Chinese Pharmacopoeia 2015 Vol. I

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Research of Quality Standard and Augment &

Amendment of Traditional Chinese Medicine in

Chinese Pharmacopoeia 2015 edition

Pengfei Tu, Ph.D.

Professional Committee of TCM Prepared Slices, China Pharmacopoeia Committee

Introduction

Chinese medicine as the major remedy for preventing and curing disease made great contribution to Chinese nation for its reproduction and prosperity.

Sheng Nong’s herbal classic (Eastern Han Dynasty, from 1st to 2nd century A.D.) records 365 kinds of Chinese medicines.

Tang Materia Medica (Tang Dynasty in 659 A.D.) is the first pharmacopeia in China, with 884 Chinese medicines recorded.

Compendium of Materia Medica (Ming Dynasty in 1578 A.D.), describes 1,892 Chinese medicines.

China Pharmacopoeia (2015 Edition) includes 616 Chinese material medica (CMM).

The 3rd census of traditional Chinese medicine (TCM) resource) showed existence of 12807 CMM.

There are about 1200 CMM used commonly.

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Introduction

Traits Complex origin: multi

origins, synonym, or homonym

Natural resources: easily affected by various factors

Difficulty in effective control during producing process.

Comprehensive and chemical constituents and treating functions.

QS

Quality Uniformity

Bioequivalence

Safe Usage灰毡毛忍冬

Lonicera macranthoides

忍冬Lonicera japonica

参环毛蚓Pheretima aspergillum

CMM

1. Idea of QS Research and Systematic

Construction of TCM

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1.1. For CMM and its Prepared Slices (PS)

CMM and PS

Chemical research

Clarification of chemical constituents

Biological or literature research

Effective constituents

TLC Identification

Characteristic spectrum

Main constituents determination

Fingerprint

TCM QS database

₰ Morphological identification₰ Microscopic identification of tissues and powders

₰ Water₰ Total ashes₰ Acid insoluble ashes₰ heavy metal and

deleterious element ₰ pesticide residue₰ SO2 residue₰ Mycotoxins₰ Limit examination

of toxic and harmfulsubstance

• Name

• Source

• Character

• Identification Microscopic

Chromatographic

• Identification—Real or Faked—Real Medicine

• Examination—Safety—Medicine with Less Toxicity

• Fingerprint & Determination—Quality—High-quality Medicine

1.1. For CMM and its Prepared Slices (PS)

• Examination

Water

Total ashes

Acid insoluble ashes

Heavy metal and deleterious element

Pesticide residue

SO2 residue

Mycotoxins

Limit examination of toxic and harmful substance

QS systems of CCM and PS in Chinese Pharmacopoeia

• Fingerprint/characteristic spectrum

• Extracts• Content determination• Processing• Properties, flavors and

channels• Functions and indications• Usage and dosage• Notice• Storage

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1.2. For Proprietary Chinese Medicine

CCM

PS

Fixed origin Fixed production places Fixed cultivation techniques Fixed harvest processing Fixed processing technology

Extract, solid-liq. separation, purification, concentration, drying, molding

Producingcontrol

Proprietary Chinese medicine

₰ Morphological identification

₰ Microscopic identification

₰ Examination Provision and inspection of

dosage-form. Heavy metal and

deleterious element Pesticide residue Endogenous poisonous and

harmful ingredients

Literature investigation

Analysis of chemical components

Analysis of effective components

Effective or major components

Fingerprint Multicomponent Determination

TLC/characteristic spectrum

Material control

TCM QS database

• Name

• Prescription

• Preparation

• Character

• Identification

Microscopic identification

chromatographic identification

Provision and inspection of dosage form

Heavy metal and deleterious element

Pesticide residue

Endogenous poisonous and harmful ingredients

QS system of Proprietary Chinese Medicine in Chinese Pharmacopeia

• Fingerprint/characteri

stic spectrum

• Content determination

• Function and

indication

• Usage and dosage

• Attentions

• Specification

• Storage

1.2. For Proprietary Chinese Medicine

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2 Research of QS System and

Enlargement & Amendment of TCM in Chinese

Pharmacopoeia 2015 edition

2.1. CCM Identification—Real vs. Faked

Character Based on

Sense or Experience

The stigmas of Crocus sativus (西红花)

The stems of Cistanche tubulosa (管花肉苁蓉)

Cordyceps sinensis冬虫夏草

Cordyceps gunnii古尼虫草

A: Bombyx BatryticatusB: fakes

The stems of Cistanchedeserticola (荒漠肉苁蓉)

The barks of Eucommia ulmoides(杜仲)

Gutta-percha

A

B

Chinese Pharmacopeia 2015 edition: some cultivating character modified

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on Basis of Tissue or Powder

starch grain

phelloderm

phloem

resin duct

cambium

wood ray

xylem

clusters of calcium oxalate

12

3

4

5

1. resin cana

2. clusters of

calcium oxalate

3. cork cell

4. Conduit

5. starch grain

Microscopic features of transverse section of Panax ginseng root

Microscopic traits of Panaxginseng root powder


Fakes: made of flour

Cordyceps sinensis

Cordyceps gunnii

味连(Coptis chinensis ) 雅连(Coptis deltoidea ) 云连(Coptis teeta )

Microscopic features of transverse section of Coptis rhizoma

35% 43% 48% 57%72%

61%71,6% 78%

0%

50%

100%

1997 1985 1990 1995 2000 2005 2010 2015

The rate of microscopic identification of crude drugs in ChP

editionChinese Pharmacopeia 2015 edition: microscopic identification of 50 CCM added.



on Basis of Tissue or Powder

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TLC identification including

Current: reference substance,

controlled medicine, and

Prospective: TLC identification of

CCM compared to reference extract.

Advantages by using the reference extract:

• Multi-component control, a higher specificity

• Large-scale production, higher consistency, uniformity, but lower cost

• Trace, chips, easy to use and carry, but lower cost

• Manual with TLC photo, more accurate and clear

S RE 1 2 3 4 5 6 7 8

S1

S2

HPTLC images of the roots of Polygala tenuifolia（远志）

1～8: P. tenuifolia roots; S1: 3,6-disinapoyl sucrose; S2: polygalaxanthone III; RE: reference extract

HPTLC of the barks of Magnolia officinalis (厚朴) (by Z. T. Wang, et al)

A B

A: Visualized by 1% vanillic aldehyde and 10% sulfuric acid ethanol solution; B: By 0.04% DPPH ethanol solution


TLC identification

S1 S2 S3 S4 1 2 3 4 5 6 7 8 S5 S6

HPTLC images of the rhizomes of Coptis (by Z. T. Wang, et al.）

1,2,7: Coptis chinensis; 3,4,8: Coptis deltoidea; 5,6: Coptis teetaS1: palmatine; S2: berberine; S3: epiberberine; S4: coptisine; S5:

jatrorrhizine; S6: columbamine

HPTLC images of the roots of Bupleurum (by P. S. Xie, et al.）

1,2,5: B. chinensis; 3,6: B. scorzonerifolium; 4,12: B. falcatum; 7: B. longiradiatum; 8: B. bicule; 9: B. polyclonum;10: B. wenchuanense; 11: B. marginatum; 13: B. yinchowense; 14: B. simithii var. parvifolia; 15: B. tenue

S1: saikosaponin f

S2: saikosaponin b2

S3: saikosaponin a

S4: saikosaponin d

Chinese Pharmacopeia 2015 edition: all herbal medicines possesses TLC identification.


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By Characteristic Spectra

Advantages higher separation abundant information higher specificity peak area ratio and semi-

quantitative control realized

Analytical method HPLC GC HPCE

Evaluation methods The characteristic peak of retention

time and relative retention time The characteristic peak of the peak

area ratio Contrast of reference crude drugs and

reference extract

Applicative TCM

Precious medicinal

materials

Easy mixed herbs

Chinese herb extracts

formula granule

Chinese patent medicine

Characteristic chart of the cortexes of Dictamnus dasycarpus （白鲜皮）

dictamnine obacunone fraxinellone


Case 1: Characteristic Spectrum and QS Establishment of Aquilariae Lignum Resinatum (Agarwood)

Aquilaria sinensis (Lour.) in Thymelaeaceae

Valuable medica with action of moving qi and ceasing pain, stopping vomiting and asthma, used for thoracic abdominal distension stuffy pain, cold stomach vomiting, hiccups, kidney empty reversed flow of qi for urgent.

Precious spices and perfumes.

Luxury handicraft raw materials and collections.

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R1 R2 R3 R4 R5 R6 R7 R8

1* H OCH3 OCH3 H H H H OH2* OH OCH3 OCH3 H H H OCH3 H3 H OCH3 H H H H OH H4 H OH H OH H OH OCH3 H5 H OH H H H OH OCH3 H6 H H H H OH H H H7 H OH H H H H OCH3 H8 H OH H H H H H H9 H OCH3 H H H OCH3 OH H10 H OCH3 OCH3 H H H OCH3 H11 H OH H H H OCH3 OH H12 H OCH3 OCH3 H H OCH3 H H13 H OCH3 H H H H H H14 H OCH3 H H H OCH3 H H15 H OH H OH H H H H16 H H OH H H H H H17 OH H H OH H H OCH3 H18 OH H H OH H H H H19 H OCH3 OH H H OH OCH3 H20 H H H H H H H H21 H OH H Cl H H H H22 H OH H H H H H H23 H H OCH3 H H H H H24 OH OCH3 H H H H H H26 H H H H H H OCH3 H27 H OCH3 H H H H OCH3 H28 H OCH3 OH H H OCH3 OH H

Chen D, et al. Eur. J. Org. Chem. 2012, 5389–5397

Phytochemical Studies

2-(2-phenylethyl)chromone （PEC, 28）

Chen D, et al. Eur. J. Org. Chem. 2012, 5389–5397

Tetrahydro-2-(2-phenylethyl)chromone (THPEC, 24)


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Huo HX, et al. Fitoterapia 2015, 106, 115-121.

Sesquiterpenes（29）


20

Fingerprint Analysis of Agarwood

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0 20 40 60 80 100 Time [min]0

2

4

6

7x10

Intens.

TIC +All MS

0 20 40 60 80 100 Time [min]0.0

0.5

1.0

1.5

2.0

2.5

7x10

Intens.

TIC +All MS

min0 20 40 60 80 100

mAU

0

50

100

150

200

250

300

数数 DAD1 B, Sig=240,16 Ref=360,100 (E:\HPLC \CD\CXYP2#.D)

min0 20 40 60 80 100

mAU

0

5

10

15

20

25

30

35

数数 DAD1 B, Sig=240,16 Ref=360,100 (E:\HPLC \CD\BMX.D)

HPLC Fingerprint of agarwood and Aquilaria wood

Agarwood

HPLC –UV Chromatograms

HPLC-MS TIC Chromatograms

Agarwood

Aquilaria wood

Aquilaria wood

7 months after artificial induction

Natural sources

Common peaks of natural and artificial sources

Highly abundant peaks in artificial sources

HPLC–UV chromatogram



Comparison of HPLC fingerprints of different types of agarwoods

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23

XIC: m/z 357, 337, 335, 333, 317, 303, 267

XIC: m/z 383, 341, 319, 311, 301, 283, 297

XIC: m/z 367, 365, 349, 347, 343, 331, 327, 313, 281

Identification of 2-(2-phenylethyl)chromones in agarwood by HPLC-ESI-MSn

70 of 2-(2-phenylethyl)chromones, including 28 kinds of simple types, 35

species of four hydrogen types, 5 five hydrogen types and 2 six hydrogen

types were identified.

24

HPLC characteristic chromatogram of agarwood included in CP (2015 ed.)

agarotetrol

agarotetrol

Comparison of characteristic chromatograms of 17 batches

Reference characteristic chromatogram

Agarwood reference

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Reference

Real

Fakes

0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 m

-50000

0

50000

100000

150000

200000

250000

300000

350000

400000

Characteristic chromatogram of Chinese agarwoods and fakes

fak

es

Chinese agarwoods

fakes

Origin of Plant: Notopterygium incisum Ting ex H. T.

Chang and N. forbesii H. de Boiss.

Properties: NR is used to treat a strong cold, headache,

rheumatism, paralysis, shoulder pain and other symptoms.

Chemical Components: Coumarins, Phenolic acids,

Polyacetylenes, Essential oil, and Terpenoids etc

OH

OH

Isoimperatorin Notopterol Falcarindiol

O

O

O

OH

Phenethyl ferulate

Notopterygium franchetii

Case 2: Characteristic Spectrum and QS Establishment of Notopterygii Rhizoma et Radix (NR)

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Chemical Constituents from NR

The reference characteristic chromatogram of Notopterygii

Rhizoma et Radix

1: Notopterol; 2: Phenethyl ferulate; 3: Isoimperatorin ; 4: falcarindiol

The characteristic chromatogram of the roots of N. incisum

The characteristic chromatogram of the roots of N. franchetiiComparison of the characteristic chromatogram of Notopterygii Rhizoma et Radix(QH) and fakes (WPQH)

HPLC characteristic chromatogram of NR included in CP (2015 ed.)

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Case 3: Characteristic Chromatogram for Identification of Multi Origin——Clematidis Radix

C. manshuricaC. hexapetalaC. chinensis

λ＝320nm

C. manshuricaC. chinensis

λ＝320 nmλ＝320nm

Specific LC-MS Identification of Animal Gelatin TCM－Chinese

Pharmacopoeia (2015 ed.)

T im e5 .0 0 1 0 .0 0 1 5 .0 0 2 0 .0 0 2 5 .0 0 3 0 .0 0 3 5 .0 0 4 0 .0 0

%

- 5

9 5

5 .0 0 1 0 .0 0 1 5 .0 0 2 0 .0 0 2 5 .0 0 3 0 .0 0 3 5 .0 0 4 0 .0 0

%

- 5

9 5

5 .0 0 1 0 .0 0 1 5 .0 0 2 0 .0 0 2 5 .0 0 3 0 .0 0 3 5 .0 0 4 0 .0 0

%

- 5

9 5

5 .0 0 1 0 .0 0 1 5 .0 0 2 0 .0 0 2 5 .0 0 3 0 .0 0 3 5 .0 0 4 0 .0 0

%

- 5

9 5

5 .0 0 1 0 .0 0 1 5 .0 0 2 0 .0 0 2 5 .0 0 3 0 .0 0 3 5 .0 0 4 0 .0 0

%

- 5

9 5

6 4 1 .3 0 6 5

0 1 4 7 9. 0 7 02M +H

5 0 0 1 0 0 0

Inte

ns

ity

(c

oun

ts)

0

5 8 9 3

C on tro l: G E A G P A G P A G P IG P V G A R

G E A G P A P A P I P V G A RR A G V P I P G P A P E G

5 5 6 . 31 9 5y 6

4 99 . 29 7 6y 5

1 2 1 6 .6 9 3 7y 14

1 0 4 8. 5 9 9 6y 1 2

7 6 5 . 35 7 2b 1 0

1 27 . 0 8 5 94 0 2 . 24 4 5

y 4

7 6 5 .8 5 5 6

0 1 40 8 .6 7 19M +H

5 00 1 0 0 0

Inte

nsity

(co

unts

)

0

2 95 74

C on tro l: G E A G P S G P A G P T G A R

G E A G P S G P A G P TG A R b M a xR A G T P A P S P A E G y M a x

1 28 1 . 6 3 11y 15

6 89 .3 4 6 7 1 0 9 5 . 5 5 68y 13

5 58 . 29 4 9y 6

9 67 .5 0 0 0y 11

0 2 03 3 . 7 3 38M +H

1 00 0

Inte

ns

ity

(c

ou

nts

)

0

2 15 34

C on t ro l: G E P G P T G V Q G P P G P A G E E G K

G E P G P T G V Q G P P G P A G E E G K b Ma xK G E E G A P G P Q V G T P G P E G y Ma x

1 84 9. 9 0 48y 20 *

1 66 3. 8 3 29y 18 *

8 57 . 40 6 1y 9* 1 20 1. 5 0 79

9 2 5 .4 3 2 6

7 3 2 .8 2 8 2

7 5 8 .3 5 3

( A )

( B )

( C )

(D )

(E )

Colla Corii Asini－Donkey skin

Oxhide gelatin－Cowhide

New Colla Corii Asini－Pigskin

Tortoise-shell glue－ tortoise-shell

Deerhorn Gelatin－ Antlers

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Biological Identification

(1) DNA identification

(2) DNA barcode

1. positive CK 2-11. Zaocys dhumnade

12. negative CK 13. Blank

(3) SDS-PAGE: animal drugs(4) Rapid TCM identification based on monoclonal antibody technique: for TCM with unique components

Tianyi Xin, Shilin Chen, et al. Acta Pharm Sin. 2012, 47(8)

2.1 Identification of TCM—Real or Fake

2.2 Examination—Safety Guaranteed

• Impurities and foreign matter：

• Heavy metals and harmful element: Atomic absorption spectrometry, plasma mass spectrometry

• Pesticide residues: detected by GC, GC-MS, LC-MS

• Mycotoxins: detected by HPLC, LC-MS

• SO2 Residue: detected by IC, GC

• Endogenous toxic and harmful substances: detected by HPLC, LC-MS, GC, GC-MS

Efficient and sensitive detection methods： Monoclonal antibodies Chemical reactions

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Heavy metals and harmful element Pretreatment Method: Microwave Digestion, Wet Digestion, Drying Digestion,

High Pressure Digestion.

Pb, Cd, As, Hg, Cu: Atomic Absorption Spectrophotometry (AAS), Inductively Coupled Plasma Mass Spectrometry (ICP-MS), Inductively Coupled Plasma Atomic Emission Spectrometry (ICP-AES).

Speciation and valence determination of As and Hg: HPLC-ICP-MS

TCM collected in Chinese Pharmacopoeia (2015 ed.):

17 herbs:Astragali Radix, Lonicerae, Panacis Quinquedolii Radix, Paeoniae Radix Alba, Salviae Miltiorrhiazae Radix et Rhizoma, Glycyrrhizae Radix et Rhizoma, Lycii Fructus, Crataegi Fructus, AsiniCorii Colla, Hirudo, Ostrese Concha, Meretricis Conacha & CyclinaeConcha, Margarita, Laminariae Thallus & Eckloniae Thallus, Sargassum, Sepiae Endoconcha, Propolis;

Seven extractions：Total Ginsenoside of Ginseng Stems and Leaves, Notoginseng Total Saponins, Gapillary Wormwood Extract, Berviscapine, Ccosides, Entella Total Glucosides, Menthol

All of the TCM injections.


Heavy metals and harmful element

Focus on the element valence state

Toxcity of 6 valences of As：Arsenious acid (As3+) ＞Arsenic acid (As5+)

＞ MMA ＞ DMA, AsB and AsC are almost innoxious.

Toxcity of 4 valences of Hg ：Methylmercury＞Ethylmercury＞ Hg2+ ＞

Hg＋, The toxcity of methylmercury and ethylmercury are hundreds-fold

higher than inorganic mercury, which might be attributed to the

stronger hydrophobicity of heptachlor.

6 valences of As 3 valences of Hg

HPLC-ICP-MS Chromatogram

Department of Traditional Chinese Medicine, Shanghai Institute for Food and Drug Control, Shanghai, China


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Pesticide residues Chinese Pharmacopoeia (2015 ed.): A total of 24 pesticides including

organic chlorine, organic phosphorus and pyrethroids, etc, are required.

Chinese Pharmacopoeia (2015 ed.): GC/MS/MS(76);LC/MS/MS (153)；227 kind of pesticide residues in total.

Chinese Pharmacopoeia (2015 ed.): Glycyrrhizae Radix et Rhizoma, Astragali Radix, Ginseng Radix et Rhizoma, Panacis Quinquefolii, Total ginsenosides of ginseng stems and leaves, and root.

TCM formula granule: Cultivated TCM herbs are required to be investigated on pesticide residues, and standards of these carrying serviouspesticide residues should be established.

Multi reaction monitoring ion superposition of 153 pesticides （LC/MS/MS）

Multi reaction monitoring ion superposition of 74pesticides （GC/MS/MS）

Department of Traditional Chinese Medicine, Shanghai Institute for Food and Drug Control, Shanghai, China


Mycotoxins

Mycotoxins: Aflatoxins G2, G1, B2, B1, Fumonisins B1, B2, T-2 toxin,

Ochratoxin A, Voitoxin, Zearalenone.

Methods: HPLC, LC-MS methods.

TCM collected in Chinese Pharmacopoeia (2015 ed.): Persicae Semen,

Ziziphi Spinosae Semen, Bombyx Batryticatus, Pericarpium Citri

Reticula, Sterculiae Lychnophorae Semen, Platycladi Semen ,

Nelumbinis Semen, Quisqualis Fructus, Arecae Semen, Hordei

Fructus Germinatus, Myristicae Semen, Cassiae Semen, Polygalae

Radix, Coscis Semen, Jujubae Fructus, Pheretima, Scolopendra,

Hirudo, scorpio.

Limits: Aflatoxin B1 ≤ 5 μg/kg; Total aflatoxin G2, aflatoxin G1

and aflatoxin B2 ≤ 10 μg/kg.


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SO2 ResidueMethods: Acid-base titration, IC and GC.

Limits: SO2 residue of 10 Chinese herbal medicine and conventional pieces including

Dioscoreae Rhizoma, Asparagi Radix , Trichosanthis Radix, Gastrodiae Rhizom, Achyranthis Bidentatae Radix, Bletilla sfriata, Atractylodis Macrocephalae Rhizoma, Paeoniae Alba Radix, Codonopsis Radix, and Puerariae Thomsonii Radix, which were traditionally deal with sulphur fumigation, should not be more than 400 mg/kg;

SO2 residue of others TCM 150 mg/kg not be more than 400 mg/kg.

The shapes and properties were revised.

Color changes of Codonopsis Radix dealed with sulphur fumigation


2.3 Fingerprint Combined Content

Determination of Multiple-components

—Quality Evaluation

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2.3.1 Establishment of Fingerprint and

Determination Standards

• Studies on Establishment of Fingerprint and Determination Standards for ChinseseMaterial Medica (CMM) and Prepared Pieces (PP)

• LC-DAD-MSn Fingerprint and TCM Chemical Constituents Database

2.3.1 Establishment of Fingerprint and Determination Standards

Research of Determination Standards of Fingerprints

Origin Species

Medicinal Part

Locality of Growth

Cultivation Techniques

Productive Processing

Processing procedure

Preparing method for test solution, control and

references

Methodology: stability, precision and

reproducibility.

Identification of major chromatographic peaks

Establishment of the control fingerprint

Determination and evaluation of samples

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Research Case: Rhei Radix Et Rhizoma

Rhei Radix Et Rhizoma is one of the most famous

TCM herbs, using to treat diarrhea attack plot,

clear heat and detoxifying, remove blood stasis, and

dredging.

Its origin resources include Rheum palmatum L., R.

tanguticum Maxim.ex Balf. and R. officinale Baill.

Major components: anthraquinones and its

glycosides, tetrahydroxystilbene-glucosides,

tannins, and chromones, etc.

Rheum palmatum

Rheum tanguticum

Rheum officinale

Rheum tanguticum

Chemical Types in Rhei Radix Et Rhizoma

Anthraquinones

Anthrones

Tannins

Tetrahydroxystilbene-glucosides

n-butyrophenones

Acyl glycosides

Chromones

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HPLC-UV Fingerprint of Rhei Radix Et Rhizoma

A

B

C

D

A. R. palmatum; B. R. tanguticum; C. R. offcihale; D. Comparison of these 3 species.

Evaluation of Fingerprint Similarity

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Evaluation of Fingerprints Similarity of R. tanguticum

Fingerprints of 42 batches of R. tanguticum

Evaluation of Fingerprints Similarity of R. tanguticum

No Similarity No Similarity No Similarity No Similarity

1 0.936 12 0.688 23 0.513 34 0.530

2 0.969 13 0.816 24 0.545 35 0.533

3 0.911 14 0.857 25 0.636 36 0.533

4 0.842 15 0.884 26 0.781 37 0.876

5 0.868 16 0.544 27 0.718 38 0.808

6 0.876 17 0.958 28 0.755 39 0.940

7 0.938 18 0.953 29 0.887 40 0.867

8 0.782 19 0.867 30 0.887 41 0.894

9 0.797 20 0.946 31 0.851 42 0.843

10 0.877 21 0.841 32 0.898

11 0.965 22 0.765 33 0.880

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Identification Fingerprint Chromatograms of Rhei Radix Et Rhizoma by LC-DAD-MSn

Proposed MS fragmentation for [M-H]- ions of anthraquinones

Proposed MS fragmentation pathway for the [M-H]- ions of phenylbutanone glucopyranosidesProposed MS fragmentation pathway for the [M-H]- ions of stilbenes

A

B

C

D

E

TIC chromatograms of

Rhei Radix Et Rhizoma

A: Anthraquinones; B: Anthrones ;

C: Tannins; D: Two styrene and

butyl benzene ketone; E: Acyl

indicant, original ketone and other

constituents。

271 chemicals Identified

including 34 anthraquinones, 83

anthrones, 46 tannins, 17 stilbenes,

24 benzyl butylketones, 26 acyl

indicans, 26 original ketones, and

15 other constituents.

Characterization of Fingerprint Chromatograms of Rhei Radix Et Rhizoma by LC-DAD-MSn

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TCM Chemical Constituents Databasebased on LC-DAD-MSn

2.3.2 Multicomponent Determination of TCM

Methodology

HPLC

LC-MS

GC

GC-MS

HPCE

SFC

NMR

Solution to lack references One marker for multi

components evaluation Multi-components

determination using control extract

Selection of chemical markers Therapeutic constituents Biological constituents Characteristic constituents

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Multi-components Determination by Markers

Determination of 14 constituents in Rhei Radix Et Rhizoma

A

B

A. HPLC chromatograms of 14 mixed references; B. HPLC chromatogram of

Rhei Radix Et Rhizoma

1. gallic acid; 2. (+)-catechin; 3. (-)-epicatechin-3-O-gallate; 4. isolindleyin; 5. 4-(4'-

hydroxyphenyl)-2- butanone; 6. lindleyin; 7.sennoside B; 8.sennoside A; 9. aloe-emodin; 10. 4-

(4'-hydroxyphenyl)-2- butanone-4'-O-β-D-(2''-O- galloyl-6''-O-cinnamoyl)-glucopyranoside; 11.

rhein; 12. emodin; 13. chrysophanol; 14. physcion

Multicomponent Determination by Control Extract

Simple preparation, low cost

Quantitative packing, convenient use

Save reference substance

Specification with chromatograms, chromatographic

peak easily identified

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Stem of Mahonia bealei (Fort.) Carr. or M. fortune

(Lindl.) Fedde. has effect of clearing heat, drying

wet, purging fire, and detoxification.

Multi-component determination by control extract,

One marker for multi components evaluation, and

qNMR conducted

Multi-component determination by control extract

was record in Chinese Pharmacopoeia 2015 ed. It is

first record in Chinese Pharmacopoeia for this

determination method

W. G. Wang, et al. Journal of Chromatography A, 2015, 1412, 100

A Research Case: Mahonia Stems

Preparation Protocol

reference extractbatch

Crude medicine

inventory yield productivityThe transfer rate of total

alkaloid

content of total

alkaloids

022101 贵州 200g 4.69g 2.34% 51.4% 44.11%

031101 贵州 5kg 132g 2.64% 50.3% 40.82%

031102 贵州 5kg 137g 2.74% 50.1% 40.37%

Material

70% Ethanol reflux, 3times 1h each Dry to the concentration of 1.1g/ml

Ethanol extract

D101 macroporous resin CC; 2BV water elution, abandon; 2BV 10%ethanol elution, abandon; 2BV50% ethanol elution, collected

50% Ethanol eluent

concentration freezer dryer

Control extract

HPLC chromatograms

A. Hybrid reference substance; B. Standard extract; C. Mahonia

C. Columbamine; J. jateorhizine; P. palmatine; B. berberine

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Quality Standard of Mahonia Stems by control extract

Source: Mahonia is the stem of Mahonia bealei (Fort.) Carr. or M. fortune (Lindl.) Fedde. beloning to berberidaceae, produced for standard extraction.

PreparationCharacter Yellowish-brown powder, slightly acid odour, taste bitterSolubleness Soluble in methanol, ethanol and water, insoluble in chloroform and ethyl acetateIdentify

ExaminationFingerprintAssay Calculated by dry goods, including columbamine, jateorhizine, palmatine,berberine, should more than 35.0 %.Package and storageApplication For Mahonia stems analysis use.

365 nm 254 nm fingerprint

Determination data Mahonia Stems by control extraction

No Place

columbamine jateorhizine palmatine berberine

外标法

（mg/g）

CEMeth.

RSD（%）

外标法

（mg/g）

CEMeth.

RSD（%）

外标法

（mg/g）

CEMeth.

RSD（%）

外标法

（mg/g）

CEMeth.

RSD（%）

1 GZ-1 1.03 1.05 2.24 6.52 6.68 2.39 7.51 7.78 3.44 5.12 5.33 4.01

2 GZ-2 0.98 0.99 1.41 6.55 6.66 1.74 7.28 7.49 2.77 4.99 5.16 3.38

3 FJ-1 0.85 0.86 1.44 6.20 6.34 2.12 5.59 5.77 3.02 3.31 3.44 4.00

4 SC 0.86 0.87 1.62 5.65 5.76 2.00 6.11 6.31 3.28 3.51 3.66 4.14

5 HB 0.89 0.91 1.69 6.96 7.14 2.53 7.73 8.00 3.43 3.07 3.20 4.10

6 JS 0.96 0.98 1.56 7.97 8.17 2.45 8.59 8.86 3.11 3.38 3.51 3.67

7 YN 0.69 0.69 0.64 6.58 6.74 2.46 6.01 6.21 3.26 7.81 8.13 3.98

8 GX-1 1.24 1.27 2.55 7.73 7.93 2.58 7.34 7.57 3.18 4.29 4.46 3.81

9 ZJ 0.87 0.88 1.16 5.65 5.73 1.46 6.34 6.52 2.79 4.02 4.16 3.58

10 FJ-2 0.93 0.95 1.69 6.19 6.32 2.04 7.10 7.33 3.19 4.28 4.44 3.85

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No Place

columbamine jateorhizine palmatine berberine

外标法（mg/g）

CEMeth.

RSD（%）

外标法

（mg/g）

CEMeth.

RSD（%）

外标法

（mg/g）

CEMeth.

RSD（%）

外标法

（mg/g）

CEMeth.

RSD（%）

11 AH 0.83 0.84 1.01 6.09 6.20 1.74 7.64 7.87 3.01 3.42 3.55 3.67

12 GZ-3 1.29 1.32 2.49 8.96 9.21 2.74 7.04 7.26 2.98 4.15 4.31 3.66

13 HN 1.19 1.21 2.18 7.81 8.00 2.36 7.46 7.68 2.95 2.82 2.93 3.69

14 HB 1.32 1.35 2.50 7.19 7.35 2.20 7.03 7.24 2.94 3.87 4.01 3.63

15 SX 1.06 1.08 1.92 7.29 7.46 2.28 7.37 7.60 3.02 2.86 2.97 3.77

16 JX 1.16 1.18 2.27 7.15 7.32 2.31 7.02 7.23 3.06 3.61 3.75 3.77

17 HN 1.44 1.48 3.12 8.00 8.23 2.87 9.56 9.91 3.59 3.39 3.53 4.07

18 HB 0.85 0.86 1.41 7.52 7.72 2.64 7.64 7.90 3.34 2.51 2.62 4.09

19 GD 1.26 1.29 2.35 8.21 8.41 2.47 8.83 9.10 3.09 3.82 3.96 3.60

20 Guilin 1.36 1.40 2.62 7.48 7.66 2.34 9.67 9.99 3.24 5.00 5.18 3.62

21 Yulin 0.93 0.94 1.47 5.05 5.10 1.16 6.32 6.50 2.87 7.27 7.53 3.55

22 GX-2 0.55 0.54 1.19 4.40 4.42 0.49 6.78 6.97 2.82 11.00 11.38 3.39

以Determination data Mahonia Stems by control extraction

3 High Efficiency Analysis Technology oF

Complex TCM System

Sample preparation

LC Separaton MS Detection

PWWE online SPE

2D LC system column switching LC

IT-TOF-MS: MDFIF, etc. Qtrap-MS: stepped MRM,

sMRM, stepped MIM, etc. Qtof-MS: UNIFITM, etc. Multi-platform combined,

overall strategy

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Traditional extraction method of TCM

Direct Analysis of Constituents from TCM Based on Online Pressurized Solvent Extraction Technique

Solvent, dosage of herb use much

Sample preparation time is long

May cause degradation compounds

Unable realize the direct analysis of the

sample

Online pressurized solvent extraction

Solvent, dosage of medicine use little Water or low proportion of organic solvent

extraction, analysis environmental Automatic extraction and analysis online

switch, high flux Avoid light, extraction time is short, avoid

degradation compounds LC - MS online connection, analysis

directly

PWWEASE vs.

Mixed standards

C. deserticola

online PWWE-TFC-LC-DAD apply on C. deserticola constituent analysis

online PWWE-TFC-LC-MS/MS apply on Polygala constituent analysis

Polygalae Radix PWWE-TFC-LC-MS/MS Chemical analysis

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Chemical analysis of Complex System Based on multi-column Chromatography

1D LC-MS/MS

HILIC-TFC-LC-MS/MS

Large polarity diversity

Separate of HILIC or RPLC cannot achieve full retention

Several compounds outflow cannot be accurate quantitative

difficult to achieve HILIC and RPLC connect directly

conventional capture column caused serious peak broadening

HILIC-TFC-LC-MS/MS method established

Comprehensive analysis for plasma samples

chemical composition after Baoyuan decoction

administration

Research Case 1: Ginseng saponin by LC-Qtrap-MS

ginseng saponins are main active

ingredient in ginseng.

It produces [M+HCOO]- and [M-H]- ion;

[M+HCOO]- broken into [M-H]- most

appropriate collision to -32eV;

Establish a step-wise MRM-IDA-EPI

method, achieve comprehensive

chemical constituent analysis

221ginseng saponins

UHPLC-sMRM method established, and

achieved all peaked saponins

quantitative analysis

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The primary metabolites,

phenylethanol, iridoid glycosides and

lignin glycosides show different

behavior in MS

Through PI, NL and predefined

MRM realized the detection of all

ingredients, and through the EPI

identification of each structure

Introduction of sMRM method for all

513 constituents relative quantitative

analysis

Using multivariate statistical analysis

to distinguish two original plants in

quantitative chemical groups

Betaine and tubuloside B are the

biomarkers

Research Case 2: Cistanche by LC-Qtrap-MS

New Strategy for TCM Quantitative Metabolites of non-Target Group

Peucedanum

（quasi-content）

PLME

(UMS)

Offline 2D LC-IT-TOF-MS

MS DATA gathering

Accurate analytical compound structure

MS fragmentation rule

Based on the OCE isomers

Online optimization

Standard curve of UMS

Step 1

Step 2

Step 3

TCM reference substance is difficult to

get comprehensively

Difficult to achieve compound detection

MS data matrix containing adduct

fragment ions, redundant information

Isomers are difficult to distinguish

MS quantitative parameter difficult to

optimize

Peak intensity how to translate into

content

Large scale chemical constituent range

Difficult to achieve large compound

quantitative all

Current Difficulties

“Three Steps”

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Peucedani Radix Offline 2D LC-IT-TOF-MS

Spectrum quantitative metabolite group（quasi-content）

mass fragmentation ruleMass spectrum parameters online optimizationUsing the best collision

energy distinguish isomers (OCE)

metabonomicsLinear range extension

Rapid Characterization of Carthamus tinctorius L. Extract Metabolites by UFLC/IT-TOF-MSn and UPLC/Qtrap-MS

Carthamus tinctorius L., also known as 'Hong hua' in

TCM, belongs to Asteraceae

Total flavonoids from C. tinctorius L. have anti-

myocardial ischemia effect;

137 metabolites and 19 prototypes were identified using

diagnostic ion strategy, including 63 in plasma; 73 in

urine;

50 in bile; 17 in faeces；

UPLC/Qtrap-MS used to quantitatively and semi-

quantitatively analyze 156 in vivo components

Typical metabolic pathways

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Establishment of Fingerprints and in vitro Metabolic profiles of Bao-Yuan-Tang based on LC-MS Global Strategy

Establishment of fingerprint chromatogram of Bao-yuan-tang based on a global strategy of LC-MS

TIC: from Sample 6 (byt0619-spe-0701) of 2014101601.wiff (Turbo Spray), Smoothed, Smoothed, Smoothed Max. 3.6e9 cps.

2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44Time, min

0.0

2.0e8

4.0e8

6.0e8

8.0e8

1.0e9

1.2e9

1.4e9

1.6e9

1.8e9

2.0e9

2.2e9

2.4e9

2.6e9

2.8e9

3.0e9

3.2e9

3.4e9

3.6e9

Intensity, cps

5.73

11.14

20.91

18.81

9.39 21.2817.13

11.47 26.6237.67

21.998.737.5227.57

35.4125.2316.85 40.245.35 23.150.73 29.36 30.2512.18 31.61 32.997.20 15.213.80

1.96

Flavonoids Saponins

LC/ESI-Q-Trap-MS EMS of Baoyuan decoction

Step-wise pMRM/IDA/EPIStep-wise MIM/IDA/EPIStep-wide MIM/IDA/EPI

XIC of -MRM (133 pairs): Exp 1, 201.500/201.500 Da ID: HT from Sample 2 (P-BYT-45) of 2014120201.wiff (Turbo Spray) Max. 6000.0 cps.

2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44Time, min

0.0

5000.0

1.0e4

1.5e4

2.0e4

2.5e4

3.0e4

3.5e4

4.0e4

4.5e4

5.0e4

5.5e4

6.0e4

Intensity, cps

Flavonoids

XIC of -MRM (133 pairs): Exp 1, 201.500/201.500 Da ID: HT from Sample 10 (p-byt31min) of 2014112501.wiff (Turbo Spray) Max. 4500.0 cps.

2 4 6 8 10 12 14 16 18 20 22 24 26 28 30Time, min

0.0

5000.0

1.0e4

1.5e4

2.0e4

2.5e4

3.0e4

3.5e4

4.0e4

4.5e4

5.0e4

5.5e4

6.0e4

6.5e4

7.0e4

7.5e4

8.0e4

Intensity, cps

27.39

0.64 19.98 23.4622.37 25.21 28.06 29.7412.82 20.9610.31 17.0916.04 17.937.081.17 13.596.28 12.498.02 26.395.191.85 9.363.34 3.67

Saponins

XIC of -MRM (133 pairs): Exp 1, 201.500/201.500 Da ID: HT from Sample 10 (p-byt31min) of 2014112501.wiff (Turbo Spray) Max. 4500.0 cps.

2 4 6 8 10 12 14 16 18 20 22 24 26 28 30Time, min

0.0

5000.0

1.0e4

1.5e4

2.0e4

2.5e4

3.0e4

3.5e4

4.0e4

4.5e4

5.0e4

5.5e4

6.0e4

6.5e4

7.0e4

7.5e4

8.0e4

Intensity, cp

s

27.39

0.64 19.98 23.4622.37 25.21 28.06 29.7412.82 20.9610.31 17.0916.04 17.937.081.17 13.596.28 12.498.02 26.395.191.85 9.363.34 3.67

XIC of -MRM (134 pairs): Exp 1, 945.500/945.500 Da ID: GC from Sample 43 (P-BYT) of 2014110901.wiff (Turbo Spray) Max. 5.8e4 cps.

2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44Time, min

0.0

5000.0

1.0e4

1.5e4

2.0e4

2.5e4

3.0e4

3.5e4

4.0e4

4.5e4

5.0e4

5.5e4

6.0e4

6.5e4

Intensity, cp

s

10.22

20.13

Scan range: 150-465

Scan Range: 945.5-1255.5Scan range:467-731

Scan Range: 535.5-943.5

The method UPLC/Q

TOF-MS, HPLC/Qtrap-

MS and UNIFI software

was adopted to construct

the LC-MS overall

strategy in order to

establish a fast and

comprehensive

identification of the

fingerprint of Bao-yuan-

Tang.

A total of 236 compounds

including 139 saponins, 83

flavonoids, 6 procyanidins,

4 lignans, and 4

diterpenoids were defined

from Bao-yuan-Tang.

Metabolic profiles analysis of Bao-yuan-tang

On the basis of fingerprints of Bao-yuant-tang, HPLC/IT-TOF-MS and

UPLC/Qtrap-MS were used to analyze the metabolic fingerprints of it.

A total of 286 metabolites were detected and identified, among which

77 were plasma, 117 in bile, 139 in urine, and 4 in faeces, including 178

flavonoids, 105 saponins and 3 small molecular phenolic compounds.

Proposed metabolic pathways of liquiritigenin-4-O-glucoside in rats

Proposed metabolic pathways of Licorice-saponin G2in rats

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THANKYOU！

Tu Pengfei, Prof., Ph.D.

Department of natural medicine,

Peking University Health Science Center

38 xueyuan road, Beijing 100191,

P.R. China

Phone(fax): 010-82802750

email: [email protected]

Chinese Pharmacopoeia 2015 Vol. I

Documents

Transcript of Chinese Pharmacopoeia 2015 Vol. I