Application Of NMR Based Metabolomics and Multivariate Data Analysis For Quality Control of Herbal Material

editor:

Ratih Dewanti-Hariyadi

Lilis Nuraida

Desty Gitapratiwi

Nelis Immaningsih

Purwiyatno Hariyadi

A/ERFR/T

W

-

CENTER-Southeast Asian Food and Agricultural Science and Technology (SEAFAST) Center

CURRENT ISSUES AND

CHALLENGES' IN FOOD SAFETY

SCIENCE-BASED APPROACH FOR FOOD SAFETY MANAGEMENT

Proceeding of The International Seminar 'Current Issues and Challenges in Food Safety: Science-Based Approach for Food Safety Management'

Bogor, December 2-3, 2009

Organized by:

Southeast Asian Food & Agricultural Science & Technology (SEAFAST) Center Bogor Agricultural University

International Commision on Microbiological Specifications for Foods (ICMSF) Norman Borlaug Institute for International Agriculture

Texas A & M University

Editor:

Ratih Dewanti-Hariyadi Lilis Nuraida Desty Gitapratiwi Nelis Immaningsih Purwiyatno Hariyadi

Southeast Asian Food & Agricultural Science & Technology (SEAFAST) Center, Bogor Agricultural University

Current Issues and Challenges in Food Safety

CURRENT ISSUES AND CHALLENGES IN FOOD SAFETY

SCIENCE-BASED APPROACH FOR FOOD SAFETY MANAGEMENT

Proceeding of The International Seminar 'Current Issues and Challenges in Food Safety: Science-Based Approach for Food Safety Management'

Bogor, December 2-3, 2009

Editor

Ratih Dewanti-Hariyadi Lilis Nuraida Desty Gitapratiwi Nelis Immaningsih Purwiyatno Hariyadi

Publisher

Southeast Asian Food & Agricultural Science & Technology (SEA FAST) Center Bogor Agricultural University

Bogor-Indonesia

National Library Republic of Indonesia ISBN 978-602-96665-3-3

Acknowledgement

Thanks to Leo Wibisono Arifin, Vesica Dwi Ariesta, Amelinda Angela, Vita Ayu Puspita, Palestina Santana, Virna Berliani Putri, Zulaikhah and Nurwandi NC

for preparing manuscript of this proceeding

Copyright©2010

Southeast Asian Food & Agricultural Science & Technology (SEAFAST) Center, Bogor Agricultural University

ii

I

Proceeding of the International Seminar

CONTENT

Preface ... iii Welcome Speech of Rector of Bogor Agricultural University ... xi Organizers ... xv

Keynote Speech

Cold Chain: Managing Food Safety for Fresh Produce and Frozen Food Product ... 1

Derk Van Mackelenbergh

PLENARY PRESENTATION

Microbiological Food Safety

A Modern Approach to Food Safety Management ... 9

Martin Cole

Use of Epidemiological Data for The Improvement of Food Safety ... 21

Fu miko Kasuga

Implementation of Food Safety Management at Industry Level in Developing Countries: Is GmpjHaccp Confusing? ... 29

Ratih Dewanti-Hariyadi

Chemical Food Safety

Controlling Allergens: Industry Perspective ... 41

Soo Chuah

Food Safety Issues in Food Packaging - Aseptic Technology

Approach ... 51

Hari Yanto Tekno Yuwono

Science-based Approach to Food Safety

Predictive Microbiology ... 61

Tom Ross

Ri sk Analysis: Using Risk Assessment to Establish Risk Based

Metrics ... 75

Leon Gorris

Role of Microbiology Criteria and Limitations of Sampling ... 89

Tom Ross

Current Issues and Challenges in Food Safety

Safety of Food Biotechnology

Safety of Food Biotechnology Products ... 1 03

James Maryanski

Safety Assessment of Genetically Modified Food in Indonesia ... 111

Maggy Thenawidjaja Suhartono

TECHNICAL PRESENTATION

Managing the Safety and Quality of Fresh Produce

Risk Assessment Of Listeria Monocytogenes In Raw Vegetables ... 121 Son Radu

Good Agricultural Practice: A Vital Program for Ensuring

Microbial Safety of Fresh Produce ... 139

Maria Auxilia T. Siringan

Biosafety of Vibrio Parahaemolyticus in Raw Salad Vegetables at Retail and Farm Level ... 147

Tunung Robin

The Effect of Blanching Method, Storage and Temperature to the Charcteristics of Cowpea Tempeh Subtituted by 40% Soybean ... 167

Mery Tambaria Damanik Ambarita

Microbiological Risk Assessment of Fresh Water Aquaculture

Fish in Malaysia: from Farm to Table ... 187

Rizal Damanik

Implementation of Food Safety and Quality Management at Industry Level

Control of Salmonella in Nut Processing ... 199

Chaven Suchart

Integrated Food Safety Management-Kraft Approach ... 213

Soo Chuah

Shelf-Life Prediction of Seasoning Powder Made from Whole

Fermented Fish (Peda) by Using Arrhenius Method ... 221

Meta Mahendradatta

Chemoreaction Drying and its Effect on Black Pepper Quality ... 235

Nur Wulandari

Proceeding of the International Seminar

Application of NMR Based Metabolomics and Multivariate Data Analysis for Quality Control of Herbal Material ... 243

ancy Dew; Yuliana

anaging Food Safety and Quality in Food Services and Retail Industries

anaging Food Safety in Food Retail: A Lesson Learnt ... 259

Andi Nur'aida

Food Safety Management in Aerofood Angkasa Citra Sarana

(ACS) Catering Service ... 265

Hoerip Satyagraha

Formalin Contamination in Children's Street Foods at Schools in Surakarta, Central Java, Indonesia ... 273

Indrias Tri Purwanti

Isolation Of Enterobacter sakazakii (Cronobacter Spp) from Powdered Infant Formula and Other Dry Foods Obtained from

Bogor Area, Indonesia ... 281

Rlltih Dewanti-Hariyadi

POSTER PRESENTATION

Inhibition of Duku (Lansium domesticum) Spoilage using Ozone ... 287

Anny Yanuriati

utrition Properties and the Prospect of Six Amorphophallus

Species of Tubers in Java ... 299

Yuzammi

IGewang (Corypha utan lam.) as Local Food in Timor Island and

Its Nutritional Properties ... 307

Joko Ridho Witono

Two Stages Tea Chemical Withering during Peak Season ... 321

lJadan Rohdiana

The Physiology of Organs and Organism of Mus Musculus

Induced Repeatedly with Formalin-Contaminated Fish and

Chlorophyllin ... 329

Alfonds Andrew Maramis

Current Issues and Challenges in Food Safety

The Occurrence of Pseudomonas sp in Grouper Fish Fillets

(Plectropoma leopardus) at Bone Pante ... 345

Rieny Sulistijowati

HACCP Plan for Green Tea Product of PT GT and its

Implementation ... 359

Raniayu

Establishment and Implementation Sanitation Standard

Operating Procedures (SSOP) in Green Tea Processing ... 369

Ratna Handayani

Utilization of Leaves of Kunyit (Curcuma domestica Val.), Ruku-Ruku (Ocimum gratissimum. L) and Mangkokan (Nothopanax

cutellarium Merr.) in Steamed Fish (Rastrelliger Sp) Processing ... 383

Novelina

Effect Of Time-Temperature and Amylose Content of Rice on

the Color and Texture of Rice-Based Emergency Canned Food ... 391

Elvira Sya msir

Safety of School Children Foods in Indonesia ... 399

Nuri Andarwulan

The Role of Women in Community Management of Biosecurity

and Food Safety ... 415

Eka Martiningsih

APPLICATION OF NMR BASED METABOLOMICS

AND MULTIVARIATE DATA ANALYSIS FOR

QUALITY CONTROL OF HERBAL MATERIAL

Nancy Dewi Yuliana1.2, Hye Kyong Kim2, Frank van der Kooy2,

Henrie Korthourt3, Young Hae ChoP, and Rob Verpoorte2

1 Dept. Food Science and Technology, Bogor Agricultural University,

Bogor, Indonesia

2 Dept. Pharmacognosy, Sect. Metabolomics, Institute of Biology,

Leiden University, The Netherlands

3 Fytagoras BV Plant Science, Leiden, The Netherlands

ABSTRACT

Despite of lack of solid scientific proof for its efficacy and safety, up to now herbal medicines are widely used both in developed and developing countries. Standardization by focusing on active constituents or marker compounds is important but sometimes it is not sufficient to guarantee the quality of herbal preparations. NMR based metabolomics is a reliable tool to provide a wealth of information concerning plant metabolites since it is able to comprehensively detect a diverse groups of plant metabolite in a single run. In combination with multivariate data analysis, the obtained NMR data can be applied to determine any adulterants or substitutes in commercial herbal preparation which cannot be detected by other chromatographic methods. Here we reviewed three projects of our lab in Leiden, focusing on the application of NMR based metabolomics and multivariate data analysis, particularly principal component analysis for quality control of herbal medicines.

INTRODUCTION

Herbal medicine has been used since ancient times to treat a wide range of diseases and to improve the health status of human beings. Despite the advanced development of modern medicine, herbal preparations are still widely used both in developed and developing countries. The reasons are various; the belief that herbals are made from natural sources therefore safer than chemical drugs

l

Science-Based Approach/or Food Safety Management

(which is not always true); diminishing trust to modern medicine due to its side effects; or lack of access to modern medicine (due to poverty or geographical factors) (Calixto, 2000; Elvin Lewis and Steve, 2005). Two important issues concerning the use of herbal medicine are proof of efficacy and safety. The last issue is the focus of this paper, highlighting on the possibility of adulteration or substitution with other plant which may not contain the reported active principles or even worse, contain toxic substances. As an example; in 1992 many young women in Belgium suffered from progressive permanent kidney damage after consuming a Chinese slimming herbal which consisted of Stephania tetrandra and

Magnolia officinalis. It is supposed that Stephania tetrandra was substituted with A ristolochia fangchi which contains aristolochic acid, a nephrotoxin and carcinogen (van Herweghem, 1998).

To address such issues, chemical standardization on the basis of active compounds or other marker compounds in case the active

compounds are unknown, is necessary. Chemical standardization involves the use of chromatographic or spectroscopic methods to identify active/marker compounds (Ong, 2004). Many analytical methods such as TLC, HPLC, LC/MS, and GC/MS can be used for that purpose. However, sometimes only targeting the marker compound is not sufficient to detect adulterants/substitutes, especially when chemical marker compounds are spiked into adulterants/substitutes while the adulterants/substitute may contain toxicant instead. In such a case, information concerning a wide range of metabolites present in the herbal preparation becomes necessary. This could be a hurdle since plants have a large number and wide variety of compounds, and herbal medicine may consist of two or more different plant materials. A wide spectrum of analytical techniques which are rapid, reproducible, and stable over time without an elaborative sample preparation is required (Choi et al., 2005). The aforementioned analytical methods have limitations to provide information about all metabolites due to the wide structural variation, as well as physical properties and chemical characteristics of compounds present in plant material (Jahangir et af., 2008).

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Proceeding of International Seminar Current Issues and ChaLLenges in FOOd Safety

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ｾ＠ QUALITY CONTROL OF HERBAL MATERIAL WITH NMR-BASED

.<:' METABOLOMICS AND MULTIVARIATE DATA ANALYSIS

The term metabolome represents the collection of all metabolites in a biological organism as the end product of their gene expression (van der Kooy et a/., 2009). One of the first application of nuclear magnetic spectroscopy (NMR) for ,fingerprinting of plant extracts was conducted in our lab, that was for characterizing plant cell cultures and quantitative analysis of sugars in the cell culture extracts (Schripsema and Verpoorte, 1991). Although the sensitivity is lower than other methods such as mass spectrometry (MS), NMR has some unique advantages since it is a quick non-destructive method which simultaneously detects diverse groups of plant metabolites in a single run (Choi et al., 2004). It is able to detect all molecules containing NMR-active nuclei and no selective ionization (like in MS) or the presence of chromophores (such as in ultraviolet spectroscopy) are required (van der Kooy et al., 2009). The huge data set reSUlting from NMR measurement need multivariate data analysis (MVA) methods for interpretation. The most commonly used MVA method is unsupervised principal component analysis (PCA). In PCA, the data are transformed into a new coordinate system so that the biggest variance by any projections of the data lie on the first coordinate (PC1), the second coordinate (PC2), and so on. The dimensionality of the data is reduced while the characteristics of the data set that contribute most to it are preserved (Eriksson et al.,

2006; van der Kooy et al., 2009). By using PCA it is possible to classify the data when only little information about the data is available (Eriksson et

a/.,

2006).

In our lab, several studies related to quality control of botanicals by using NMR based metabolomics coupled with multivariate data analysis, in particular PCA, have been conducted. Herewith we give three examples of those studies.

1. Metabolic Fingerprinting of Ephedra Species Using lH-NMR Spectroscopy and PCA (Kim et a/., 2005)

Ephedra or Ma Huang, with ephedrine type alkaloids (e.g.

ephedrine, pseudoephedrine, methylephedrine and norephedrine) as the primary active ingredients, is used as a weight loss agent and to boost performance of athletes (Haller

Science-Based Approach/or FoodSa/ety ｍ｡ｮ｡ｧ･ｭｾｮｴ＠

and Benowitz, 2000; Calfee and Fadale, 2006). Ephedra sinica

considered as the main source of the active ingredients the ephedrine alkaloids, although other Ephedra species may also contain these alkaloids though in different concentrations. In this paper, the use of IH NMR coupled with PCA for the metabolite fingerprinting of Ephedra species was reported. The discrimination between 3 different species of Ephedra (E. sinica,

E. intermedia, E. distachya var. distachya) and nine commercial

Ephedra samples was shown.

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A. Score Plot of PCl and PC2 of organic fraction of Ephedra species

B. Loading plot of PCl of organic fraction of Ephedra species

C. Score plot of PCl and PC2 of aqueous fraction of Ephedra species D. Loading plot of PCl of aqueous fraction of Ephedra species

E. Score Plot of PCl and PC2 of aqueous fraction for Ephedra including mixture of E. sinica and E. intermedia

(S=E. sinica; I=E. intermedia; D=E. distachya var. distachya. 1-9, commercial

Ephedra herbs, SI12=mixture of E. sinica and E. intermedia (1 : 2, wjw),

SI11=mixture of E. sinica and E. intermedia (1 : 1, wjw), SI21=mixture of E. sinica

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The solvent mixture of CHCb-MeOH-HzO-NH40H was used to extract metabolites from Ephedra species. Organic and aqueous fractions were measured by NMR separately. The most alkaloids were present in the organic solvent phase. The PCA score plots and loading plots for each fractions are presented in Fig. 1.A-D. Figure 1.A. shows that the three species of Ephedra are separated from each other. The loading plot of PCl was analyzed further since it is the main PC for the separation. It was found that characteristic NMR signals for ephedrine alkaloids such as CH3 at 8 0.9-0.8 ppm, N-CH3 at 8 2.60 ppm, and the aromatic signal at 8 7.3 ppm were responsible for the separation. These alkaloids are higher in E. sinica while ephedrine was not detected

in E. distachya var. distachya, therefore this species was located

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outside of the 95% Hotelling T2 confidence eclipse. Analysis of aqueous fractions was also performed since there was no clear separation between commercial Ephedra samples. From fig. LC-D, it is obvious that all commercial samples clustered close to E.

intermedia except sample number 6. Analysis of the aromatic

area showed typical signals of benzoic acid analogues at 8.08 (d, 1-7.2 Hz), 7.70 (t, 1-7.5 Hz), and 7.56 (t, 1-7.8 Hz) and monosubstituted phenolic compounds like phenylalanine at d 7.46 (m) which are higher in E. intermedia. Sample 6 was located between E. sinica and E. intermedia. PCA analysis of a mixture of

E. sinica and E. intermedia (fig.1.E.) confirmed that sample 6 was

indeed the mixture of both species.

This study showed that standardization by focusing on alkaloids content alone is not efficient and it is not possible to detect if synthetic ephedrine alkaloids were added to the commercial herbs. This is the advantage of applying NMR spectroscopy coupled to MVA since the analysis is based on total metabolic fingerprinting.

2. Classification of !lex Species Based on Metabolomic Fingerprinting Using NMR and Multivariate Data Analysis (Choi

et al., 2005)

I1ex paraguariensis var. paraguariensis, or yerba mate, is a

popular beverage in South America, besides it is also used as tonic, diuretic, to reduce fatigue and to improve gastric function (Gonzalez et al., 1993). This study was conducted to see the differentiation between !lex paraguariensis var. paraguariensis.

and some other I1ex species which are commonly used as adulterants or substitutes, (Le. I. argentina, I. brasiliensis, I. brevicuspis, I. dumosa var. dumosa, I. dumosa var. guaranina, I.

integerrima, I. microdonta, I. pseudobuxus, I. taubertiana, and I.

theezans). Moreover, the difference between these 10

adulterants/substitutes was analyzed as well. An Extraction method similar to the one used in the Ephedra study was performed and NMR analysis was carried out for both organic and aqueous fractions

From visual observation of the IH-NMR spectra (Fig. 2), only

!lex paraguariensis var. paraguariensis was observed to contain

caffeine and theobromine. This was confirmed by score plot of

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Proceeding of International Seminar Current Issues and Challenges in Food Safety

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Science-Based Approach for Food Safety Management

3. Metabolomic quality control of claimed anti-malarial Artemisia afra herbal remedy and A.afra and A. annua plant extract (van der Kooy et al., 2008)

Artemesia annua L. has been used for centuries in traditional Chinese medicine to treat malaria and fever with artemisinin as

active constituent (Klayman, 19'85). Artemisia afra jacq. Ex Willd. is originally from South Africa and commonly used as a medicine for a wide range of illness such as cough, colds, fever, loss of appetite, and also malaria, although there is no report that A. afra

contains artemisinin or its derivetives (van der Kooy et al., 2008).

In this study, a commercial anti-malaria capsule, which was claimed to contain 400 mg of pure A. afra without any additives or excipients, was investigated. The producer claimed that artemisinin is the active ingredient in these capsules. For NMR analysis, 100 g of each sample (plant material removed from capsules, powder of A. annua, powder of A. afra) were extracted with deuterated chloroform, sonicated, filtered, then transefered to NMR tubes. The lH NMR data was subjected to PCA analysis, to determine whether the capsule contained A. annua, A. afra, or a combination of the two.

From the PCA score plot ｩｾ＠ is clear that A. annua and A. afra are separated from each other in PC1, but the commercial sample could not be differentiated as A. annua or A. afra The loading plot

revealed that artemisinin is an important marker for the differentiation in PCl (methyl signals at 0.99 ppm, 1.21 ppm, 1.44

ppm). LC-MS analysis of three samples, confirmed that A. annua

contains 0.078 - 0.84% artemisinin but it is not present in A. afra

or the commercial sample. It could be concluded that the commercial capsules contain A. afra.

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CONCLUSION

NMR based metabolomics in combination with multivariate data analysis can be applied as a finger printing tool for the quality control of herbal medicine. Adulterants or substitutes in commercial herbal preparations can be identified very quickly with a simple sample preparation.

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