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ベンジルイソキノリンアルカロイド、ヒゲナミンおよびベルベリン検出のためのバイオアッセイの開発

ポームラフィ, ヌンタウォン POOMRAPHIE, NUNTAWONG 九州大学

2023.03.20

概要

九州大学学術情報リポジトリ
Kyushu University Institutional Repository

DEVELOPMENT OF BIOASSAYS FOR THE DETECTION OF
BENZYLISOQUINOLINE ALKALOIDS,HIGENAMINE AND
BERBERINE
ポームラフィ, ヌンタウォン

https://hdl.handle.net/2324/6787541
出版情報:Kyushu University, 2022, 博士(臨床薬学), 課程博士
バージョン:
権利関係:

(様式5)




: ポームラフィ

論 文 題 名

ヌンタウォン

: DEVELOPMENT OF BIOASSAYS FOR THE DETECTION OF
BENZYLISOQUINOLINE

ALKALOIDS,HIGENAMINE,

AND

BERBERINE (ベンジルイソキノリンアルカロイド、ヒゲナミンおよびベル
ベリン検出のためのバイオアッセイの開発)




: 甲















Benzylisoquinoline alkaloids (BIA) are one classes of the natural compounds which are well
known as the pharmacologically active substances. Thus far, there are various kinds of medicines and dietary
supplements which are derived from the plant containing BIA in the market. The specific and reliable
analytical methods for determination of the individual BIA content in plant samples and plant-containing
samples are required for quality control of these products.
Higenamine (HM) is the BIA which are well-known as β-adrenoreceptor agonist. HM is recognized
as the prohibited compounds in sport games according to World Anti-Doping Agency (WADA). Here, the
immunological approaches for (S)-HM determination in plant samples were developed (Figure 1). The
antibody against HM was detected after immunization of mouse using HM conjugated with bovine serum
albumin (BSA) as an antigen. The monoclonal antibody against (S)-HM (MAb E8) was produced from the
systematically screened hybridoma (colony E8) obtained by fusing the mouse splenocytes with myeloma
(SP2/0). The MAb E8 was characterized and utilized for indirect competitive enzyme-linked immunosorbent
assay (icELISA). The detectable range of the developed icELISA was 7.81–125 ng/mL with the limit of
detection (LOD) of 4.41 ng/mL. Various validation methods were conducted to ensure the precision and
accuracy of the developed icELISA. The plant samples analysis revealed that the (S)-HM content detected
from icELISA were correlated to those obtained from HPLC-UV. The results confirmed that this developed
icELISA can be a useful tool for screening of (S)-HM-containing plants to prevent the unintentional used of
the HM in athletes [1].
Apart from that, the usefulness of MAb E8 was expanded as it was used for development of lateral
flow immunoassay (LFA) for (S)-HM detection. The detection sensor of the system was colloidal gold
nanoparticle conjugated with MAb E8. The paper-made device comprised of rabbit anti-mouse IgG
antibodies and HM-γ-globulin conjugates as control and test zones, respectively. The bounded HM on the
strip and free (S)-HM in the samples were competitively reacted with the detection sensor. The results of
these microenvironment phenomena were represented as the pink spot on the test zone, where the intensity
of the spot inversely relate to the content of free (S)-HM in the samples. The LOD of the developed LFA was
156 ng/mL. The sensitivity, selectivity, repeatability, and accuracy of the assay were systemically assessed to
ensure the reliability of the LFA. As a result, the fast and reliable HM determination assay, LFA, was
successfully developed [2]. The developed system could expand the versatility of detection to be possible in
more scenarios.

5

Figure 1 schematic diagram of determination methods development of (S)-HM.
Berberine (BBR) was also the BIA of interest in this study. BBR was contained in over 10 Kampo
medicine preparations. Here, the aptamer-based fluorometric assay for BBR detection in Kampo medicine
preparations was developed (Figure 2). The assay based on the fluorescent enhancement of BBR when it
binds with aptamer. The specific aptamer against BBR was selected using colloidal gold nanoparticle-based
systematic evolution of ligands by exponential enrichment (GOLD-SELEX). The aptamer candidate with
highest fluorescent intensity was used as the template for structure evolution of the aptamer. By truncation,
mutation, and polymerization of the candidate aptamer, the 80-mer aptamer was successfully obtained. The
aptamer-based fluorometric assay was developed with determination range of 0.780–25.0 µg/mL with LOD
of 0.431 µg/mL. The validation processes were implemented to confirm the reliability of the assay. Moreover,
the BBR content in Kampo medicine preparations determined by developed aptamer-based fluorometric
assay and those determined by HPLC-UV were correlated. This can be the useful tool for BBR determination
in Kampo medicine samples.
In conclusion, this study successfully developed the assay for BIA detection based on the binding
affinity of the macromolecule and ligand. These immunological assays and aptamer-based assay could be
applied for BIA detection in plant samples as an alternative method or use alongside with conventional
detection methods.

Figure 2 schematic diagram of determination methods development of BBR.
[1] Nuntawong P. Tanaka H. Sakamoto S. Morimoto S. Planta Med., 86 (11) 760-766, 2020.
[2] Nuntawong P. Ochi A. Chaingam J. Tanaka H. Sakamoto S. Morimoto S. Drug Test Anal., 13 (4)
762-769, 2021.

6

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ACKNOWLEDGEMENTS

On behalf of my advisors, Professor Satoshi Morimoto, Professor Hiroyuki Tanaka, and

Associate Professor Seiichi Sakamoto, I would like to express my profound and sincere gratitude.

They have always inspired, advised, encouraged, and trusted me throughout the course of my

research and preparation of this thesis. With their assistance and opportunities, the purpose of my

life is to grow.

I am grateful to Professor Tomofumi Miyamoto of the Graduate School of Pharmaceutical

Science at Kyushu University for his insightful comments and ideas on my dissertation.

Additionally, I would like to express my profound thanks and gratitude to Associate

Professor Boonchoo Sritularak of the Faculty of Pharmaceutical Sciences, Chulalongkorn

University, Thailand and Assistant Professor Gorawit Yusakul of the School of Pharmacy,

Walailak University, Thailand who inspired me and instilled in me a passion for study.

In addition, I would like to convey my thanks and admiration to Kanta Noguchi, Kei

Minami, and Hiroki Uchiyama for their everyday assistance, emotional support, and

encouragement.

I'd also want to thank the members of our department and the Thai exchange students for

their friendship and support.

Lastly, I would want to thank my family for their love and support, which have allowed

me to reach my objective.

Poomraphie

100

Nuntawong

...

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