Anti-inflammatory constituents from Psychotria prainii H. Lév

Article abstract of DOI:10.1080/14786419.2017.1408095

One new and three known compounds were isolated from the ethanol extract of Psychotria prainii aerial parts. By means of spectroscopic methods, their structures were elucidated to be deacetylasperulosidic acid 6-ethyl ether (1), asperulosidic acid (2), asperuloside (3) and obtucarbamates C (4). The isolated compounds were evaluated for their inhibitory effect on NO production in LPS-stimulated RAW264.7 cells. Among them, compounds 2 and 4 exhibited strong effect with the IC₅₀ values of 5.75?±?0.85 and 6.92?±?0.43?μM, respectively. This is the first report for the chemical composition and biological activity of P. prainii.

Full text of DOI:10.1080/14786419.2017.1408095

Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: http://www.tandfonline.com/loi/gnpl20
Anti-inflammatory constituents from Psychotria
prainii H. Lév
Phi Hung Tran, Viet Dung Le, Thi Ha Do, Thi Luyen Nguyen, Phuong Thao
Nguyen, Trong Thong Nguyen & Tien Dat Nguyen
To cite this article: Phi Hung Tran, Viet Dung Le, Thi Ha Do, Thi Luyen Nguyen, Phuong Thao
Nguyen, Trong Thong Nguyen & Tien Dat Nguyen (2017): Anti-inflammatory constituents from
Psychotria prainii H. Lév, Natural Product Research, DOI: 10.1080/14786419.2017.1408095
To link to this article: https://doi.org/10.1080/14786419.2017.1408095
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Date: 07 December 2017, At: 00:08

Natural Product research, 2017
ꢁꢁpꢂ://ꢃꢄi.ꢄꢅg/10.1080/14786419.2017.1408095
ꢀ
Anti-inflammatory constituents from Psychotria prainii H. Lév
a,b
a
a
c,d
Phi Hung Tran , Viet Dung Le , Thi Ha Do , Thi Luyen Nguyen ,
c
e
d
Phuong Thao Nguyen , Trong Thong Nguyen and Tien Dat Nguyen
ꢆ
b
dꢇpꢆꢅꢁmꢇnꢁ ꢄf Pꢀyꢁꢄꢈꢀꢇmiꢂꢁꢅy, Nꢆꢁiꢄnꢆꢉ Inꢂꢁiꢁꢊꢁꢇ ꢄf Mꢇꢃiꢈinꢆꢉ Mꢆꢁꢇꢅiꢆꢉꢂ, hꢆnꢄi, Viꢇꢁnꢆm; dꢇpꢆꢅꢁmꢇnꢁ
ꢈ
ꢄf Pꢀꢆꢅmꢆꢈy, Nꢆꢁiꢄnꢆꢉ hꢄꢂpiꢁꢆꢉ ꢄf tꢅꢆꢃiꢁiꢄnꢆꢉ Mꢇꢃiꢈinꢇ, hꢆnꢄi, Viꢇꢁnꢆm; Inꢂꢁiꢁꢊꢁꢇ ꢄf Mꢆꢅinꢇ Biꢄꢈꢀꢇmiꢂꢁꢅy,
Viꢇꢁnꢆm aꢈꢆꢃꢇmy ꢄf sꢈiꢇnꢈꢇ ꢆnꢃ tꢇꢈꢀnꢄꢉꢄgy (Vast), hꢆnꢄi, Viꢇꢁnꢆm; cꢇnꢁꢇꢅ fꢄꢅ rꢇꢂꢇꢆꢅꢈꢀ ꢆnꢃ tꢇꢈꢀnꢄꢉꢄgy
tꢅꢆnꢂfꢇꢅ, Vast, hꢆnꢄi, Viꢇꢁnꢆm; dꢇpꢆꢅꢁmꢇnꢁ ꢄf Pꢀꢆꢅmꢆꢈꢄꢉꢄgy, hꢆnꢄi Mꢇꢃiꢈꢆꢉ univꢇꢅꢂiꢁy, hꢆnꢄi, Viꢇꢁnꢆm
ꢃ
ꢇ
ABSTRACT
ARTICLE HISTORY
One new and three known compounds were isolated from the ethanol
extract of Psychotria prainii aerial parts. By means of spectroscopic
methods, their structures were elucidated to be deacetylasperulosidic
acid 6-ethyl ether (1), asperulosidic acid (2), asperuloside (3) and
obtucarbamates C (4). The isolated compounds were evaluated for
their inhibitory effect on NO production in LPS-stimulated RAW264.7
cells. Among them, compounds 2 and 4 exhibited strong effect with
the IC values of 5.75 ± 0.85 and 6.92 ± 0.43 μM, respectively. This is
rꢇꢈꢇivꢇꢃ 11 apꢅiꢉ 2017
aꢈꢈꢇpꢁꢇꢃ 10 Nꢄvꢇmbꢇꢅ 2017
KEYWORDS
Psychotria prainii; ꢆnꢁi-
inflꢆmmꢆꢁiꢄn; iꢅiꢃꢄiꢃ;
ꢆꢂpꢇꢅꢊꢉꢄꢂiꢃiꢈ ꢆꢈiꢃ;
ꢄbꢁꢊꢈꢆꢅbꢆmꢆꢁꢇꢂ c
5
0
the first report for the chemical composition and biological activity
of P. prainii.
1
. Introduction
The genus Psychotria is the largest genus within the Rubiaceae family, distributed throughout
tropical and subtropical regions of the world, with approximately 1500 species (Yang et al.
2016). To date, 41 species of the Psychotria genus have been chemically investigated, including
alkaloids of indole, quinoline and benzoquinolizidine, terpenoids, steroids, phenolics and
aliphatic compounds (Yang et al. 2016). Those compounds exhibit several biological prop-
erties, such as antioxidant (Matsuura and Fett-Neto 2013), cytotoxicity (Adjibadé et al. 1989;
CONTACT tiꢇn dꢆꢁ Ngꢊyꢇn
ngꢁiꢇnꢃ@imbꢈ.vꢆꢂꢁ.vn; Viꢇꢁ dꢊng lꢇ
ꢉꢇviꢇꢁꢃꢊng75imm@yꢆꢀꢄꢄ.ꢈꢄm
sꢊppꢉꢇmꢇnꢁꢆꢉ ꢃꢆꢁꢆ fꢄꢅ ꢁꢀiꢂ ꢆꢅꢁiꢈꢉꢇ ꢈꢆn bꢇ ꢆꢈꢈꢇꢂꢂꢇꢃ ꢆꢁ ꢀꢁꢁpꢂ://ꢃꢄi.ꢄꢅg/10.1080/14786419.2017.1408095.
©
2017 Infꢄꢅmꢆ uK limiꢁꢇꢃ, ꢁꢅꢆꢃing ꢆꢂ tꢆyꢉꢄꢅ & Fꢅꢆnꢈiꢂ Gꢅꢄꢊp

2
P. H. TRAN ET AL.
Mahmud et al. 1993), antimicrobial (Khan et al. 2001; Jayasinghe et al. 2002), antiviral (Khan
et al. 2001; Kuo et al. 2001) and antiparasitic activities (Kato et al. 2012). In Vietnamese tradi-
tional medicine, P. prainii is often used for the treatment of a variety of diseases as an anti-
flatulent or antitussive agent. It exhibits antifungal, appetite-inducing and counter-irritant
properties and has been used to treat digestive and intestinal disorders. However, the studies
of chemical constituents and biological effects of P. prainii are still unknown.
In the course of the screening programme for biologically active natural products, P. prainii
has been a subject of extensive investigation due to its biological activities. This paper
describes the isolation and structural elucidation of four compounds (1–4), including one
new iridoid derivative (1) and three known compounds (2–4) from the aerial parts of P. prainii
(
Figure 1). Their anti-inflammatory effects were also examined via the inhibition of nitric
oxide (NO) production.
2
. Results and discussion
One new iridoid derivative (1) and three known compounds (2–4) were isolated from the
water layer of an EtOH extract using combined chromatographic methods. Detailed analysis
of the spectroscopic data (1D, 2D NMR and MS) and comparison with previously reported
values led to the elucidation of the known as asperulosidic acid (2) (Tzakoua et al. 2007),
asperuloside (3) (Otsuka et al. 1991) and obtucarbamate C (4) (Huang et al. 2012).
Compound 1 was obtained as a white solid. A C H O molecular formula was deduced
1
8
26 11
+
1
from the ion peak at m/z 441.1401 [M + Na] observed in the HR-ESI-MS. The H NMR
spectrum of 1 showed two olefinic signals [δ 7.57 and 6.11 (each singlet, H-3 and H-7)],
H
along with one hemiacetal signal [δ 5.04 (d, J = 8.5 Hz, H-1)], one oxymethylene [δ 4.24
H
H
(
d, J = 15.5 Hz, H-10a) and 4.49 (d, J = 15.5 Hz, H-10b), one oxymethine [δ 4.51 (br s, H-6)]
H
Figure 1. tꢀꢇ ꢂꢁꢅꢊꢈꢁꢊꢅꢇ ꢄf ꢈꢄmpꢄꢊnꢃꢂ 1–4.

NATURAL PRODUCT RESEARCH
3
1
and two methines [δ 3.09 (m, H-5) and 2.52 (dd, J = 8.5, 7.5 Hz, H-9)]. Moreover, the H NMR
H
spectrum also displayed an anomeric signal at δ 4.75 (d, J = 8.0 Hz, H-1′) arising from a
H
1
3
β-glucopyranose. These findings, taken together with the corresponding C NMR data,
revealed that 1 was an iridoid glycoside (Miyagoshi et al. 1987; Otsuka et al. 1991; Tzakoua
1
et al. 2007). However, H NMR spectrum of 1 exhibited additional signals of an ethyl group
1
3
at δ 1.08 (t, J = 6.0 Hz, H-13) and 3.52 (m, H-12). The C NMR spectrum of 1 contained 18
H
carbon signals, 12 of which were assigned to an iridoid aglycone and the remaining 6 carbons
to a β-glucopyranosyl moiety. These NMR data suggested that 1 was an iridoid glycoside
with a skeleton similar to that of deacetylasperulosidic acid (Tzakoua et al. 2007), except for
the presence of an additional ethanolyl group at C-6. This suggestion was further confirmed
by the HMBC correlations from H-12 (δ 3.52) to C-6 (δ 83.4) and from H-6 (δ 4.51) to C-12
H
C
H
(
δC 66.2).
The relative configuration of 1 was deduced based on the NOESY correlations and other
NMR data comparisons with a previous report. The relative configurations in 1 were estab-
lished based on the NOESY experiment and 13C chemical shifts. The NOE cross-peak between
H-5 (δ 3.09) and H-9 (δ 2.52) indicated the cis-fused iridoid skeleton. The NOESY correlation
H
H
between H-5 (δ 3.09) and H-6 (δ 4.51) suggested the α-orientation of the ethanolyl group.
H
H
Therefore, compound 1 was determined to be deacetylasperulosidic acid 6-ethyl ether. Since
the sample extraction by ethanol might produce ethyl ether adducts, the ethanol and meth-
anol extracts of P. prainii aerial parts were prepared and analysed by HPLC. Compound 1 was
detected in both extracts, which confirmed that compound 1 was not an artefact formed
during the extraction process (see Supplemental).
Nitric oxide (NO) plays an important role in the regulation of various physiological pro-
cesses in mammals. Overproduction of NO causes numerous human diseases including
inflammation, arthritis, asthma and cancer. Therefore, an assay for inhibition of NO produc-
tion is one of the possible ways to screen anti-inflammatory agents (Luiking et al. 2010). The
inhibitory effect of the isolated compounds on the NO production was evaluated in LPS-
stimulated RAW264.7 cells. Compounds 2 and 4 strongly inhibited NO production with the
IC values of 5.75 ± 0.85 and 6.92 ± 0.43 μM, respectively. The MTT assay showed that those
5
0
compounds had no significant toxicity to RAW264.7 cells up to 30 μM (data not shown)
indicating that the inhibitory effect on NO production was not due to cytotoxicity. Compounds
1
and 3 were inactive at concentration up to 30 μM.
3
. Experimental
3
.1. General procedures
Optical rotations were read on a JASCO P-2000 digital polarimeter. NMR experiments were
performed on a Bruker AM500 FT-NMR spectrometer. The HR-ESI-MS data were obtained
from an API Q-STAR PULSAR I of Applied Biosystem. Thin layer chromatography (TLC) was
performed using precoated Kiesel gel 60 F254 and visualised by UV light 254 nm and 10%
H SO reagent by heating. Column chromatography (CC) was performed using silica gel 60
2
4
(
Merck, 70–230 mesh). HPLC analysis was carried out in an Agilent 1260 Series Single
Quadrupole LC/MS Systems.

4
P. H. TRAN ET AL.
3
.2. Plant materials
The aerial parts of P. prainii H. Lév were collected in Son La, Vietnam in July 2013, and iden-
tified by Dr. Pham Thanh Huyen, National Institute of Medicinal Materials and Assoc. Prof.
Tran Van On, Hanoi University of Pharmacy. The voucher specimens (TB-9938 and HUP-
1572013SL) were deposited at the Herbarium of the National Institute of Medicinal Materials
and Hanoi University of Pharmacy.
3
.3. Extraction and isolation
The air-dried materials (10 kg) were refluxed with ethanol for 2 h (20 L × 3 times). The com-
bined extracts were concentrated to give 339.3 g of crude extract, which was then resus-
pended in water (500 mL) and successively extracted with n-hexane and ethyl acetate (each
5
0
00 mL × 3 times). The water residue was filtered through a Diaion HP-20 column eluted by
, 25 and 100% methanol in water. The 100% methanol-eluted fraction was concentrated
and fractionated by a silica gel column using a gradient of 0–100% methanol in dichlo-
romethane to afford five fractions (F-1–F-5). Fraction F-2 was passed through a silica gel
column eluted by n-hexane–acetone (1.8:1 v/v) to obtain 4 (11.8 mg). Compound 3 (7.0 mg)
were purified from fraction F-3 using RP-C column eluted with methanol–water (1:4 v/v)
1
8
followed by a Sephadex LH-20 column (methanol–water, 1:3 v/v). Fraction F-4 was fraction-
ated on a RP-C column eluted with methanol–water (1:3 v/v) to give seven subfractions,
1
8
(
F-4.1–F-4.7). A RP-C column chromatography using methanol–water (1:5 v/v) was applied
18
for F-4.4 to give 1 (25.0 mg). Compound 2 (241.4 mg) was obtained from fractions F-4.4 and
F-4.7 by a similar column with methanol–water (1:3 v/v).
3
.3.1. Deacetylasperulosidic acid 6-ethyl ether (1)
24
1
White solid; [ꢀ] = +4.0 (c 0.1, MeOH). H NMR (500 MHz, CD OD): δ 1.08 (3H, t, J = 6.0 Hz,
D
3
H-13), 2.52 (1H, dd, J = 8.5, 7.5 Hz, H-9), 3.09 (1H, m, H-5), 3.27 (1H, m, H-2′), 3.30 (1H, m, H-5′),
3
5
.35 (1H, m, H-4′), 3.43 (1H, dd, J = 9.0, 8.5 Hz, H-3′), 3.52 (2H, m, H-12), 3.70 (1H, dd, J = 12.0,
.5 Hz, H-6′a), 3.85 (1H, dd, J = 12.0, 2.0 Hz, H-6′b), 4.24 (1H, d, J = 15.5 Hz, H-10a), 4.49 (1H,
d, J = 15.5 Hz, H-10b), 4.51 (1H, br s, H-6), 4.75 (1H, d, J = 8.0 Hz, H-1′), 5.04 (1H, d, J = 8.5 Hz,
1
3
H-1), 6.11 (1H, s, H-7), 7.57 (1H, s, H-3). C NMR (125 MHz, CD OD): δ 101.6 (C-1), 154.8 (C-3),
3
108.9 (C-4), 42.2 (C-5), 83.4 (C-6), 128.4 (C-7), 152.1 (C-8), 45.9 (C-9), 61.7 (C-10), 171.2 (C-11),
6
6.2 (C-12), 15.9 (C-13), 100.6 (C-1′), 74.9 (C-2′), 77.8 (C-3′), 71.4 (C-4′), 78.2 (C-5′), 62.6 (C-6′);
+
and HR-ESI-MS (positive): m/z 441.1401 [M + Na] (calcd for 441.1373, C H NaO ).
1
8
26
11
3
.4. Acid hydrolysis of 1
Compound 1 (0.5 mg) was heated in 1 N HCl (400 μL) at 80 °C for 2 h, then the solution was
extracted with ethyl acetate (1 mL × 3). The aqueous layer was neutralised with NH OH and
4
then dried under reduced pressure. The obtained residue was redissolved in 150 μL pyridine
containing 10 μmol of L-cysteine methyl ester and heated at 80 °C for 1 h. 6 μl o-tolyl isothi-
ocyantate was added, and the solution was heated for another hour. The reaction solution
was then analysed by HPLC using Cosmosil 5C18-MS-II column (4.6 × 150 mm), mobile phase
of 20% acetonitrile in 0.2% TFA water, UV detection at 254 nm. The sugars were identified
as d-glucose (t 9.02 min).
R

NATURAL PRODUCT RESEARCH
5
3
.5. HPLC analysis of 1
The HPLC analysis was performed to confirm that compound 1 was not an artefact formed
during the extraction process. Two parts of P. prainii materials (each 1 g) were individually
extracted with 2 mL of ethanol and methanol in a sonic bath for 10 min. Each extracts were
diluted by 8 mL of water and then mixed with 5 mL of hexane. The aqueous solutions were
filtered through a 0.45-μm filter unit and injected to the HPLC system. The separation was
done a Zorbax Eclipse XDB C18 column (250 × 4.6 mm, 5 μm) using a gradient of 10–90%
acetonitrile in water for 25 min, flow rate: 0.5mL/min. Compound 1 was detected in the
tested solutions by retention time (16.76 min) and mass ion peak (m/z 419.1).
3
.6. Assay for inhibition of NO production
Determination of NO production in LPS-induced RAW264.7 cells was performed as previously
described (Dang et al. 2017). Cardamonin was used as a positive control (IC , 2.24 μM).
5
0
Acknowledgement
We thank Dr. Marie-Lise Bourguet-Kondracki (Musée National d’Histoire Naturelle, Paris, France) for
providing HRMS data.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This work is supported in part by a grant from the Institute of Marine Biochemistry [grant number
HSB17-CS03].
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