1. Gene isolation related to
biosynthesis of benzylisoquinoline alkaloid in
Nelumbo nucifera Gaertn. (Lotus)
Thitirat Meelaph
Department of Biochemistry and
Microbiology, Biomedicinalchemistry
Faculty of Pharmaceutical Science
2. Purpose
• To isolate gene which involved in Biosynthesis of
Benzylisoquinoline alkaloids in Lotus
6. Benzylisoquinoline Alkaloid
• Benzyllisoquinoline
alkaloids (BIAs)
A large and
structurally diverse
group of plant-
metabolites derived
from aromatic
amino acid tryrosine
via a complex array
of biosynthetic
enzymes. (David K.
Liscombe er al., 2009)
Staniek A, et al., Natural product-modifying
pathways in plant, Biotech.J 2013;8: 1159-1171-
DOI10.1002/biot201300224
8. Coclaurine N-methyl transferase
• Coclaurine N-methyltransferase (CNMT)
: S-adenosyl-L-methionine (SAM)
: CNMT catalyzes the transfer of a methyl group from S-adenosyl-L-methionine to
the amino group of the tetrahydrobenzylisoquinoline alkaloid coclaurine. (This is a
unique N-methyltransferase in the biosynthesis of benzylisoquinoline alkaloids)
(Inui, T., et al., 2007)
10. Isolation of novel N-methyltransferases from
three benzylisoquinoline alkaloid-producing species
Plant species Study topic Investigator Year
Coptis japonica Molecular Cloning and
Characterization of Coclaurine
N-Methyltransferase
Kum-Boo Choi., et al. 2002
Coptis japonica
Thalictrum minus
Overexpression of Coptis
japonica Norcoclaurine 6-O-
Methyltransferase in Cultured
Eschscholzia californica
Inui T., et al. 2007
Eschscholzia californica
Papaver bracteatum
Thalictrum flavumcell
Metabolite profiling
(CNMT genes already isolated)
Liscombe., K.D., et al. 2009
11. LOTUS
Lotus is perennial aquatic plant that is consumed around
the world (600 cultivars to date)
1. Nelumbo nucifera Gaertn : widely distributed in East
Asia, especially in China and India,
2. Nelumbo lutea (Willd.) Pers. : from North America.
Lotus has been cultivated based on its use :
- flower production,
- seed production, and for
- rhizome production (Guo 2009)
Taxonomy Hierarchy
Kingdom Plantae
Subkingdom Viridiplantae
Division Tracheophyta – vascular plant
Subdivision Spermatophytina – seed plants
Class Magnoliopsida
Order Proteales
Family Nelumbonaceae
Genus Nelumbo
12. The research of Separation and
Purification of Alkaloid in Lotus
13. Bioactive compounds in Lotus
• Lotus plants provide several bioactive compounds
Alkaloids FlavonoidsTherpenes Phenolics
(T C M D, Vo., et al.,2013)
(Akinjokunla, OJ., et al.,2010)
(Shidhar, KR., et al.,2007)
14. LC/MS guided isolation of alkaloids from Lotus leaves
by pH-Zone Refining counter current-chromatography
15. Preparative seperation of alkaloids from Nelumbo
Nucifera leaves by conventional and pH-zone-refining
Counter-Current Chromatography
16. Determination of Lotus leave alkaloids by solid phase extraction
combined with high performance liquid chromatography with
diode array and tandem mass spectrometry detection
17. Bioactive alkaloid in Lotus
Yang G, et al.,Rapidsimuluneous Determination of four alkaloids in Lotus plumule by CZE with
Ephedrine Hydrochloride as an internal standard, Chromatographai 2012;75 :1295-1300-
DOI/s10337 10.1007012-2312-7
18. Pharmaceutical Value of Bioactive
alkaloids in Lotus plants
Found in Leaf, Seed and Embryo
Value : Inhibit Bacteria growth
and Anti-HIV
Akinjokunla, OJ et al. (2010)
Sridhar, K.R. and Bhat, J. (2007)
Yang G, et al., Rapidsimuluneous Determination of four alkaloids in Lotus plumule by CZE with
Ephedrine Hydrochloride as an internal standard, Chromatographai 2012;75 :1295-1300-
DOI/s10337 10.1007012-2312-7
19. Pharmaceutical Value of Bioactive
alkaloids in Lotus plants
Found in Leaves Embryo and Plumule
Value : Antibacterial and
Anti-HIV activity
Sridhar, K.R. and Bhat, J. (2007)
Yang G, et al.,Rapidsimuluneous Determination of four alkaloids in Lotus plumule by CZE with
Ephedrine Hydrochloride as an internal standard, Chromatographai 2012;75 :1295-1300-
DOI/s10337 10.1007012-2312-7
20. Pharmaceutical Value of Bioactive
alkaloids in Lotus plants
Found in Leave and Seed
Value : Anti-diabetic and HIV activity
K. Hou Nguyen, at al., Nuciferine stimulates insulin secretion from beta cells-An in vitro
comparison with glibenclamide,Journal of Ethnopharmacology 2012;142:488-495
T C M V Do, et al., Analysis of Alkaloids in Lotus(Nelumbo Nucifera Gaertn.) leaves by non-aqueous capillaryelec-
trophoresis using altraviolet and mass spectrometric detection, Journal of Chromatography A 2013;174-180
(Shidrha, KR. Bhat,R. (2007)
(Akinjokunla, OJ et all, 2010)
Nuciferine
23. Research Methodology
Gene isolation and
recombinant construction
Alignment
RNA extraction
Primer design
Recombinant DNA (pET28B)
EST database (Liscombe.,
K.D., et al.,2009)
Lotus transformation- Agrobacterium
Expression of recombinant in E.coli (BL21)
Enzyme activity
Lotus seed
germination
Explants
callus induction
Explants
Shoot induction
Subcloning to
pCambia1302
Co-transformation
with Agrobacterium
Whole plant
transformation with
Agrobacterium
Explants sampling
and gene profile
24. BamHI
SalI
Kanamycin
E.Coli BL21
induce protein
expression
(1 mM IPTG, 50mg/ml
Kana 37ᵒC, 4hr) in
shaker
Culture in LB
broth at 37ᵒC,
CO2 incubator
Ni ,Co Affinity resin
6000 rpm, 5 min
4ᵒC , lysis pellet
20 mM Tris/HCl, pH 7.5, 0.5 M KCl,
10% glycerol,10 mM b-mercaptoethanol
10 000 g20 min at 4C,
Comassie
blue dye
HPLC analysis
80 µM S-adenosyl-L-methionine, 2 µM S-adenosyl-L-
[methyl-14C]-methionine, 1–500 µM alkaloid
substrate in 100 mM Tris/HCl, pH 7.5, and 12 mM
b-mercaptoethanol, at 37C.
(Choi., et al.,2002)
-Plant secondary metabolites which is Plant Natural Product(PNP) are derived from primary metabolites have been described function in Bioactive Compounds 3 Large classes of PNP
-Alkaloids are a diverse group of low-molecular-weight, nitrogen-containing compounds found in about 20% of plant species.
Many of the »12,000 alkaloids for which structures have been described function in the defense of plants against herbivores and pathogens.
The potent biological activity of some alkaloids has also led to their exploitation as pharmaceuticals, stimulants, narcotics, and poisons.
The biosynthesis of all BIAs begins with condensation of dopamine and 4-hydroxyphenylacetaldehyde by norcoclaurine synthase (NCS) to yield (S)-norcoclaurine. Subsequently, norcoclaurine 6-O-methyltransferase (6OMT), coclaurine N-methyltransferase (CNMT) and 3-hydroxy-N-methylcoclaurine 4-O-methyltransferase (4OMT) convert (S)-norcoclaurine to the central branch-point intermediate (S)-reticuline
S-adenosyl-L-methionine (SAM)-dependent NMTs occur in diverse metabolic pathways and are common in plant specialized
metabolism, mostly associated with alkaloid biosynthesis.
Isolation of novel N-methyltransferases from benzylisoquinoline alkaloid-producing species
Most research has targeted the isolation of biosynthetic enzymes and corresponding genes involved in the formation of relatively few compounds in a limited number of species
- Metabolic engineering is broadly defined as the improvement of cellular activities by the manipulation of enzymatic, transport, or regulatory functions using recombinant DNA technology. Several alkaloid biosynthetic genes have been used to genetically alter the production of various secondary metabolites in plants. Transgenic plants with altered or novel enzyme activities have also become a powerful tool to study the metabolic control architecture of alkaloid pathways.
Lotus is an important perennial aquatic plant that is consumed around the
world. There are more than 600 cultivars to date, comprising two species Nelumbo
nucifera Gaertn and N. lutea (Willd.) Pers. from two different isolated geographical
regions (Ni 1987). N. nucifera are widely distributed in East Asia, especially in China
and India, and N. lutea are from North America. N. nucifera is also called Chinese
lotus because it originated in China and has been cultivated a long time as an aquatic
crop. Based on its use, Chinese lotus can be classified into three types: for flower
production, for seed production, and for rhizome production (Guo 2009)
This topic presents Bioactive alkaloid in Lotus and Pharmaceutical Value leads The research of Separation and Purification of Alkaloid in Lotus
Eventually found in leave and Lotus leaves are popular remedy in Asia
Object : To develop efficient method for the preparative separation and purification of relatively minor bioactive component
Object : To separate alkaloids from crude extract of Nelumbo nucifera leaves by pH-Zone-refining CCC
Object : To develop and validate and analytical method for the identification and quantitation of bioactive alkaloids of extracts from lotus leaf and
benzylioquinoline alkaloids, bioactive compound mostly found
