-Based Approach for Food Sujety Munagement Proceeding uf l t r i e rn~~~una l Seminar Current Issues and L'hallengcs~ in Food S u f e ~

igher the ratios of CaO to fresh pepper, the shorter the drying ~eeded. Chetnoreaction drying can be used to substitute or to ~ u e the sun drying process.

:hemoreaction drying had no effect on the volatile oil content of pepper produced compared to the fresh pepper. This drying ises resulted black pepper with volatile oil content between - 2.70% [dry basis]. The color of the oil was clear greenish r with good flavor quality.

wledgements

Ne would like to say thank to URGE Batch V I'roject for rting this research, and to SEAFAST Center II'H for the tuni ty to present this paper.

ences

:] Association of Official Analytical Chemists. 1995. Official viethods of Analysis of the Association of Official Analytical :hernists, 14'" ed. AOAC, Inc. Arlington, Virginia.

her E. 1990. Minyak Atsiri. Jilid I . Terjemahan oleh Ketaren S. 'enerbit Universitas Indonesia (UI Press), Jakarta.

B. 1995. Pengeringan biji lada [Piper nigrum [,inn) secara lbsorpsi dengan kaput- api (CaO). Skripsi. Furusan Teknologi 'angan dan Gizi, Fateta, IPB, Bogor.

ii W. 1990. llmu Kimia Analitik Dasar. PT Gramedia, Jakarta.

;love JW, Brown EC, Green CL, Robin SR]. 1981. Spices. lolume I. Longman, London.

lr Nasional Indonesia. 1995. Standar Mutu Lada Hitam S N I 0 1 - I005 - 1995, Jakarta: BSN

APPLICATION OF NMR BASED METABOLOMICS AND MULTIVARIATE DATA ANALYSIS FOR QUALITY CONTROL OF HERBAL MATERIAL

Nancy Dewi YulianaiJ, Hye Kyong Kimz, Frank van der Kooy2, Henrie Korthourt3, Young Hae Choiz, and Rob Verpoorte2

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 i ts efficacy and s a f ~ t y , u p 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. N M R based metabolomics is a reliable tool t o provide a wealth of information concerning pIant 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 bc 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, pdrticularly prin

c

ipal 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

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:he organic fractions (Fig.3.) which shows that Ilex paroguariensis Jar. poroguariensis is dearly separated from other species.

2. 1H spectra of organic fractions of llex argentina leaves (a), I paraguariensis leaves @), I. pseudobuxus leaves (c), I. touberionaleaves [d). Peaks: 1= H-8 of theobromine; 2 = H- 8 of caffeine (Reproduced from (Choi et aL, 2005). with permission)

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Proceeding ofInrurn*~~untlf Srm~nur Current Issues and Challenges in Food Sajev

The use of NMR and MVA made it possible to further analyze the difference between 10 other species beside Ilex paraguoriensis by using the same data. For this purpose, Ilex poraguoriensis was omitted from the score plot (Fig. 4.A.). llex clurt~osa var. dumosa (4) and I. tauberianu (10) wcre separated from other specially since I . dumosa var. dumosa contains more tri terpenoids while I , touberiana contains more fatty acids (Fig.4.B.). Further analysis of the aqueous fractions was performed by adding another principal component (PC3) (Fig.5.A.). As a result, it was shown that ilex paraguariensis was separated from other species since it has a high amount of phenylpropanoids and sucrose (Fig.5.B-C).

Figure 3. Score plot of organic fractions of llex species (1=L argentina; 2= 1, brasiliensis; 3= 1. brevicuspis; 4= 1. dumosa var, dumosa; 5.1. dumosa var. guaronina; 6= 1. integerrima; 7= 1. microdonta; 8=1, pa raguoriensis var. paraguariensis; 9 . 1. pseudobuxus; 10. 1. tuubertiana; 11= I. theezans) (reproduced from [Choi et ab, 2005), with permission)

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Figure 4. (reproduced from (Choi et a)., 2005), with permission) a. Score plot of organic solvent fractions of 10 species of llex [without 1. paraguariensis) b. Loading plot of organic solvent fractions of 10 species of Ilex (without I. paraguariens!~)

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Figure 5. (reproduced from (Choi et al., 20051, with permissi a. Score plot of PC1, PC2, PC3 of aqueous fractions of llex sp b. Loading plot of PC2 c. Loading plot of PC3

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3. Metabolomic quality control of claimed anti-malarial Artemisia afra herbal rcmedy and A.aJra and A. unnua plant extract (van der Kooy et a/., 2 008)

Artemesia annua I. . has been used for centuries i n traditional Chinese medicine to treat malaria and fever with artemisinin as active constituent (Klayman, 1985). Artemisio 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 a / , 2008). I n 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 artcmisinin is the active ingredient in these capsules. For N M K analysis, 100 g of each sample (plant material removed From capsules, powder of A , annrra, powder of A. nfra) were extracted with deuternted chloroform, sonicated, filtered, thcn transefered to N M R tubes. The H 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, it is clear that A. annua and A. nfra are separated from each other in PCl, 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 P C 1 (methyl signals at 0.99 ppm, 1 . 2 1 ppm, 1.44 pprnj. LC-MS analysis of three samples, confirmed tha t A. onntia contains 0.078 - 0.84% artemisinin but it is not present in A. afra or t he commercial sample. It could be concluded tha t the commercial capsules contain A. arra.

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Figure. 6. Score plot of the Artemisia samples showing the differentiation in PC 1 between the A. annuo (A) sa and the A. afra (B) and con~rnercial herb (C) (repro from [van der Kooy et al., 20081, with permission)

CONCLUSION

N M R based metabolomics in combination with multiv data analysis can be applied as a finger printing tool for the q control of herbaI medicine. Adulterants or substitutes in comm herbal preparations can be identified very quickly with a s sample preparation.

References

Calfee, R. and Fadale, P. (2006). "Popular ergogenic drl~gs supplements in young athletes." Pediatrics 11713): e577-51

Calixto, j. (2000). "Efficacy, safety, quality control, marketing regulatory guidelines for herbal medicines [phytothera~ agents)." Brazilian lournal of Medical and Biolo~ical Resl 33: 179-189.

Chni, Y . H . , Kim, H. K., Hazekamp, A., Erkelens, C., Lefeber, A. W. M Verpoorte, R, [2004). "Metabolomic differentiation of Can sativa cultivars using lH NMR spectroscopy and Prin

2 5 4 I t e c h n i c a l p r e s e n t a t i o n t e c h n i c a l p r e s e n t a t i o n

r- Basc J ilpproacl~ fi)r FooJ Saj>n, Management

letabolomic quality control of claimed anti-malarial Arfert~isio - -

fro herbal remedy and A.afro and A. annua plant extract (van der ooy et al., 2008)

Artemesio annua L. has been used for centuries i n traditior~al ~inese medicine to treat malaria and fever with artctnisitlin as : t h e constituent (Klayrnan, 1985). Arternhio ufro jacq. Eu Wilid. originally from South Africa and commonly used as a medicine r a wide range of illness such as cough, colds, fever, loss of petite, and also malaria, although there is no report t h a t A. afru ntains artemisinin or its derivetives (van der Kooy et oL, 2008). this study, a commercial anti-malaria capsule, which was

limed to contain 400 mg of pure A. afra without any additives excipients, was investigated. The producer claimed that

:ernisinin is the active ingredient in these capsules. For N M R alysis. 100 g of each sample (plant material removed from ~sules, powder of A. onnuo, powder of A. u J r ~ ) were extracted :h deuteroted chloroform, sonicated, filtered, then transefered VMR tubes. The 'H N M R data was subjected to PCA analysis, to ermine whether the capsule contained A. annual A. o/io, or a nbination of the two.

+om the PCA score plot, it is clear that A. onnuo and A. afru are arated from each other in PCI, but the commercial sample Id not be differentiated as A. annuo or A. o/ro The loading plot saled that artemisinin is an important marker for the srentiation in PC1 (methfl signals at 0.99 ppm, 1.21 pprn. 1.44 I). LC-MS analysis of three samples, confirmed that A, anrllia

rains 0.078 - 0.84% artemisinin but it is not present in A. ujku the commercial sample. I t could be concluded t h a t the mercial capsules contain A. afra

n i c a l p r e s e n t a t i o n

Proceeding ,![International Semrnor Currrnl Issues und C'hallenge.~ in Food Safety

Figure. 6. Score plot of the Artemisia samples showing t h e clear differentiation in PC 1 between the A. annua [A) samples and the A. ofra (B) and commercial herb (C) (reproduced from (van der Kooy et o l , 2008), with permission)

CONCLUSION

N M R 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.

References

Calfee. R. and Fadale, P. (2006). "Popular ergogenic drugs and supplements in young athletes." Pediatrics 117(3): eS77-589.

Calixto, J , (2000). "Efficacy, safety, quality control, marketing and regulatory guidelines for herbal medicines (phytotherapeutic agents)." Brazilian lournal of Medical and Biological Research 33: 179-189.

Choi, Y. H., Kim, H. K., Hazekamp, A., Erkelens, C.. Lefeber. A. W. M. and Verpoorte, R. (2004). "Metabolomic differentiation of Cannabis sativo cultivars using lH NMR spectroscopy and Principal

t e c h n i c a l p r e s e n t a t i o n 1255

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Component Analysis." Lournal of Natural Products 67161: 953- Schripsema, J. and Verpoorte, R. 11991). "Investigation of extri 957. plant cell cultures by proton nuclear magnetic resc

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Elvin Lewis, M. and Steve, L. T. (2005). "Safety issues associated with herbal ingredients." Advances in Fond and Nutrition Research

Eriksson, L., Johansson, E., Kettaneh-Wold, N. and Wold, S. (2006). Multi-and Me~avariate Data Analysis. Umea, Sweden, Umetrics AB.

Gonzilez, A., Fel-I-eira, F., Vizquez, A., Moyna, P. and Paz, E. A. 11993). "Biological screening of Uruguayan medicinal piants." Journal of Ethnopharrnaj- 39[3): 217-220.

Hailer, C. A. and Benowitz, N . L. (2000). "Adverse cardiovascular and central nervous system events associated with dietary supplements containing Ephedra alkaloids." New England J o e d o f c c i 343[25]: 1833-1838.

jahangir, M., Kim, H. K., C'hoi, Y. H. and Verpoorte, K. (2008). "Metabolomic response of Brnssica rapa submitted to pre- harvest bacterial contamination." Food Chemistry 107C1): 362- 3 68.

Kim, H. K., Choi, Y. H., Erkelens, C., Lefeber, A. W. M. and Verpoorte, R. (2005). "Metabolic fingerprinting of Ephedra species using 1H- NMR spectroscopy and Principal Component Analysis." -Pharn~aceutical Bul le t in 53(1): 105-109.

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van der Kooy, F., Maltese, F. and Choi, Y. (2009). "Quality con herbal material and phytopharrnaceuticals with MS and based metabolic fingerprinting." Planta Medica 7 5171: 763

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t e c h n i c a l p r e s e n t a t i o m

Based Approach for Food Safew Miinagemen1 PrrIceeding of lnternariond Stminur Curr~') i t Issuc'~ utld C'htilletrx~-.\ I I I Focld SuJef~'

omponent Analysis." journal of Natural Products 6716): 953- S,-hripsema, J , and Verpoorte, R. (1991). "Investigatior~ of extracts of 57. plant cell cultures by proton nuclear magnetic resonance

H., Sertic, S., Kim, H. K., Wilson, E. G., Michopoulos, F., Lefebel-, spectroscopy." Phvtochemical Analysis 2 (4): 1 5 5 - 162.

W. M., Erkelens, C., Prat Kricun, S. D. a n d Verpoorte, R. (2005). van der Kooy, F., Maltese, F. and Choi, Y. (20093. "Quality control of :lassification of l lex species based on ~ne t a bolomic herbal and phytopharmaceuticals with MS and NMR ~gerprinting using Nuclear Magnetic Resonance a n d based metabolic fingerprinting." Planta Medica 7517): 763-775. ultivariate Data Analysis." Journal of A~ricultural and Fuud

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343125): 1833-1838.

; M., Kim, H. K., Choi, Y. H. and Verpoorte, R. (2008). 'etabolomic response of ,Brassica rap0 submitted to pre- rvest bacterial contamin~tion." Food 107(1): 362- 8.

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