Acridone alkaloids from the rhizomes of Luvunga scandens (Roxb.) Buch. Ham.

Article abstract of DOI:10.1080/14786419.2018.1492577

The ethyl acetate extract of the rhizomes of Luvunga scandens (Roxb.) Buch. - Ham. ex Wight & Arn (Rutaceae) delivered one new acridone alkaloid named Luvungaside A (1) together with three known acridone alkaloids, namely 1,3-dihydroxy-2-methoxy-10-methyl-9-acridone (2), arborinine (3) and 1-hydroxy-3-methoxy-10-methyl-9-acridone (4). Compounds were reported for the first time from the species L. scandens applying various chromatography methods. Chemical structures were elucidated by IR, UV, HR-ESI-MS, NMR 1D & 2D experiments and comparison with the literature. The cytotoxicity and hepatoprotective activity of compounds 1–4 in human hepatoma cell line HepG2 was measured by MTT assay. At 10–100 μM, compounds expressed significant hepatoprotective effect with prevention percentage ranging from 81.1% to 194.3%, compared to the positive control quercetin displaying 49.0%.

Full text of DOI:10.1080/14786419.2018.1492577

Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: http://www.tandfonline.com/loi/gnpl20
Acridone alkaloids from the rhizomes of Luvunga
scandens (Roxb.) Buch. Ham.
Nguyen Minh An Tran, Thi Hong Tuoi Do, Luu Hong Truong, Dung Tien Le,
Minh Nhat Phan, Nguyen Kim Tuyen Pham, Dinh Tri Mai & Tan Phat Nguyen
To cite this article: Nguyen Minh An Tran, Thi Hong Tuoi Do, Luu Hong Truong, Dung Tien Le,
Minh Nhat Phan, Nguyen Kim Tuyen Pham, Dinh Tri Mai & Tan Phat Nguyen (2018): Acridone
alkaloids from the rhizomes of Luvungaꢀscandens (Roxb.) Buch. Ham., Natural Product Research,
DOI: 10.1080/14786419.2018.1492577
To link to this article: https://doi.org/10.1080/14786419.2018.1492577
View supplementary material
Published online: 10 Nov 2018.
Submit your article to this journal
View Crossmark data
Full Terms & Conditions of access and use can be found at
http://www.tandfonline.com/action/journalInformation?journalCode=gnpl20

NATURAL PRODUCT RESEARCH
https://doi.org/10.1080/14786419.2018.1492577
RESEARCH ARTICLE
Acridone alkaloids from the rhizomes of Luvunga
scandens (Roxb.) Buch. Ham.
Nguyen Minh An Trana, Thi Hong Tuoi Dob, Luu Hong Truongc, Dung Tien Led,
Minh Nhat Phane, Nguyen Kim Tuyen Phamf, Dinh Tri Maie,g and
Tan Phat Nguyene,g
aFaculty of Chemical Engineering, Industrial University of Ho Chi Minh City, Ho Chi Minh City, Viet
b
Nam; Faculty of Pharmacy, University of Medicine and Pharmacy, Ho Chi Minh City, Viet Nam;
cSouthern Institute of Ecology, Vietnam Academy of Science and Technology, Ho Chi Minh City,
d
Vietnam; Institute of Applied Materials Science, Vietnam Academy of Science and Technology,
e
Ho Chi Minh City, Viet Nam; Institute of Chemical Technology, Vietnam Academy of Science and
f
Technology, Ho Chi Minh City, Viet Nam; Faculty of Environmental Science, Sai Gon University, Ho
g
Chi Minh city, Viet Nam; Faculty of Chemistry, Graduate University of Science and Technology,
Vietnam Academy of Science and Technology, Hanoi, Viet Nam
ABSTRACT
ARTICLE HISTORY
Received 4 May 2018
Accepted 18 June 2018
The ethyl acetate extract of the rhizomes of Luvunga scandens
(Roxb.) Buch. - Ham. ex Wight & Arn (Rutaceae) delivered one
new acridone alkaloid named Luvungaside A (1) together with
three known acridone alkaloids, namely 1,3-dihydroxy-2-methoxy-
10-methyl-9-acridone (2), arborinine (3) and 1-hydroxy-3-methoxy-
10-methyl-9-acridone (4). Compounds were reported for the first
time from the species L. scandens applying various chromatog-
raphy methods. Chemical structures were elucidated by IR, UV,
HR-ESI-MS, NMR 1D & 2D experiments and comparison with the
literature. The cytotoxicity and hepatoprotective activity of com-
pounds 1–4 in human hepatoma cell line HepG2 was measured
by MTT assay. At 10–100lM, compounds expressed significant
hepatoprotective effect with prevention percentage ranging from
81.1% to 194.3%, compared to the positive control quercetin
displaying 49.0%.
KEYWORDS
Luvunga scandens;
Luvungaside A; alkaloid;
cytotoxicity;
hepatoprotective activity
CONTACT Tan Phat Nguyen
ntphat@ict.vast.vn
Supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2018.1492577.
ß 2018 Informa UK Limited, trading as Taylor & Francis Group

2
N. M. A. TRAN ET AL.
1. Introduction
L. scandens has been used traditionally in Vietnam for the treatment of cirrhosis,
rheumatism, scorpion bite, hepatitis, sinusitis and pain in bone joints and muscles
(Luu 2007). Chemical constituents farnesyl acetate, farnesol, carvotanacetone, methyl
eugenol, caryophyllene oxide, myristicin, 1,8-cineole, carvone, a-pinene, camphene,
phellandrene, terpinolene, a-terpineol, camphor, caryophyllene, b-sitosterol and
sesamin were reported from the essential oil of the berries as well as cytotoxic,
antioxidative, antiinflammatory and antibacterial properties of stem extracts (Aggarwal
et al. 1985; Sirinut et al. 2014). Furthermore, the tirucallane triterpenes flindissol and
3-oxotirucalla-7,24-dien-21-oic-acid were identified from the dichloromethane extract
of the stem showing potent cytotoxicity against MCF-7 cell line with IC50 values of
13.8 lM and 27.5 lM, respectively. (Al-Zikri et al. 2014). As part of our continuing study
on hepatoprotective activity of bioactive compounds from plant origin (Nguyen,
Mai, et al. 2017; Nguyen, Tran, et al. 2017) and phytochemical constituents from the
rhizomes of L. scandens (Nguyen, Phan, et al. 2017), we report herein the isolation and
structure elucidation of one new acridone alkaloid and three known ones which
indentified for the first time from the rhizomes of L. scandens collected in Ta Kou
Mountain, Binh Thuan province, Viet Nam.
2. Results and discussion
The ethyl acetate extract from the dried rhizomes of L. scandens was subjected to
column chromatography over silica gel normal-phase and reversed-phase RP-18 to
give one acridone alkaloid, named Luvungaside A (3-O-b-[D-apiofuranosyl-(1!6)]-b-D-
glucopyranosyl-1,3-dihydroxy-2-methoxy-10-methyl-9-acridone) (1), together with three
known acridone alkaloids, 1,3-dihydroxy-2-methoxy-10-methyl-9-acridone (2)
(Ngoumfo et al. 2010), arborinine (3) (Bergenthal et al. 1979), and 1-hydroxy-3-
methoxy-10-methyl-9-acridone (4) (Ngoumfo et al. 2010) (Figure 1).
5
4
1
R2
R1
N
a
a
5
4
3
6
7
a
9
a
9
2
8
8
H
O
=
O
--
=
,
:
:
:
:
e
>
c
l
6
G
)
1
R1 OM R2 Api 1
( )
(
=
=
e
,
e
2
R1 OM R2 OH
( )
=
=
R1 R2
M
3
O
( )
=
=
,
e
4
R1 H R2 OM
( )
Figure 1. Structure of compounds 1–4.

NATURAL PRODUCT RESEARCH
3
Compound 1 was obtained as a yellow amorphous powder. The molecular formula
was established as C26H31NO13 by HR-ESI-MS data ([M þ H]þ m/z 566.1821, calcd.
566.1864). The IR spectrum of 1 showed absorptions of hydroxyl (3446 cmꢀ1) and car-
bonyl (1641 cmꢀ1) groups. The 13C-NMR and DEPT spectrum showed twenty six car-
bons including one carbonyl carbon, seven quaternary aromatic carbons, five methine
aromatic carbons, two anomeric carbons, one oxygenated quaternary carbon, five oxy-
genated methine carbons, three oxygenated methylene carbons, one oxygenated
methyl carbon and one nitrogenated methyl carbon (Table S1). The presence of one
carbonyl carbon at dC 180.2 (C-28), two nitrogenated aromatic carbons at dC 139.8 (C-
4a), 142.0 (C-5a), two quaternary aromatic carbons at dC 105.4 (C-9a), 119.6 (C-8a),
three oxygenated aromatic carbons and five methine aromatic carbons indicated an
acridone aglycone (Ngoumfo et al. 2010; Wouatsa et al. 2013). Thus 1 was identified
1
as an acridone alkaloid bearing two methyl groups and two sugar units. The H-NMR
spectrum showed two methyl groups at dH 3.91 (s, N-CH3), 3.78 (s, O-CH3), five aro-
matic protons at dH 6.82 (s, H-4), 7.85 (m, H-5, H-6), 7.38 (td, 2.0, 8.0, H-7) and 8.32 (d,
8.0, H-8), respectively (Table S1). Moreover, two anomeric protons at dH 5.24 (d, 7.5, H-
10), 4.81 (d, 3.5, H-100) corresponding to two anomeric carbons at dC 100.1 (C-10), 109.1
(C-100) were assigned to b-D-glucose (Glc) and b-D-apiose (Apio) units, respectively.
The COSY, HSQC and ROESY spectrum allowed analysis of their spin systems and
assignment of their proton resonances to determine sugar units. Moreover, acidic
hydrolysis and TLC comparision of the hydrolysate with authentic sugars verified sugar
moieties as D-glucose and D-apiose, see also under 3.5. Acid hydrolysis, Experimental
part. In the HMBC spectrum, one methyl group at dH 3.91 (N-CH3) correlated with two
carbons at dC 139.8 (C-4a), 142.0 (C-5a) and one methyl group at dH 3.78 (O-CH3) cor-
related with carbon at dC 129.8 (C-2) (Figure S1). In addition, the HMBC spectrum
showed correlations between anomeric proton at dH 5.24 (H-10) of Glc and carbon at
dC 157.3 (C-3) of acridone skeleton; between anomeric proton at dH 4.81 (H-100) of
Apio and carbon at dC 67.9 (C-60) of Glc; between oxygenated methylene proton at dH
3.96 (d, 10.0, H-60a) and acetal carbon at dC 109.1 (C-100). The connectivity was further
confirmed by correlations observed in the ROESY spectrum between H-60b (dH 3.44) of
Glc and H-10 (dH 4.81) of Apio (Figure S1).
Table 1. The cytotoxicity activity of 1–4 in HepG2 cell line.
Sample
Concentration (mM)
OD570 nm SD
Percentage of control (%)
1
10
50
100
10
50
100
10
50
100
10
50
100
10
50
100
10
0.566 0.029
0.610 0.009
0.765 0.033
0.632 0.015
0.690 0.025
0.760 0.039
0.698 0.029
0.668 0.015
0.578 0.036
0.672 0.021
0.740 0.029
0.782 0.080
0.711 0.037
0.674 0.019
0.616 0.011
0.317 0.023
79.6
90.5
124.2
88.9
102.4
123.3
98.2
99.0
93.7
94.5
109.7
126.9
2
3
4
Control
Paclitaxel
44.6

4
N. M. A. TRAN ET AL.
Table 2. The hepatoprotective activity using HepG2 of 1–4.
Sample
Concentration (mM)
OD570 SD
Prevention percentage (%)
1
10
50
100
10
50
100
10
50
100
10
50
100
10
50
100
10
50
0.346 0.026
0.314 0.014
0.379 0.039
0.735 0.036
0.831 0.016
0.587 0.007
0.759 0.027
0.750 0.029
0.569 0.032
0.954 0.021
0.911 0.029
0.775 0.031
0.711 0.037
0.674 0.019
0.616 0.011
0.453 0.037
0.412 0.019
0.368 0.011
0.579 0.009
ꢀ41.5
ꢀ37.5
4.4
109.3
159.8
88.2
118.5
128.8
81.1
194.3
190.3
163.7
2
3
4
DMSO
Control
100
10
Quercetin
49.0
Based on HR-ESI-MS, 1D, 2D-NMR data and comparision with published data, the
structure of 1 was identified as 3-O-b-[D-apiofuranosyl-(1!6)]-b-D-glucopyranosyl-1,3-
dihydroxy-2-methoxy-10-methyl-9-acridone and named Luvungaside A.
The cytotoxicity and hepatoprotective effect of compounds (1-4) on human hepa-
toma cell line HepG2 was measured by MTT assay (Table 1–2). While at 10-100 mM
cell growth was not affected, a significant hepatoprotective effect on CCl4-induced
hepatotoxicity in HepG2 cells was observed, with prevention percentage ranging from
81.1% to 194.3% compared to the positive control quercetin displaying 49.0%.
In summary, one new acridone alkaloid (1) together with three known acridone alka-
loids (2-4) were isolated from the ethyl acetate extract of the rhizomes of L. scandens, the
latter three reported here for the first time from this species. Compounds 1-4 exhibited
significant hepatoprotective activity, surmounting that of the positive control quercetin.
Structures were elucidated by intensive spectroscopic and spectrometric analyses.
3. Experimental part
3.1. General experimental procedures
The high resolution electrospray ionisation mass spectroscopy (HR-ESI-MS) was
recorded on a Bruker MicrOTOF-QII spectrometer. The 1H-NMR (500 MHz), 13C-NMR
(125 MHz), DEPT, COSY, HSQC and HMBC spectra were recorded on a Bruker
AM500 FT-NMR spectrometer using tetramethylsilane (TMS) as internal standard.
Column chromatography was carried out using Merck Silica gel normal-phase
(230–240 mesh) and reversed-phase C18 (Merck). Analytical TLC was carried on in silica
gel plates (Merck DC-Alufolien 60 F254). Compounds were visualized by spraying with
aqueous 10% H2SO4 and heating for 3–5 min.
3.2. Chemicals
Eagle’s Minimum Essential Medium (EMEM), fetal calf serum (FCS) and trypsin-EDTA
were purchased from Gibco, USA; L-glutamine, penicillin-streptomycin, phosphate

NATURAL PRODUCT RESEARCH
5
buffer, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), paclitaxel,
quercetin and carbon tetrachloride (CCl4) from Sigma-Aldrich, USA and dimethylsulfox-
ide (DMSO) as well as isopropanol from Merck, Germany. All chemicals met cell cul-
ture standards.
3.3. Plant material
The rhizomes of Luvunga scandens were collected in Ta Kou Mountain, Binh Thuan
province, Viet Nam and identified by Dr. Hong Truong Luu, Southern Institute of
Ecology, Vietnam Academy of Science and Technology. A voucher specimen (No LS-
128) was deposited in Bioactive Compounds Laboratory, Institute of Chemical
Technology, Vietnam Academy of Science and Technology, Viet Nam.
3.4. Extraction and isolation
Powdered rhizomes of L. scandens (5.0 kg) were extracted three times with 30 L of 96%
EtOH each at room temperature. The residue was filtered and solvents evaporated
under low pressure to obtain the crude extract (350 g). The crude extract was dis-
solved in water and successively partitioned into n-hexane (120 g), CHCl3 (80 g), EtOAc
(30 g) and the aqueous portion (110 g). The CHCl3 extract (80 g) was fractionated on
silica gel with n-hexane–EtOAc (50:1) increasing the amount of EtOAc to 100% to give
seven fractions. Fraction C.III (12 g) was eluted with n-hexane–EtOAc (50:1!1:1, v/v) to
give three subfractions C.III.1–C.III.3. Subfraction C.III.2 (4.6 g) was chromatographed
repeatedly over a silica gel column using n-hexane–EtOAc (10:1) to afford 3 (48 mg)
and 4 (8 mg). Subfraction C.III.3 (3.0 g) was chromatographed over silica gel column
and eluted with CHCl3 MeOH (10:1) to obtain compound 2 (10 mg). The EtOAc extract
(30 g) was subjected to silica gel column chromatography and eluted with a gradient
solvent system of chloroform - methanol (50:1-1:1) to afford fractions M1-M5. Fraction
M5 (5,3 g) was chromatographed over silica gel column and eluted with CHCl3-MeOH
(5:1!1:1) to give four subfractions M5.1-M5.4. Subfraction M5.2 was further purifed by
RP-18 with MeOH-H2O (1:2, v/v) to give compound 1 (12 mg).
3.4.1. Luvungaside A (1)
Yellow amorphous powder; IR ꢀmax (KBr): 3446, 1641, 1506, 1253 cmꢀ1; UV (MeOH)
kmax: 231 and 274 nm; HR-ESI-MS: m/z 566.1821 [M þ H]þ (calcd for C26H32NO13,
1
566.1864); H and 13C NMR data (DMSO-d6), see Table S1.
3.5. Acid hydrolysis
Compound 1 (2 mg) was refluxed with 2 N aq. CH3COOH (5 mL) for 2h at 100 ꢁC. After
extraction with CHCl3 (3 ꢂ 5 mL), the aqueous layer was repeatedly evaporated to
dryness with MeOH until neutral, and then analyzed by TLC over silica gel
(MeCOEt–isoPrOH–Me2CO–H2O 20:10:7:6) by comparison with authentic sugars of
D-apiose Rf 0.47 and D-glucose Rf 0.40 according to Nguyen et al. 2015, 2016.

6
N. M. A. TRAN ET AL.
3.6. The cytotoxicity and hepatoprotective assay
HepG2 cells were seeded and cultured in EMEM containing 10% FCS, 2 mM L-
glutamine, 100 IU/mL penicillin, 100 mg/mL streptomycin at 5% CO2 at 37 ꢁC to attain
confluency, then harvested and seeded into 96-well-plates at 2.5 ꢂ 104 cells/cm2. Cells
were treated with EMEM containing 2 mM CCl4 and compounds alone or combined
at different concentrations. Cell viability was evaluated as mitochodrial succinate
dehydrogenase activity, a marker of viable cells using MTT test according to Nguyen,
Mai, et al. 2017; Nguyen, Tran, et al. 2017. Doxorubicin was used as positive control
for cytotoxicity and quercetin for hepatoprotective effect.
Disclosure statement
No potential conflict of interest was reported by the authors.
References
Aggarwal VK, Aggarwal SG, Thappa RK, Mehra MS, Dhar KL. 1985. Chemical constituents of
Luvunga scandens Buch & Ham. Indian Perfumer. 27:163–165.
Al-Zikri PNH, Taher M, Susanti D, Rezali MF, Read RW, Sohrab MH, Hasan CM, Ahmad F. 2014.
Cytotoxic tirucallane triterpenes from the stem of Luvunga scandens. Rev Bras Farmacogn.
24:561–564.
Bergenthal D, Mester I, Rozsa Z. 1979. 13C-NMR-Spektren einiger acridonalkaloide.
Phytochemistry. 18:161–163.
Luu HT. 2007. Some useful plants on Mt. Ta Kou, Ta Kou Nature Reserve. Publisher of General
Ho Chi minh, Viet Nam; p. 36–37.
Ngoumfo RM, Jouda J-B, Mouafo FT, Komguem J, Mbazoa CD, Shiao TC, Choudhary MI, Laatsch
H, Legault J, Pichette A, Roy R. 2010. In vitro cytotoxic activity of isolated acridones alkaloids
from Zanthoxylum leprieurii Guill. et Perr. Bioorg Med Chem. 18:3601–3605.
Nguyen TP, Mai DT, Do THT, Phan NM. 2017. Flavonoids with hepatoprotective activity from the
leaves of Cleome chelidonii. Nat Product Commun. 12:1061–1063.
Nguyen, T.P., Le, T.D., Phan, N.M., Bui, T.D., Mai DT. 2016. Triterpene saponins with a-glucosidase
inhibition and cytotoxic activity from the leaves of Schefflera sessiliflora. J Asian Nat Products
Res. 18:542–549.
Nguyen TP, Le TVH, Mai DT, Le TD, Phan, NM, Bui TD. 2015. Two new oleanane-type triterpene
saponins from the leaves of Schefflera sessiliflora De. P. V. Phytochem Lett. 11:102–105.
Nguyen TP, Phan NM, Bui TD, Le TD, Tran NMA, Pham TNT, Mai DT. 2017. Limonoid from the
rhizomes of Luvunga scandens (Roxb.) Buch. Ham. Nat Product Res. 31:2281–2285.
Nguyen, TP, Tran, C.L., Vuong, C.H., Do, T.H.T., Le, T.D., Mai, D.T., Phan NM. 2017. Flavonoids with
hepatoprotective activity from the leaves of Cleome viscosa L. Nat Product Res. 31:2587–2592.
Sirinut P, Petkongkeaw A, Thongyoo P. 2014. Biological activities of stem extracts from Luvunga
scandens. In: Proc. the 5th International Conference on Natural Products for Health and
_
Beauty, Thai Lan; pp. 61–65.
Wouatsa, V.N.A., Misra, L., Kumar, S., Prakash, O., Khan, F., Tchoumbougnang, F., and Venkatesh,
R.K. 2013. Aromatase and glycosyl transferase inhibiting acridone alkaloids from fruits of
Cameroonian Zanthoxylum species. Chem. Central J. 7:125.