number
r
tR (min)
Assigned identity
Molecular formula
Mean measured mass (Da)
Theoretical exact mass (Da)
Mass accuracy (ppm)
1
−0.9811
4.95
Paeoniflorinsulfonate
C23H28O13S
543.1194
544.5256
3.3714
2
−0.9799
12.20
Benzoyloxy paeoniflorin
C30H32O13
599.1785
600.1843
0.3046
3
−0.9793
9.77
Galloyl paeoniflorin
C30H32O15
631.1725
632.1741
0.3856
4
−0.9737
14.79
Paeonilacto-ne B
C17H18O6
317.1750
318.1103
3.9209
5
−0.9736
0.56
Paeonilacto-ne C
C10H12O4
195.0496
196.0736
5.3192
6
−0.9734
8.09
Tetragalloyglucose
C34H28O22
787.1008
788.5729
5.6607
7
−0.9728
2.30
Vanillic acid
C7H6O5
169.0161
170.1195
3.7471
8
−0.9716
13.91
Desbenzoyl paeoniflorin
C16H24O10
375.1868
376.1369
4.4505
9
−0.9708
13.52
Benzoyl paeoniflorin
C30H32O12
583.1848
584.5679
0.6258
10
−0.9684
4.15
6-O-β-D-Glucopyranosyllactinolide
C16H26O9
361.1537
362.3722
9.3435
11
−0.9670
10.37
Paeoniflorin; albiflorin R1; albiflorin; mudanpioside I
C23H28O11
519.1261
480.4618
1.5284
12
−0.9655
11.67
Gallic acid
C9H10O2
151.0750
150.1745
5.9590
13
−0.9640
4.93
Paeonol
C9H10O3
165.0545
166.1739
4.0676
14
−0.9625
11.67
Lactiflorin
C23H26O10
480.1872
462.4465
0.4449
15
−0.9609
6.67
Isomaltopaeoniflorin
C29H38O16
643.2221
642.6024
2.6569
16
−0.9589
8.52
Albiflorin
C23H27O11
957.3118
479.4539
9.3723
17
−0.9582
6.79
1′-O-Benzoylsucrose
C19H26O12
464.1757
446.4025
2.3668
18
−0.9567
10.57
Pentagalloylglucose
C41H32O26
939.1166
940.6772
6.6292
19
−0.9562
8.02
Oxypaeoniflorin
C23H28O12
514.1934
496.4612
1.8992
20
−0.9544
11.58
Ethyl gallate
C9H10O5
221.0433
198.1727
3.2445
21
−0.9478
10.11
Eugeniin
C41H30O26
937.1010
938.6613
6.7086
22
−0.9395
5.85
Paeonilactinone
C9H14O2
153.0904
154.2063
7.5750
23
−0.9352
1.13
Lactinolide
C10H16O4
201.1111
200.2316
7.8907
24
−0.9173
13.73
Palbinone
C22H30O4
357.2047
358.4712
5.2115The concentrations of these components increased with increasing amounts of RPA. Paeoniflorin sulfonate, benzoyloxy paeoniflorin, galloyl paeoniflorin, paeonilacto-neB, paeonilacto-neC, and tetragalloyglucose were the major RPA components that were identified, with Pearson’s correlation coefficients of −0.9811, −0.9799, −0.9793, −0.9737, −0.9736, and −0.9734, respectively. The changes in content of the 24 components that were highly negatively correlated (r
≤
-
0.9) with acute toxicity are shown in Figure 7.Ion intensity trend plots for the chemical components that were highly negatively correlated with toxicity (r
≥
0.9).
(a)
(b)


### 3.3. Cell Proliferation Assay

To further validate the toxicity of the combination ofV. nigrum L. and RPA on neurons, we used veratridine and paeoniflorin, the major pharmacologically active components ofV. nigrum L. and peony forin vitro experiments. The effect of these two chemical components on the viability of SH-SY5Y cells was tested using MTS assays. As shown in Figures 8(a) and 8(b), veratridine had an LD50 of 450, whereas paeoniflorin exerted no significant toxicity at doses of 0–2000 μM. Therefore, the dose of veratridine was fixed at 400 μM, and the RPA dose was varied from 50 to 2000 μM. SH-SY5Y cell cytotoxicity was increased when the concentration of paeoniflorin was decreased in the combination (Figure 8(c)). Therefore, paeoniflorin could offset the cytotoxicity caused by veratridine in SH-SY5Y cells [25].SH-SY5Y cell viability: SH-SY5Y cells were cultured with (a) veratridine (25–800μM), (b) paeoniflorin (50−2000 μM), and (c) 400 μM veratridine in combination with 50−2000 μM paeoniflorin for 24 h. Cell viability was then assessed using MTS assays. Data are presented as mean
±
S
.
D. Means of three independent experiments, Asterisks indicate a statistically significant difference compared with untreated cells. P
*
<
0.05; P
*
*
<
0.01; P
*
*
*
<
0.001 versus controL.
(a)
(b)
(c)


## 3.1. Mouse Acute Toxicity

Mice in the 12 groups were administered the decoctions by intragastric administration. Death occurred 5−10 min after administration. Toxicity was manifested predominantly as trembling, convulsions, and spasms. The major organs showed no obvious lesions at necropsy by the naked eye [6, 17, 18]. Mouse mortality is shown in Table 2.Table 2
Mice mortality after the intragastric administration of different proportion decoctions ofV. nigrumL. and RPA.
Group
V. nigrumL. dose (g)
RPA dose (g)
Crude drug concentration ratio
Female mortality
Male mortality
Total mortality
A
6.415
0.6415
10 : 1
100%
90%
95%
B
6.415
0.802
8 : 1
100%
100%
100%
C
6.415
1.069
6 : 1
100%
60%
80%
D
6.415
1.604
4 : 1
100%
60%
80%
E
6.415
3.208
2 : 1
90%
70%
80%
F
6.415
6.415
1 : 1
90%
100%
95%
G
6.415
12.83
1 : 2
50%
70%
60%
H
6.415
25.66
1 : 4
60%
60%
60%
I
6.415
38.49
1 : 6
0
20%
10%
J
6.415
51.32
1 : 8
0
0
0
K
6.415
64.15
1 : 10
0
0
0
L
6.415
0
0
70%
70%
70%Group L (with aV. nigrum L. dose of the LD
50 2.566 g/kg) had a mortality of 70%. Groups A−F were treated with the same dose ofV. nigrum L., but the groups fed a lower ratio of RPA had a higher mortality than group L. Groups G−K had a higher proportion of RPA and lower mortality than group L, suggesting that toxicity was minimized. Groups B and F were the most toxic with mortalities of 100% and 95%, respectively. Overall analyses showed that mortality increased when the dose ofV. nigrum L. was less than the dose of RPA. Conversely, toxicity decreased when the proportion of RPA increased. The trends in mortality are shown in Figure 1.Figure 1
Total mortality of mice after the intragastric administration of decoctions containing different proportions ofV. nigrum L. and RPA. The groups represent the combinations shown in Table 1.Female mouse mortality decreased with increasing proportions of RPA, whereas males mortality exhibited two peaks at groups B and F, which both had 100% (Figure2).Figure 2
Mortality of male and female mice after the intragastric administration of decoctions containing different proportions ofV. nigrum L. and RPA. The groups represent the combinations shown in Table 1.


## 3.2. Identification and Quantification of the Chemical Components ofV. nigrum L. and RPA

To explain the cause of acute toxicity of the combined use ofV. nigrum L. and RPA, we compared the chemical composition of the 12 groups using a supervised orthogonal partial least squared discriminant analysis (OPLS-DA). After Pareto scaling with mean centering, the data from both the positive and negative ion modes were displayed as scores plots (Figure 3). The scores plots clearly revealed that the samples were clustered into 12 groups; replicates of same group were comparable, but the 12 groups could be distinguished easily. This suggests that the changes in chemical compositions of the 12 groups were consistent, and the experiment was reproducible.BBThe OPLS-DA score plot of 36 samples obtained using Pareto scaling with mean centering. (a) Positive ion mode. (b) Negative ion mode. The same score plots represent the same group. Both the positive and negative ion modes showed that the samples were clearly clustered into 12 groups. Replicates of the 12 groups could be distinguished easily.
(a)
(b)UPLC-TOF-MS is a rapid, specific, and sensitive method used to identify and quantify individual components. Representative BPI chromatograms of the 36 samples in the positive and negative mode ESI are shown in Figure4.Representative BPI chromatograms. (a) Positive ion mode. (b) Negative ion mode. Some of identified constituents (1–10) are labeled in the BPI chromatogram, and the numbers correspond to Tables3 and 4.
(a)
(b)The contents of some special alkaloids change significantly in different proportion decoctions. To determine which constituents contributed to the differences in toxicity observed with the 12 decoctions, statistical analyses were performed using Pearson’s correlation, and the correlation coefficient (r) described the degree of linear correlation between two variables: total mouse mortality and the chemical composition of the 12 decoctions. The r values ​​were between −1 and +1; r
>
0 indicates that two variables were positively correlated, whereas r
<
0 indicates a negative correlation. The larger the absolute value, the higher the correlation; therefore we defined r
=
0.90
–
1.00 as extremely related and r
=
0.80
–
0.89 as highly related. We extracted the relevant chemical component data when r
≥
0.8 and identified the individual components using the RPA andV. nigrum L. chemical composition databases.According to Pearson’s correlation coefficient, 131 compositions had an extremely positive relationship (r
≥
0.9) with toxicity. However, only 14 of these could be identified and all came fromV. nigrum L. In addition, 35 components were highly positively correlated (r
≥
0.8) to acute toxicity, and these constituents contributed most of the toxicity identified in the 12 decoctions. Therefore, we hypothesized that these components might play important roles in the acute toxicity of this drug combination. We identified that these compounds were all veratrum alkaloids that were detected in each sample in various ionized forms. The chemical components with r
≥
0.8 are shown in Table 3 [7, 19–24].Table 3
The chemical components that were highly positively correlated with toxicity (r
≥
0.8).
Number
r
tR (min)
Assigned identity
Molecular formula
Mean measured mass (Da)
Theoretical exact mass (Da)
Mass accuracy (ppm)
1
0.9779
12.98
3-Veratroylgermine
C36H53NO11
676.3667
675.8061
0.1805
2
0.9661
12.39
Jervine
C27H39NO3
426.3004
425.6035
2.3624
3
0.9589
12.61
Veretramine
C27H39NO2
410.3042
409.6041
2.9751
4
0.9581
13.43
Germanitrine
C39H59NO11
718.4169
717.8859
2.5015
5
0.9402
12.20
Germidine
C34H53NO10
636.3412
635.7853
0.5755
6
0.9393
13.40
Germerine
C37H59NO11
716.3999
693.8645
0.3408
7
0.9390
12.91
3-Angeloylzygadenine
C32H49NO8
576.3557
575.7334
1.2708
8
0.9311
10.35
11-Deoxojervine
C27H41NO2
412.3189
411.6199
6.4392
9
0.9240
1.57
1β,3α-Dihydroxy-5β-jervanin-12-en-11-one
C27H41NO4
444.3088
443.6187
2.4040
10
0.9237
6.96
Jervinone
C27H37NO3
424.2860
423.5876
2.0140
11
0.9182
11.25
Neogermbudine
C37H59NO12
732.3971
709.8639
4.9169
12
0.9143
4.25
Germine
C27H43NO8
510.3059
509.6322
1.5549
13
0.9131
4.18
Neogermine, Veramanine
C27H43NO5
462.3213
461.6340
1.4522
14
0.9034
11.58
Veraline B
C27H45NO3
432.3465
431.6511
2.9646
15
0.8978
11.16
Verdine
C27H41NO5
460.3056
459.6181
1.5249
16
0.8903
11.63
Stenophylline A
C36H51NO11
674.3474
673.7902
9.8655
17
0.8870
10.35
Germitrine
C39H61NO12
736.4278
735.9011
0.8288
18
0.8805
9.14
Polydatin
C20H22O8
429.0937
390.3839
3.4849
19
0.8738
10.81
Cervdine
C32H49NO9
592.3498
591.7328
2.0608
20
0.8675
12.34
3-Veratrum acyl protoveratrine
C36H51NO12
690.3482
689.7896
1.0609
21
0.8671
12.89
Zygadenitic acidδ-lactone-16-angelate
C32H47NO8
574.3408
573.7175
4.8884
22
0.8652
12.32
Maackinine
C39H59NO11
718.4171
717.8859
0.6797
23
0.8641
12.38
Rubijervine; rubivirine; etioline; epirubijervine; Isorubijervine
C27H43NO2
414.3357
413.6358
3.6827
24
0.8639
10.49
Solanidine
C27H43NO3
430.3308
429.6352
3.0494
25
0.8492
10.57
Pseudojervine
C32H49NO8
588.3525
587.7441
1.9710
26
0.8444
11.81
Neojerminalanine
C39H61NO13
752.4244
751.9005
3.0014
27
0.8379
8.43
Zygacine
C29H45NO8
536.3223
535.6695
0.0000
28
0.8358
13.20
Neoverataline B
C27H42N2O9
556.3260
538.6304
4.079
29
0.8201
12.80
3-Veratroylzygadenine
C36H51NO10
696.3187
657.7908
5.3469
30
0.8176
5.83
Zygadenine
C27H43NO7
494.3110
493.6328
1.5434
31
0.8176
0.54
γ-Aminobutyric acid
C4H9NO2
246.0978
103.1198
1.6121
32
0.8160
12.73
Veratrosine
C33H49NO7
616.3528
571.7447
6.8329
33
0.8128
13.22
Stenophylline A
C37H55NO10
674.3901
673.8333
0.5430
34
0.8119
13.34
7-Acetyl-15-methylbutyryl-3- veratroylgermine
C43H61NO13
838.3791
799.9433
1.3104
35
0.8017
12.13
Vanilloylzygadenine
C35H49NO10
644.3444
643.7643
1.51563-Veratroylgermine, jervine, veratramine, germanitrine, germidine, and germerine were the main chemical components that were associated with acute toxicity, with Pearson’s correlation coefficients of 0.9779, 0.9661, 0.9589, 0.9581, 0.9402, and 0.9393, respectively. These compounds are all veratrum alkaloids, and the changes in their content are shown in Figure5. The other 29 components that were highly positively correlated (r
≥
0.8) with acute toxicity are shown in Figure 6.Figure 5
Ion intensity trend plots for 3-veratroylgermine, jervine, veratramine, germanitrine, germidine, and germerine, which were highly positively correlated (r
≥
0.9393) with toxicity.Ion intensity trend plots for the remaining 29 chemical components that were highly positively correlated (r
≥
0.8) with toxicity.
(a)
(b)Interestingly, Pearson’s correlation coefficient identified 24 RPA components that were extremely negatively correlated (r
≤
-
0.9) with toxicity. The chemical components with r
≤
-
0.9 are shown in Table 4 [25–33].Table 4
The chemical components that were highly negatively correlated with toxicity (r
≤
-
0.9).
Number
r
tR (min)
Assigned identity
Molecular formula
Mean measured mass (Da)
Theoretical exact mass (Da)
Mass accuracy (ppm)
1
−0.9811
4.95
Paeoniflorinsulfonate
C23H28O13S
543.1194
544.5256
3.3714
2
−0.9799
12.20
Benzoyloxy paeoniflorin
C30H32O13
599.1785
600.1843
0.3046
3
−0.9793
9.77
Galloyl paeoniflorin
C30H32O15
631.1725
632.1741
0.3856
4
−0.9737
14.79
Paeonilacto-ne B
C17H18O6
317.1750
318.1103
3.9209
5
−0.9736
0.56
Paeonilacto-ne C
C10H12O4
195.0496
196.0736
5.3192
6
−0.9734
8.09
Tetragalloyglucose
C34H28O22
787.1008
788.5729
5.6607
7
−0.9728
2.30
Vanillic acid
C7H6O5
169.0161
170.1195
3.7471
8
−0.9716
13.91
Desbenzoyl paeoniflorin
C16H24O10
375.1868
376.1369
4.4505
9
−0.9708
13.52
Benzoyl paeoniflorin
C30H32O12
583.1848
584.5679
0.6258
10
−0.9684
4.15
6-O-β-D-Glucopyranosyllactinolide
C16H26O9
361.1537
362.3722
9.3435
11
−0.9670
10.37
Paeoniflorin; albiflorin R1; albiflorin; mudanpioside I
C23H28O11
519.1261
480.4618
1.5284
12
−0.9655
11.67
Gallic acid
C9H10O2
151.0750
150.1745
5.9590
13
−0.9640
4.93
Paeonol
C9H10O3
165.0545
166.1739
4.0676
14
−0.9625
11.67
Lactiflorin
C23H26O10
480.1872
462.4465
0.4449
15
−0.9609
6.67
Isomaltopaeoniflorin
C29H38O16
643.2221
642.6024
2.6569
16
−0.9589
8.52
Albiflorin
C23H27O11
957.3118
479.4539
9.3723
17
−0.9582
6.79
1′-O-Benzoylsucrose
C19H26O12
464.1757
446.4025
2.3668
18
−0.9567
10.57
Pentagalloylglucose
C41H32O26
939.1166
940.6772
6.6292
19
−0.9562
8.02
Oxypaeoniflorin
C23H28O12
514.1934
496.4612
1.8992
20
−0.9544
11.58
Ethyl gallate
C9H10O5
221.0433
198.1727
3.2445
21
−0.9478
10.11
Eugeniin
C41H30O26
937.1010
938.6613
6.7086
22
−0.9395
5.85
Paeonilactinone
C9H14O2
153.0904
154.2063
7.5750
23
−0.9352
1.13
Lactinolide
C10H16O4
201.1111
200.2316
7.8907
24
−0.9173
13.73
Palbinone
C22H30O4
357.2047
358.4712
5.2115The concentrations of these components increased with increasing amounts of RPA. Paeoniflorin sulfonate, benzoyloxy paeoniflorin, galloyl paeoniflorin, paeonilacto-neB, paeonilacto-neC, and tetragalloyglucose were the major RPA components that were identified, with Pearson’s correlation coefficients of −0.9811, −0.9799, −0.9793, −0.9737, −0.9736, and −0.9734, respectively. The changes in content of the 24 components that were highly negatively correlated (r
≤
-
0.9) with acute toxicity are shown in Figure 7.Ion intensity trend plots for the chemical components that were highly negatively correlated with toxicity (r
≥
0.9).
(a)
(b)


## 3.3. Cell Proliferation Assay

To further validate the toxicity of the combination ofV. nigrum L. and RPA on neurons, we used veratridine and paeoniflorin, the major pharmacologically active components ofV. nigrum L. and peony forin vitro experiments. The effect of these two chemical components on the viability of SH-SY5Y cells was tested using MTS assays. As shown in Figures 8(a) and 8(b), veratridine had an LD50 of 450, whereas paeoniflorin exerted no significant toxicity at doses of 0–2000 μM. Therefore, the dose of veratridine was fixed at 400 μM, and the RPA dose was varied from 50 to 2000 μM. SH-SY5Y cell cytotoxicity was increased when the concentration of paeoniflorin was decreased in the combination (Figure 8(c)). Therefore, paeoniflorin could offset the cytotoxicity caused by veratridine in SH-SY5Y cells [25].SH-SY5Y cell viability: SH-SY5Y cells were cultured with (a) veratridine (25–800μM), (b) paeoniflorin (50−2000 μM), and (c) 400 μM veratridine in combination with 50−2000 μM paeoniflorin for 24 h. Cell viability was then assessed using MTS assays. Data are presented as mean
±
S
.
D. Means of three independent experiments, Asterisks indicate a statistically significant difference compared with untreated cells. P
*
<
0.05; P
*
*
<
0.01; P
*
*
*
<
0.001 versus controL.
(a)
(b)
(c)


## 4. Discussion

The eighteen incompatible medicaments (Shi Ba Fan) is a well-known traditional Chinese medicine (TCM) theory. It basically listed eighteen pairs of TCM herbal medicine that are not to be used in combination as they may cause fatal consequences. This principal has been used as guidance for general TCM practices for hundreds of years with little chemical comprehension. In this work, for the first time, we used the UPLC-TOF MS coupled with multivariate statistical analysis in an effort to gain scientific understanding for the causes of the incompatibility.We previously reported that theLD
50 of aqueous extracts ofV. nigrum L. and RPA were 2.566 g/kg and 160 g/kg, respectively, after intragastric administration [11].V. nigrum L. extract was severely toxic, whereas RPA had minimal toxicity. Although the amount ofV. nigrum L. in each group was unchanged, the mortality changed significantly with altered ratios ofV. nigrum L. to RPA. When the doses ofV. nigrum L. were equal to or higher than RPA, particularly ratios of 8 : 1 and 1 : 1, mouse mortality increased significantly. Therefore, components ofV. nigrum L. were the major causative factors of toxicity. In addition, RPA modulated the toxicity ofV. nigrum L. Therefore, clinical applications should be monitored carefully.Mice died 5−10 min after drug administration. Toxicity manifested predominantly as trembling, convulsions, and spasms, but the major organs showed no obvious lesions at necropsy by the naked eye. Based on the toxic reactions and the time of death in the current study and according to the guidelines for Chinese natural medicine acute toxicity testing, we speculate that the toxicity was caused predominantly by suppression of the central nervous system and thatV. nigrum L. was the major cause of death.V. nigrum L. water extracts exert toxic effects on the digestive tract mucosa, nucleus nervi vagi, and central nervous system [6, 17, 18]. Pharmacological studies of veratrum alkaloids have been performed previously and reported that these alkaloids stimulate the central nervous system and inhibit the brain. This results in spasms, convulsions, coma, drowsiness, and other symptoms of consciousness in animals, which is consistent with the symptoms of the mice in the current study [6, 17].Veratrum alkaloid is the toxic component ofV. nigrum L. We previously investigated the differently expressed genes in SH-SY5Y cells treated with veratridine using cDNA microarrays [34]. Data revealed that veratridine treatment altered the expression of MMP, caused Ca
2
+ concentration overload, increased reactive oxygen species production, enhanced LDH release, damaged the cell membrane, reduced SH-SY5Y cell viability, and induced apoptosis. Under oxidizing conditions, the MAPK signaling pathway is activated, which increases apoptosis. This study illustrates the neurotoxicity of the combination ofV. nigrum L. and reveals the molecular mechanism behind the neurotoxic effects ofV. nigrum L. (data not shown).The amount ofV. nigrum L. in each group was the same; the dose of RPA is in ascending order, and the chemical components of RPA are also increased, but the mortality of each group changed significantly with the increase of RPA; so we conclude that higher amount of RPA can have protection effect, which can offset the toxicity of combination ofV. nigrum L. and RPA. And pharmacological studies have demonstrated that RPA exerts a wide range of pharmacological activities, including analgesic, sedative, anticonvulsant, and antispasmodic effects on the central nervous system. RPA could also inhibit writhing reactions and antagonize pentylenetetrazol-induced convulsions. The most important active ingredient in RPA is paeoniflorin, which has significant neuroprotective effects [26–34].In this study, mouse acute toxicity experiments revealed that the mortality of mice changed significantly when the dose ofV. nigrum L. was higher than or equal to that of RPA, particularly at ratios ofV. nigrum L.: RPA of 8 : 1 and 1 : 1. UPLC-Q-TOF/MS with automated data analysis used to identify and quantify the specific chemical components. According to Pearson’s correlation coefficient, most of the chemical components that were positively correlated with toxicity were fromV. nigrum L. We conclude that the major veratrum alkaloids ofV. nigrum L. caused acute toxicity and death in mice. When the doses ofV. nigrum L. were higher than or equal to RPA, the main chemical components ofV. nigrum L. and the toxicity of the decoctions were increased. This suggests thatV. nigrum L. and RPA have opposing roles. Additional experiments revealed that the cytotoxicity of veratridine in SH-SY5Y cells was decreased with increasing concentrations of paeoniflorin. Therefore, the clinical use of these agents should be considered carefully. This study also provides information to help improve the incompatibility theory of TCM and introduces novel ideas for further studies on the development and application of TCM.When preparingV. nigrum L. and RPA decoctions, a lower amount of RPA contributes to the dissolution of veratrum alkaloids. Conversely, higher amounts of RPA can exert neuroprotective effects, which can offset the toxicity caused by the combination ofV. nigrum L. and RPA. Under these conditions, dissolution is the most important factor forV. nigrum L. toxicity. The results of cell-based experiments also demonstrated that paeoniflorin could offset the neurotoxic effects of veratridine when treated in combination, which excluded the factor of dissolution in preparation.



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*Source: 892797-2014-06-12.xml*