DMS Page for Acknowledgement · 2019-09-19 · Figure 3. HPTLC chromatogram of spray dried aqueous extract of hempedu bumi (Andrographis paniculata (Burm. f.) Nees.) documented under

SIRIM/MOA 4-4:2019

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Herbal extracts - Specification - Part 4: Hempedu bumi (Andrographis paniculata (Burm. f.) Nees) spray dried

aqueous extract

© Copyright 2019 SIRIM Berhad &

Ministry of Agriculture and Agro-Based Industry Malaysia

SIRIM/MOA STANDARD

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MOA The Ministry of Agriculture and Agro-based Industry (MOA), is a ministry of the Government of Malaysia that is responsible for agriculture, agro-based industry, agritourism, livestock, veterinary services, fisheries, quarantine, inspection, agricultural research, agricultural development, agricultural marketing, pineapple industry, agribusiness, botanical garden, food security and food sovereignty.

SIRIM SIRIM Berhad is a premier total solutions provider in quality and technology innovations that helps industries and businesses to compete better through every step of the business value chain. SIRIM Berhad is the centre of excellence in standardisation, facilitating industries and businesses in enhancing their production and competitiveness, protecting consumers’ health and safety, and giving them the choice for quality products and services.

SIRIM/MOA STANDARD SIRIM/MOA Standard is developed according to SIRIM standardisation procedures, which are in line with international practices that ensure appropriate notification of work programmes and participation of interested parties. As a standards development organisation, SIRIM Berhad has extensive expertise in standards research and consultancy which helps industries and businesses meet local and international requirements and practices. SIRIM/MOA Standard is developed through collaboration with SIRIM which provides requirements, specifications, guidelines or characteristics that can be used to ensure that materials, products, processes and services are fit for their purpose. SIRIM/MOA Standard is developed through consensus by established committee, which consists of experts in the subject matter. The use of this standard is voluntary, and it is open for adoption by regulators, government agencies, associations, industries, professional bodies, etc. © Copyright 2019 For further information on SIRIM/MOA Standards, please contact: Standard Department SIRIM STS Sdn Bhd 1, Persiaran Dato’ Menteri Seksyen 2, Peti Surat 7035 40700 Shah Alam Selangor Darul Ehsan Phone: 60 3 5544 6314/6909 Fax: 60 3 5510 8830 Email: [email protected] Website: http://www.sirimsts.my

OR Ministry of Agriculture and Agro-Based Industry Malaysia Blok 4G1 Wisma Tani, No. 28, Persiaran Perdana Presint 4, Pusat Pentadbiran Kerajaan Persekutuan 62624 Wilayah Persekutuan Putrajaya Phone: 60 3 8870 1200/1400 Fax: 60 3 8888 6906 Email: [email protected] Website: www.moa.gov.my

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Contents

Page

Foreword ................................................................................................................... ii 1 Scope ............................................................................................................ 1 2 Normative references .................................................................................... 1

3 Terms and definitions .................................................................................... 1 4 Extraction method .......................................................................................... 1 5 Drying technique ............................................................................................ 2 6 Requirements ................................................................................................ 2 7 Packaging and labelling ................................................................................. 6 8 Legal requirements ........................................................................................ 6 Annex A Detection of andrographolide in hempedu bumi (Andrographis

paniculata (Burm.f.) Nees) spray dried aqueous extract using high-performance liquid chromatography (HPLC) .......................................... 7

Annex B Qualitative analysis for identification of hempedu bumi (Andrographis

paniculata (Burm.f.) Nees) spray dried aqueous extract using high-performance thin-layer chromatography .............................................. 10

Bibliography ............................................................................................................. 13

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ii © SIRIM Berhad and Ministry of Agriculture and Agro-Based Industry Malaysia 2019 - All rights reserved

Foreword

SIRIM/MOA 4 standard series was developed by the Project Committee on Industry Standards for Herbs established by SIRIM Berhad. This standard consists of the following parts, under the general title, Herbal extracts - Specification: Part 1: Belalai gajah (Clinacanthus nutans (Burm.f.) Lindau) spray dried aqueous extract Part 2: Dukung anak (Phyllanthus niruri L.) spray dried aqueous extract Part 3: Halia (Zingiber officinale Roscoe) spray dried aqueous extract Part 4: Hempedu bumi (Andrographis paniculata (Burm.f.) Nees) spray dried aqueous extract Part 5: Kacip fatimah (Marantodes pumilum (Blume) Kuntze syn (Labisia pumila (Blume) Fern.-Vill) spray dried aqueous extract Part 6: Mas cotek (Ficus deltoidea Jack var kunstleri (King) Corner) spray dried aqueous extract Part 7: Misai kucing (Orthosiphon aristatus (Blume) Miq.) spray dried aqueous extract Part 8: Mengkudu (Morinda citrifolia L.) spray dried aqueous extract Part 9: Pegaga (Centella asiatica (L.) Urb.) spray dried aqueous extract Part 10: Roselle (Hibiscus sabdariffa L.) spray dried aqueous extract Part 11: Tongkat ali (Eurycoma longifolia Jack) spray dried aqueous extract This standard will be reviewed periodically to ensure that it reflects current needs and conditions. Users and other interested parties may submit comments on the contents of this standard for consideration in future versions. Compliance with this standard does not by itself grant immunity from legal obligations.

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© SIRIM Berhad and Ministry of Agriculture and Agro-Based Industry Malaysia 2019 - All rights reserved 1

Herbal extracts - Specification - Part 4: Hempedu bumi (Andrographis paniculata (Burm.f. Nees)) spray dried aqueous extract

1. Scope

This standard specifies the requirements for the production of spray dried aqueous extract derived from leaves of hempedu bumi (Andrographis paniculata (Burm. f.) Nees). which includes extraction method, drying technique, quality and safety tests and packaging and labelling requirements of the herbal extract. The herbal extract is not intended for direct consumption. It can be used as an ingredient in food, health supplements, cosmetics and traditional medicines. NOTE. Incorporating the extracts into a finish product requires an establishment of potency data.

2. Normative references

The following normative references are indispensable for the application of this standard. For dated references, only the edition cited applies. For undated references, the latest edition of the normative reference (including any amendments) applies. MS 1860:2015, Herbal products - Vocabulary SIRIM/MOA 1:2017, Good practice for primary processing of post-harvested herbs SIRIM/MOA 2:2017, Good practice for the production of herbal extracts - Aqueous and/or ethanolic extraction SIRIM/MOA 3:2017, Recommended test methods for herbs, herbal substances and herbal extracts Malaysian Herbal Monograph 2015

3. Terms and definitions For the purposes of this standard, the terms and definitions given in MS 1860:2015 and SIRIM/MOA 2:2017 apply.

4. Extraction method The extraction process shall be in accordance with 4.9, SIRIM/MOA 2: 2017. NOTE. For the purpose of this standard, the extraction conditions are as follows.

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2 © SIRIM Berhad and Ministry of Agriculture and Agro-Based Industry Malaysia 2019 - All rights reserved

a) Herbal substance to water ratio: 1:10.

b) Temperature: 70 °C 5 °C.

c) Duration of extraction process: 3 h.

5. Drying technique The herbal extract can be dried by means of spray drying techniques as described in Annex G of SIRIM/MOA 2:2017.

6. Requirements 6.1 General 6.1.1 The raw material should comply with the Malaysian Herbal Monograph 2015 or SIRIM/MOA 1:2017. 6.1.2 The herbal extract shall be evaluated based on the results of the quality tests (for the presence of designated marker compound) and safety tests as specified in 6.2, 6.3 and 6.4. 6.1.3 The amount of maltodextrin added in the herbal extract shall not be more than 30 % w/w. 6.2 Chemical composition requirements 6.2.1 The herbal extract shall comply with the chemical composition limits specified in Table 1.

Table 1. Chemical composition limits for spray dried aqueous extract of hempedu bumi (Andrographis paniculata (Burm.f.) Nees)

Chemical composition Limit a Test method

(Annex of SIRIM/MOA 3:2017)

Protein, (% w/w) > 20.0 B

Glycosaponin, (% w/w) > 20.0 C

Total polysaccharide, (% w/w) < 40.0 D

Total phenolic content, (mg GAE/g) > 30.0 E

Total alkaloid, (% w/w) < 1.0 F

Total flavonoids, (mg RE/g extract) > 20.0 J

a Limits are based on 30 % w/w maltodextrin content.

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6.2.2 Where the end product requires determination of biological properties specified in Table 2, the herbal extract shall be tested according to the specified test method and should comply with the specified limits.

Table 2. Biological property limits for spray dried aqueous extract of hempedu

bumi (Andrographis paniculata (Burm.f.) Nees)

Biological property Limit a Test method

(Annex of SIRIM/MOA 3:2017)

Antioxidant (ABTS, IC50), (mg/ml) < 2.0 G

Antioxidant (DPPH, IC50), (mg/ml) < 15.0 H

a Limits are based on 30 % w/w maltodextrin content.

6.3 Marker compound requirement 6.3.1 The herbal extract shall comply with the marker compound requirements specified in Table 3.

Table 3. Marker compound requirements for spray dried aqueous extract of hempedu bumi (Andrographis paniculata (Burm.f.) Nees)

Marker compound Requirements Test method

Andrographolide Present HPLC (refer Annex A)

Andrographolide Present HPTLC (refer Annex B)

6.3.2 Typical HPLC profiling of andrographolide standard is as shown in Figure 1.

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0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 min

-200

-150

-100

-50

0

50

100

150

200

250

300

350

400

450

500

550

mAU230nm,4nm (1.00)

And

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Figure 1. HPLC chromatogram of standard andrographolide, 100 mg/l at RT 12.38 min

6.3.3 Typical high-performance liquid chromatography (HPLC) profiling of spray dried aqueous extract of hempedu bumi (Andrographis paniculata (Burm.f.) Nees) is as shown in Figure 2.

2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 min

-200

-150

-100

-50

0

50

100

150

200

250

300

350

400

450

500

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mAU230nm,4nm (1.00)

And

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Figure 2. HPLC chromatogram of spray dried aqueous extract of hempedu bumi (Andrographis paniculata (Burm.f.) Nees)

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6.3.4 Qualitative determination of marker compound using high-performance thin-layer chromatograpy (HPTLC) is shown in Figure 3.

Key Lane 1 Andrographolide standard Lane 2 Hempedu bumi (Andrographis paniculata (Burm. f.) Nees.) spray dried aqueous extract

Figure 3. HPTLC chromatogram of spray dried aqueous extract of hempedu

bumi (Andrographis paniculata (Burm. f.) Nees.) documented under white light after derivatisation

6.4 Safety requirements 6.4.1 Permissible heavy metal and microbial contaminant limits allowed in the herbal extract are as given in Tables 4 and 5.

Table 4. Permissible heavy metals limits

Parameters Limit

(mg/kg) Test method

Arsenic (As) < 5.0

Annex L of SIRIM/MOA 3:2017 Cadmium (Cd) < 0.3

Lead (Pb) < 10.0

Mercury (Hg) < 0.5

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Table 5. Permissible microbial limits

Parameters Limits a Test method

Total bacteria count < 105 CFU/g

Annexes M and N of SIRIM/MOA 3:2017

Total yeast and mould count <104 CFU/g

Bile-tolerant gram negative bacteria

< 104 CFU/g

Salmonella spp Absent in 25 g of sample

Escherichia coli Absent in 1 g of sample

Staphylococcus aureus Absent in 1 g of sample

Pseudomonas aeruginosa Absent in 1 g of sample

a Malaysian Herbal Monograph, 2015.

6.4.2 Additional safety testing shall be performed based on the intended use of the end products.

7. Packaging and labelling The requirements for packaging and labelling of the herbal extract shall be as specified in 4.17 of SIRIM/MOA 2:2017.

8. Legal requirements 8.1 The manufacturer shall ensure that the safety requirements stipulated by relevant statutes and regulations for the products in which the herbal extracts are used, are met. NOTE. Products that incorporate herbal extracts such as food, cosmetics, health supplements and traditional medicines are subject to Food Act 1983, Food Regulations 1985, ASEAN Cosmetic Directive, Poison Act 1952 and Sale of Drugs Act 1952, where relevant.

8.2 The manufacturer shall also ensure that all other relevant statutory and regulatory requirements enforced in Malaysia, including those related to the operation of the facilities used for the production of herbal extracts, are complied with.

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Annex A (normative)

Detection of andrographolide in hempedu bumi (Andrographis paniculata (Burm.f.) Nees) spray dried aqueous extract using high-

performance liquid chromatography (HPLC)

A.1 Scope This annex describes the detection of the marker compound in spray dried aqueous extract of hempedu bumi (Andrographis paniculata (Burm.f.) Nees) using high-performance liquid chromatography (HPLC).

A.2 Summary of test method Andrographolide is detected using high-performance liquid chromatography (HPLC) method. Peak is identified on the basis of their retention times and standards.

A.3 Apparatus Use the usual laboratory apparatus and, in particular, the following. A.3.1 Membrane filter, pore size 0.45 µm. A.3.2 HPLC, consisting of pump, sample injector, UV detector, temperature regulated column oven at 30 °C and integrator. A.3.3 Analytical column, C18 column (5 µm, 150 mm × 4.6 mm). A.3.4 Analytical balance, accurate to 0.001 g.

A.4 Reagents

Unless otherwise specified, use reagents of recognised analytical grade and the following. A.4.1 Formic acid, 0.1 %. A.4.2 Acetonitrile, HPLC grade. A.4.3 Methanol, HPLC grade. A.4.4 Andrographolide standard, CAS No. [5508-58-7] (purity ≥ 98%).

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A.4.5 Ultra-pure water or water of equivalent purity, for example double distilled deionised water.

A.5 Procedure

A.5.1 Preparation of andrographolide standard stock solution Weigh 1.0 mg of andrographolide standard, add 1 ml of methanol and sonicate the mixture to dissolve the standard material. Label as stock solution of 1 000 mg/l. A.5.2 Preparation of andrographolide standard working solution Dilute the stock standard solution to make 100 mg/l standard solution. A.5.3 Preparation of test sample for hempedu bumi spray dried aqueous extract Weigh 200 mg of hempedu bumi spray dried aqueous extract into 5 ml volumetric flask and mark to volume with methanol. Sonicate the sample solution for 20 min at room temperature and evaporate to dryness. Dissolve the concentrate with 5 ml methanol. Sonicate for 20 min at room temperature, filter and transfer to a HPLC vial for analysis. A.5.4 Chromatographic condition A.5.4.1 Detector, UV 230 nm. A.5.4.2 Column, C18 (5 µm, 150 mm x 4.6 mm). A.5.4.3 Column oven temperature, 30 °C. A.5.4.4 Flow rate, 1.0 ml/min. A.5.4.5 Injection volume, 10 µl. A.5.4.6 Run time, 25 min. A.5.5 Mobile phase (gradient mode) solution preparation for HPLC A.5.5.1 Solution A: Prepare 0.1 % of formic acid in water, filter through 0.45 µm membrane filter followed by 15 min of sonication. A.5.5.2 Solution B: Acetonitrile, filter through 0.45 µm membrane filter followed by 15 min of sonication.

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Table A.1 Mobile phase (gradient mode) solution preparation for HPLC

Time (min)

Solution A acetonitrile

(%)

Solution B 0.1 % formic acid

(%)

0 5 95

7 35 65

15 35 65

20 5 95

25 5 95

A.5.6 Standard andrographolide Inject 10 µl of standard (A.5.2) into HPLC system employing the above condition to obtain retention time. A.5.7 Detection of andrographolide in sample Inject 10 µl of sample into HPLC system. Record the retention time of the andrographolide in the sample. A.5.8 System suitability requirements Perform at least six replicate injections using (A.5.2) the prepared concentration of andrographolide standard. The requirements of the system suitability parameters are as follows. a) Symmetric factor (As) shall not be more than 1.5. b) Percentage of relative standard deviation (RSD) of the retention time (RT) for

andrographolide shall not be more than 2.0 %.

A.6 Acceptance criteria The acceptance criteria set as retention time (RT) of andrographolide of the tested sample and shall be similar to RT of the standard.

A.7 Expression of results The presence of andrographolide in the sample is detected based on the retention time (RT) of the standard.FOR STAKEHOLD

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Annex B (normative)

Qualitative analysis for identification of hempedu bumi (Andrographis paniculata (Burm. f.) Nees) using high-performance thin-

layer chromatography (HPTLC)

B.1 Scope This annex describes a procedure for use in a herbal material identification test using high-performance thin layer chromatography (HPTLC).

B.2 Summary of test method It is applicable for authentication and identification of herbal extract by chromatographic fingerprinting.

B.3 Apparatus B.3.1 Sample applicator, e.g. auto sampler injector or semi automatic injector or microcapillary tube for manual injection. B.3.2 Automatic development chamber or a manual development tank. B.3.3 TLC sprayer or automatic dipping device. B.3.4 TLC plate heater or hot plate or oven. B.3.5 TLC visualiser or UV lamp for TLC.

B.4 Reagents B.4.1 Standard solution Dissolve 1 mg andrographolide standard in 1 ml methanol to produce 1 000 mg/l solution.

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B.4.2 Sample solution Weigh 200 mg of hempedu bumi spray dried aqueous extract into 5 ml volumetric flask and mark to volume with methanol. Sonicate the sample solution for 20 min at room temperature and evaporate to dryness. Dissolve the concentrate with 5 ml methanol. Sonicate for 20 min at room temperature, filter. B.4.3 Derivatisation reagent Mix 170 ml of ice cooled methanol with 20 ml acetic acid, followed by 10 ml sulphuric acid and 1ml of anisaldehyde.

B.5 Chromatographic system B.5.1 HPTLC plates HPTLC plates of suitable dimension coated with silica gel 60 F254 with fluorescent indicator as absorbent. B.5.2 Application volume

6 l of standard solution and 10 µl of sample solution, as 8 mm bands. B.5.3 Developing mobile phase A mixture containing toluene, ethyl acetate and formic acid at a ratio of 5:3.5:1.5 B.5.4 Developing distance 8 cm from the bottom of the plate.

B.6 Procedure B.6.1 Preconditioning of the plate If an automated development chamber is available, condition the plate to a relative humidity of 33 % for a minimum of 10 min with a saturated solution of magnesium chloride and report the humidity and temperature. If this is not available, proceed to B.6.2 B.6.2 Preparation of development chamber Use a flat or a twin trough chamber to develop the chromatogram. Place filter paper inside the chamber and saturate for 20 min with the mobile phase (B.5.3).

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B.6.3 Analysis B.6.3.1 Apply 6 µl standard solution and 10 µl sample solution as bands to a suitable HPTLC plate and air dry. B.6.3.2 Insert the plate in the saturated chamber (B.6.2), develop until the developing distance reaches 8 cm from the bottom of the plate, remove the plate from the chamber, and air dry.

B.6.3.3 Treat with derivatisation reagent, heat the resultant plate at 100 °C for 3 min and examine under white light.

B.7 Acceptance Criteria B.7.1 Under white light after derivatization, the chromatogram of the sample solution exhibits a purple band in the middle third of the plate due to andrographolide, corresponding in color and Rf to that in the standard solution.

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Bibliography [1] Food Act 1983 [2] Food Regulations 1985 [3] Poison Act 1952 [4] Sale of Drugs Act 1952 [5] Malaysian Herbal Monograph, 2015 [6] ISO 14502-1 Determination of substances characteristic of green and black tea

- Part 1: Content of total polyphenol in tea - Colorimetric method using Folin-Ciocalteu reagent

[6] British Pharmacopoeia (BP) 2014 appendix XVI B. Microbiological Examination

of Non-sterile Products [8] EPA SW 846 7473 using NIC Mercury Analyser [9] Reyes et al. (2016) Pretreatment for hydrolysis Bio resources 11(1), 612-625 [10] Standard Methods for the Examination of Water and Wastewater, 21st Edition

2005 APHA, 3030 E. Nitric Acid Digestion [11] USP 233: Elemental Impurities - Procedures/Chemical Test [12] Quality control methods for medicinal plant materials, 1998, WHO [13] ASEAN Cosmetics Directive

Downloadable from http://www.hsa.gov.sg/content/hsa/en/Health Products Regulation/Cosmetic Products/Overview/ASEAN Cosmetic Directive.html

[14] US Pharmacopoeia chapter 203, High-Performance Thin-Layer

Chromatography Procedure for Identification of Articles of Botanical Origin Downloaded from https://hmc.usp.org/sites/default/files/documents/HMC/GCs-

Pdfs/c203.pdf [15] Standard Operating Procedure for HPTLC, Version 03, 26 July 2018, The

International Association for the Advancement of High-Performance Thin-Layer Chromatography (HPTLC Association)

Downloaded from file:///C:/Users/norazian/Downloads/SOP_for_HPTLC_ZHAW_new_14_Feb_2017.pdf

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Acknowledgements

SIRIM Berhad would like to thank the members of the Project Committee on Industry Standards for Herbs who have contributed their ideas, time and expertise in developing this standard Ms Sarifah Rejab (Chairman) SIRIM Berhad (Industrial Biotechnology

Research Centre) Nor Azian Duriat (Technical Secretary)

SIRIM STS Sdn Bhd

Mohd Suhaifizan Arifin Bioalpha R&D Sdn Bhd

Ms Jean Wong Hoi Jin Biotropics Malaysia Berhad

Mr Mohd Khairul Nizam Mazlan Centre for Herbal Standardisation, Universiti Sains Malaysia

Ms Fauziah Abdullah/ Dr Ling Sui Kiong

Forest Research Institute of Malaysia

Mr Mohd Isa Wasiman Institute for Medical Research

Dr Norma Hussin Malaysian Agricultural Research and Development Institute (MARDI)

Ms Syalwati Asnawi Ministry of Agriculture and Agro-Based Industry Malaysia

Mr Mohd Helme Mohd Helan Ministry of Energy, Science Technology, Environment and Climate Change

Ms Zakiah Abd Ghafar/ Ms Azrina Hassan/ Mr Mohamed Shahrizan Shahril/ Mr Muhammad Shahariz Mohamad Adzib/ Ms Nur Azra Mohamad Pauzi

National Pharmaceutical Regulatory Agency

Mr Salman Zhari Orchid Life Sdn Bhd

Ms Siti Aishah Asmah Yusob/ Mr Anuar Nordin/ Ms Karimah Muhamad

SIRIM Berhad (Environmental Technology Research Centre)

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Acknowledgements (continued)

Dr Theanmalar Masilamani/ Mr Lim Chuan Gee/ Ms Noorrasyidah Mohd Sarmin/ Ms Nurul Farah Athirah Shamsuri/ Ms Nurul Hammizah Hamidon/ Ms Thavamanithevi Subramaniam/ Ms Aidawati Mohamed Shabery/ Ms Siti Kasmarizawaty Suboh/ Ms Badariah Abdullah/ Ms Nurul Husna Abdullah/ Ms Dahlia Daud/ Ms Siti Hamizah Abdul Rahman/ Ms Norhafizah Mat Zan/ Ms Norfaeza Zainuddin/ Mr Mohd Mahayuddin Hussin Mr Mohd Khairul Azwan Ahmad Ms Faridah Rosdi Ms Murni Ismail Mr Mohd Hafiz Abdul Ghani/ Dr Zanariah Ujang/ Dr Ahmad Hazri Ab Rashid

SIRIM Berhad (Industrial Biotechnology Research Centre)

Prof Dr Zhari Ismail Universiti Sains Malaysia

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© Copyright 2019

All rights reserved. Unless otherwise specified, no part of this standard may be reproduced or utilised in any form or by any means, electronic or mechanical, including photocopying, recording or otherwise, without prior written permission from SIRIM Berhad and Ministry of Agriculture and Agro-Based Industry Malaysia.

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Documents

DMS Page for Acknowledgement · 2019-09-19 · Figure 3. HPTLC chromatogram of spray dried aqueous extract of hempedu bumi (Andrographis paniculata (Burm. f.) Nees.) documented under