Post on 26-Jun-2020
Comparison of the toxicities of raw and
processed Pinelliae Rhizoma in rats
PhD candidate: Su Tao
Hong Kong Baptist University
2015.10.27
The dried tuber of Pinellia ternata, first recorded in Sheng Nong’s herbal classic (a book
published 2,000 years ago).
Pinellia ternata Pinelliae Rhizoma (BX)
Pinelliae Rhizoma (Banxia, BX)-1
http://www.kmzyw.com.cn/pages/channel_906/1140731/906.1406769866000.4354.shtml
Pinelliae Rhizoma (Banxia, BX)-2
Distribution
>2000 pharmaceutical companies using BX as the raw material in the development of new
drugs or CM for the Chinese market;
In 2014, the output of BX in Mainland China is more than one billion RMB;
The demand reaches 4500-5000 tons/year for the pharmaceutical companies and various
medical units in China;
In Japan, BX is not only used as drugs (syrup, granule, pill, tablet), but also can be prepared
as nutrition, such as porridge, beverage.
Pinelliae Rhizoma (Banxia, BX)-3
Great demand of BX:
irritant,
reproductive and
organ toxicities
Chemical study: alkaloids, organic acids, proteins, etc.
Pharmacological study: antitussive, expectorant, antiemetic, antitumor,
antibacterial, anti-inflammatory, antioxidant properties, etc.
Toxic
Processing can reduce the toxicity
Pinelliae Rhizoma (Banxia, BX)-4
Processing methods of BX
Processing is a traditional pharmaceutical technology.
The main aims of processing: efficacy ↑, toxicity↓.
Many traditional ways for processing BX: processing with rice wine, vinegar, wheat
bran, alumen, licorice, ginger juice, etc.
Chinese Pharmacopoeia : Jiangbanxia (JBX) --ginger juice, alumen
(2010 edition) Qingbanxia--alumen
Fabanxia--quick lime, licorice
Preparation of the JBX sample
Comprehensive comparison of the chemical profiles of raw BX and JBX is lacking.
Alkaloids: Raw BX > JBX (Acid dye colorimetry)
Proteins: Raw BX > JBX (Coomassie brilliant blue staining)
Organic acids: Raw BX < JBX (Potentiometric titration)
Reducing sugar: Raw BX < JBX (Dinitrosalicylic acid colorimetric method)
Processing alters different constituents:
Chemical studies of raw BX and JBX
Irritant substances
?
Homogentisic acid
Glucoside of protocatechualdehyde
Raphides of calcium oxalate
Acute toxicity, LD50: Raw BX: i.p. 0.325 g/kg in mice; i.g. 42.7 g/kg in mice;
JBX: i.p. 13.35 g/kg in mice; i.g. cannot be detected.
Irritant toxicity (debatable)
Chemical basis
Molecular mechanisms
Toxicity in different body parts
Toxicological
Data
Not fully
available
Toxicological studies of raw BX and JBX
Metabolomics
Tissue
serum/urine
Bio-
markers
PL
S-D
A
PC
A
IPA
It has advantages to solve the complex issue,
in particular, the herb-induced toxicity.
1) Compare the toxicities of raw BX and JBX in rats;
-----Try to decipher the mechanisms of BX-induced toxicity and the
detoxifying effect of processing.
2) Compare the chemical profiles of raw BX and JBX;
-----Try to explore the chemical basis behind the reduced toxicity caused
by processing.
Aims of this study
Toxicological
basis
Cheimcal
basis
Methods and Results
Comparison of the toxicities of BX and JBX in rats
Mute Diarrhea
Raw √ √
Processed × ×
1) Smaller body weight gain
2) Mute
3) Diarrhea
Raw BX:
Clinical symptoms of raw BX- and JBX-treated rats
Control
JBX
Raw BX
Raw BX: CK↑, CK-MB ↑, LDH ↑ Markedly increased cardiac enzymes are important indicators of myocardial injury
and myocardial infarction.
Processing reduced the cardiotoxicity of raw BX
--- biochemical assays
*p<0.05, **p<0.01 vs. Control; &p<0.05, &&p<0.01 vs. Raw BX
JBX
Processing reduced the heart toxicity of raw BX
---histopathological assays
Processing altered the serum metabolic profiles
Total ion chromatograms (TIC)
Control
Raw BX
JBX
Time (min)
Top 200 significant ions were selected for metabolite identification;
Total of 34 metabolites were identified from the serum samples;
10 metabolomic metabolites were found to be most significant.
Control
JBX
Raw BX
Processing altered the serum metabolites
---PLS-DA score plot
Examination of the metabolic changes:
PLS-DA score plot readily divided into three clusters
On the basis of the metabolic changes as revealed by TIC→adopted the pattern
recognition method (partial least squares discriminant analysis, PLS-DA).
Significantly altered metabolites
Molecular network in serum of BX-treated rats
1. Amino acid metabolism;
2. Lipid metabolism;
3. Small molecule biochemistry;
4. Cell-to-cell signaling and interaction;
5. Vitamin and mineral metabolism.
Five top canonical pathways:
1. Proline degradation
2. Uracil degradation II
3. Serotonin and melatonin biosynthesis
4. Lysine degradation II
5. Leucine degradation I.
To further understand the correlation among the candidate biomarkers, bioinformatics
analyses were performed using the ingenuity pathway analysis (IPA), leading to the
identification of biological association networks.
Molecular network in serum of JBX-treated rats
1. Amino acid metabolism;
2. Post-translational modification;
3. Small molecule biochemistry;
4. Lipid metabolism;
5. Free radical scavenging.
Five top canonical pathways:
1. Tyrosine biosynthesis IV;
2. Proline degradation ;
3. Uracil degradation II ;
4. Tryptophan degradation X;
5. Lysine degradation II.
Different metabolites and corresponding pathways in BX-
and JBX-treated rats
Leucine↑ and 5-HT↑ maybe associated with the toxicity;
Tryptophan↑, PAPB↑ and Tyrosine↑ maybe associated with detoxifying effect of processing
UPLC/Q-TOF-MS analysis
To explore the chemical basis behind the reduced toxicity caused by processing, we
compared the chemical profiles of raw BX and JBX using a rapid and highly sensitive
UPLC/Q-TOF-MS method developed by us.
Agilent 1200 system;
Column: ACQUITY UPLC T3 C18 ;
Temperature: 35 ℃;
Mobile phase: A (0.1% FA in water);
B (0.1% FA in ACN);
Flow rate: 0.35 ml/min;
Injection vol.:5 μl.
Conditions:
Agilent 6540 Q-TOF mass
Mass range: m/z 100-1700;
Gas temperature: 300 ℃;
drying gas (N2) flow rate: 8 L/min;
Sheath gas temperature: 350 ℃;
Sheath gas flow: 8 L/min;
Capillary voltage: 4500 V;
Fragmentor: 175 V;
Skimmer voltage: 65 V;
OctopoleRFPeak: 600 V.
Total: 8 peaks ↓, 5 peaks ↑, 5 peaks only detected in JBX;
13 compounds tentatively identified;5 peaks: unidentified;
6 peaks confirmed by reference compounds;
Processing altered the chemical profile of BX
Compounds identified from raw Banxia and Jiangbanxia extracts
Peak
no. tR (min) Assigned identity Molecular
Mean
measured
mass(Da)
Mass
accurac
y (ppm)
Theoretica
l exact
mass(Da)
Quasi-
molecular ion
Change
trend after
processing
1 0.931 Citrulline C6H13N3O3 177.1191 0.76 176.1035 [M+H]+ ↓
2a 1.423 Tyrosine C9H11NO3 183.0809 2.12 182.0817 [M+H]+ ↓
3a 1.873 Succinic acid C4H6O4 120.0331 7.52 119.0344 [M+H]+ ↓
4a 2.324 Aminobutyric acid C4 H9NO2 229.1161 1.04 103.1164 [2M+Na]+ ↓
5a 3.266
5-
Hydroxymethylfur
fural
C6H6O3 128.0386 4.70 127.0395 [M+H]+ ↑
6 3.617 L-Valyl-L-valine
anhydride C10H18N2O2 245.1388 3.42 198.1124 [M+H+2Na]+ ↓
7 4.501 Trigonelline C7H7NO2 275.1185 3.36 137.1401 [2M+H]+ ↑
8a 11.974 shogaol C17 H24 O3 278.1820 2.54 277.1804 [M+H]+ ↑
9 12.151 S-Gingerol C17H26O4 362.3061 1.21 294.1831 [M+HCOONa]+ ↑
10 12.976
N,2-dimethyl-3-
hydroxy-6-(9-
phenylnonyl)
piperidine
C22H37NO 332.2999 3.21 331.2875 [M+H]+ ↑
11a 14.036 6-gingerol C17H26O4 317.1757 9.64 294.1831 [M+Na]+ ↑
12 15.427 Monpalmitin C19H38O4 354.2687 8.38 353.2668 [M+H]+ ↓
13 17.279 Paracoumaryl
alcohol C9H10O2 301.1432 -1.87 150.1745 [2M+H]+ ↑
Compounds identified from raw BX and JBX extracts
Summary
1. Processing reduced the toxicity of raw BX (Body weight gain, mute, heart damage).
2. The mechanisms of BX-induced toxicity maybe associated with ↑leucine and ↑5-HT;
and the detoxifying effect of processing maybe associated with ↑tryptophan, ↑PAPB
and ↑tyrosine.
3. Processing altered the chemical profile of raw BX.
Further study will be performed to establish
the relationship between the reduced toxicity
and changed chemical profiles.
Significance
1) Shed new light on the mechanisms of BX-induced toxicity
and the detoxifying effect of processing.
2) Help us to optimize the processing procedure, and to
facilitate the rational and safe use of BX.
Acknowledgements
Thank You!
L9(34) Orthogonal Test
The Hong Kong PhD Fellowship provides an annual
stipend of HK$240,000 (approximately US$30,000) and a
conference and research-related travel allowance of
HK$10,000 (approximately US$1,300) per year to each
awardee for a period of up to three years. Fellowships will
be awarded in the 2016/17 academic year.
APPLICATION DEADLINE: Dec. 1, 2015 https://cerg1.ugc.edu.hk/hkpfs/index.html
Welcome to join us ! Dr YU Zhiling
Expert in Pharmacology & Processing of Chinese Medicines
zlyu@hkbu.edu.hk
Selected Publications:
Hong Kong Baptist University