Departemen Kimia FMIPA

Advance Research Laboratory

Metabolomik: Konsep Dasar

dan Prospek Aplikasi di
Bidang Fitopatologi

Mohamad Rafi

Workshop PFI Seri 1 Tahun 2021

Rabu, 31 Maret 2021
Biosketch

Dosen dan peneliti di Departemen Kimia FMIPA dan Pusat

Studi Biofarmaka Tropika LPPM IPB. Saat ini diberi
amanah sebagai Kaprodi S2/S3 Kimia IPB, Ketua Editor
Jurnal Jamu Indonesia, dan Ketua Himpunan Kimia
Indonesia 2021.
Penelitian yang dilakukan banyak di bidang metabolomik
dan kemometrik untuk pengembangan metode kendali
mutu, identifikasi senyawa penciri dan standardisasi http://mra.staff.ipb.ac.id/
tumbuhan untuk bahan baku obat herbal/pangan. Email:
mra@apps.ipb.ac.id Education
• B.Sc - IPB University, Indonesia
• M.Sc – IPB University, Indonesia
• Ph.D – Gifu University, Japan
 2021
Identifikasi senyawa bioaktif antikanker baru menggunakan pendekatan
Aktivitas Penelitian metabolomik berbasis LC-MS/MS sebagai upaya mendukung
penanggulangan penyakit kanker (PDUPT)
Pendekatan metabolomik untuk standardisasi sidaguri (Sida
rhombifolia) melalui pemrofilan metabolit (Spektrum FTIR, KLT, LC-
MS/MS) serta aktivitas antioksidan dan inhibisi xantin oksidase (RKI)

2020
Standardisasi simplisia tempuyung (Sonchus arvensis) menggunakan
pendekatan metabolomik: pemrofilan fitokimia, aktivitas biologis, dan
unsur logam (PDUPT)
Varietas unggul temu ireng: pengembangan berdasarkan aktivitas
farmakologi dan kandungan metabolit (PTUPT)

2019
Pengembangan obat herbal antidiabetes dengan pendekatan
metabolomik berbasis kepada konsep multikomponen-multitarget dan
sinergestik (PDUPT)
Metode identifikasi dan autentikasi bahan baku obat herbal
menggunakan pendekatan metabolomik berbasis kromatografi cair
kinerja tinggi dan spektroskopi UV-Vis (PDUPT)
 PUBLIKASI

01
Feasibility of UV-Vis spectral
03
An HPLC-DAD Method to
fingerprinting combined with
Quantify Flavonoids in Sonchus
chemometrics for rapid arvensis and Able to Classify the
detection of Phyllanthus niruri Plant Parts and Their
adulteration with Leucaena Geographical Area through
leucocephala (accepted in Sains Principal Component Analysis
Malaysiana. 2021) (Separations. 2021)

02 04
Untargeted Metabolomics
FTIR-based fingerprinting and
Analysis Using FTIR and UHPLC-
chemometrics for rapid Q-Orbitrap HRMS of Two
investigation of antioxidant Curculigo Species and Evaluation
activity from Syzygium of their Antioxidant and α-
polyanthum extracts (Indones. Glucosidase Inhibitory Activities.
J. Chem. 2021) (Metabolites 2021)
 PUBLIKASI

05 07
Metabolite Profiling Quantitative Analysis of
of Andrographis Multi-components
paniculata Leaves and Stem in Curcuma xanthorrhiza by
Extract Using UHPLC-Orbitrap- Single Marker (Indones. J.
MS/MS (Nat. Prod. Res. 2020) Pharm. 2019)

06 08
Multiple spectroscopic HPLC fingerprinting coupled
fingerprinting platforms for rapid
with linear discriminant
characterization of α-glucosidase
inhibitors and antioxidants from
analysis for the detection of
some commonly consumed adulteration in Orthosiphon
Indonesian vegetables and spices (J. aristatus (J. Liq. Chrom. Rel.
Food Meas. Charact. 2020) Tech. 2019)
Materi

Apa itu metabolomik?

Strategi dalam analisis

metabolomik

Aplikasi metabolomic dalam

bidang Fitopatologi
Tren metabolomik
Metabolomik tumbuhan

Plant metabolomics is a
powerful tool to study the
metabolic mechanisms
underlying plant responses
to adverse environmental
conditions (abiotic stress)
and/or plant–attacker
interactions (biotic stress)
that adversely affect plant
growth and development.

Resistance, Tolerant, or
Susceptible

Jorge TF, Mata AT, António C. 2016. Phil.

Trans. R. Soc. A 374: 20150370
Metabolomik di bidang fitopatologi
metabolomik
Metabolomik

.....identifikasi dan kuantifikasi sistematis molekul kecil/metabolit*

hasil metabolisme (metabolom)$ dari suatu sistem biologis (sel,
jaringan, organ, cairan biologis, atau organisme) pada titik waktu
tertentu (https://www.nature.com/subjects/metabolomics)

* Metabolit: setiap molekul organik yang terdeteksi dalam tubuh dengan MW

<1500 Da

$ Metabolom: koleksi lengkap metabolit/molekul kecil di sel, organ, jaringan

atau organisme
Metabolomik

Nothing new in metabolomics!

Just:
• Different direction
• Combination of present technology
Metabolomik

Quantify everything =
metabolomics
Metabolomik

Metabolomik
menyediakan sebuah
pandangan fungsional
dari organisme,
sebagaimana
ditentukan oleh
keseluruhan dari
gennya, RNA, protein,
dan faktor lingkungan
(functional genomic)
Metabolomik

Ghatak A., Chaturvedi P., Weckwerth W.

(2018) Metabolomics in Plant Stress
Physiology. In: Varshney R., Pandey M.,
Chitikineni A. (eds) Plant Genetics and
Molecular Biology. Advances in Biochemical
Engineering/Biotechnology, vol 164. Springer,
Cham. https://doi.org/10.1007/10_2017_55
Metabolomik

… Identifikasi dan kuantitasi sistematis dari semua molekul kecil atau yang
relevan secara biologis dalam kompartemen, sel, jaringan atau cairan tubuh
tertentu → analisis global pada tingkat metabolit

Apa yang sedang

mereka kerjakan?
Metabolomik

Hanya analisis pada level global yang dapat

menjelaskan keseluruhan cerita

Claudio Luchinat, CERM, Università

di Firenze
Metabolomik
Strategi 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

Strategi metabolomik

Metabolite fingerprinting Selection of technology is a

Pattern recognition to classify compromise between
samples by shifts in “global” speed, selectivity and
metabolite composition sensitivity.
Metabolite target Metabolite profiling
analysis Detection, identification and
Analysis of specific/known Four approximate quantification of a
metabolites. Approaches large set of target compounds
Particular interest in within a specific metabolite
low molecular weight pathway
compounds – the Metabolomics
substrates and products Unbiased identification of
in pathways. all metabolites in sample. (Fiehn, 2001)
Strategi metabolomik

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

Strategi metabolomik
Different approaches and respective techniques: pitfalls and strengths
APPROACH TARGETED ANALYSIS METABOLITE METABOLITE FINGERPRINTING
PROFILING

STRENGHT • Quantitative Global image of the • Global image of the samples

• Low detection limit samples • Directly applicable to
• High troughput pattern recognition
• Highest throughput

PITFALL • Limited number of targeted • Semi-quantitative No compound identification

compounds • Difficulties in
• No detection of untargeted informatics
compounds • Medium throughput
• Need for the pure targeted
compounds for the calibration
• Expensive

TECHNIQUES Chromatography and spectroscopy

P Vernocchi et al. 2012. Frontiers in Cellular and Infection Microbiology. 2: Article 156
Strategi metabolomik
Metabolite fingerprinting
Metabolite fingerprinting

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

Z. cassumunar (C)
Metabolite fingerprinting

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

LC-MS/MS analysis
UHPLC tandem Q Exactive Plus Orbitrap-HRMS
Ionization source ESI(+), mass analyzer: Q-orbitrap
Column: AccucoreTM Phenyl Hexyl (100×2.1 mm, 2.6 µm)
Ionization energy:18, 35, 53 eV
Mobile phase: 0.1% FA in MeOH)

Sample extraction Identification of metabolite

Powdered sample of leaves and stem
Software Compound Discoverer 2.1
were extracted with 70% EtOH (1:10)
with local databse
using sonication method in room
temperature for 30 minutes

Sample Classiication using

preparation PCA
3-month-old of AP was Software The Unscrambler
used and separate between X 10.4.
leaves and stem

1 2 3 4 5
Metabolite profiling

a. 0.1% FA in water
b. 0.1% FA in ACN
c. 0.1% FA in MeOH

Modifikasi dari metode yang dikembangkan oleh Song et al. 2013. Molecules. 18(10):12192–12207.
Metabolite profiling

TIC of stem
extract

TIC of leaves
extract
Metabolite profiling
Ditepene lactone
1. Paniculide C
2. Paniculide B
3. 14-deoxyandrographiside
4. 14-deoxy-11-oxoandrographolide
5. 12S-hydroxyandrographolide
6. 14-deoxy-17-hydroxyandrographolide
7. Andrographic acid
8. Andrographolide
9. Neoandrographolide
10. Paniculide A
11. Andrographanin
12. 14-deoxyandrographolide
13. Andrographolactone
14. Dehydroandrographolide
15. Bis-andrographolide A
16. 14-acetylandrographolide
Flavonoid Phenolic acid
1. Luteolin 1. Cinamic acid
2. Apigenin 2. Caffeic acid
3. 5,7-dihydroxy-8-methoxyflavone 3. 3-O-Caffeoylcuinic acid
4. Apigenin-7-O-β-D-glucoronide
5. Dihydroskullcapflavone I
6. 5,4’dihydroxy-7,8-dimethoxy flavone
7. 5-hydroxy-7,8,2’-trimethoxy flavone 5-glucoside
8. 5-hydroxy-3,7,8,2’-tetramethoxy flavone
9. Apigenin-7,4’-dimethylether
10. 5-hydroxy-7,8-dimethoxy flavanone
11. 5-hydroxy-7,2’,6’-trimethoxy flavone
12. Andrographidin A
Metabolite profiling

Variabel: luas pita dari 18 komponen mayor

Metabolite profiling

Variabel yang berkontribusi pada pengelompokan yaitu senyawa

nomor 26, 27, dan 28 untuk ekstrak daun sambiloto dan nomor 17,
18, 19, dan 21 untuk ekstrak batang sambiloto.

17. Andrographolide ,
18. Apigenin-7,4’-dimethylether,
19. 5-hydroxy-7,8-dimethoxy flavanone,
21. Andrographidin A,
26. Andrographanin,
27. 14-deoxyandrographolide ,
28. Andrographolactone
Targeted analysis

curcumin

demethoxycurcumin

bisdemethoxycurcumin

xanthorrhizol

E Erpina et al. Journal of Liquid Chromatography and Related Techniques 40(12): 635-639.
 Strategi metabolomik --- workflow

Ghatak A., Chaturvedi P., Weckwerth W.

(2018) Metabolomics in Plant Stress
Physiology. In: Varshney R., Pandey M.,
Chitikineni A. (eds) Plant Genetics and
Molecular Biology. Advances in Biochemical
Engineering/Biotechnology, vol 164. Springer,
Cham. https://doi.org/10.1007/10_2017_55
Strategi metabolomik --- workflow

A typical analytical workflow for metabolomics studies

 Strategi metabolomik --- workflow

Flowchart detailing the steps

involved in plant metabolomics.
There are three main steps in
the metabolomic analysis,
which include sample
preparation, data acquisition,
and data analysis, which lead to
the biochemical interpretation
Ghatak A., Chaturvedi P., Weckwerth W. of the identified metabolite
(2018) Metabolomics in Plant Stress
Physiology. In: Varshney R., Pandey M.,
Chitikineni A. (eds) Plant Genetics and
Molecular Biology. Advances in Biochemical
Engineering/Biotechnology, vol 164. Springer,
Cham. https://doi.org/10.1007/10_2017_55
Strategi metabolomik --- workflow
Strategi Metabolomik --- Instrumentasi
Strategi Metabolomik --- Instrumentasi

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

Kebutuhan dalam studi metabolomik

1. How to extract all metabolites?

2. How to separate or detect the metabolites extracted?
3. How to reduce the huge data set obtained from
analytical detection?
4. How to extract hidden information from raw data?
5. How can identify metabolites from the mixture?
Dr. Young H. Choi, Workshop on Metabolomics, 2011
 Aplikasi metabolomik dalam bidang fitopatologi

Typical LC-MS based metabolomics workflow in

phytopathological studies. (A) Plant tissues (here,
tomato leaves) are inoculated )i.e., mock) or with
the pathogenic microbes (here, a fungus causing
necrotic lessions) or subjected to another relevant
biotic stress (e.g., elicitors). Following a time course
expressed as day post-inoculation (DPI), plant tissues
are sampled in liquid nitrogen for metabolic
quenching and stored until targeted (B) and/or
untargeted metabolomics can be performed
Aplikasi metabolomik dalam bidang fitopatologi
Aplikasi metabolomik dalam bidang fitopatologi
Aplikasi metabolomik dalam bidang fitopatologi
Aplikasi metabolomik dalam bidang fitopatologi
Aplikasi metabolomik dalam bidang fitopatologi
Aplikasi metabolomik dalam bidang fitopatologi
Aplikasi metabolomik dalam bidang fitopatologi
Aplikasi metabolomik dalam bidang fitopatologi
Ringkasan

• Metabolomik merupakan multidisciplinary science

• Aplikasi metabolomik dalam bidang fitopatologi telah luas digunakan

dalam identifikasi tanaman yang terkena penyakit, plant-pathogen
interaction, dlsb

• Multi-omics (genomics, transcriptomics, proteomics, dan metabolomics)

akan menjadi tren ke depan untuk dapat lebih memahami plant-pathogen
interaction sehingga dapat mengembangkan suatu tanaman yang lebih
tahan atau toleran terhadap patogen
1 2 3 4 5
TERIMA KASIH

• Pusat Studi Biofarmaka Tropika LPPM

• Departemen Kimia FMIPA