SIRIM/MOA STANDARD ONLY · MOA . The Ministry of Agriculture and Agro-based Industry (MOA), is a...

SIRIM/MOA 4-2:2019

ICS: 71.100.99

Herbal extracts - Specification - Part 2: Dukung anak (Phyllanthus niruri L.) 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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mailto:[email protected]

SIRIM/MOA 4-2:2019

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

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 corilagin in dukung anak (Phyllanthus niruri L.) spray dried aqueous extract using high-performance liquid ................................ chromatography (HPLC)………………………………………………..……...7 Annex B Qualitative analysis for identification of dukung anak (Phyllanthus niruri L.) Urb.) spray dried aqueous extract using high-

performance thin-layer chromatography (HPTLC).………………….…….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 2: Dukung anak (Phyllanthus niruri L.) spray dried aqueous extract

1. Scope This standard specifies the requirements for the production of spray dried aqueous extract derived from leaves of dukung anak (Phyllanthus niruri L.) which includes extraction method, drying technique, quality and safety tests and packaging and storage 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 of SIRIM/MOA 2:2017.

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NOTE. For the purposes of this standard, the extraction conditions are as follows.

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 technique 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 dukung anak (Phyllanthus niruri L.)

Chemical composition Limit a Test method

(Annex of SIRIM/MOA 3:2017)

Protein, (% w/w) > 25 B

Glycosaponin, (% w/w) > 15 C

Total polysaccharide, (% w/w) < 40 D

Total phenolic content (TPC), (mg GAE/g) > 100 E

Total alkaloid, (% w/w) < 1 F

Total flavonoids, (mg RE/g extract) > 0.3 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 dukung

anak (Phyllanthus niruri L.)

Biological property Limit a Test method

(Annex of SIRIM/MOA 3:2017)

Antioxidant (ABTS, IC50), (mg/ml) < 0.4 G

Antioxidant (DPPH, IC50), (mg/ml) < 1 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 dukung anak (Phyllanthus niruri L.)

Marker compound Requirements Test method

Corilagin Present HPLC (refer Annex A)

Corilagin Present HPTLC (refer Annex B)

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

Figure 1. HPLC chromatogram of corilagin standard 100 mg/l at 10.683 min

5.0 10.0 15.0 20.0 25.0 30.0 min

0

25

50

75

100

125

150

175

200

225

250

mAU270nm,4nm (1.00)

Co

rila

gin

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6.3.3 Typical high-performance liquid chromatography (HPLC) profiling of spray dried aqueous extract of dukung anak (Phyllanthus niruri L.) is as shown in Figures 2a and 2b.

Figure 2a. HPLC chromatogram of spray dried aqueous extract of dukung anak (Phyllanthus niruri L.)

Figure 2b. HPLC chromatogram of dukung anak (Phyllanthus niruri L.) aqueous extract (through zooming showing peaks corresponding to

corilagin).

0.0 5.0 10.0 15.0 20.0 25.0 30.0 min

-200

-100

0

100

200

300

400

500

600

700

800

900

1000

1100

1200

1300

1400

mAU270nm,4nm (1.00)

Cor

ilagi

n

2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 min

0

25

50

75

100

125

150

175

200

225

250

mAU270nm,4nm (1.00)

Cor

ilagi

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

Key Lane 1. Corilagin standard Lane 2. P. niruri spray dried aqueous extract (North), 0.2 g/mL methanol Lane 3. P. niruri spray dried aqueous extract (South), 0.2 g/mL methanol Lane 3. P. niruri spray dried aqueous extract (South), 0.2 g/mL 70% ethanoll

Figure 3. HPTLC chromatogram of spray dried aqueous extract of dukung anak (Phyllanthus niruri L.) documented under white light

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 metal limits

Parameter 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

Parameter Limit Test method a

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 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 corilagin in dukung anak (Phyllanthus niruri L.) spray dried aqueous extract using high-performance liquid chromatography (HPLC)

A.1 Scope This annex describes the determination of corilagin content in spray dried aqueous extract of dukung anak (Phyllanthus niruri L.) by using high-performance liquid chromatography (HPLC).

A.2 Summary of test method Corilagin content is identified using high-performance liquid chromatography (HPLC) with UV detector. Peaks are 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, temperature-regulated UV detector, temperature regulated column oven at 30 °C and integrator. A.3.3 Analytical column, C18 (5 µm, 250 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 Acetic acid, 0.1 %. A.4.2 Acetonitrile, HPLC grade. A.4.3 Ethanol, HPLC grade. A.4.4 Corilagin standard, CAS No [23094-69-1] (purity ≥ 98 %)

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A.4.5 HPLC water or water of equivalent purity, for example double distilled deionised water

A.5 Procedure A.5.1 Preparation of corilagin standard stock solution Weigh 1 mg of corilagin standard, add 1 ml of 70 percent v/v solution of ethanol and sonicate the mixture to dissolve the standard material. Label as stock solution of 1 000 mg/l. A.5.2 Preparation of corilagin standard working solution Dilute the stock standard solution to make 100 mg/l standard solution. A.5.3 Preparation of test sample for dukung anak spray dried aqueous extract Weigh 100 mg of dukung anak spray dried aqueous extract into a 10 ml volumetric flask and mark to 10 ml with 70 percent v/v solution of ethanol. Sonicate the mixture for 20 min at room temperature, filter the solution and transfer to a HPLC vial for analysis.

A.5.4 Chromatographic conditions

A.5.4.1 Detector, UV 270 nm.

A.5.4.2 Column, C18 (5µm, 250 mm × 4.6 mm,). A.5.4.3 Column oven temperature, 30 °C. A.5.4.4 Flow rate,1.5ml/min. A.5.4.5 Injection volume, 20 µl.

A.5.4.6 Run time, 34 min. A.5.5 Mobile phase (gradient mode) solution preparation for HPLC A.5.5.1 Solution A: Prepare 0.02 % of acetic 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 0.02 % of acetic acid

(%)

Solution B acetonitrile

(%)

0 100 0

4 80 20

30 70 30

34 20 80

A.5.6 Standard corilagin chloride Inject 20 µl of standard (A.5.2) into HPLC system employing the above conditions to obtain retention time. A.5.7 Detection of corilagin in sample Inject 20 µl of sample into HPLC system. Record the retention time of the corilagin in the sample. A.5.8 System suitability requirements Perform at least six replicate injections using (A.5.2) the prepared concentration of corilagin 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

corilagin shall not be more than 2.0 %.

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

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

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Annex B (normative)

Qualitative analysis for identification of dukung anak (Phyllanthus niruri L.) Urb.) spray dried aqueous extract 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 extracts by chromatographic fingerprinting.

B.3 Apparatus B.3.1 Sample applicator, e.g. automatic TLC sampler or semi-automatic sampler or microcapillary tube for manual loading. 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 corilagin in 1 ml methanol to produce 1 mg/ml solution B.4.2 Sample solution Weigh 0.2 g of dukung anak (Phyllanthus niruri L.) spray dried aqueous extract, add 1.0 ml of methanol, vortex for 1 min then sonicate for 45 min at 40 ºC, and filter through a 0.45 µm membrane filter.

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Or Weigh 0.2 g of dukung anak (Phyllanthus niruri L.) spray dried aqueous extract, add 1.0 ml of 70 % ethanol, vortex for 1 min then sonicate for 45 min at 40 ºC, and filter through a 0.45 µm membrane filter. B.4.3 Derivatisation reagent 3 % ferric chloride in ethanol. B.5 Chromatographic system B.5.1 HPTLC plates HPTLC plates of suitable dimension coated with silicagel 60 F254 with fluorescent indicator as absorbent. B.5.2 Application volume 4 µl of standard solution and 8 µl of sample solution, as 8 mm bands. B.5.3 Mobile phase A mixture containing ethyl acetate, formic acid, glacial acetic acid and water at a ratio of 100:11:11:26. 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 the developing 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 developing solvent (B.5.2). B.6.3 Analysis B.6.3.1 Apply the standard solution and the sample solution as bands to a suitable HPTLC plate and air dry.

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B.6.3.2 Develop the chromatograms in a saturated chamber, remove the plate from the chamber, and dry. B.6.3.3 Examine under white light. B.6.3.4 Treat with derivatisation reagent, heat the resultant plate at 100 oC for 3 min and examine under white light. B.7 Acceptance criteria Under white light after derivatisation, the chromatogram of the sample solution exhibits a brown band in the middle third of the plate due to corilagin, corresponding in colour and Rf in the standard solution.

Under white light after derivatisation, the chromatogram of the sample solution exhibits additional brown bands above the band due to corilagin.

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Bibliography [1] Food Act 1983 [2] Food Regulations 1985 [3] Poison Act 1952 [4] Sale of Drugs Act 1952 [5] ASEAN Cosmetics Directive

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

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

Chromatography Procedure for Identification of Articles of Botanical Origin Downloadable from

https://hmc.usp.org/sites/default/files/documents/HMC/GCs-Pdfs/c203.pdf [7] Wagner, H.; Bladt, S. Plant Drug Analysis: A Thin Layer Chromatography Atlas,

2nd edition, Springer 2001 [8] Kunle, O.F.; Egharevba, H.O.; Ahmadu, P.O. Standardization of herbal

medicines - A review, International Journal of Biodiversity and Conservation, 2012, 4, 101-112

[9] J Garg, V.; Dhar, V.J.; Sharma, A.; Dutt, R. Facts about standardization of

herbal medicine: A review, Journal of Chinese Integrative Medicine: 2012, 10, 1077-1083

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http://www.hsa.gov.sg/content/hsa/en/Health

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

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) Ms Nor Azian Duriat (Technical Secretary)

SIRIM STS Sdn Bhd

Mohd Suhaifizan Arifin Bioalpha R&D Sdn Bhd

Ms Wong Hoi Jin, Jean 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)

Ms Thavamanithevi Subramaniam/ Dr Theanmalar Masilamani/ Mr Lim Chuan Gee/ Ms Noorrasyidah Mohd Sarmin/ Ms Nurul Farah Athirah Shamsuri/ Ms Nurul Hammizah Hamidon/ 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 Jamia Azdina Jamal Universiti Kebangsaan Malaysia 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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