Abstract
Dysosma pleiantha (Hance) Woodson, (Berberidaceae) is a herbaceous,
rhizomatous perennial, highly prized by the mountain tribes of Taiwan for its
medicinal properties. It has long been used as a traditional Chinese medicine for
general remedies including treatment of snakebite, condyloma, weakness,
lymphadenopathy and tumors. It contains lignans, flavonoids and steroids and these
compounds are accumulated abundant in rhizome. These lignans and flavonoids
have potential application in anticancer activities. In recent years, natural
populations of D. peliantha have declined considerably due to anthropogenic
activities such as habitat destruction and over exploitation for medicinal
applications. Thus, the present study focused on the quantification of anticancer
compounds in D. pleiantha collected from different geographical regions, in vitro
callus culture and production of anticancer compounds under the influence of
organic additives, isolation, cloning, characterization and expression analysis of
lignan biosynthetic pathway genes and anticancer activity of biologically
synthesized metal nanoparticles from D. pleiantha rhizome.
At first, we have quantified the podophyllotoxin, quercetin and kaempferol in
different parts and rhizome of D. pleiantha collected from different geographical
regions using RP-HPLC. Initially we have used different mobile phase in various
combinations for the simultaneous detection of podophyllotoxin, quercetin and
kaempferol. Among the different solvent used, water and methanol in the ratio of
50:50 gave symmetric and high peak area. The quantitative analysis of different
parts of D. pleiantha showed that the rhizome was the best source for the extraction
of medicinally important podophyllotoxin as compared to other plant parts, whereas
both quercetin and kaempferol were mainly distributed in stem, roots and rhizome
as compared to leaf and petioles. On the other hand, the rhizome of D. pleiantha collected from different geographical area result showed that the rhizome collected
from Hsinchu, Miaoli, Nantou and Yangminshan showed maximum
podophyllotoxin accumulation than the rhizome grown in Alishan. In the case of
quercetin, Alishan and Miaoli chemotypes possess maximum accumulation than
other chemotypes, whereas, kaempferol were abundantly accumulated in all the
chemotypes with slightly lower in Hsinchu and Yangminshan.
The second major part of the study is focused on in vitro callus culture of D.
pleiantha and production of important anticancer compounds such as
podophyllotoxin, quercetin and kaempferol. Among the different explants used, the
leaf produced 100 % callus frequency and the leaf callus was further investigated
for mass multiplication and secondary metabolite content. We found that the callus
grown on B5 medium (Gamborg et al., 1968) supplemented with 1 mg/L 2,4-D
under dark condition produced maximum biomass of callus. Further, the callus was
cultured under different concentration of organic additives. Among the different
concentration of various additives used, the leaf callus cultured on B5 medium
supplemented peptone (1-4 g/L) is most suitable for the callus biomass induction
and production of lignan and flavonoids.
The third part of the thesis aims on isolation, cloning, characterization and
expression of lignan biosynthetic pathway genes in D. pleiantha. We have
successfully isolated two genes namely secoisolariciresinol dehydrogenase (DpSD)
and pinoresinol synthase (DpPS) from D. pleiantha using PCR based approach. The
expression of DpSD and DpPS genes in different parts of D. pleiantha were
analyzed using qRT-PCR. We found that both DpSD and DpPS mRNA accumulates
predominantly in rhizome.
The fourth part of the study investigates the anti-metasatic activity of
biologically synthesized gold nanoparticles on human fibrosarcoma cell line HT-
1080. The biosynthesized gold nanoparticles were confirmed and characterized by
ultraviolet–visible spectroscopy, Fourier transform infrared spectroscopy,
transmission electron microscopy, and scanning electron microscopy equipped with
energy dispersive spectroscopy. The results revealed that aqueous extract of D.
pleiantha rhizome has potential to reduce chloroauric ions into gold nanoparticles
and the synthesized gold nanoparticles were showed spherical in shape with an
average size of 127 nm. Further, we investigated the anti-metastatic activity of
biosynthesized gold nanoparticles against human fibrosarcoma cancer cell line HT-
1080. The results showed that the biosynthesized gold nanoparticles were non-toxic
to cell proliferation and, also it can inhibit the chemo-attractant cell migration of
human fibrosarcoma cancer cell line HT-1080 by interfering the actin
polymerization pathway.
Final part of the thesis covered anticancer activity of biologically synthesized
silver nanoparticles on breast cancer and AGS colon cancer cell lines. In this study,
silver nanoparticles were successfully synthesized by a rapid biological synthetic
method from aqueous rhizome extract of Dysosma pleiantha. The synthesized Dp-
AgNPs were characterized by ultraviolet–visible spectroscopy, Fourier transform
infrared spectroscopy, transmission electron microscopy, and scanning electron
microscopy equipped with energy dispersive spectroscopy. Further, we tested for its
efficiency as a potent cytotoxic agent against breast and colon cancer cell lines. It
was observed that the synthesized silver nanoparticles induced concentration
dependent inhibition of cell proliferation in both breast and colon cancer cells.

Contents
Abstract i
Contents iv
Contents of figures and tables vi
Chapter 1. Introduction
1.1 Medicinal plants 01
1.2 Occurrence of Lignans and flavonoids in plants 02
1.3 Dysosma pleiantha (Hance) Woodson 03
1.4 Toxicology of Dysosma pleiantha 08
1.5 Lignan biosynthetic pathway 18
1.6 Biotechnological approach for the production of secondary
metabolites 19
1.7 Biogenic synthesis of metal nanoparticles and its uses in medicine 21
1.8 Rationale of the thesis 22
Chapter 2. Quantitative analysis of anti-cancer compounds from different chemo
types of Dysosma pleiantha
2.1 Introduction 24
2.2 Materials and methods 26
2.3 Results and discussion 29
2.4 Conclusion 36
Chapter 3. Production of anti-cancer compounds (lignan and flavonoids) via
callus culture of Dysosma pleiantha
3.1 Introduction 37
3.2 Materials and methods 38
3.3 Results and discussion 42
3.4 Conclusion 50
v
Chapter 4. Isolation, cloning and expression of genes in different tissues of
Dysosma pleiantha
4.1 Introduction 51
4.2 Materials and methods 53
4.3 Results and discussion 64
4.4 Conclusion 80
Chapter 5. Anti-metastatic activity of biologically synthesized gold nanoparticles
on human fibrosarcoma cell line HT-1080
5.1 Introduction 81
5.2 Materials and methods 84
5.3 Results and discussion 89
5.4 Conclusion 102
Chapter 6. Anticancer activity of biogenic silver nanoparticles on breast cancer
cell lines and colon cancer
6.1 Introduction 103
6.2 Materials and methods 106
6.3 Results and discussion 110
6.4 Conclusion 117
Summary and future perspectives 118
References 121
Publication 151

Contents of figures and tables
Chapter 1
Figure 1 Dysosma pleiantha (Hance) Woodson 04
Figure 2 Structure of chemical constituents present in the Dysosma
pleiantha 08
Figure 3 A general schematic representation of podophyllotoxin
toxicity 12
Figure 4 Podophyllotoxin biosynthetic pathway 19
Table 1 List of chemical constituents of Dysosma pleiantha 07
Table 2 List of 24 patients of Dysosma pleiantha poisoning and
initial diagnosis 13
Chapter 2
Figure 1 The standard (reference) peaks of podophyllotoxin 31
Figure 2 The standard (reference) peaks of quercetin 31
Figure 3 The standard (reference) peaks of Kaempferol 32
Figure 4 Podophyllotoxin, quercetin and kaempferol content in
different parts of D. pleiantha 34
Figure 5
The content of podophyllotoxin, quercetin and
kaempferol in rhizome of D. pleiantha collected from
different geographical sources
35
Table 1 The least square regression analysis of podophyllotoxin,
quercetin and kaempferol calibration graph 30
Chapter 3
Figure 1 Influence of additives on leaf callus culture for the
production of anti- cancer compounds 49
Table 1 Callus induction efficiency in different explants of
Dysosma pleiantha 43
Table 2 Influence of different parameters in callus biomass
production 44
Table 3 Influence of additives on leaf callus biomass production 48
vii
Chapter 4
Figure 1
Hypothetical biosynthetic pathway leading from
coniferylalcohol to podophyllotoxin and 6-
methoxypodophyllotoxin
52
Figure 2 Total RNA isolated from leaf tissue of D. pleiantha 64
Figure 3 Multiple sequence alignment of SD gene nucleotide
sequences 65
Figure 4 PCR amplification of 837 bp DpSD gene 66
Figure 5 Map of pGEM-T easy vector 67
Figure 6 Nucleotide and deduced amino acid sequence of DpSD 68
Figure 7 Multiple sequence alignment and Conserved Domain
Database (CDD) search on NCBI server 69
Figure 8 Kyte-Doolittle Hydropathy plot 70
Figure 9 Phylogenetic tree constructed by MEGA 4 software 71
Figure 10 PCR amplification of ~582 bp DpPS gene 72
Figure 11 Nucleotide and deduced amino acid sequence of DpPS 73
Figure 12 ClustalW alignment of DpPS with other plant PS amino
acid sequences and CDD search on NCBI server 74
Figure 13 Kyte-Doolittle Hydropathy plot and transmembrane
domain (Amino acid residue 7-29) 75
Figure 14 Phylogenetic tree constructed by MEGA 4 software 76
Figure 15 Differential expression of DpSD and DpPS gene 78
Chapter 5
Figure 1 UV - Visible spectrum of Dp-AuNps 90
Figure 2 FT-IR spectra of Dp-AuNPs 92
Figure 3 TEM and SEM micrographs of the synthesized Dp-AuNPs 94
Figure 4 Cell viability studies at different concentrations of Dp-
AuNPs on HT-1080 at 6, 12 and 24 h period 96
Figure 5 Actin and DAPI staining to reveal actin and nucleus
structure 97
Figure 6
The inhibitory effect of the synthesized Dp-AuNPs (120
μM) on cell motility of human fibrosarcoma cell line HT-
1080
99
Figure 7 Rac1 GTP pull down assay 101
viii
Chapter 6
Figure 1 UV-visible spectrum of surface plasmon resonance of Dp-
AgNps 111
Figure 2 FT-IR analysis of Dp-AgNps 112
Figure 3 TEM micrographs of Dp-AgNps 113
Figure 4 SEM and EDS micrographs of Dp-AgNps 114
Figure 5 Cytotoxicity of different concentrations of Dp-AgNPs on
breast cancer cell line MDA-MB-231 and MDA-MB-453 115
Figure 6 Cytotoxicity of different concentrations Dp-AgNPs on
AGS Colon cancer cells

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