Antimicrobial and antiquorum-sensing activity of Ricinus communis extracts and ricinine derivatives

Article abstract of DOI:10.1080/14786419.2017.1423306

Ricinine (1), a known major alkaloid in Ricinus communis plant, was used as a starting compound for the synthesis of six ricinine derivatives; two new and four known compounds. The new derivatives; 3-amino-5-methyl-1H-pyrazolo[4,3-c]pyridin-4(5H)-one (2),

Full text of DOI:10.1080/14786419.2017.1423306

Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: http://www.tandfonline.com/loi/gnpl20
Antimicrobial and antiquorum-sensing activity of
Ricinus communis extracts and ricinine derivatives
Mai H. El-Naggar, Abdelaziz Elgaml, Fatma M. Abdel Bar & Farid A. Badria
To cite this article: Mai H. El-Naggar, Abdelaziz Elgaml, Fatma M. Abdel Bar & Farid A. Badria
(2018): Antimicrobial and antiquorum-sensing activity of Ricinus communis extracts and ricinine
derivatives, Natural Product Research, DOI: 10.1080/14786419.2017.1423306
To link to this article: https://doi.org/10.1080/14786419.2017.1423306
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Natural Product research, 2018
https://doi.org/10.1080/14786419.2017.1423306
Antimicrobial and antiquorum-sensing activity of Ricinus
communis extracts and ricinine derivatives
Mai H. El-Naggara,b, Abdelaziz Elgamlc, Fatma M. Abdel Barb and Farid A. Badriab
adepartment of Pharmacognosy, Faculty of Pharmacy, sohag university, sohag, egypt; bdepartment of
c
Pharmacognosy, Faculty of Pharmacy, Mansoura university, Mansoura, egypt; department of Microbiology
and Immunology, Faculty of Pharmacy, Mansoura university, Mansoura, egypt
ABSTRACT
ARTICLE HISTORY
received 28 october 2017
accepted 26 december 2017
Ricinine (1), a known major alkaloid in Ricinus communis plant, was
used as a starting compound for the synthesis of six ricinine derivatives;
two new and four known compounds. The new derivatives; 3-amino-
5-methyl-1H-pyrazolo[4,3-c]pyridin-4(5H)-one (2), and 3-amino-5-
methyl-1-(phenylsulfonyl)-1H-pyrazolo[4,3-c]pyridin-4(5H)-one (3),
as well as the previously prepared derivatives (4–7) were subjected
for antimicrobial and antiquorum-sensing evaluation in comparison
to different R. communis extracts. Acetyl ricininic acid derivative (5)
showed the highest antimicrobial activity among all tested derivatives
against Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae,
Pseudomonas aeuroginosa and Candida albicans. However, compound
7 (4-methoxy-1-methyl-2-oxo-1,2-dihydropyridine-3-carboxamide)
showed the highest antiquorum-sensing activity among all tested
compounds and extracts. These findings proved the usefulness of
ricinine as a good scaffold for the synthesis of new antimicrobial and
antiquorum-sensing derivatives in spite of its poor contribution to
the antimicrobial activity of the plant extracts.
KEYWORDS
antimicrobial; antiquorum-
sensing; ricinine alkaloid;
Ricinus communis l.; ricinine
derivatives; semisynthesis
CONTACT Farid a. Badria
badri002@mans.edu.eg
supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2017.1423306.
© 2018 Informa uK limited, trading as taylor & Francis Group

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M. H. EL-NAGGAR ET AL.
1. Introduction
The aggravating global threat of the development of antimicrobial resistance is leading to
a continuous demand for the discovery of new antimicrobial drugs and anti-virulence agents.
Bacterial cell to cell communication termed as‘quorum-sensing’represents a signalling sys-
tem that is important for the regulation of several cellular processes such as virulence factors
expression, biofilm formation and competence (Bassler 1999; Miller and Bassler 2001; Kalia
2013). Therefore, targeting the bacterial pathogenic potential through quorum-sensing
inhibition is one of the new strategies that are used to combat the microbial resistance
(Bhardwaj et al. 2013).
Plants represent a valuable sustainable source of antimicrobial drugs and resistance mod-
ifying agents (Cowan 1999; Abreu et al. 2012). Ricinus communis L. (Castor oil plant) is an
important industrial crop belonging to the spurge family, Euphorbiaceae. It is a fast-growing
plant with a great environmental and economic value and it was reported to have different
biological activities (Ribeiro et al. 2016). The antimicrobial activity of R. communis extracts
is one of the most studied activities of this plant. The plant extracts, especially its leaf extract,
were reported to have antimicrobial activity against different microorganisms; however, the
pure compound(s) responsible for this activity was not identified (Ribeiro et al. 2016). It is
important to identify the pure compound(s) responsible for the antimicrobial activity since
the use of total plant extracts as antimicrobial drugs is not preferable because of their com-
plicated chemical nature and difficulty of standardisation (Cos et al. 2006; Abreu et al. 2012).
Plant alkaloids, either natural or semisynthetic, represent a historical opulent source of anti-
microbial and antibiotic enhancing agents (Cushnie et al. 2014). Ricinine, the predominant
alkaloid in R. communis plant, is a simple and moderately toxic α-pyridone alkaloid. Several
α-pyridone nucleus-containing compounds, either natural or synthetic, were reported to
have antimicrobial activity (Li et al. 2000; Jessen and Gademann 2010; Wu et al. 2014). Despite
the reported antimicrobial activity of R. communis extracts and compounds containing the
α-pyridone ring, the antimicrobial activity of pure ricinine and most of its derivatives was
not previously investigated. It is worth to note that the antimicrobial activity for only the
3-carboxamide derivative of ricinine (7) was previously investigated against Staphylococcus
aureus normal and drug-resistant strains (Swarupa et al. 2017). They reported that the 3-car
-
boxamide derivative of ricinine showed potent activity against S. aureus and caused inhibi-
tion of biofilm formation in all tested strains (Swarupa et al. 2017).
Castor meal, the residual waste left after defatting the castor seeds, contains about 0.77%
w/w of ricinine and it represents a good source for isolation of this alkaloid (De Melo Cazal
et al. 2009; Li et al. 2013). Though, castor meal has about 34–36% protein content, it is used
with caution as organic fertilizer and cannot be used as an animal feed because of the pres-
ence of the extremely toxic protein (ricin), other toxic allergens and ricinine alkaloid (Anandan
et al. 2005; Dubois et al. 2013). The production of large amounts of this waste is one of the
problems associated with biodiesel industry from castor oil on large scale (Li et al. 2013).
Therefore, the use of this waste for isolation of large amounts of ricinine alkaloid may reduce
the environmental hazards of biodiesel industry from castor oil.
The aim of this research was the isolation of ricinine (1) and testing its antimicrobial and
antiquorum-sensing activities in comparison to different R. communis extracts and investi-
gation of its contribution to the antimicrobial activity of these extracts. Furthermore, the
antimicrobial and antiquorum-sensing activities of some synthesised ricinine derivatives

NATURAL PRODUCT RESEARCH
3
(2–7) were determined and compared to ricinine activities in an attempt to study their
structure–activity relationship.
2. Results and discussion
2.1. Chemistry
Ricinine (1) was isolated in a high yield from R. communis seed extract (0.11% w/w). Its
structure was identified by comparing its spectroscopic data with that reported in literature
(Sule and Sani 2008; De Melo Cazal et al. 2009; Li et al. 2013). Several studies reported the
isolation of ricinine from R. communis leaves and seeds (Kang et al. 1985; De Melo Cazal
et al. 2009; Li et al. 2013). However, this method of isolation of ricinine showed to be more
suitable for isolation of ricinine from large quantities of the castor meal that is left after
obtaining the castor oil for biodiesel production.
Compounds 2–7 (Figure 1) were prepared via chemical derivatization of ricinine and
subjected to antimicrobial and antiquorum-sensing evaluation. Compound 2 was obtained
by the reaction of ricinine with hydrazine hydrate. 1H-NMR spectrum (Table S1) of 2 showed
close resemblance to that of ricinine except for the presence of two additional proton signals
at δ 5.13 (2H, br. s, NH2) and δ 11.69 (1H, br. s, 1-NH) and the absence of the methoxyl group
signal. The 13C-NMR spectrum (Table S2) revealed the presence of only seven carbon signals
instead of eight and showed the absence of the methoxyl group and the nitrile carbon
signals. In addition, the upfield shift of the carbon signal at δ 144.3, C-4c and the appearance
of imine carbon at δ 153.2, C-3 indicated reaction of ricinine with hydrazine hydrate and
formation of pyrazolo-pyridine structure. Therefore, 2 was concluded to be a new compound
(3-amino-5-methyl-1H-pyrazolo[4,3-c]pyridin-4(5H)-one).
Compound 3 was obtained by reaction of 2 with benzene sulfonyl chloride. Analysis of
its HR-FAB+-MS data suggested the molecular formula C13H12N4O3S with 10 degrees of
Figure 1. structures of compounds 1–7.

4
M. H. EL-NAGGAR ET AL.
unsaturation indicating presence of extra phenyl moiety. This was confirmed by the presence
of extra signals for 5 aromatic protons at δ 7.83 (2H, d, J = 7.6 Hz, H-2′/6′), 7.73 (1H, overlapped
1
dd, H-4′) and 7.61 (2H, dd, J = 8.0, 7.6 Hz, H-3′/5′) in its H-NMR spectrum (Table S1). Its
13C-NMR spectrum (Table S2) also showed the presence of extra signals for 6 aromatic carbons
at δ 136.9 (qC, C-1′), 135.2 (CH, C-4′), 130.2 (CH, C-3′/5′) and 127.5 (CH, C-2′/6′). The assignment
of the carbons of this compound was confirmed by HMBC spectrum. Consequently, com-
pound 3 was concluded to be the new compound, 3-amino-5-methyl-1-(phenylsulfon-
yl)-1H-pyrazolo[4,3-c]pyridin-4(5H)-one and confirmed the incidence of a reaction between
benzene sulfonyl chloride and the NH group of (2).
The known compounds 4–7 were identified as ricininic acid (4), the acetyl derivative of
ricininic acid (5), 5-bromoricinine (6), 3-carboxamide derivative of ricinine (7) when compared
to their reported spectroscopic data (Robinson and Hook 1964; Yuldashev 2001; Zhao et al.
2015).
2.2. Antimicrobial activity
Ricinine (1), its derivatives (2–7), total MeOH extracts of leaves and seeds of R. communis,
EtOAc and aqueous fractions of the seed extract and castor oil were subjected for in vitro
agar disc-diffusion antibacterial assay. In this assay, the MICs were determined for the most
active compounds and/or extracts using Gram-positive bacteria (S. aureus), and Gram-
negative bacteria (E. coli, K. pneumoniae and P. aeuroginosa) as presented in Table S3.
Ampicillin and gentamicin were used as standard antibacterial drugs (positive control), while
DMSO alone was used as a negative control. The obtained results indicated that 5 showed
the highest activity among the tested ricinine derivatives and R. communis extracts. It showed
moderate activity against K. pneumoniae and P. aeruginosa, while, strong activity against
E. coli and S. aureus. However, ricinine itself was found to be completely inactive against any
of the tested bacterial strains. Derivative 2 showed moderate activity against E. coli and weak
activity against K. pneumoniae. Derivative 3 showed moderate activity against E. coli,
K. pneumoniae, P. aeruginosa and S. aureus. In addition, derivative 4 showed moderate activity
against K. pneumoniae and P. aeruginosa, while, weak activity against E. coli. Moreover, deriv-
ative 6 exhibited moderate activity against K. pneumoniae. In accordance with the results
of Swarupa et al. (2017), derivative 7 showed antibacterial activity against S. aureus. Although,
its potency against S. aureus was found to be moderate, and lower than that reported by
Swarupa et al. (2017). In accordance with literature (Ribeiro et al. 2016), the leaf extract of
R. communis showed reasonable antibacterial activity against all tested bacterial strains. The
seed extract showed strong activity against P. aeruginosa and moderate activity against
E. coli and S. aureus. Moreover, the castor oil and the EtOAc fraction of the seed extract
showed reasonable antibacterial activity against P. aeruginosa bacterial strain and to lesser
extent towards E. coli.
The antifungal activity of the same compounds and extracts was also tested against the
yeast-like pathogenic fungus C. albicans and fluconazole was used as a standard antifungal
drug and DMSO as a negative control. Leaf extract showed the highest antifungal activity
among all tested compounds and extracts. In addition, MeOH extract of the seed, 4 and 5
showed potential antifungal activity. However, the parent compound, ricinine, showed no
antifungal activity. In all antimicrobial assays, the negative control, DMSO, did not show any
noticeable activity.

NATURAL PRODUCT RESEARCH
5
2.3. Antiquorum-sensing activity
Besides the antimicrobial testing, all compounds and extracts were further evaluated for
their antiquorum-sensing activity against Ch. violaceum using catechin as a positive and
DMSO as a negative controls. The antiquorum-sensing activity of the compounds and
extracts was determined by testing their ability for inhibiting the release of a violet pigment
(violacein), which is released in response to acyl homoserine lactones signals (McLean et al.
2004; Chu et al. 2011). The pigment inhibition radius in this assay, revealed that 7, showed
the highest antiquorum-sensing activity among the tested compounds and extracts. On the
other hand, 5 showed moderate activity, and 2 and 3 exhibited weak activity as shown in
Table S4. However, the parent compound, ricinine showed no activity at all. Furthermore,
the leaf extract showed moderate and much better activity than that of the total seed extract.
2.4. Structure–activity relationship of ricinine derivatives as antimicrobial agents
Based on the obtained results of antimicrobial and antiquorum-sensing activities for ricinine
derivatives (1–7), it could be inferred that conversion of the cyano-pyridone nucleus of
ricinine into a pyrazolo-pyridone nucleus imparted moderate and weak antibacterial activ-
ities to 2 against Gram-negative bacteria E. coli and K. pneumonia, respectively, and weak
antiquorum-sensing activity. Interestingly, the presence of the benzene sulfonyl moiety in
3 increased the antibacterial activity of 2 against E. coli and K. pneumonia and improved its
activity against P. aeuroginosa and S. aureus. Meanwhile, the removal of the O-methyl group
of ricinine and the presence of a phenolic group at position 4 as in ricininic acid (4) caused
moderate antimicrobial activity against P. aeuroginosa, K. pneumoniae and Candida albicans,
while weak antibacterial activity against E. coli. The replacement of the O-methyl group of
ricinine with acetyl group greatly affected its antimicrobial activity. The presence of the acetyl
group imparted antimicrobial activity to the most active ricinine derivative (5) against all
tested bacterial strains and the pathogenic fungus, C. albicans. The presence of this group
also caused reasonable inhibition of quorum-sensing in comparison to the inactive parent
compound (ricinine). Bromination of ricinine at position 5 as in derivative 6 did not greatly
affect its antimicrobial activity and only caused moderate antibacterial activity against
K. pneumonia. The conversion of the cyanide group of ricinine into a carboxamide group as
shown in 7 caused weak antibacterial activity against E. coli and K. pneumoniae, while, mod-
erate antibacterial activity against S. aureus. However, the presence of the carboxamide
group imparted the highest antiquorum-sensing activity to compound 7 among the other
derivatives.
3. Conclusion
This study described a simple method for isolation of ricinine alkaloid in high yield from
R. communis seeds. The isolated ricinine was used as a synthon for production of valuable
compounds with better and diverse biological activities. Six compounds; two new and four
known were prepared from ricinine. The antimicrobial testing of the prepared compounds
showed that although ricinine does not contribute to the antimicrobial activity of the leaf
and seed extracts of R. communis plant, it represents a good scaffold for antimicrobial and
antiquorum-sensing derivatives. Castor oil and the components of the EtOAc fraction may

6
M. H. EL-NAGGAR ET AL.
partially contribute to the antimicrobial activity of the seed extract and they may exhibit a
synergistic effect.
It is worth to be noted that chemical treatment of castor meal with NaOH is one of the
most efficient methods used for detoxification of castor meal (Anandan et al. 2005). Treating
castor meal with NaOH may be used for its detoxification to be used as an animal feed and
for preparation of ricininic acid (4) (Yuldashev 2001) to be used as a synthon for preparation
of ricinine derivatives. This may contribute to minimise the environmental hazards associated
with biodiesel industry from castor oil and providing additional medical and economic ben
-
efits for R. communis plant via preparing new antimicrobial and antiquorum-sensing
derivatives.
Acknowledgements
We do acknowledge and appreciate the great help and support of Prof Dr Kuniyoshi Shimizu, Division of
Systematic Forest and Forest Products Sciences, Department of Agro-Environmental Sciences, Graduate
School of Bioresource and Bioenvironmental Sciences, Kyushu University, Fukuoka, Japan, for carrying
out the HR-MS analysis for the prepared compounds.
Disclosure statement
No potential conflict of interest was reported by the authors.
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