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