ISO/DIS 19047:2024(en)

ISO/TC 249 /WG1

Date: 2024-07-29

Secretariat: SAC

Traditional Chinese medicine –Polygonum multiflorum root

(French title)

© ISO 2024

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Contents Page

Foreword iv

Introduction v

1 Scope 1

2 Normative references 1

3 Terms and definitions 1

4 Descriptions 1

5 Requirements 2

6 Sampling 4

8 Test report 5

9 Packaging, storage and transportation 5

10 Marking and labeling 6

Annex A (informative) Thin-layer chromatogram (TLC) identification 7

Annex B (informative) Determination of 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside contents 9

Annex C (informative) Determination of total anthraquinone glycosides content 11

Annex D (informative) Reference values of Polygonum multiflorum root in different national and regional standards 14

Bibliography 17

Foreword

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Introduction

Polygonum multiflorum root (also called Fallopia multiflora root) is the dried root of Polygonum multiflorum Thunb. (Fam. Polygonaceae), which is known as Heshouwu (何首乌 in Chinese) in China. It has a long history of medicinal use in East Asian countries and is traditionally valued and reported for hair-blacking, liver and kidney-tonifying and anti-aging effects as well as low toxicity.

To date, although Polygonum multiflorum root and its related prescriptions are limited in the international market owing to the safety of liver injury, there is still a large demand for Polygonum multiflorum root in the Chinese Materia Medica market at home and abroad, 200~300 tons per year of Polygonum multiflorum root are exported to Japan, Korean, Hongkong of China, and other counties and regions. In addition, Polygonum multiflorum root is ranked as No. 18 in ISO 23975:2019 which has urgent need for developing international standard.

To date, a unified international standard of Polygonum multiflorum root has not yet come into being. The regulatory authorities in many countries have not adequately differentiated the adulterants of Polygonum multiflorum root. Additionally, the quality of Polygonum multiflorum root provided from different areas varies a lot. Therefore, an international standard for Polygonum multiflorum root in terms of quality control of this herb and its products is imminently required to ensure the safe use of these medicinal materials.

As national implementation may differ, national standards bodies are invited to modify the values given in 5.4 and 5.5 in their national standards. Examples of national and regional values are given in Annex D.

Traditional Chinese medicine –Polygonum multiflorum root

This document specifies the quality and safety requirements and test methods of Polygonum multiflorum root, which is derived from the dried root of Polygonum multiflorum Thunb. (Fam. Polygonaceae).

This document is applicable to raw and processed Polygonum multiflorum root that are sold and used as natural medicines in international trade, including Chinese materia medica (whole medicinal material) and decoction pieces derived from this plant.

The following referenced documents are indispensable for the application of this document. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies.

ISO 18664, Traditional Chinese Medicine — Determination of heavy metals in herbal medicines used in Traditional Chinese Medicine

ISO/TS 21310, Traditional Chinese medicine — Microscopic examination of medicinal herbs

ISO 21371, Traditional Chinese medicine — Labelling requirements of products intended for oral or topical use

ISO 22217, Traditional Chinese medicine —Storage requirements for raw materials and decoction pieces

ISO 22258, Traditional Chinese medicine — Determination of pesticide residues in natural products by gas chromatography

ISO 23723, Traditional Chinese medicine — General requirements for herbal raw material and materia medica

For the purposes of this document, the following terms and definitions apply.

ISO and IEC maintain terminological databases for use in standardization at the following addresses:

— ISO Online browsing platform: available at https://www.iso.org/obp

— IEC Electropedia: available at https://www.electropedia.org/

3.1

Polygonum multiflorum root

dried root of Polygonum multiflorum Thunb (Fam. Polygonaceae), and also called Fallopia multiflora root

Note 1 to entry: syn. Reynoutria multiflora (Thunb.) Moldenke; Fallopia multiflora (Thunb.) Haraldson

In this document, the structure of Polygonum multiflorum Thunb. (Fam. Polygonaceae). and the dried root are shown in Figure 1.

Key

Figure 1 — Structure of the plant and root of Polygonum multiflorum Thunb.

The following requirements shall be met before the sampling refer to Clause 6.

a) Polygonum multiflorum root shall be clean and free from leaf, stem and foreign matter.

b) The presence of living insects, mouldy fruit and external contaiminants which are visible to the naked eye shall not be permitted.

The root is briquette or irregular spindle.

The external surface of the root is reddish-brown with irregular wrinkles, transverse elongated lenticel-like protrusion, and with fine rootlet scars.

The root is 6 cm to 15 cm in length measured from the base to the end of the root, and 4 cm to 12 cm in diameter measured at the middle of the root.

The texture is dense, compact, and not easily broken.

The fracture is pale yellowish-brown or reddish-brown. The drug is powdery when it is fractured. The phloem exhibits 4 to 11 subround allotype vascular bundles arranged in a ring, forming brocaded patterns; the xylem in the central part is larger with an occasionally woody core.

Odour is slight and characteristic, and the taste is slightly bitter and astringent.

As shown in Figure 2, the microscopical characteristics of transverse section and powder are shown as follows:

Transverse section: the cork consists of several layers of cork cells (key 1) filled with brown contents. The phloem is relatively broad, scattered with 4–11 subrounded allotype vascular bundles of collateral type, vessels are rare. The central cambium of the root is in a ring, with few vessels in the xylem, surrounded by some tracheids and a few lignified fibres. The parenchymatous cells contain starch granules (key 2) and clusters of calcium oxalate (key 3).

Powder: The powder is yellowish-brown, and it shows simple or 2 to 9 compound starch granules, the simple granules are sub-rounded with 4 to 50 μm in diameter and a V-shaped, stellate or Y-shaped hilum, the large granules show clearly visible layers. Cluster crystals of calcium oxalate is obtuse angles and 10-80 μm in diameter, sometimes up to 160 μm. Fragments of parenchyma consisting of thin-walled, sub-rounded or rectangular cells, sometimes containing brown, yellowish-brown or reddish-brown inclusions (key 4). Pitted annular vessels (key 5) are 15 μm to 180 μm in diameter. Scattered brown masses are varied in shape, size and colour.

Key

Figure 2 — Structure of powdered Polygonum multiflorum root

The moisture content in percentage mass should not be more than 14,0 %.

The total ash content in percentage mass should not be more than 5,5 %.

The mass fraction of water-soluble extracts should be determined.

The identification of extracts of Polygonum multiflorum root with thin-layer chromatogram (TLC) shall present the specific spot or band to Polygonum multiflorum root.

The content of marker compound, such as 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside (C₂₀H₂₂O₉), total anthraquinone glycosides, calculated as the sum of emodin-8-O-β-D-glucoside (C₂₁H₂₀O₁₀) and physcion-8-O-β-D-glucoside (C₂₂H₂₂O₁₁), should be determined.

The contents of heavy metals such as arsenic, mercury, lead and cadmium shall be determined.

The contents of pesticide residues, such as DDT, endrin, BHC, aldrin, dieldrin, etc., shall be determined.

Sampling of raw and processed Polygonum multiflorum root shall be in accordance with ISO 23723, 8.

Samples not less than 500 g are taken from each batch randomly. These samples are examined by naked eyes observation in sunlight, smell and taste.

The testing method specified in ISO/TS 21310 shall apply.

The testing method specified in ISO 23723, Clause 7.2.3 shall apply.

The testing method specified in ISO 23723, Clause 7.2.3 shall apply.

The testing method specified in ISO 23723, Clause 7.2.5 shall apply.

See Annex A for additional information on thin-layer chromatogram identification.

See Annex B and Annex C for additional information on determination of 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside contents and total anthraquinone glycosides content.

The testing method specified in ISO 18664 shall apply.

The testing method specified in ISO 22258 shall apply.

For each test method, the test report shall specify the following:

a) all information necessary for the complete identification of the sample;

b) the sampling method used;

c) the test method used, with reference to this international standard;

d) the test result(s) obtained;

e) all operating details not specified in this international standard, or regarded as optional, together with details of any incidents which may have influenced the test result(s);

f) any unusual features (anomalies) observed during the test;

g) the date of the test.

The packaging and transportation shall not transmit any odour or flavour to the product and shall not contain substances which may damage the product or constitute a health risk. The packaging shall be strong enough to withstand normal handling and transportation.

The storage requirements for Polygonum multiflorum root shall be refered to ISO 22217.

The products shall be protected from light, moisture, pollution and entry of foreign substances during long distance delivery. Carriers should be well ventilated to keep dry and moisture-proof.

See the method specified in ISO 21371. The following items shall be marked or labeled on the packages:

a) all quality features indicated in Clause 5, determined in accordance with methods specified in Clause 7;

b) gross weight and net weight of the package;

c) country of origin and province or state of the products;

d) date of production and expiry date of the products;

e) storage method.

1. 
   (informative)
   
   Thin-layer chromatogram (TLC) identification
   1. Preparation of test solution

Weigh 1,0 g of sample to grind and pass it through a 24 mesh or coarser sieve, add 50 mL ethanol and extract with reflux in a water bath for 60 min, fliter and concentrate the flitered solution to 3 mL as the test solution.

• 1. Preparation of refernce drug solution

Weigh 0,25 g of reference drug of Polygonum multiflorum root to grind and pass it through a 24-mesh or coarser sieve and treat it in the same manner as in B.1 as the reference drug solution.

• 1. Preparation of refernce substance solution

Dissolve a quantity of 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside, emodin, and physcion Chemical Reference Substance (CRS)with ethanol in a brown cliometric flask to produce a solution containing 0,5 mg of each per mL as the reference solution.

• 1. Developing solvent system

Prepare a mixture of hexane—ehtyl acetate—formic acid (30 : 10 : 0,1, v/v/v) as the mobile phase A, and dichloromethane—methanol (8 : 2, v/v) as the mobile phase B.

• 1. Procedure

Apply 2,0 μL each of the reference drug solution and the test solutions on the same TLC plate(silica gel H) previously dried at 110 °C for 15 min in the oven. Develop firstly the plate in the mobile phase A decribed in A.4 to 5,0 cm, subsequently develop the same plate in mobile phase B to 8,0 cm. Then move the plate and dry in air to determine the colour of spots. Examine the plate under UV light at 366 nm. Identify the spots of the test solutions by comparing the positions and colours with those of the reference drug solution.

Typical reference TLC chromatograms are shown in Figure A.1.

Key

Figure A.1 —Typical TLC chromatograms of Polygonum multiflorum root

1. 
   (informative)
   
   Determination of 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside contents
   1. Principle of the test method

The high-performance liquid chromatography (HPLC) method is employed to determine the content of 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside.

The HPLC system consists of a quaternary pump, continuous vacuum degasser, thermostated auto-sampler and column compartment coupled to a variable wavelength diode-array detector

• 1. Preparation of reference standard solution

Dissolve a quantity of 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside CRS with ethanol in a brown cliometric flask to produce a solution containing 0,2 mg of each per mL as the reference solution.

• 1. Preparation of test solution

Weigh 250 g of sample to grind and pass it through an 80 mesh or finer sieve. Weigh accurately 0,2 g of the powder in a stopper conical flask. Accurately add 25 ml of ethanol. Extract under reflux in a water bath for 30 min. Cool and weigh again. Replenish the loss of mass with ethanol and mix well. Filter and use the successive filtrate. Filter the supernatant solution through a 0,45 μm Millipore filter unit prior to the HPLC analysis.

• 1. Chromatographic system

Column.

a) Stationary phase: octadecylsilane bonded silica gel as analysing column or equivalent;

b) Size: l = 250 mm, Φ = 4,6 mm;

c) Theoretical plates: not less than 2,000.

Mobile phase:

a) Mobile phase A: water for chromatography R.

b) Mobile phase B: acetonitrile for chromatography R.

c) Isocratic elution: a mixture of mobile phases A and B (75: 25).

Flow rate: 1,0 ml/min.

Detector: 320 nm.

Column temperature: 30 °C.

Injection volume: 10 μl.

• 1. Content calculation of geniposide

B.5.1 The content of betaine, C (%) is calculated with Formula B.1.

(B.1)

where

B.5.2 A typical reference HPLC chromatogram is shown in Figure B.1.

a) 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside

b) Polygonum multiflorum root

Figure B.1 —Typical HPLC chromatograms of Polygonum multiflorum root

1. 
   (informative)
   
   Determination of total anthraquinone glycosides content
   1. Principle of the test method

The high-performance liquid chromatography (HPLC) method is employed to determine the content of total anthraquinone glycosides, calculated as the sum of emodin-8-O-β-D-glucoside (C₂₁H₂₀O₁₀) and physcion-8-O-β-D-glucoside (C₂₂H₂₂O₁₁).

A quantitative analysis of multi-components by single-marker (QAMS) method is used for the simultaneous determination of emodin, emodin-8-O-β-D-glucoside and physcion-8-O-β-D-glucoside. The conversion factors (f)of emodin-8-O-β-D-glucoside and physcion-8-O-β-D-glucoside were established by HPLC method with emodin as internal standard, which were used to calculate the content of other two anthraquinone glycosides.

The HPLC system consists of a quaternary pump, continuous vacuum degasser, thermostated auto-sampler and column compartment coupled to a variable wavelength diode-array detector

• 1. Preparation of reference standard solution

Dissolve a quantity of emodin, emodin-8-O-β-D-glucoside and physcion-8-O-β-D-glucoside. CRS with ethanol in a brown cliometric flask to produce a solution containing 0,02 mg of each per mL as the reference solutions.

• 1. Preparation of test solution

Weigh 250 g of sample to grind and pass it through an 80 mesh or finer sieve. Weigh accurately 0,5 g of the powder in a stopper conical flask. Accurately add 25 ml of methanol. Extract with ultrasound for 30 min. Cool and weigh again. Replenish the loss of mass with methanol and mix well. Filter and use the successive filtrate. Filter the supernatant solution through a 0,45 μm Millipore filter unit prior to the HPLC analysis.

• 1. Chromatographic system

Column.

a) Stationary phase: octadecylsilane bonded silica gel as analysing column or equivalent;

b) Size: l = 250 mm, Φ = 4,6 mm;

c) Theoretical plates: not less than 2 000.

Mobile phase:

a) Mobile phase A: methanol for chromatography R.

b) Mobile phase B: 0,1 % formic acid-water (v/v) for chromatography R.

c) Gradient elution: see Table C.1.

Table C.1 —the information of gradient elutions

Flow rate: 1,0 ml/min.

Detector: 254 nm.

Column temperature: 30 °C.

Injection volume: 10 μl.

• 1. Content calculation of total anthraquinone glycosides

C.5.1 Using the chromatograms of reference standard solutions and the reference chromatogram of Polygounm multiflorum root, identify the retention times of the peaks corresponding to emodin, emodin-8-O-β-D-glucoside, and physcion-8-O-β-D-glucoside in the Sample solution. The approximate relative retention times and conversion factor of the analytes is provided in Table C.2.

Table C.2 — The approximate relative retention times of the analytes

C.5.2 A typical reference HPLC chromatogram is shown in Figure C.1.

Key

Figure C.1 —Typical HPLC chromatograms of Polygonum multiflorum root

1. 
   (informative)
   
   Reference values of Polygonum multiflorum root in different national and regional standards

Different countries and regions have their own limits for Polygonum multiflorum root, as shown in Table D.1.

Table D.1 — The test items and reference values of Polygonum multiflorum root in diffrernert national and regional standards

Bibliography

[1] China Pharmacopoeia Commission. Pharmacopoeia of the People's Republic of China. Part 1. Beijing: Chinese medicines and Technology Press, 2020

[2] Committee on Japanese pharmacopoeia. The Japanese Pharmacopoeia. 18th ed. Ministry of Health, Labour and Welfare, 2021

[3] Ministry of Food and Drug Safety. The Korean Pharmacopoeia. 12th ed. Chungcheong: The Ministry of Food and Drug Safety, 2019

[4] European Pharmacopoeia Commission. European Pharmacopoeia. 10th ed. Strasbourg: European Directorate for the Quality of Medicines & Health Care, 2019

[5] The U.S. Pharmacopoeia Commission. USP Pharmacopoeia-Herbal medicines compendium, 2013