A new hexenoic acid glycoside with cytotoxic activity from the leaves of Psychotria luzoniensis

Article abstract of DOI:10.1080/14786419.2020.1765345

A new hexenoic acid glycoside (1) together with known compounds, flavonol glycosides (2–4), iridoid glycoside (5), megastigmane glycoside (6), and amino acid (7) were isolated from the leaves of P. luzoniensis by resin column chromatography and preparative HPLC. Their structures were determined based on spectroscopic analysis, including HRFABMS and NMR (¹H and ¹³C, ¹H?¹H COSY, HMQC, and HMBC) data. All compounds tested for cytotoxicity were active (IC₅₀ < 50 μM) with IC₅₀ values ranging from 1.97 to 32.85 μM against human colon adenocarcinoma cell line, compared to etoposide (IC₅₀ 1.19 μM).

Full text of DOI:10.1080/14786419.2020.1765345

Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: https://www.tandfonline.com/loi/gnpl20
A new hexenoic acid glycoside with cytotoxic
activity from the leaves of Psychotria luzoniensis
Rhian Jaymar D. Ramil, Ma. Danica I. Ramil, Taisuke Konno, Toshihiro
Murata, Kyoko Kobayashi, Buyanmandakh Buyankhishig, Shirley C. Agrupis
&
Kenroh Sasaki
To cite this article: Rhian Jaymar D. Ramil, Ma. Danica I. Ramil, Taisuke Konno, Toshihiro
Murata, Kyoko Kobayashi, Buyanmandakh Buyankhishig, Shirley C. Agrupis & Kenroh Sasaki
(2020): A new hexenoic acid glycoside with cytotoxic activity from the leaves of Psychotria
luzoniensis, Natural Product Research, DOI: 10.1080/14786419.2020.1765345
To link to this article: https://doi.org/10.1080/14786419.2020.1765345
View supplementary material
Published online: 13 May 2020.
Submit your article to this journal
View related articles
View Crossmark data
Full Terms & Conditions of access and use can be found at
https://www.tandfonline.com/action/journalInformation?journalCode=gnpl20

NATURAL PRODUCT RESEARCH
https://doi.org/10.1080/14786419.2020.1765345
A new hexenoic acid glycoside with cytotoxic activity
from the leaves of Psychotria luzoniensis
a
a
c
c
Rhian Jaymar D. Ramil , Ma. Danica I. Ramil , Taisuke Konno , Toshihiro Murata ,
c
c
b
Kyoko Kobayashi , Buyanmandakh Buyankhishig , Shirley C. Agrupis and
c
Kenroh Sasaki
a
b
Pharmacy Department, Mariano Marcos State University, Batac, Philippines; Biological Sciences
c
Department, Mariano Marcos State University, Batac, Philippines; Pharmacognosy Department,
Tohoku Medical and Pharmaceutical University, Sendai, Japan
ABSTRACT
ARTICLE HISTORY
A new hexenoic acid glycoside (1) together with known com-
pounds, flavonol glycosides (2–4), iridoid glycoside (5), megastig-
mane glycoside (6), and amino acid (7) were isolated from the
leaves of P. luzoniensis by resin column chromatography and
preparative HPLC. Their structures were determined based on
Received 16 December 2019
Accepted 25 April 2020
KEYWORDS
Psychotria luzoniensis;
hexenoic acid glycoside;
cytotoxicity
1
13
spectroscopic analysis, including HRFABMS and NMR ( H and C,
1
1
Hꢀ H COSY, HMQC, and HMBC) data. All compounds tested for
cytotoxicity were active (IC50 < 50 lM) with IC50 values ranging
from 1.97 to 32.85 lM against human colon adenocarcinoma cell
line, compared to etoposide (IC50 1.19 lM).
1. Introduction
Phytochemical and biological investigations from the genus Psychotria have resulted
to the identification of many compounds (indole, monoterpene indole, quinolone, and
isoquinoline alkaloids as well as flavonoids, coumarins, terpenoids, and cyclic peptides)
with cytotoxic, analgesic, antiviral, antifungal and antibacterial activities (Calixto et al.
2016; Yang et al. 2016). The species-rich genus Psychotria comprises approximately
2000 species and considered to be the largest genera within the Rubiaceae family
(Turner and Kumar 2018), of which 112 species can be found in the Philippines
CONTACT Rhian Jaymar D. Ramil
jaymar_1329@yahoo.com.ph
Supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2020.1765345.
ß 2020 Informa UK Limited, trading as Taylor & Francis Group

2
R. J. D. RAMIL ET AL.
(Parungao et al. 2018). Plants from this genus have collective folkloric use in traditional
medicine for the treatment of various illnesses such as bronchial and gastrointestinal
disorders (Soares et al. 2017). Psychotria luzoniensis (Cham. & Schltdl.) Fern.-Vill, an
endemic plant to the Philippines, is a terrestrial shrub growing to a height of 1.5 to 5
meters (Pelser et al. 2011). P. luzoniensis, locally known as tagpong-gubat, is used trad-
itionally by Filipinos through leaf decoction for treating dysentery and ulcerated
wounds (Carag and Buot 2017; Parungao et al. 2018). On the contrary, there is no pub-
lished data on the phytochemical constituents and limited information as to the bio-
logical activities of P. luzoniensis.
The global burden of cancer is progressing unabated (Bray et al. 2018). Recently,
the WHO reported that colorectal cancer remains as one of the leading causes of inci-
dence and deaths worldwide affecting both sexes (WHO 2018). In the Philippines,
rd
among all types of cancer, colorectal cancer ranked 3 as to incidence with 10,167
th
cases and 4 in terms of mortality with 6,004 deaths (Ferlay et al. 2018). In the search
for new drug leads for colon cancer, the study presented the isolation and structural
elucidation of bioactive compounds from the leaves of P. luzoniensis via direct phyto-
chemical isolation approach. Herein, for the first time, we disclose the identification of
one new hexenoic acid glycoside (1) and six known compounds, flavonol glycosides
(2–4), one iridoid glycoside (5), one megastigmane glycoside (6), and amino acid (7).
These isolated compounds except the amino acid were tested against HT29 cancer
cells to assess their cytotoxic activities using the WST-8 assay.
2. Results and discussion
An EtOH extract of P. luzoniensis leaves was subjected to solvent partitioning and the
H2O sub-extract was initially purified to open column chromatography using a Diaion
HP-20, a macroporous resin. Further purifications were applied using preparative HPLC
with reversed phase columns. The chemical structure of a new compound (1) was
1
13
determined based on the spectroscopic data, including H and C NMR and MS.
Other isolated known compounds, quercetin 3-O-rutinoside (2) (Kazuma et al. 2003),
quercetin-3-O-glucopyranoside (3) (Kazuma et al. 2003), kaempferol 3-O-b-D-apiosyl
(
9
1!2)-b-D-glucopyranoside (4) (Wu et al. 2007), asperuloside (5) (Li et al. 2006), (6S,
R)-roseoside (6) (Yamano and Ito 2005), and tryptophan (7) were identified by com-
paring their spectroscopic data with those reported in the literature (Figure 1).
Compound 1, was isolated as a colorless amorphous powder, with a molecular for-
þ
mula C H O based on HRFABMS m/z 461.1636 [M þ Na] (calcd for C H O Na,
1
8
30 12
18 30 12
1
4
61.1634). The H NMR spectrum of 1 showed signals of a triplet methyl (d 0.96, 3H,
J ¼ 7.0 Hz, H -6), two methylene (d 1.52, 2H, m, H -5; 2.23, 2H, m, H -4), and two ole-
3
2
2
finic (d 7.10, 1H, dt, J ¼ 16.0, 7.0 Hz, H-3; 5.90, 1H, dt, J ¼ 16.0, 1.0 Hz, H-2) proton reso-
nances. Their corresponding carbons assigned as d 152.5 (C-3), 121.7 (C-2), 35.4 (C-4),
22.4 (C-5), and 14.0 (C-6) and a carbonyl carbon at d 166.7 (C-1) suggested the pres-
ence of a 2-hexenoyl moiety (Kon et al. 2009). Furthermore, the correlations from H-2
1
1
to H-3, H-3 to H -4, H -4 to H -5, and from H -5 to H -6 were confirmed by the H- H
2
2
2
2
3
COSY spectrum of 1. The coupling constant between H-2 and H-3 (J ¼ 16.0 Hz) sug-
13
gested their E configuration (Kon et al. 2009). In the C NMR spectrum of 1, the other

NATURAL PRODUCT RESEARCH
3
Figure 1. Structures of compounds 1–7.
0 0 0 0 0
2 oxygenated carbon resonances (d 95.7, C-1 ; 73.9, C-2 ; 77.9, C-3 ; 71.0, C-4 ; 77.8, C-5 ;
9.5, C-6 ;104.6, C-1 ; 75.1, C-2 ; 78.0, C-3 ; 71.5, C-4 ; 78.0, C-5 ; 62.7, C-6 ) and two
1
6
0
00
00
00
00
00
00
0
00
anomeric proton resonances (d 5.50, 1H, d, J¼ 8.0 Hz, H-1 ; 4.32, 1H, d, J¼ 7.5 Hz, H-1 )
revealed the presence of two glucopyranosyl moieties (Yu et al. 2010). In the HMBC spec-
0
0
0
0
trum (Fig. S5), the H-1 resonance was long-range coupled with C-6 and H-1 coupled
with C-1, which showed the (6-O-glucopyranosyl)-glucopyranosyl sugar moiety bonded to
C-1 in the (E)-2-hexenoyl moiety. Sugar identification using HPLC indicated that the two
glucosyl moieties were D-glucose (Tanaka et al. 2007), and the coupling constants J¼ 7.0
and 8.0 Hz showed their b-orientation. From these data, compound 1 was therefore estab-
lished as 6-O-b-D-glucopyranosyl-b-D-glucopyranosyl (E)-hex-2-enoate (Figure 1).
Compounds 1–6 showed active (<50 lM) cytotoxicity against HT29 cancer cells
with IC50 values ranging from 1.97 to 32.85 lM (Table S1) with reference to etoposide
(IC50 1.19 lM). The cytotoxicity of the new hexenoic acid glycoside (1) can be linked to
the presence of (6-O-glucopyranosyl)-glucopyranosyl sugar moiety in its structure.
Although the individual effects of 2-hexenoyl group and two glucopyranosyl moieties
of the new compound against colorectal cancer cells are unknown, previous studies of
some compounds containing two glucopyranosyl moieties reported to possess cyto-
toxicity against HL-60 and JB6 cell lines (Liu et al. 2001; Akihisa et al. 2012) while a
recent study indicated no cytotoxic effect of a compound with 2-hexenoyl group (Di
et al. 2017). The structurally related cytotoxic effects of the isolated flavonoids (2–4)
can be attributed to the presence of a double bond between C-2 and C-3 in ring C,
0
0
and the hydroxy moieties at the C-3 and C-4 positions on the B-ring (Chang et al.
008; Jaramillo-Carmona et al. 2014). Besides, previous studies reported that rutin
2
found to display selective cytotoxicity against CHME, Neuro-2a, U-118, LN-229, and SK-
N-SH cancer cells with IC50 values between 15-20 lM (Yan et al. 2019) and isoquercitrin

4
R. J. D. RAMIL ET AL.
showed cytotoxicity on T24 cancer cells with IC50 values between 20-80 lM (Wu et al.
017). Similarly, the cytotoxic activities were previously reported for asperuloside with
IC50 values of 16.5 lM on HL-60 and 26.9 lM on HCT-15 (Wang et al. 2018) and (6S,
R)-roseoside indicated an IC50 value of 21.3 lM and 44.2 lM on HepG2 and MCF7,
respectively (Fan et al. 2017).
2
9
3. Experimental part
Detailed general experimental procedures, plant material, extraction and isolation,
sugar identification and cytotoxicity testing are available as Supplementary Material.
3.1. 6-O-b-D-glucopyranosyl-b-D-glucopyranosyl (E)-hex-2-enoate (1)
2
5
þ
Colorless amorphous powder, [a]
– 13.3 (c 0.12, MeOH); HRFABMS m/z [M þ Na]
1
D
4
61.1636 (Calcd for C H O Na, 461.1634); H NMR (400 MHz, CD OD) d 7.10 (1H, dt,
18
30 12
3
0
J¼ 16.0, 7.0 Hz, H-3), 5.90 (1H, dt. J¼ 16.0, 1.0 Hz, H-2), 5.50 (1H, d, J¼ 8.0 Hz, H-1 ), 4.32
0
0
0
(
6
5
3
1H, d, J¼ 7.5 H, H-1 ), 4.15 (1H, dd, J¼ 11.0, 2.0 Hz, H-6 ), 3.84 (1H, dd, J¼ 12.0, 2.0Hz, H-
0
0
0
0
00
), 3.76 (1H, dd, J¼ 11.0, 5.0 Hz, H-6 ), 3.66 (1H, dd, J¼ 12.0, 5.5 Hz, H-6 ), 3.55 (1H, m, H-
0 00 0 0 00 00
), 3.43 (overlapping, H-4 and H-3 ), 3.25-3.40 (overlapping, H-2 , H-3 , H-4 , and H-5 ),
0
0
.20 (1H, dd, J¼ 9.0, 7.5 Hz, H-2 ), 2.23 (2H, m, H -4), 1.52 (2H, m, H -5), 0.96 (3H, t,
2
2
13
J¼ 7.0 Hz, H -6); C NMR (100MHz, CD OD) d 166.7 (C-1), 152.9 (C-3), 121.7 (C-2), 104.6
3
3
0
0
0
00
00
0
0
00
0
(
C-1 ), 95.7 (C-1 ), 78.0 (C-3 ), 78.0 (C-5 ), 77.9 (C-3 ), 77.8 (C-5 ), 75.1 (C-2 ), 73.9 (C-2 ),
00 0 0 00
7
1.5 (C-4 ), 71.0 (C-4 ), 69.5 (C-6 ), 62.7 (C-6 ), 35.4 (C-4), 22.4 (C-5), 14.0 (C-6).
4. Conclusion
A new hexenoic acid glycoside, together with other known compounds, quercetin 3-
O-rutinoside, quercetin-3-O-glucopyranoside, kaempferol 3-O-b-D-apiosyl (1!2)-b-D-
glucopyranoside, asperuloside, (6S, 9 R)-roseoside, and tryptophan were isolated from
the leaves of P. luzoniensis. The new compound showed active cytotoxicity against
HT29 cell line with IC50 of 3.17 lM, hence a potential anti-cancer lead compound.
Disclosure statement
No potential conflict of interest was reported by the authors.
Acknowledgment
The authors gratefully acknowledge the fund and support given to RJD Ramil and MDI Ramil by
the Mariano Marcos State University and Tohoku Medical and Pharmaceutical University.
References
Akihisa T, Tochizawa S, Takahashi N, Yamamoto A, Zhang J, Kikuchi T, Fukatsu M, Tokuda H,
Suzuki N. 2012. Melanogenesis-inhibitory saccharide fatty acid esters and other constituents
of the fruits of Morinda citrifolia (Noni). Chem Biodivers. 9(6):1172–1187.

NATURAL PRODUCT RESEARCH
5
Bray F, Ferlay J, Soerjomataram I, Siegel R, Torre L, Jemal A. 2018. Global cancer statistics 2018 :
GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries.
CA Cancer J Clin. 68(6):394–424.
Calixto NO, Pinto MEF, Ramalho SD, Burger MCM, Bobey AF, Young MCM, Bolzani VS. Pinto Ac
2016. The genus Psychotria: Phytochemistry, chemotaxonomy, ethnopharmacology and
biological properties. J Braz Chem Soc. 27:1355–1378.
Carag H, Buot I. 2017. A checklist of the orders and families of medicinal plants in the
Philippines. Sylvatrop Tech J Philipp Ecosyst Nat Resour. 27:39–83.
Chang H, Mi M, Ling W, Zhu J, Zhang Q, Wei N, Zhou Y, Tang Y, Yuan J. 2008. Structurally
related cytotoxic effects of flavonoids on human cancer cells in vitro. Arch Pharm Res. 31(9):
1137–1144.
Di T-M, Yang S-L, Du F-Y, Zhao L, Xia T, Zhang X-F. 2017. Cytotoxic and hypoglycemic activity of
triterpenoid saponins from Camellia oleifera Abel. seed pomace. Molecules. 22(10):1562–1569.
Fan JJ, Liu X, Zheng XL, Zhao HY, Xia H, Sun Y. 2017. A novel cytotoxic physalin from Physalis
angulata. Nat Prod Commun. 12:1589–1591.
Ferlay J, Ervik M, Lam F, Colombet M, Mery L, Pi n~ eros M, Znaor A, Soerjomataram I, Bray F.
2018. Global cancer observatory: cancer today. [accessed 2019 Sep 11]. https://gco.iarc.fr/
today.
Jaramillo-Carmona S, Lopez S, Abia R, Rodriguez-Arcos R, Jimenez A, Guillen R, Muriana F. 2014.
Combination of quercetin and kaempferol enhances in vitro cytotoxicity on human colon
cancer (HCT-116) cells. Rec Nat Prod. 8:262–271.
Kazuma K, Noda N, Suzuki M. 2003. Malonylated flavonol glycosides from the petals of Clitoria
ternatea. Phytochemistry. 62(2):229–237.
Kon Y, Imao D, Nakashima T, Sato K. 2009. Palladium(II)-catalyzed selective oxidation
of a,b-unsaturated aldehydes to a,b-unsaturated carboxylic acids with hydrogen peroxide.
Chem Lett. 38(5):430–431.
Li B, Zhang DM, Luo YM, Chen XG. 2006. Three new and antitumor anthraquinone glycosides
from Lasianthus acuminatissimus Merr. Chem Pharm Bull. 54(3):297–300.
Liu G, Bode A, Ma WY, Dong Z, Sang S, Ho CT. 2001. Two novel glycosides from the fruits
of Morinda citrifolia (noni) inhibit AP-1 transactivation and cell transformation in the mouse
epidermal JB6 cell line. Cancer Res. 61(15):5749–5756.
Parungao AM, Sena GL, Penaranda SZ, Salvan AJ, Marie C, Solis A, Rio PC, Tengco GSC, Hilario
AL. 2018. Gastroprotective property of Psychotria luzoniensis leaf decoction on HCl/ethanol-
induced gastric ulcers in mice. Int J Med Sci Innov Res. 3:353–358.
Pelser P, Barcelona J, Nickrent D. 2011. Co’s Digital Flora of the Philippines. [Internet]. [cited
2019 Jun 26]. https://phillippineplants.org/Families/Rubiaceae.html.
Soares DBS, Duarte LP, Cavalcanti AD, Silva FC, Braga AD, Lopes MTP, Takahashi JA, Vieira-Filho
SA. 2017. Psychotria viridis: Chemical constituents from leaves and biological properties.
An Acad Bras Ci ^e nc. 89(2):927–938.
Tanaka T, Nakashima T, Ueda T, Tomii K, Kouno I. 2007. Facile discrimination of aldose
enantiomers by reversed-phase HPLC. Chem Pharm Bull. 55(6):899–901.
Turner IM, Kumar VS. 2018. Flora of Singapore precursors, 4. A summary of scandent Psychotria
(Rubiaceae) in Singapore and Peninsular Malaysia. Phytotaxa. 361(2):183–197.
Wang C, Xin P, Wang Y, Zhou X, Wei D, Deng C, Sun S. 2018. Iridoids and sfingolipids from
Hedyotis diffusa. Fitoterapia. 124:152–159.
WHO. 2018. Latest global cancer data : Cancer burden rises to 18. 1 million new cases and 9. 6
million cancer deaths in 2018 Latest global cancer data : Cancer burden rises to 18. 1 million
new cases and 9. 6 million cancer deaths in 2018. Geneva, Switzerland.
Wu P, Liu S, Su J, Chen J, Li L, Zhang R, Chen T. 2017. Apoptosis triggered by isoquercitrin in
bladder cancer cells by activating the AMPK-activated protein kinase pathway. Food Funct.
8(10):3707–3722.
Wu B, Takahashi T, Kashiwagi T, Tebayashi SI, Kim CS. 2007. New flavonoid glycosides from the
leaves of Solidago altissima. Chem Pharm Bull. 55(5):815–816.

6
R. J. D. RAMIL ET AL.
Yamano Y, Ito M. 2005. Synthesis of optically active vomifoliol and roseoside stereoisomers.
Chem Pharm Bull. 53(5):541–546.
Yan X, Hao Y, Chen S, Jia G, Guo Y, Zhang G, Wang C, Cheng R, Hu T, Zhang X, et al. 2019.
Rutin induces apoptosis via P53 up-regulation in human glioma CHME cells. Transl Cancer
Res. 8(5):2005–2013.
Yang H, Zhang H, Yang C, Chen Y. 2016. Chemical constituents of plants from the genus
Psychotria. Chem Biodivers. 13(7):807–820.
Yu Y, Gao H, Dai Y, Wang Y, Chen HR, Yao XS. 2010. Monoterpenoids from the fruit of Gardenia
jasminoides. Helv Chim Acta . 93(4):763–771.