Bioactive luteoloside produced by Myroides odoratimimus, solvent-tolerant bacterium form the rhizosphere of Lonicera japonica

Article abstract of DOI:10.1080/14786419.2018.1481838

Luteoloside (luteolin-7-O-glucoside), the biomarker of Lonicera japonica, was efficiently bio-synthetized from its cheaper precursor luteolin. The structure of luteoloside was characterized by LC-MS and NMR analyses. Compared to the significant inhibitory effect of luteolin on human hepatocyte cell line LO2 at high doses, luteoloside did not show obvious cytotoxic effects at any test dose. Moreover, luteoloside exhibited obvious promotive effects on human hepatocyte cells, suggesting a potential application in hepatoprotective therapies.

Full text of DOI:10.1080/14786419.2018.1481838

Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: http://www.tandfonline.com/loi/gnpl20
Bioactive luteoloside produced by Myroides
odoratimimus, solvent-tolerant bacterium form
the rhizosphere of Lonicera japonica
Y. X. Zhao, J. J. Wang, L. Zhang, S. Zhang, S. L. Su, J. A. Duan, Z. Z. Yao & S. K.
Xu
To cite this article: Y. X. Zhao, J. J. Wang, L. Zhang, S. Zhang, S. L. Su, J. A. Duan, Z. Z.
Yao & S. K. Xu (2018): Bioactive luteoloside produced by Myroides odoratimimus, solvent-
tolerant bacterium form the rhizosphere of Lonicera japonica, Natural Product Research, DOI:
10.1080/14786419.2018.1481838
To link to this article: https://doi.org/10.1080/14786419.2018.1481838
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Natural Product research, 2018
https://doi.org/10.1080/14786419.2018.1481838
SHORT COMMUNICATION
Bioactive luteoloside produced by Myroides odoratimimus,
solvent-tolerant bacterium form the rhizosphere of Lonicera
japonica
Y. X. Zhao, J. J. Wang, L. Zhang, S. Zhang#, S. L. Su, J. A. Duan#, Z. Z. Yao and S. K. Xu
Jiangsu collaborative Innovation center of chinese Medicinal resources Industrialization, Jiangsu Key
laboratory for high technology research of tcM Formulae, state Key laboratory cultivation Base for tcM
Quality and efficacy, Nanjing university of chinese Medicine, Nanjing, Pr china
ABSTRACT
ARTICLE HISTORY
received 24 January 2018
accepted 21 May 2018
Luteoloside (luteolin-7-O-glucoside), the biomarker of Lonicera
japonica, was efficiently bio-synthetized from its cheaper precursor
luteolin. The structure of luteoloside was characterized by LC-MS and
NMR analyses. Compared to the significant inhibitory effect of luteolin
on human hepatocyte cell line LO2 at high doses, luteoloside did not
show obvious cytotoxic effects at any test dose. Moreover, luteoloside
exhibited obvious promotive effects on human hepatocyte cells,
suggesting a potential application in hepatoprotective therapies.
KEYWORDS
Myroides odoratimimus;
rhizosphere bacteria;
Lonicera japonica;
biotransformation;
luteoloside;
hepatocytotoxicity
1. Introduction
Lonicera japonica, ‘Jinyinhua’ in Chinese, is an important herbal plant that has been widely
used in Chinese medicine for thousands of years. This important herb is used to treat various
diseases, such as severe acute respiratory syndromes, H1N1 influenza, and hand-foot-and-
mouth disease (Yuan et al. 2014). Luteoloside as well as chlorogenic acid is the biomarker
used by the Chinese Pharmacopoeia for evaluating the quality of Lonicera japonica.
Pharmacological experiments have shown that luteoloside has a spectrum of biological
CONTACT s. Zhang
zhangsci@njucm.edu.cn; J. a. duan
dja@njucm.edu.cn
#current address: 138 Xianlin road, college of Pharmacy, Nanjing university of chinese Medicine, Nanjing 210023, china.
supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2018.1481838.
© 2018 Informa uK limited, trading as taylor & Francis Group

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Y. X. ZHAO ET AL.
Figure 1. efficient biosynthesis of luteoloside by Myroides odoratimimus Xt02 from the rhizosphere of
Lonicera japonica.
activities, particularly anti-inflammatory (Francisco et al. 2014) and hepatoprotective prop-
erties (Zheng et al. 2004).
To obtain plenty of luteoloside, a number of methods have been developed, such as plant
extraction (He et al. 2012; Wang et al. 2017) and aqueous biocatalysis from cheaper precursors
(Hyung Ko et al. 2006; Palmeri et al. 2017), but it is still difficult to satisfy the commercial
demand for luteoloside for pharmaceutical production. Glycosylation of flavonoid aglycones
in aqueous miscible organic media has long been proven to be an ideal way to both enhance
value and reduce cytotoxicity (Wu et al. 2013; Chu et al. 2014). In this study, we reported an
efficient biosynthesis of luteoloside by the newly isolated Myroides odoratimimus XT02 from
the rhizosphere of Lonicera japonica. The hepatocytotoxicities of luteoloside and its aglycone
luteolin were also investigated.
2. Results and discussion
2.1. Establishment of efficient biotransformation system
Efficient glycosylation of luteolin was carried out in aqueous miscible organic media by the
Lonicera japonica rhizosphere strain M. odoratimimus XT02 (Figure 1). Retention times of
HPLC spectrograms for luteolin and its derivative were 18.7 min and 14.9 min, respectively
(Figure S1). In 15% dimethyl sulfoxide (DMSO, v/v) reaction system for 15 h, over 95% of
luteolin (1.5 g /L) was converted to the product, which was much higher than that in the
water phase (approximately 8% yield of 0.2 g/L luteolin in 15 h). DMSO, considered as a green
solvent and catalyst, is usually used as the solvent or cosolvent for the biotransformation of
natural products (Cao et al. 2017). However, organic solvents, especially with a log P value
below 0 (DMSO has a log P value of -1.35), are extremely toxic to enzymes as well as microbes
(Zhang et al. 2013). Thus, the use of solvent tolerant strains is considered to be a key suc-
cessful factor for efficient biotransformation in natural product research.
2.2. Purification and characterization of the bio-transformed product
The high resolution ESI-MS gave a molecular ion [M − H]− at m/z 447.0219 (Figure S2), sug-
gesting an empirical molecular formular of C21H20O11. The m/z of the product was 162 (equal
to the mass of one hexose) higher than that of luteolin, presumably corresponding to a
glycosyl luteolin derivative. Since the 1H NMR and 13C NMR results (Figure S3) were identical
to those of an authentic luteolin in the literature (Wang et al. 2017), the product was

NATURAL PRODUCT RESEARCH
3
Figure 2. effects of luteoloside and luteolin on the proliferation of human hepatocyte cell line lo2.
identified as luteoloside, which was further confirmed by HMBC and HSQC correlations
(Figure S4). Although the glycosylation of luteolin by plant cells (El Riachy et al. 2011) and
UDP-glucosyltransferase (Hyung Ko et al. 2006; Palmeri et al. 2017) have been elaborated,
our report is the first demonstration of the efficient glycosylation of luteolin by bacteria.
2.3. Hepatocytotoxicity tests of luteolin and luteoloside
The effects of luteolin and luteoloside on the proliferation of human hepatocyte cell line
LO2 were evaluated by methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay. As shown
in Figure 2, higher doses of luteolin (0.067 μM and 0.200 μM) exhibited significant inhibitory
effects on human hepatocyte cell line LO2 proliferation in a dose-dependent manner, while
its glycosides showed the opposite effect.
Luteolin is known to have a protective effect on liver cells, and could be a promising
auxiliary drug for the treatment of liver cancer (Chei et al. 2017). However, fewer hydrophilic
groups leads to a low bioavailability which limits its clinical application (Ross and Kasum
2002). Moreover, according to previous reports (Galati and O'Brien 2004), higher dose of
flavonoids may cause an increased toxicity. Some studies have found that glycosyl can
increase the bioavailability of flavonoids (Hyung Ko et al. 2006; Ajish et al. 2015; Palmeri et
al. 2017). However, there are few reports on the toxic effects of the flavonoid glycoside
compared to its aglycone. In this study, we found that luteoloside did not show obvious
cytotoxic effect compared to luteolin at a higher dose. Moreover, luteoloside exhibited obvi-
ous promotive effect on human hepatocyte cells.
3. Conclusions
This study reported a rhizosphere bacterium isolated from Lonicera japonica, which showed
efficient glycosylation activity for the production of luteoloside, a biomarker of Lonicera
japonica. Compared to its aglycone, luteloside not only showed lower hepatotoxicity, but
also exhibited promotive effect on human hepatocyte cells. In this context, the efficient
production of luteoloside and its application in hepatoprotective drugs could be an exciting
prospect.

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Y. X. ZHAO ET AL.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This work was supported by the Natural Science Foundation of Jiangsu Province (BK 20161048), the
Environmental Research Project of Jiangsu Province (2015022), the China Agriculture Research System
(CARS-21) and the State Project of TCM standardization ‘standardization construction of Re-Du-Ning
injection’(ZYBZH-C-JS-31).
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