Biochemical Systematics and Ecology 39 (2011) 888–892

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Biochemical Systematics and Ecology

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Anthraquinones and an iridoid glycoside from the roots of Morinda

pandurifolia
Thanatip Ruksilp a, Jirapast Sichaem a, Suttira Khumkratok b, Pongpan Siripong c,
Santi Tip-pyang a, d, *
a
Natural Products Research Unit, Department of Chemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand
b
Walai Rukhavej Botanical Research Institute, Mahasarakham University, Mahasarakham 44000, Thailand
c
Natural Products Research Section, National Cancer Institute, Bangkok 10440, Thailand
d
Center for Petroleum, Petrochemicals and Advanced Materials, Chulalongkorn University, Bangkok 10330, Thailand

a r t i c l e i n f o

Article history:
Received 6 September 2010
Accepted 16 July 2011
Available online 16 August 2011

Keywords:
Rubiaceae
Morinda pandurifolia
Anthraquinones
Iridoid glycoside

1. Subject and source

Thirteen species of the genus Morinda, belonging to the Rubiaceae family, can be found in Thailand. Morinda pandurifolia,
locally known as “Yotime”, is a shrub found scattered in the moist upper mixed deciduous forest, in the Central and Peninsula
regions of the country. The leaves, bark and wood produce a yellowish-red pigment for dyeing clothes (Smitinand, 2001;
Chayamarit, 2005). The roots, bark, stems, leaves and fruits of several Morinda species have been used as a traditional folk
medicine for the treatment of many diseases including diabetes, hypertension and cancer (Kamiya et al., 2005; Su et al., 2005).
The roots of this plant were collected from Mahasarakham Province of Thailand in June 2007 and identified by Ms. Suttira
Khumkratok, a botanist at the Walai Rukhavej Botanical Research Institute, Mahasarakham University, where a voucher
specimen (Khumkratok no. 103–09) is deposited.

2. Previous work

Phytochemical reports on the genus Morinda have revealed the occurrence of anthraquinones (Kamiya et al., 2005; Pawlus
et al., 2005; Deng et al., 2007; Akihisa et al., 2007), anthraquinone glycosides (Kamiya et al., 2009), benzophenones (Deng
et al., 2007), flavonol glycosides (Sang et al., 2001), iridoids (Kamiya et al., 2005), iridoid glycosides (Su et al., 2005; Sang
et al., 2001; Akihisa et al., 2007), coumarin (Wu et al., 2009) and phenolic glycoside (Kanchanapoom et al., 2002). There
are no reports on the phytochemical of M. pandurifolia.

* Corresponding author. Natural Products Research Unit, Department of Chemistry, Phayathai Road, Pathumwan, Bangkok 10330, Thailand. Tel.: þ66 02
2187625; fax: þ66 02 2187598.
E-mail address: Santi.Ti@chula.ac.th (S. Tip-pyang).

0305-1978/$ – see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bse.2011.07.003
 T. Ruksilp et al. / Biochemical Systematics and Ecology 39 (2011) 888–892 889

O R1 HO COOH
8
H
1
8a 9a R2
7 9
2
A B
10 O
R4 6 10a 4a 3 R3 AcO
5 4 H
O OH O

R1 R2 R3 R4 HO O

1 OH CH2OCH3 OH H HO
2 OCH3 CHO OH H OH
3 OH CHO OH H 12
4 OH CH3 H OH
5 OH CH2 OH OH H
6 OH CH2OCH2CH3 OH H
7 OH OCH3 OCH3 H
8 OH OH H OH
9 OCH3 CH3 H H
10 OCH3 CH2 OCH3 OH H
11 OH OH OH H

Fig. 1. Structure of compounds 1–12 isolated from M. pandurifolia.

3. Present study

Air-dried and powdered roots of M. pandurifolia (1.2 kg) were successively extracted in a Soxhlet with CH2Cl2, EtOAc and
MeOH. Removal of solvents from each extract under reduced pressure gave CH2Cl2 (19.30 g), EtOAc (14.99 g) and MeOH
(64.31 g) crude extracts, respectively. The MeOH extract was suspended in water and partitioned successively with n-BuOH

Table 1
Occurrence of anthraquinones in some species of Morinda.

Compound Plant part Source

Unsubstituted A-ring anthraquinones
2-Methyl anthraquinone (Tectoquinone) Roots M. officinalis (Yoshikawa et al., 1995)
Roots M. umbellata (Burnett and Thomson, 1968)
2-Methoxy anthraquinone Roots M. officinalis (Wu et al., 2009)
Roots and stems M. umbellata (Burnett and Thomson, 1968)
2-Hydroxymethyl anthraquinone Rhizome and M. parvifolia (Chang and Lee, 1984)
roots
2-Hydroxy anthraquinone Roots and stems M. umbellata (Burnett and Thomson, 1968)
2-Formyl-1-hydroxy anthraquinone Roots M. elliptica syn. M. citrifolia var. elliptica (Ismail et al.,
1997)
1-Hydroxy-2-methyl anthraquinone Roots M. elliptica syn. M. citrifolia var. elliptica (Ismail et al.,
1997)
Roots and stems M. umbellata (Burnett and Thomson, 1968)
2-Hydroxy-1-methoxy anthraquinone (Alizarin-1-methyl ether) Roots M. elliptica syn. M. citrifolia var. elliptica (Ismail et al.,
1997)
Roots M. officinalis (Yoshikawa et al., 1995)
Roots M. officinalis (Wu et al., 2009)
Rhizome and M. parvifolia (Chang and Lee, 1984)
roots
Fruits M. citrifolia (Pawlus et al., 2005)
1,2-Dihydroxy anthraquinone (Alizarin) Roots and stems M. umbellata (Burnett and Thomson, 1968)
1-Hydroxy-2-methoxy anthraquinone (Alizarin-2-methyl ether) Roots and stems M. umbellata (Burnett and Thomson, 1968)
1-Methoxy-2-methyl anthraquinone Roots and stems M. umbellata (Burnett and Thomson, 1968)
1,2-Dioxymethylene anthraquinone (Morindaparvin-A) Rhizome and M. parvifolia (Chang and Lee, 1984)
roots
1-Hydroxy-2-hydroxymethyl anthraquinone (Digiferruginol) Rhizome and M. parvifolia (Chang and Lee, 1984)
roots
1-Hydroxy-3-hydroxymethyl anthraquinone Roots M. officinalis (Yoshikawa et al., 1995)
1,3-Dihydroxy anthraquinone (Xanthopurpurin) Roots and stems M. umbellata (Burnett and Thomson, 1968)
3-Hydroxy-2-hydroxymethyl anthraquinone Roots M. officinalis (Wu et al., 2009)
(continued on next page)
890 T. Ruksilp et al. / Biochemical Systematics and Ecology 39 (2011) 888–892

Table 1 (continued )

Compound Plant part Source

1,3-Dihydroxy-2-methyl anthraquinone (Rubiadin) Roots M. citrifolia (Kamiya et al., 2010)
Roots M. angustifolia (Xiang et al., 2008)
Roots M. elliptica syn. M. citrifolia var. elliptica (Ismail et al.,
1997)
3-Hydroxy-1-methoxy-2-methyl anthraquinone (Rubiadin-1-methyl Roots and stems M. umbellata (Burnett and Thomson, 1968)
ether) Roots M. citrifolia (Kamiya et al., 2010)
Roots M. elliptica syn. M. citrifolia var. elliptica (Ismail et al.,
1997)
roots M. officinalis (Wu et al., 2009)
1,3-Dihydroxy-2-hydroxymethyl anthraquinone (Lucidin) Roots and stems M. umbellata (Burnett and Thomson, 1968)
Roots M. citrifolia (Kamiya et al., 2010)
3-Hydroxy -2-hydroxymethyl -1-methoxy anthraquinone Roots M. umbellata (Burnett and Thomson, 1968)
(Damnacanthol) Roots M. citrifolia (Kamiya et al., 2010)
1,3-Dihydroxy-2-methoxymethyl anthraquinone (Lucidin-u-methyl Roots M. angustifolia (Xiang et al., 2008)
ether) Roots M. citrifolia (Kamiya et al., 2010)
Roots M. elliptica syn. M. citrifolia var. elliptica (Ismail et al.,
1997)
Rhizome and M. parvifolia (Chang and Lee, 1984)
roots
1,3-Dihydroxy-2-ethoxymethyl anthraquinone (Lucidin-u-ethyl ether) Roots M. officinalis (Yoshikawa et al., 1995)
Rhizome and M. parvifolia (Chang and Lee, 1984)
roots
1,3-Dihydroxy-2-butoxymethyl anthraquinone (Lucidin-u-buthyl ether) Roots M. angustifolia (Xiang et al., 2008)
3-Hydroxy-1-methoxy-2-methoxymethyl anthraquinone Roots M. angustifolia (Xiang et al., 2008)
Roots M. citrifolia (Kamiya et al., 2010)
2-Formyl-1,3-dihydroxy anthraquinone (Nordamnacanthal) Roots M. citrifolia (Kamiya et al., 2010)
Roots M. elliptica syn. M. citrifolia var. elliptica (Ismail et al.,
1997)
2-Formyl-3-hydroxy-1-methoxy anthraquinone (Damnacanthal) Roots M. citrifolia (Kamiya et al., 2010)
Roots M. elliptica syn. M. citrifolia var. elliptica (Ismail et al.,
1997)
1-Hydroxy-2-hydroxymethyl anthraquinone-3-O–b-primeveroside Roots M. angustifolia (Xiang et al., 2008)
(Lucidin-3-O–b-primeveroside)
1,2-Dihydroxy-3-methyl anthraquinone Roots M. officinalis (Wu et al., 2009)
1-Hydroxy-2,3-dimethyl anthraquinone Roots M. officinalis (Yoshikawa et al., 1995)
1,3-Dihydroxy-2-methoxy anthraquinone Fruits M. citrifolia (Pawlus et al., 2005)
1,3-Dihydroxy anthraquinone-2-carboxylic acid (Munjistin) Roots M. umbellata (Burnett and Thomson, 1968)
Substituted A-ring anthraquinones
1-Hydroxy-6-hydroxymethyl anthraquinone Rhizome and M. parvifolia (Chang and Lee, 1984)
roots
1,6-Dihydroxy-2-methyl anthraquinone (Soranjidiol) Roots M. citrifolia (Kamiya et al., 2010)
Roots M. elliptica syn. M. citrifolia var. elliptica (Ismail et al.,
1997)
1,5-Dihydroxy-2-hydroxymethyl anthraquinone (Morindaparvin-B) Rhizome and M. parvifolia (Chang and Lee, 1984)
roots
1,8-Dihydroxy-6-methoxy-3-methyl anthraquinone (Physcion) Roots and stems M. umbellata (Burnett and Thomson, 1968)
Roots M. officinalis (Wu et al., 2009)
1,5,6-Trihydroxy-2-methyl anthraquinone (Morindone) Roots M. citrifolia (Kamiya et al., 2010)
Roots M. elliptica syn. M. citrifolia var. elliptica (Ismail et al.,
1997)
1,6-Dihydroxy-5-methoxy-2-methyl anthraquinone Roots M. elliptica syn. M. citrifolia var. elliptica (Ismail et al.,
(Morindone-5-methyl ether) 1997)
1,3,8-Trihydroxy-2-methoxy anthraquinone Roots M. officinalis (Wu et al., 2009)
2-Methoxy-1,3,6-trihydroxy anthraquinone Fruits M. citrifolia (Pawlus et al., 2005)
1,8-Dihydroxy-2-hydroxymethyl-5-methoxy anthraquinone Fruits M. citrifolia (Pawlus et al., 2005)
1,6-Dihydroxy-5-methoxy-2-methyl anthraquinone Fruits M. citrifolia (Pawlus et al., 2005)
1,8-Dihydroxy-2-methyl-3,7-dimethoxy anthraquinone Roots M. angustifolia (Xiang et al., 2008)

giving n-BuOH extract (37.17 g). The CH2Cl2 extract (19.30 g) was fractionated by vacuum liquid chromatography (VLC) over
silica gel (Merck Art. 7730), eluting with n-hexane, CH2Cl2, EtOAc and MeOH with increasing polarity to provide five fractions
(C1-C5). Fraction C2 was subjected to silica gel column chromatography (CC) and eluted with a gradient system of n-hexane-
CH2Cl2, then CH2Cl2-EtOAc (from 1:0 to 0:1) to afford soranjidiol (4, 44 mg, Adesogan, 1973). Fraction C4 was further subjected
to silica gel CC (n-hexane-CH2Cl2, 0.5:0.5) and radial chromatography (chromatotronÒ), using n-hexane-EtOAc (4:1) to yield
lucidin-u-methyl ether (1, 640 mg, Banthorpe and White, 1995) and damnacanthal (2, 410 mg, Zhou et al., 1994).
The initial EtOAc extract (14.99 g) was similarly chromatographed on silica gel VLC, eluting with CH2Cl2-n-hexane (1:2, 1:1,
3:2, 4:1 and 1:0), followed by EtOAc-CH2Cl2 (1:19, 1:9, 1:4 and 1:3) to yield nine fractions (E1–E9). Fraction E4 was
rechromatographed on CC over silica gel and eluted with gradient mixtures of n-hexane, CH2Cl2, EtOAc and MeOH with
 T. Ruksilp et al. / Biochemical Systematics and Ecology 39 (2011) 888–892 891

increasing polarity followed by chromatotronÒ, using n-hexane-EtOAc (4:1) to obtain lucidin (5,10 mg, Jegorov et al., 2005),
lucidin-u-ethyl ether (6, 8 mg, Chang, P. and Lee, K., 1984) and anthragallol-2,3-dimethyl ether (7, 7 mg, Roberge and Brassard,
1981). Fraction E5 was further fractionated over silica gel CC, using CH2Cl2, EtOAc and MeOH with increasing polarity and
chromatotronÒ, using n-hexane-EtOAc (4:1) to furnish nordamnacanthal (3, 170 mg, Zhou et al., 1994). Fraction E8 was
subsequently purified on silica gel CC, using a stepwise gradient elution of CH2Cl2-EtOAc and EtOAc-MeOH, yielding a new
natural anthraquinone, flavopurpurin (8, 10 mg, Auerbach and Crookes, 2008). Fraction E7 was rechromatographed on silica
gel CC, using CH2Cl2, EtOAc and MeOH with increasing polarity followed by chromatotronÒ (eluted with n-hexane-EtOAc
(4:1)), and then preparative TLC (100% CH2Cl2) to obtain 1-methoxy-2-methyl anthraquinone (9, 3 mg, Zembower et al., 1992),
3-hydroxy-1-methoxy-2-methoxymethyl anthraquinone (10, 3 mg, Kamiya et al., 2010) and anthragallol (11, 5 mg,
Dhananjeyan et al., 2005). 1H and 13C NMR data of 8 have not previously been reported yet these data are presented.
Flavopurpurin (8): brown-yellow amorphous powder; C14H8O5; ESI-MS m/z 225.288 [M  H]; 1H NMR (400 MHz, CDCl3-
CD3OD): d 13.10 (s, 1H, OH-1), 7.41 (d, J ¼ 7.6 Hz, 1H, H-3), 7.62 (d, J ¼ 7.6 Hz, 1H, H-4), 7.49 (s, 1H, H-5), 7.12 (d, J ¼ 8.0 Hz, 1H,
H-7), 8.12 (d, J ¼ 8.0 Hz, 1H, H-8); 13C NMR (100 MHz, CDCl3-CD3OD): d 160.6 (C-1), 160.0 (C-2), 135.0 (C-3), 119.0 (C-4), 131.0
(C-4a), 112.9 (C-5), 163.2 (C-6), 135.8 (C-7), 130.0 (C-8), 125.2 (C-8a), 188.0 (C-9), 115.0 (C-9a), 183.2 (C-10), 121.2 (C-10a).
The n-BuOH soluble extract (37.17 g) was dissolved in water and subjected to Diaion HP-20 column and successively eluted
with water, MeOH and acetone. The MeOH eluent was subjected to silica gel CC, using EtOAc-MeOH-H2O (9:1:0, 40:10:1 and
70:30:3, respectively) to afford five fractions (B1-B5). Fraction B4 was further purified using Sephadex LH-20 column and
eluting with n-hexane-CH2Cl2-MeOH (5:3:2), to yield asperulosidic acid (12, 5 mg, Wang et al., 1999) (Fig. 1).
Compounds 1–8 were subjected to in vitro cytotoxicity studies against human cervical carcinoma (HeLa) and human
mouth epidermal carcinoma (KB) cell lines, using the standard MTT colorimetric method (Kongkatip et al., 2003). Compounds
2, 3 and 4 showed moderate cytotoxicity against KB cells with IC50 values of 6.35, 5.99 and 7.67 mg/mL, and showed weak
cytotoxicity against HeLa cells with IC50 values of 13.37, 12.26 and 16.65 mg/mL, respectively. The IC50 value of adriamycin
(standard) in the both cell lines was 0.018 mg/mL.

4. Chemotaxonomic significance

The present study reports the isolation of eleven anthraquinones (1–11) and one iridoid glycoside (12) for the first time
from the roots of M. pandurifolia (Fig. 1). It is noteworthy that 30 of the 41 listed anthraquinones reported to be isolated from
the genus Morinda as summarized in Table 1 are unsubstituted A-ring anthraquinones (15 of 1,2,3-trisubstituted B-ring, 11 of
1,2-, 1,3-, and 2,3-disubstituted B-ring and 4 of 2-monosubstituted B-ring), while 11 of the substituted A-ring anthraquinones
have substituents at the 5, 6 or 8-positions. This can be considered as a characteristic chemotaxonomic feature typical of the
genus Morinda. In the case of our investigation, 1,2,3-trisubstitued B-ring anthraquinones were found in 1–3, 5–7 and 10–11
and 6- substituted A-ring anthraquinones were also found in 4 and 8 from M. pandurifolia. Compound 1 was found in Morinda
angutifolia (Xiang et al., 2008), Morinda citrifolia (Kamiya et al., 2010), Morinda elliptica (Ismail et al., 1997) and Morinda
parvifolia (Chang and Lee, 1984), while iridoid glycoside 12 was detected in M. citrifolia (Sang et al., 2001; Pawlus et al., 2005),
M. elliptica (Noiarsa et al., 2006), Morinda officinalis (Yoshikawa et al., 1995) and Morinda coreia (Kanchanapoom et al., 2002).
Compounds 1 and 12 may be considered as a chemotaxonomic marker for the genus level. This is the first report of the
occurrence of flavopurpurin (8) as a new natural anthraquinone, though it has been artificially prepared from a-bisul-
phanthraquinonic acid (Auerbach and Crookes, 2008). The presence of compounds 1–3 as the major components may
possibly be utilized for identification of M. pandurifolia, which is a significant chemotaxonomic finding.

Acknowledgements

T. Ruksilp is grateful to Loei Rajabhat University for supporting the doctoral scholarship. The Graduate School of Chula-
longkorn University for the fellowship, the Higher Research Promotion and National research University of Thailand, Office of
the Higher Commission (FW645A) and Center for Petroleum, Petrochemical and Advanced Materials also partially supports
this project.

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