A quinoline alkaloid rich Quisqualis indica floral extract enhances the bioactivity

Article abstract of DOI:10.1080/14786419.2019.1634709

A volatile alkaloid quinoline-4-carbonitrile (QCN) was isolated from the floral extract of Quisqualis indica. Major compounds were trans-linalool oxide (1.0, 4.5%), methyl benzoate (1.0, 4.0%), 2,2,6-trimethyl-6-vinyl-tetrahydropyran-3-one (7.4, 17.8%), 2,2,6-trimethyl-6-vinyl-tetrahydropyran-3-ol (1.0, 1.2%), (E,E)-α-farnesene (29.1, 16.1%), QCN (5.7, 1.3%) in live and picked flowers, respectively. Flower compositions were altered due to change in enzymatic reaction at the time of picking. Some rearrangements of oxygenated terpenoids occurred in the process of hydrodistillation to obtain essential oil. Chemical synthesis of QCN and its selectively reduced products derived from QCN were prepared through green reaction process. The catalytic modification of QCN has produced quinoline-4-methylamine; the later compound has shown enhanced bio-activities. QCN and floral extract (absolute) have shown potential anti-inflammatory and antioxidant activities. Besides, floral absolute has shown significant anti-inflammatory and antioxidant activities due to improved QCN (19.7%) content to synergize amongst terpenoids and benzenoids as compared to the essential oil with 1.1% of QCN.

Full text of DOI:10.1080/14786419.2019.1634709

Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: https://www.tandfonline.com/loi/gnpl20
A quinoline alkaloid rich Quisqualis indica floral
extract enhances the bioactivity
Prasant Kumar Rout, Prashant Kumar, Y. Ramachandra Rao, Anant Kumar,
Dnyaneshwar U. Bawankule, Ruchi Singh, Kijay Bahadur Singh, Chandan
Singh Chanotiya & S. N. Naik
To cite this article: Prasant Kumar Rout, Prashant Kumar, Y. Ramachandra Rao, Anant Kumar,
Dnyaneshwar U. Bawankule, Ruchi Singh, Kijay Bahadur Singh, Chandan Singh Chanotiya & S.
N. Naik (2019): A quinoline alkaloid rich Quisqualisꢀindica floral extract enhances the bioactivity,
Natural Product Research, DOI: 10.1080/14786419.2019.1634709
To link to this article: https://doi.org/10.1080/14786419.2019.1634709
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NATURAL PRODUCT RESEARCH
https://doi.org/10.1080/14786419.2019.1634709
A quinoline alkaloid rich Quisqualis indica floral
extract enhances the bioactivity
Prasant Kumar Routa,b , Prashant Kumara , Y. Ramachandra Raoc,
Anant Kumarb,d , Dnyaneshwar U. Bawankuleb,d , Ruchi Singha,
Kijay Bahadur Singha , Chandan Singh Chanotiyaa,b , and S. N. Naike
aChemical Sciences Division, CSIR-Central Institute of Medicinal and Aromatic Plants, Lucknow, Uttar
b
c
Pradesh, India; Academy of Scientific and Innovative Research (AcSIR), New Delhi, India; Forest and
Marine Products Department, CSIR-Regional Research Laboratory (IMMT), Bhubaneswar, Odisha,
d
India; Molecular Bioprospection Department, CSIR-Central Institute of Medicinal and Aromatic
Plants, Lucknow, Uttar Pradesh, India; Center for Rural Development and Technology, Indian
e
Institute of Technology Delhi, Hauz Khas, New Delhi, India
ABSTRACT
ARTICLE HISTORY
Received 10 October 2018
Accepted 18 June 2019
A volatile alkaloid quinoline-4-carbonitrile (QCN) was isolated
from the floral extract of Quisqualis indica. Major compounds
were trans-linalool oxide (1.0, 4.5%), methyl benzoate (1.0, 4.0%),
2,2,6-trimethyl-6-vinyl-tetrahydropyran-3-one (7.4, 17.8%), 2,2,6-tri-
methyl-6-vinyl-tetrahydropyran-3-ol (1.0, 1.2%), (E,E)-a-farnesene
(29.1, 16.1%), QCN (5.7, 1.3%) in live and picked flowers, respect-
ively. Flower compositions were altered due to change in enzym-
atic reaction at the time of picking. Some rearrangements of
oxygenated terpenoids occurred in the process of hydrodistilla-
tion to obtain essential oil. Chemical synthesis of QCN and its
selectively reduced products derived from QCN were prepared
through green reaction process. The catalytic modification of QCN
has produced quinoline-4-methylamine; the later compound has
shown enhanced bio-activities. QCN and floral extract (absolute)
have shown potential anti-inflammatory and antioxidant activities.
Besides, floral absolute has shown significant anti-inflammatory
and antioxidant activities due to improved QCN (19.7%) content
to synergize amongst terpenoids and benzenoids as compared to
the essential oil with 1.1% of QCN.
KEYWORDS
Quisqualis indica; HS-SPME;
Essential oil; Absolute;
Quinoline-4-carbonitrile; Pd/
Ac Catalyst; Anti-
inflammatory
CONTACT Prasant Kumar Rout
pk.rout@cimap.res.inprasantrout@gmail.com
Supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2019.1634709.
ß 2019 Informa UK Limited, trading as Taylor & Francis Group

2
P. ROUT ET AL.
1. Introduction
Quisqualis indica L syn. Q.densiflora (family Combretaceae) is a large woody scandent
shrub and a hardy creeper to provide profuse shade around the house (Anon 1991).
This plant is native to Indo-Malaysian region as well as in tropical Africa. In general,
it is being cultivated throughout the India as well as in China (Yan et al. 2018).
It is planted in gardens for the white-red colored flowers and particularly blooming
generously in pleasant weather during the months of March–May (Spring) and
September–November (Autumn). Flowers blossom in the evening as white colored
flowers and gradually changing to red by afternoon. Flowers have a distinguishing
mild refreshing fragrance soothing into the environment during evening hours to late
night. The floral aqueous-alcoholic extract had shown 90.2% of antioxidant activity in
500 mg/L solution with 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical (Afify and Hassan
2016). Recently, Yan et al. (2016) reported 35 compounds based on the computer
matching of mass spectra. Rout et al. (2008) had reported the comparison of concrete,
absolute and essential oil of Q.indica flowers. There are 34 compounds identified
including quinoline-carbonitrile through GC/MS analysis. Presently, fresh flowers are
collected from CSIR-CIMAP campus (Accession No: 83008) for preparation of pentane
extract (concrete) (Figure S1), and essential oil (Figure S2). Quinoline-carbonitrile is
extracted upto 15% in concrete as compared to 1.1% in essential oil. However, its
identification was only tentative and the exact isomer could not be ascertained. There
was only report available on isolation of quinoline-4-carbonitrile (QCN) from Fungus
Xylaria sp. BCC 9653 (Pongcharoen et al. 2007). Hence, thorough characterization work
of this compound was undertaken (Figure S3). Furthermore, the extracts, isolated
quinoline-carbonitrile and its derivatives from Q. indica flowers were investigated for
in-vitro anti-inflammatory activity. Flower volatiles were determined by headspace-solid
phase microextraction (HS-SPME) analysis. Due to its excellent fragrance profile as well
as bioactivities (anti-inflammatory and antioxidant) of floral extract or its pure
molecule, this flower may be useful in various pharmaceutical preparations.
2. Results and discussion
2.1. HS-SPME analysis of live and picked flowers
Headspace volatiles compositions of live and picked flowers contained mono-
terpenoids (9.2–9.6%, 22.3–26.2%) and sesquiterpenoids (28.3–30.6%, 15.1–17.0%),
respectively (Figure S4-S5, Table S1). In particular, trans-linalool oxide (4.2-4.8%) and 2,
2,6-trimethyl-6-vinyl-tetrahydropyran-3-one (14.7-19.2%) were significantly enhanced in
picked flowers. Linalool was not detected in live flowers, but it was found accumu-
lated upto 1.2% in picked flowers. In a similar study, Mookherjee et al. (1990) had
studied a number of flowers and reported that linalool was significantly increased (up
to 10 fold) in picked flowers. Interestingly, linalool (8.0%) was found higher percentage
in essential oil, whereas it remained very low (0.2%) in the absolute. It is predicted
that the enzymatic reaction after flowers picking followed by hydrodistillation at
higher temperature probably increases linalool percentage in essential oil (Lawrence
1995). In contrast, live flowers contained improved percentage of (E,E)-a-farnesene

NATURAL PRODUCT RESEARCH
3
(28.0–30.3%) as compared to the picked flowers (15.0–16.8%). The present findings are
in good agreement with reported work by Mookherjee et al. (1990). Importantly, QCN
was detected with higher percentage in the live flowers (5.6–5.8%). It was also
observed that the live flowers contained higher percentage of sesquiterpenes (29.5%)
and QCN (5.7%) with pleasant aroma; hence, it precisely attracts the pollinator for
healthy pollination (Yan et al. 2016). Variations in compositions of live and picked
flowers are attributed to the enzymatic reaction and also impulsive shock arises
due to detachment of flowers from the plant (Xu et al. 2018). Thus, picked flowers
possess a modified aroma profile (Mookherjee et al. 1990).
2.2. Comparison of HS-SPME with the extracts
Percentage yields of the absolute and essential oil were 0.37% and 0.015%, respect-
ively. The representative chromatogram of HS-SPME is given in Figure 5. It has been
observed that the HS-SPME composition was quite different from that of essential oil.
Pyranoid form
a linalool oxide (2,2,6-trimethyl-6-vinyl-tetrahydropyran-3-ol) was
present upto 1.3% in headspace contributing to top-note fragrance profile emitted by
the flowers. This pyranoid form is very sensitive to heat in the presence of water,
which resulted in rearrangement to linalool oxide during distillation (Figure S6). Other
compounds identified were 2,2,6-trimethyl-6-vinyl-tetrahydropyran-3-one (5.5%) and
linalool (8.0%) in the essential oil. Similarly, low linalool (1.2%) percentage in the
flower headspace was observed but 2,2,6-trimethyl-6-vinyl-tetrahydropyran-3-one was
accumulated up to 19.5%. Besides, furanoid form of trans- and cis-linalool oxides were
identified in the essential oil due to their comparatively stable structures (Table S2). It
has been reported that aromatic compounds exist in glycosidically bound form and at
a specific developmental stage of flower, the floral parts follow a controlled enzymatic
hydrolysis mechanism to release the terpenoids/benzenoids (Lawrence 1995). Thus,
essential oil was not replicated the aroma profile of live flowers and QCN was isolated
only in 1.1% in the essential oil. It was due to its lesser volatility with water in the
process of hydrodistillation; however, it was recovered to 15% in the extract (Rout
et al. 2008). Besides waxy materials, the absolute composition might be compared
with HS-SPME compositions of flowers. Absolute was improved form of the extract
contained 19.7% of QCN along with 42.1% of non-volatile wax like materials.
Interestingly, alkaloids with structure relationship with QCN were not detected
in LC-QTOF LC/MS analysis (Figures S7 and S8). Furthermore, quinoline-carbonitrile
isolation and characterization was carried out using extensive NMR experiments.
2.3. Spectroscopic analysis of QCN
The yield of quinoline-carbonitrile was 430 mg from 4 g absolute. Elemental analysis
indicated the percentage of C (79.1%), H (3.9%) and N (16.6%), the empirical molecular
formula is C10H6N2. FT-IR spectrum illustrated absorption at 2231 cmꢀ1, which confirms
the presence of characteristic CN group (Figure S9). HR-MS analysis revealed that the
molecular mass of M þ H was 155.0604 having molecular formula ([C10H6N2]þH)þ
(Figure S10). Therefore, molecular formula of the compound was C10H6N2.

4
P. ROUT ET AL.
Characteristic electron impact (EI) masses are molecular ion peak (m/z 154) along with
m/z 127 and m/z 100 arising by loss of two successive HCN units as illustrated in the
structure of quinoline-carbonitrile or isoquinoline-carbonitrile (Figure S11). 1H-NMR
(500 MHz, CDCl3) for six protons viz. doublet (d) at d 9.05 (1H), a double doublets (dd)
at d 8.22 (2H), a doublet of doublets of doublets (ddd) at d 7.88 (1H), another ddd at
d 7.79 (1H) and d at d 7.77 (1g) (Figure S12). 13C-NMR (125 MHz, CDCl3): d 115.5, 118.7,
124.8, 124.9, 125.8, 129.2, 130.4, 131.2, 148.1, 149.4 (Figure S13). Above analysis indi-
cated that the probable structure would be one of the six possible isomers of quin-
oline-2-carbonitrile, quinoline-3-carbonitrile, QCN, quinoline-5-carbonitrile, quinoline-8-
carbonitrile and isoquinoline-1-carbonitrile (Figure S14).
1H-1H-COSY shows a ddd coupling of aromatic protons indicating the presence of two
ddd adjacent protons (Figure S15). Therefore, it ruled out the probable like quinoline-5-car-
bonitrile and quinoline-8-carbonitrile. Presence of downfield signal at d 9.05 splitting to a
doublet due to adjacent aromatic proton cannot be expected in isoquinoline-1-carbonitrile
and leaving only quinoline-2-carbonitrile, quinoline-3-carbonitrile and QCN for deciding
the structure. Further, synthesized quinoline-2-carbonitrile was used for spectral compari-
1
son. Its H-NMR showed signals at d 8.32 (d, 1H, J ¼ 8.4HZ, Ar-H), d 8.18 (d, 1H, J ¼ 8.6HZ,
Ar-H) , d 7.90 (d, 1H, J ¼ 8.4HZ, Ar-H), d 7.85 (ddd, 1H, J ¼ 8.6, 6.9, 1.5HZ, Ar-H), d 7.71 (d, 1H,
J ¼ 8.4HZ Ar-H), d 7.70 (ddd, 1H, J ¼ 8.2, 6.8, 1.0HZ Ar-H) (Figure S16). 1H-NMR of quinoline-
2-carbonitrile and the expected structure are quite different. Hence, the structure may be
either quinoline-3-carbonitrile or QCN. In case of quinoline-3-carbonitrile, proton attached
at C-2 will experience electron with-drawing effect not only from the C¼ N of ring nitro-
gen but also from the CN group at 3-position. Consequently, this proton is expected at
downfield farther to d 9.05 as rather a singlet. Even if it couples with proton at C-4, J values
will be about 2 Hz. Finally, all the spectral data are compatible with QCN. In 1H-NMR, down-
field signal at d 9.05 (d, J ¼ 4.5) is due to the proton at C-2 and coupling to C-3 proton. The
other downfield signals centered at d 8.22 (dd, 2H) are due to protons at C-5 and C-8.
While the ddd at d 7.88 is due to C-7, C-5 and C-8 protons, similarly ddd at d 7.79 is due to
1
C-6, C-8 and C-5 protons and d at d 7.77 is due to C-2 proton. H-1H-COSY indicated the
proton-proton coupling of d 9.05 (d, H-2) with d 7.77 (d, H-3); d 8.22 (d, H-5) with d 7.88
(ddd, H-6); d 7.79 (ddd, H-7) with d 7.88 (ddd, H-6); d 7.79 (ddd, H-7) with d 8.20 (d, H-8)
(Figure S15). Similarly, 1H-13C-HSQC gave the proton coupling with corresponding carbons
viz. d 9.05 (d) with d 149.4 (C-2); d 8.22 (d) with d 131.2 (C-5); d 8.20 (d) with d 130.4 (C-8); d
7.88 (ddd) with d 129.2 (C-6); d 7.79 (ddd) with d 124.9 (C-7); d 7.77 (d) with d 124.8 (C-3)
(Figure S17). Finally, HMBC coupling as follows viz. H-2 (d 9.05) with C-10 (d 148.1), C-3 (d
124.8), C-4 (d 118.7); H-3 (d 7.77) with C-2 (d 149.4), C-11 (d 115.5); H-5 (d 8.22) with C-10 (d
148.1), C-7 (d 129.2), C-4 (d 118.7); H-6 (d 7.88) with C-8 (d 130.4), C-9 (d 125.8); H-7 (d 7.79)
with C-10 (d 148.1), C-5 (d 124.9); H-8 (d 8.20) with C-6 (d 131.2), C-9 (d 125.8) (Figure S18).
Thus, the isolated compound was identified as QCN.
2.4. Confirmation of compound through synthesis
The yield of QCN from quinoline-4-carboxaldehyde (2 g) was 1.4 g with m.p. 97 ꢁC
(Figure S19). Synthesized compound was identical as isolated QCN with respect to m.
p. and super-imposable FT-IR, NMR and HR-MS data. Further, reduced products are

NATURAL PRODUCT RESEARCH
5
obtained at different temperature and solvent systems using heterogeneous catalysts
(Table S3). These modified structures might be suitable for high value fragrance or
pharmaceutical applications. In particular, quinoline-4-methylamine (QCM) was taken
for the anti-inflammatory and antioxidant studies due to its different functional entity
along with aromatic character as compared to the natural product.
2.5. Anti-inflammatory and antioxidant studies
Essential oil, absolute, QCN and QCM were screened for in-vitro anti-inflammatory
activity against LPS-induced inflammation in macrophages isolated from peritoneal
cavity of mice (Yang et al. 2018). Productions of pro-inflammatory cytokines (TNF-a,
IL-1b) were quantified from cell culture supernatant using enzyme-linked immune-
sorbent assay (ELISA). Pro-inflammatory cytokines (TNF-a and IL-1b) was significantly
(p < 0.05) increased in vehicle treated LPS-stimulated cells when compared with nor-
mal un-stimulated cells. The treatment of (Essential oil, absolute, QCN and QCM) has
shown significant anti-inflammatory activity by inhibiting LPS-induced pro-inflamma-
tory cytokines production in a dose-dependent manner as compared to vehicle
treated LPS-stimulated cells. Percent inhibition of pro-inflammatory cytokines treat-
ment is depicted in Table S4. Among the treatment, QCM possessed significant
inhibition of TNF-a, IL-1b in a dose dependent manner followed by QCN, essential oil
and absolute. The absolute was contained 19.7% of QCN along with other terpenoids
and benzenoids. Therefore, absolute has been shown enhanced activity with lesser
dose with respect to QCN. Interestingly, essential oil has been shown inferior activity,
which might be correlated with least percentage of QCN (1.1%). Finally, anti-inflam-
mation activity is depended on the QCN in the extract.
Antioxidant study revealed that the extracts have potent activities as presented
in Table S5. Absolute has shown better antioxidant activity as compared to the iso-
lated QCN. Enhancing antioxidant activity of absolute may be due to the phenolics
as benzyl tiglate, (Z)-3-hexenyl benzoate, hexyl benzoate, phenyl ethyl tiglate, (Z)-
3-hexenyl phenyl acetate, benzyl benzoate, etc. It has been inferred that the
enhanced antioxidant activity of absolute due to the synergistic interaction among
phenolics (10.1%) and QCN (19.7%). Polyphenols have played the synergistic role
for enhancing antioxidant activities (Afify and Hassan 2016). Alternatively, QCM
has been shown enhanced antioxidant activity as compared to QCN. Similarly,
poor percentage of phenolics (8.3%) and QCN (1.1%) were responsible for inferior
activity of the essential oil.
3. Conclusions
Essential oil is not true representative of volatiles emitted by flowers. Application of
HS-SPME analysis gives scope for formulation through blending, which might be
closely representing the floral fragrance. Similarly, live flowers analysis also provides
scope to identify the chemical markers (particular composition) for attracting pollina-
tors. QCN (5.8%) is a major compound in the live flowers of Q. indica, which is respon-
sible for refreshing environment especially during evenings due to the presence of

6
P. ROUT ET AL.
this volatile alkaloid. Bio-efficacy study of this compound has been shown potent anti-
inflammatory activity. Similarly, absolute contained 19.7% of QCN along with other ter-
penoids and benzenoids, which are in combination, revealed enhanced anti-inflamma-
tory and antioxidant activities. This work is a complete characterization of natural
product (QCN) with its bioactivity. Besides, heterogeneous catalytic reduction had pro-
duced QCM, which was shown enhanced anti-inflammatory and antioxidant activities.
Acknowledgements
We are grateful to Director, CSIR-CIMAP, Lucknow for providing necessary facility under Aroma
Mission (HCP 0007). Authors are thankful to SERB, DST, New Delhi, India (EMR/2016/005359) for
research funding and fellowship to Prashant Kumar, to Dr. Priyabrat Mohapatra, Head, Chemistry
Department, C.V. Raman College of Engineering for catalyst characterization and Dr. Hariom
Gupta, CSIR-CIMAP for NMR interpretation.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
DST, SERB (EMR/2016/005359); INDIA.
ORCID
Prasant Kumar Rout
Prashant Kumar
https://orcid.org/0000-0003-3018-3357
https://orcid.org/0000-0001-6594-7930
Anant Kumar
Dnyaneshwar U. Bawankule
Kijay Bahadur Singh https://orcid.org/0000-0003-2179-560X
Chandan Singh Chanotiya http://orcid.org/0000-0001-9802-0162
https://orcid.org/0000-0002-5643-1286
https://orcid.org/0000-0002-0824-2368
https://orcid.org/0000-0003-0677-0981
S. N. Naik
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