IRIDOIDS FROM Tamilnadia uliginosa
Nitirat Visetkit1,*, Tanawan Kummalue2, Veena Nukoolkarn3, Weena Jiratchariyakul3,#
1
Master of Science in Pharmacy Program in Pharmaceutical Chemistry and Pharmaceutical
Phytochemistry, Department of Pharmacognosy, Faculty of Pharmacy, Mahidol University,
Bangkok, Thailand
2
Department of Clinical Pathology, Faculty of Medicine Siriraj Hospital, Mahidol University,
Bangkok, Thailand
3
Department of Pharmacognosy, Faculty of Pharmacy, Mahidol University, Bangkok,
Thailand
*e-mail: wikiterter@gmail.com, #e-mail: weena.jir@mahidol.ac.th
Abstract
Tamilnadia uliginosa (Retz.) Tirveng. & Sastre (Synonym: Catunaregam uliginosa
(Retz.) Sivar.), locally known as “Talumphuk”, is a plant in Rubiaceae family. In Thailand, it
has traditionally use as astringent, antidiarrhea, tonic, and one of ingredients in liver cancer
recipe. Phytochemical study of this plant is scarcely reported, only one report on
phytochemical screening. However, several phytochemical studies of other Catunaregam
plants found in Thailand revealed that they contained several types of iridoids. Because
iridoids have many pharmacological activities, they might be the active compounds in
Tamilnadia uliginosa. The aim of this study is to investigate iridoids in Tamilnadia uliginosa.
On thin-layer chromatogram, iridoids which were shown as blue bands, were observed in
ethanolic extract of Tamilnadia uliginosa root. The extract was then separated by column
chromatography, using gradient mobile phase of methanol/dichloromethane, giving fifteen
fractions (A1-A15). Fractions A11 and A12, which contained blue bands, were further
chromatographed on reversed-phase column chromatography, using MCI gel CHP20P as
stationary phase and gradient mobile phase of methanol/water, giving six fractions (B1-B6).
Fraction B4 was evaporated and the pure compound (TU1) was obtained. The structure of
TU1 was determined by spectroscopic analysis including UV, IR, mass, 1D and 2D NMR
spectra. Results showed that TU1 had the molecular weight of 432 (C19H28O11). The
comparison of NMR data with the literature showed the identical structure to diffusoside A,
an iridoid glucoside isolated from aerial parts of Hedyotis diffusa. It can be concluded that
Tamilnadia uliginosa contained iridoids, which one of them was identified as diffusoside A.
This is the first time to report the presence of iridoids in this plant.
Keywords: Tamilnadia uliginosa, Rubiaceae, iridoids, diffusoside A
Introduction
Tamilnadia uliginosa (Retz.) Tirveng. & Sastre (Synonym: Catunaregam uliginosa
(Retz.) Sivar.)[1], locally known as “Talumphuk”, is a plant in Rubiaceae family. It has
traditionally use as astringent, antidiarrhea and tonic. Moreover, it is one of the ingredients in
traditional recipe for liver cancer in the north-eastern region of Thailand. Phytochemical
study of this plant is scarcely reported. The plant contained many secondary metabolites,
including alkaloids, coumarins, glycosides, phenolic compounds, saponins and tannins[2].
However, several phytochemical studies of other Catunaregam plants found in Thailand
revealed that they contained several types of iridoids[3-4]. Example of iridoids found in these
Catunaregam plants is shown in Table 1.
Table 1. Example of iridoids found in Catunaregam plants.
Iridoids name
6α-hydroxygeniposide
Found in
Catunaregam tomentosa
Deacetyl asperulosidic acid methyl ester
Catunaregam spinosa
Gardenoside
Catunaregam spinosa
Catunaregam tomentosa
Geniposide
Catunaregam spinosa
Randinoside
Catunaregam spinosa
Scandoside methyl ester
Catunaregam spinosa
Catunaregam tomentosa
Shanzhiside methyl ester
Catunaregam tomentosa
Iridoids are monoterpene compounds based on their fused cyclopentapyranoid ring.
Their specific character is that they can give the decomposed blue polymer after being
hydrolysed with acid[5]. These compounds are found in some plant families, for example,
Ericaceae, Gentianaceae, Lamiaceae, Oleaceae, Plantaginaceae, Rubiaceae, Scrophulariaceae
and Valerianaceae. Iridoids have many pharmacological activities including antibacterial,
antifungal, antiprotozoal, antiviral, anticancer, antidiabetic, antihyperlipideamic, antiinflammatory, antinociceptive, antiosteoporosis and antioxidant activity[6-7]. From the facts
that iridoids have many pharmacological activities and their presence in Catunaregam plants,
they might be responsible for the activities of Tamilnadia uliginosa. The aim of this study is
to investigate the iridoids in Tamilnadia uliginosa using the chromatographic method and
spectroscopic analyses.
Methodology
Plant material
The roots of Tamilnadia uliginosa were collected from Nongbualamphu province,
Thailand. They were dried at 50๐C for 48 hr and then powdered.
Extraction and isolation
The dried powder of Tamilnadia uliginosa root (2.2 kg) was extracted with 95%
ethanol in Soxhlet apparatus. The extract was concentrated by rotary evaporation at 40๐ C,
giving 101.70 g of dry extract. The ethanolic dry extract (100.0 g) was placed in
chromatographic column, using silica gel as stationary phase and gradient mobile phase of
methanol and dichloromethane (3:97, 5:95, 7.5:92.5, 10:90, 15:85, 20:80 and 25:75), giving
fifteen fractions (A1 to A15). Each fraction was examined on thin-layer chromatogram to
monitor the separation. The combined fractions A11 and A12 (300.0 mg) were further
chromatographed on reversed-phase chromatographic column, using MCI gel CHP20P as
stationary phase and gradient mobile phase of methanol and water (20:80, 25:75, 30:70,
35:65 and 40:60), yielding six fractions (B1 to B6). Each fraction also was monitored on thinlayer chromatogram. Fraction B4 was then evaporated and the pure compound (TU1) was
obtained. Summary of isolation process was described in Figure 1.
Figure 1. TU1 isolation process
Identification
The spectroscopic analyses of compound TU1 were performed. IR and UV spectra
were obtained from SHIMADZU UV-2600 and FTIR system (NICOLET 6700), respectively.
ESI-MS data was measured on Bruker microTOF spectrometer. 1H-, 13C- and 2D-NMR
spectra were obtained from Bruker AV-500. The NMR spectral data were measured in
CD3OD, and chemical shifts were expressed in δ (ppm), referring to TMS.
Results
The isolation of Tamilnadia uliginosa roots extract was monitored on thin-layer
chromatogram. Because iridoids are unstable and decomposed into a blue polymer after
hydrolysis with acid, this property can be used for screening iridoids contained in plant
material. Chromatograms of Tamilnadia uliginosa were shown in Figure 2. According to blue
bands appearing on the chromatogram, it can be concluded that fraction A11 to A14
contained iridoids.
The chromatogram of the separated fraction (B1 to B6) from A11 and A12 was shown
in Figure 3. Four fractions (B1, B2, B3 and B4) contained blue bands indicating the presence
of iridoids. Only B4 had one blue band (Rf. = 0.30). B4 was then evaporated under reduced
pressure and the pure compound (TU1), 17.8 mg, was obtained.
Figure 2. Chromatogram of fractions A11 to A15 eluted from the silica gel column.
M = root extract chromatogram.
Adsorbent: silica gel 60 F254.
Mobile phase: ethyl acetate: methanol: water (80:18:2).
Detection: Spraying with 10% H2SO4 in methanol and heated on the hot plate (110๐C) for a few minute.
Figure 3. Chromatogram of fractions B1 to B6 eluted from the MCI gel CHP20P column.
A= the loaded sample.
TLC condition was the same as Figure 2, except the mobile phase was changed to ethyl acetate: methanol:
water (80:12:8).
TU1 was pale yellow amorphous. The UV spectrum in methanol had maximum
absorption (λmax) at 236.5 nm. The IR spectrum in methanol (HATR) showed strong bands at
3366.00, 2923.08, 1693.13 and 1285.36 cm-1. ESI-MS showed the parent peak [M+Na]+ of
the mass per charge ratio (m/z) of 455.1618. The corresponding compound had the molecular
weight of 432 (C19H28O11). 1H-, 13C- and 2D-NMR spectral data are shown in Table 2. From
the mentioned evidences, TU1 was identified as diffusoside A.
Table 2. NMR data of TU1a,b
Position
1
3
4
5
6
7
8
9
10
 Cc
 H (Hz)d
101.63 (D)
154.95 (D)
108.47 (S)
42.02 (D)
83.31 (D)
128.29 (D)
152.05 (S)
45.95 (D)
61.69 (T)
11
12
1′
2′
3′
4′
5′
6′
169.57 (S)
51.81 (Q)
100.64 (D)
74.92 (D)
77.81 (D)
71.39 (D)
78.24 (D)
62.52 (T)
1′′
66.08 (T)
2′′
15.87 (Q)
5.01 d, 8.86
7.63 d, 1.03
3.07 dt, 1.07, 6.69
4.45 s
6.61 d, 1.45
2.54 t, 8.04
3.88 m;
4.19 d, 15.79
3.74 s
4.71 d, 7.84
3.24 m
3.49 m
3.38 m
3.24 m
3.65 d, 12.14;
3.81 d, 12.14
3.49 m;
3.65 dd, 5.42, 12.14
1.04 t, 7.02
COSY
H-9
H-6, H-9
H-5, H-7
H-6
H-1, H-5
-
HMBC
H-5,
H-5
H-3, H-5
H-3, H-7
H-5, H-9, H-1′′
H-9, H-10
H-1, H-6
H-6, H-7
H-7
H-2′
H-1′, H-3′
H-2′
H-3′, H-5′
H-6′
H-5′
H-3, H-5, H-12
H-3′
H-4′
H-1′, H-5′
H-2′, H-6′
H-3′
H-3′, H-4′
H-2′′
H-6
H-1′′
-
a
CD3OD.
500 MHz 1H- NMR and 125 MHz 13C-NMR.
c
Multiplicity obtained from DEPT-135 experiment.
d
δ in ppm, J in Hz.
b
Discussion and Conclusion
Compound TU1 was obtained as pale yellow amorphous. Its IR spectra showed the
presence of hydroxyl (3366.00 cm-1; O-H stretching), carbonyl (1693.13 cm-1; C=O
stretching) and ether functional groups (1285.36 cm-1; C-O stretching). UV spectrum showed
maximum absorption at 236.5 nm, represented the iridoid skeleton. ESI-MS showed based
peak at m/z of 455.1618 [M+Na]+, indicated the molecular weight of 432 (C19H28O11).
The 1H-, 13C- and 2D-NMR spectra provided the evidence for iridoid glucoside as
follow: Nineteen carbon signals obtained from 13C-NMR spectrum. DEPT-135 spectrum
revealed two methyl (δC = 15.87 and 51.81), three methelene (δC = 61.69, 62.52 and 66.08),
ten methine (δC = 42.02, 45.95, 71.39, 74.92, 77.81, 78.24, 83.31, 101.63, 128.29 and 154.95)
and three quaternary carbons (δC = 108.47, 152.05 and 169.57). From 13C-NMR data, one
anomeric carbon (C-1′, δC = 100.64), one methylene (C-6′, δC = 62.52) and four methines (C4′, C-2′, C-3′ and C-5′; δC = 71.39, 74.92, 77.81 and 78.24, respectively) displayed a glucose
unit. The J value of anomeric proton (H-1′, 7.84 Hz) indicated the β-configuration of this
glucose unit.
According to the downfield shift of C-1 (δC = 101.63), this methine carbon could be
attached to oxygen in pyranoid ring (position 2) and O-glucosyl moiety. From above
evidence, glucose unit could be connected with pyranoid ring via C-1 position. C-1 also
connected with C-9 carbon (δC = 45.95), which connected with C-5 carbon (δC = 42.24),
confirmed by COSY. Because C-3 had downfield shift than ordinary olefinic carbon (δC =
154.95), this carbon could be attached to the other site of oxygen in position 2. Quaternary
carbon C-4 (δC = 108.47) was connected with C-3 and C-5, according from the HMBC data.
From above data, C-1, C-3, C-4, C5 and C-9 were the members of pyranoid ring.
From COSY data, C-5 was adjacent to C-6 (δC = 83.31), which was connected to C-7
(δC = 128.29. HMBA data showed the correlation between C-8 (δC = 152.05) and H-1, as well
as between C-8 and H-6. This supported the connectivity among C-7, C-8 and C-9 positions.
From the mentioned data, C-5, C-6, C-7, C-8 and C-9 were the members of cyclopentene
ring. Because C-5 and C-9 were also the members of pyranoid ring, this NMR data could be
referred to fused cyclopentapyranoid structure, which two rings connected via C-5 and C-9
positions.
Carbonyl carbon (C-11, δC = 169.57) was connected with methoxy carbon (C-12, δC =
51.81) indicated from correlation between C-11 and H-12 obtained from HMBC,
representing the acetate substituent. Due to upfield shift of C-4 and the correlation of C-11
carbon to H-3 and H-5 obtained from HMBC, this acetate moiety should attach to C-4
carbon. According to COSY data, one methyl carbon (C-2′′, δC = 15.87) was connected with
methylene carbon (C-1′′, δC = 66.08). Because of the downfield shift of C-1′′ carbon, this
carbon should attach with oxygen atom, indicating the ethoxy substituent. From the
downfield shift of C-6 carbon (δC = 83.31) and the correlation of C-1 and H-6 obtained from
HMBC, this ethoxy moiety should connect to cyclopentene ring at C-6 position. One
methylene carbon (C-10, δC = 61.69) was also attached with cyclopentene ring at C-8
position, due to correlation between C-10 and H-7 obtained by HMBC. This methylene
carbon (C-10) was attached with hydroxyl group due to downfield shift. Consequently, the
structure of TU1 was identified as the structure shown in Figure 4a.
The NMR data of TU1 was compared with previous NMR spectra of known iridoids.
TU1 was identical to diffusoside A (Figure 4b) isolated from Hedyotis diffusa[8]. This is the
first report of diffusoside A presence in Tamilnadia uliginosa.
Figure 4. Structure of TU1 (a) and diffusoside A(b)
In conclusion, T. uliginosa contained iridoid compounds. One of them was diffusoside
A, which was successfully isolated from the ethanolic extract using silica gel followed by
MCI gel CHP20P columns. This is the first report of the presence of iridoid compounds in
this plant. Further investigation of the biological activity of the isolated compound is planned.
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Acknowledgements:
The authors are thankful to the Institute of Thai Traditional Medicine for the financial
support.