2016/12/20

PENGANTAR ANALISIS
METABOLOMIK

M Rafi, R Heryanto

DIVISI KIMIA ANALITIK

2015

Metabolomik

• Metabolomik adalah teknologi yang berkembang pesat

dekade terakhir, sebagai bagian dari keluarga "omics"
yang melengkapi analisis transkrip gen (transkriptomik)
skala besar dan sidikjari protein (proteomik)

• Menjelaskan dan mengidentifikasi perbedaan antara

organisme (misalnya perbedaan genotipe dan fenotipe
dan klasifikasinya yang disebut kemotaksonomi) dan
menjelaskan faktor-faktor lingkungan yang
mempengaruhi reaksi-reaksi biokimia

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Metabolomik
(a) Skema umum
organisasi
Omics.

The general flow of

information is from
genes to transcripts,
to proteins, to
metabolites, to
function (or
phenotype); whilst
blue vertical arrows
indicate interactions
regulating
respective omic
expression.
(b) Our ‘‘traditional’’ linear view of a metabolic pathway and
‘‘scale free’’ connections in a metabolite neighbourhood.

R Goodacre. 2005. Metabolomics 1: 2

Metabolomik

The three cornerstones of metabolomics and the three main challenges

related to metabolomic data analysis.
J Boccard, S Rudaz. 2014. J. Chemometrics 28: 1

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Metabolomik

A Krastanov. 2010. Biotechnol. & Biotechnol. Eq. 24:1

Metabolomik
Strategies dalam analisis metabolomik
Term Deskripsi
Metabolomics Identifikasi non-bias dan kuantifikasi semua metabolit
dalam suatu sistem biologi. Persiapan sampel tidak
harus mengecualikan suatu kelompok metabolit, dan
selektivitas dan sensitivitas teknik analitis harus tinggi
Metabolite Identifikasi dan kuantifikasi sejumlah tertentu metabolit
profiling yang telah ada, umumnya terkait dengan jalur metabolit
tertentu. Persiapan sampel dan instrumentasi yang
digunakan dapat mengisolasi senyawa-senyawa target
tersebut dari matriks lainnya sebelum deteksi, biasanya
menggunakan teknik pemisahan kromatografi lalu
dideteksi dengan MS. Dalam industri farmasi, cara ini
secara luas digunakan untuk studi penemuan kandidat
obat baru, produk metabolisme obat dan efek
perawatan terapi

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Metabolomik

Term Deskripsi
Metabolic High-throughput, rapid, global analysis of samples to
fingerprinting provide sample classification. Quantification and
metabolic identification are generally not employed. A
screening tool to discriminate between samples of
different biological status or origin. Sample preparation
is simple and, as chromatographic separation is absent,
rapid analysis times are small (normally 1 min or less)
Metabolite Qualitative and quantitative analysis of one or a few
target analysis metabolites related to a specific metabolic reaction.
Extensive sample preparation and separation from other
metabolites is required and this approach is especially
employed when low limits of detection are required.
Generally, chromatographic separation is used followed
by sensitive MS or UV detection

Metabolomik

Term Deskripsi
Metabonomics Evaluation of tissues and biological fluids for changes in
endogenous metabolite levels that result from disease
or therapeutic treatments
WB Dunn, DI Ellis. 2005. Trends in Analytical Chemistry 24: 285

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Metabolomik

WB Dunn, DI Ellis. 2005. Trends in Analytical Chemistry 24: 285

Alur Analisis Metabolomik

Flowchart of the metabolomic study

in plants. Sample preparation steps
can be changed depending on the
analytical methods; however, in
general many steps are common.

*This step can be omitted

in a certain analysis.)

KEMOMETRIKA

HK Kim, R Verpoorte. 2010. Phytochemical Analysis 21: 4

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Teknik Analitik Dalam Analisis Metabolomik

R Goodacre et al. 2004. Trends in Biotechnology 22: 245

Teknik Analitik Dalam Analisis Metabolomik

Some standard techniques

used in metabolomic
analysis. In general, one
technology is not sufficient
for the analysis of all
compounds, but any form of
separation will inherently
introduce a bias towards
the analytes being detected.

F Tugizimana, L Piater, I Dubery. 2013. South African Journal of Science 109

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Alur Analisis Metabolomik

http://www.cial.uam-
csic.es/metabolomics/workflow.html

Pipa Saluran Metabolomik

S Moco, RJ Bino, RCH De Vos, J Vervoort. 2007. Trends in Analytical Chemistry, 26: 855

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Metabolomik Dalam Riset Obat Herbal

Key features of the

technologies used
in metabolomics for
herbal medicine
research

LF Shyur, NS Yang. 2008. Current Opinion in Chemical Biology 12: 66

Analisis Data Dalam Metabolomik

C Hu, G Xu. 2013. Trends in Analytical Chemistry 52: 36

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Kemometrika Dalam Metabolomik

Chemometrics
A science of relating measurements made on a chemical system or
process to the state of the system via application of mathematical or
statistical method (International Chemometrics Society)

Chemometrics

Pattern recognition Multivariate calibration

(qualitative) (quantitative)

Principal Cluster Discriminant Multiple Partial

component analysis analysis linear least
analysis regression square

H.A. Gad et al. 2014. Phytochemical Analysis 24: 1

Kemometrik Dalam Metabolomik

R Goodacre et al. 2004. Trends in Biotechnology 22: 245

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Kebutuhan Dalam Studi Metabolomik

1. How can we extract all metabolites?

2. How can we separate or detect the
metabolites extracted?
3. How can we reduce the huge data set
obtained from analytical detection?
4. How can we identify the metabolites?

Contoh Kajian Metabolomik

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Karakteristik Obat Herbal

Sinergi

Lingkungan
Budidaya
tumbuh
Multikomponen
Panen dan
Genetik
pascapanen

Obat Herbal

Karakteristik Obat Herbal

Produksi senyawa bioaktif

Kadar gingerol and shogaol pada tiga varietas jahe Indonesia

mg/g

M. Rafi, L.W. Lim, T. Takeuchi, L.K. Darusman. 2013. Talanta 103: 28.

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Karakteristik Obat Herbal

Metode Kendali Mutu Berbasis Metabolomik

Identifikasi Pattern-
oriented
(Fingerprint
Kendali Mutu analysis)
dengan
Diskriminasi Senyawa
Kimia Compound-
oriented
(Marker
analysis)
Autentikasi

Kemometrika
Z. Zeng et al., Chin. Med., 3 (2008) 9
S. Govindaraghavan et al., Fitoterapia, 83 (2012) 979

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Sidikjari KLT

Sidikjari KLT
Diskriminasi Kunyit, Temulawak, dan Bangle

a b c
Pola KLT sidikjari dengan visualisasi sinar tampak (a), UV 254 nm (b), dan UV
366 nm (c)

Keterangan:
CCM = kurkumin DMC = demetoksikurkumin
BDC = bisdemetoksikurkumin TMK = Temulawak
KNY = Kunyit BNGL = Bangle

M Rafi, E Rohaeti, A Miftahudin, LK Darusman. 2011. Indonesia Journal of Chemistry 11: 71

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Sidikjari KLT

a b
Pola KLT sidikjari bangle (a), kunyit (b), dan temulawak
(c) dengan visualisasi sinar tampak
Keterangan:
STD = senyawa standar kurkuminoid
NGD = Ngadirejo, Wonogiri
TMB = Tembalang, Semarang
TWN = Tawangmangu, Karanganyar
SMN = Semen, Kediri
SLH = Slahung, Ponorogo
NGR = Ngrayun, Ponorogo
TJK = Tanjung Kerta, Sumedang
RCK = Rancakalong, Sumedang
CKR = Cikembar, Sukabumi
c
DMG = Dramaga, Bogor

Sidikjari KLT

a b
Pola KLT sidikjari bangle (a), kunyit (b), dan temulawak
(c) dengan visualisasi UV 254 nm
Keterangan:
STD = senyawa standar kurkuminoid
NGD = Ngadirejo, Wonogiri
TMB = Tembalang, Semarang
TWN = Tawangmangu, Karanganyar
SMN = Semen, Kediri
SLH = Slahung, Ponorogo
NGR = Ngrayun, Ponorogo
TJK = Tanjung Kerta, Sumedang
RCK = Rancakalong, Sumedang
CKR = Cikembar, Sukabumi
DMG = Dramaga, Bogor c

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Sidikjari KLT

Pola KLT sidikjari bangle (a), kunyit (b), dan temulawak (c) dengan visualisasi UV 366 nm
Keterangan:
STD = senyawa standar kurkuminoid NGD = Ngadirejo, Wonogiri
TMB = Tembalang, Semarang TWN = Tawangmangu, Karanganyar
SMN = Semen, Kediri SLH = Slahung, Ponorogo
NGR = Ngrayun, Ponorogo TJK = Tanjung Kerta, Sumedang
RCK = Rancakalong, Sumedang CKR = Cikembar, Sukabumi
DMG = Dramaga, Bogor

Metode Kendali Mutu --- Kemometrik

Langkah-langkah dalam mengembangkan metode kendali
mutu obat herbal menggunakan kemometrik

Preparasi Sampel Koleksi sinyal dan Analisis kemometrik

prapemrosesan

Validasi

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Metode Kendali Mutu --- Kemometrik

Data spektrum Spektrum original
original dari sampel X

Prapemrosesan sinyal, data pretreatment

Pemilihan model

Set kalibrasi Set validasi Set prediksi

The pretreatments and the

Model IDA logical flow of different
calibration, validation, and
prediction sets
I. C. Yang et al., J. Food Drug Anal. 21 (2013) 268

Metode Kendali Mutu --- Kemometrik

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Diskriminasi Tiga Varietas Jahe

Diskriminasi Tiga Varietas Jahe

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Diskriminasi Tiga Varietas Jahe

mg/g

Diskriminasi Tiga Varietas Jahe

• Instrumentasi: KCKT
• Prapemrosesan
sinyal: koreksi garis
dasar
• Metode kemometrik: 4
DF-1 (72.83 %)

analisis
diskriminant/discrimin 2
ZOR-11
ZOO-12
ZOR-6 ZOR-9
ZOO-1 ZOR-1
ant analysis (DA) ZOO-6 ZOR-12ZOR-2
DF-2 (27.17 %)

ZOR-8 ZOR-7
ZOR-5
ZOO-4 ZOO-5 ZOR-3
0 ZOO-2ZOO-8 ZOR-10 ZOR-4
ZOA-13
ZOO-3 ZOA-7 ZOA-11
ZOO-10 ZOA-4 ZOA-5
ZOA-10
ZOO-9 ZOA-8
ZOO-11 ZOO-7 ZOA-2
-2
ZOA-12
ZOA-1 ZOA-9

ZOA-3 ZOA-6

-4
-4 -2 0 2 4

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Diskriminasi Bangle, Lempuyang Emprit, dan

Lempuyang Gajah

Diskriminasi Bangle, Lempuyang Emprit, dan

Lempuyang Gajah

Analisis sidikjari
+
Kemometrik

Identifikasi &
Diskriminasi

Z.
montanum

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Diskriminasi Bangle, Lempuyang Emprit, dan

Lempuyang Gajah

v
Reference peak

min
Kromatogram sidikjari KCKT
Z. montanum (a), Z. americans (b) dan Z. zerumbet (c)

Diskriminasi Bangle, Lempuyang Emprit, dan

Lempuyang Gajah
Analisis Komponen Utama

Analisis Diskriminan

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Identifikasi Kunyit, Temulawak dan Bangle

Identifikasi Kunyit, Temulawak dan Bangle

Turmeric (C. Java turmeric Cassumunar ginger

longa) (C. xanthorrhiza) (Z. cassumunar)

Turmeric??
Java turmeric??
Cassumunar ginger??

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Identifikasi Kunyit, Temulawak dan Bangle

Spektra FTIR representatif dari C. longa (A), C. xanthorrhiza (B), and

Z. cassumunar (C)

Identifikasi Kunyit, Temulawak dan Bangle

• Instrumentasi:
Spektroskopi FTIR
• Prapemrosesan
sinyal: standar normal
variate
• Metode kemometrik:
analisis variat CL
ZC
kanonik/canonical
variate analysis (CVA)
CX

Plot CVA

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Autentikasi Temulawak --- Sidikjari KCKT

Autentikasi Temulawak --- Sidikjari KCKT

Kadar kurkuminoid
mg/g
30 CUR

DMC
25
BDMC

20

15

10

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Autentikasi Temulawak --- Sidikjari KCKT

[105] 1.5

v

0.5 e
d
c
b
a
0
0 10 20 30 40 min
Kromatogram CX (a), 5% CL dalam CX (b), 25% CL dalam CX
(c), 50% CL dalam CX (d) and CL (e) (UV 254 nm)

Autentikasi Temulawak --- Sidikjari KCKT

Analisis Variat Kanonik
CV2 (1.1%)

CV1 (98.9%)

Plot CVA CX (◆), 5% CL dalam CX (▲), 25% CL dalam CX (),

50% CL dalam CX () and CL (■)

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GC-MS Based Metabolomics

GC-MS Based Metabolomics

ABSTRACT

Introduction – Metabonomic analysis is an important molecular phenotyping

method for characterising plant ecotypic variations; hence, it may become a
powerful tool for quality control and discrimination of traditional Chinese
medicine (TCM).

Objective – To discriminate and assess the quality of Curcuma phaeocaulis,

C. kwangsiensis and C. wenyujin from different ecotypes. The identification of
the compositions of essential oils from the three Curcuma species was
included in this study.

Methodology – Metabolomics analysis was carried out on all samples by gas

chromatography–mass spectrometry (GC‐MS) coupled with multivariate
statistical analysis. Characterisation of phytochemicals in essential oils was
performed by automated matching to the MS library and comparison of their
mass spectra (NIST05 database).

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GC-MS Based Metabolomics

Results – Principal component analysis (PCA) effectively distinguished the

samples from different species and ecotypes. Partial least squares
discrimination analysis (PLS‐DA) was successfully employed in classifying
the GC‐MS data of authentic, commercial and introduction cultivation
samples. Furthermore, the components contributing significantly to the
discrimination, namely curzerenone, germacrone, curdione and
epicurzerenone, were screened by PCA and PLS‐DA loading plots and
further can be used as chemical markers for discrimination and quality control
among different groups of samples.

GC-MS Based Metabolomics

Representative GC‐MS chromatograms of the essential oil from

(a) C. wenyujin, (b) C. kwangsiensis and (c) C. phaeocaulis.

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GC-MS Based Metabolomics

Score plots of
(a) PCA and (b)
PLS‐DA, and
loading plots of
(c) PCA and (d)
PLS‐DA for
the 62 samples,
using common
components as
input data.

(a) PCA and (b) PLS‐DA projection

plots for the 62 samples, using peak
areas of four chemical markers as input
data.

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NMR Based Metabolomics

NMR Based Metabolomics

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Terima Kasih
Atas Perhatiannya !

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