J Nat Med (2008) 62:369373

DOI 10.1007/s11418-008-0229-0

NATURAL RESOURCE LETTER

Two new flavonoids from Centella asiatica (Linn.)

Ravi Subban A. Veerakumar R. Manimaran
K. M. Hashim Indira Balachandran

Received: 12 October 2007 / Accepted: 20 December 2007 / Published online: 13 February 2008
The Japanese Society of Pharmacognosy and Springer 2008

Abstract Two new flavonoids named castilliferol 1 and

castillicetin 2, as well as a known compound, isochlorogenic acid 3, were isolated from the whole plant of
Centella asiatica. Isolates 1 and 2 exhibited good antioxidant activity using 2,2-diphenyl-1-picryl hydrazyl
radical solution with IC50 values of 23.10 and 13.30,
respectively. The structures of these isolates were determined by analytical and spectral data, including 1-D and 2D NMR spectra.
Keywords Apiaceae
Centella asiatica
Castilliferol

Castillicetin
Isochlorogenic acid

this controversy has been resolved and it is has been concluded that Brahmi is B. monnieri and Mandookaparni is
C. asiatica [2]. The plant is indigenous to South-East Asia,
India, Sri Lanka, parts of China, the western South Sea
Islands, Madagascar, South Africa, South-East USA,
Mexico, Venezuela, Columbia and eastern South America.
Previously, triterpenoid acids [28], volatile and fatty oils
[2, 9], alkaloids [9], glycosides [2, 3, 913], flavonoids
[2, 11], and steroids [2, 9] have been isolated from the
different parts of the plant. This paper deals with the isolation and structural elucidation of two new constituents
(1, 2) and one known constituent 3, and the antioxidant
effect of these isolates.

Introduction
Results and discussion
Centella or Indian pennywort, Centella asiatica (Linn.)
Urban syn. Hydrocotyle asiatica Linn., belonging to the
family Apiaceae, is valued in Indian systems of medicine
for improving memory and for the treatment of nervine
disorders and skin diseases [1]. It has been used extensively
as a memory enhancer. The herb is known as Brahmi in
Unani medicine, Mandookaparni in Ayurveda and Gotu
Kola in the Western world. In India, the plant was previously confused with Bacopa monnieri Wettst., as both were
sold in the market under the name Brahmi. However,

R. Subban (&)
K. M. Hashim
I. Balachandran

Phytochemistry Division, Centre for Medicinal Plants Research,
Arya Vaidya Sala, Kottakkal, Kerala, India 676503
e-mail: ravisubban@rediffmail.com
A. Veerakumar
R. Manimaran
JSS College of Pharmacy, Udhagamandalam,
Nilgiris, Tamilnadu, India 643001

Castilliferol 1 was isolated as a yellow powder which

decomposes at 274 C. The molecular formula C24H16O8
was determined from elemental analysis and from the
pseudomolecular ion peak in the ESIMS negative mode
([M-H]- m/z 431) and the ESIMS positive mode ([M+H]+
m/z 433) spectra. The IR spectrum showed absorption
bands at 3,344 (OH), 1,680, 1,664 (conjugated carbonyl),
1,517 cm-1 (Ar ring). The UV spectrum displayed bands at
260 and 366 nm and showed that compound 1 was a flavone derivative. The band at 366 nm was shifted by
+44 nm upon the addition of NaOMe and by +33 nm when
NaOAc was added, indicative of a free hydroxyl group at
the C-40 position. A bathochromic shift of 45 nm in the
band I absorption maximum with AlCl3 and AlCl3/HCl
revealed the presence of a chelated hydroxyl in C-5. The
13
C NMR spectrum of 1 (Table 1) displayed signals for 24
carbons, which were edited by HSQC and DEPT into 12
methines and 12 quaternary carbons consisting of two

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Table 1 NMR data for
compounds 1 and 2

J Nat Med (2008) 62:369373

Position

1
dH (mult., J [HZ])

2
COSY

dH (mult., J [HZ])

COSY

dC

157.8

159.2

136.5

136.4

172.0

171.7

162.7

158.4

6.52 (d, 2.1)

H-8

99.4

6.32 (d, 2.1)

H-8

99.6

164.4

6.40 (d, 2.1)

H-6

94.6

158.6
6.50 (d, 2.1)

H-6

94.4

148.2

157.8

10

104.4

104.7

10

125.6

2
30

7.12 (d, 2.1)

H-6

115.3
144.8

H-3
H-20

50

6.90 (d, 8.0)

H-60

116.0

7.00 (d, 8.0)

H-60

116.5

60

7.95 (d, 8.0)

H-50

130.6

7.75 (dd, 8.0, 2.1)

H-50 ,20

117.7

200

8.18 (d, 8.0)

H-300

132.6

7.00 (d, 2.1)

H-600

117.4

300

7.00 (d, 8.0)

H-200

115.7

130.7
116.1

125.5
0

7.95 (d, 8.0)

6.90 (d, 8.0)

40

148.8

00

148.5

133.1

00

126.4
148.6

161.6

152.3

500

7.00 (d, 8.0)

H-600

115.6

6.80 (d, 8.0)

H-600

119.5

600

8.18 (d, 8.0)

H-500

131.6

7.85 (dd, 8.0, 2.1)

H-500 ,200

117.1

00

7.95 (d, 8.0)

H-8

00

148.4

7.65 (d, 8.3)

H-800

148.6

800

6.90 (d, 8.0)

H-700

115.4

6.98 (d, 8.3)

H-700

116.6

900

carbonyls at dc 172.03 and 174.30 and seven oxygenated

carbons. The 1H NMR spectrum (500 MHz, CH3COCH3
d6) showed resonances due to four hydroxyl groups at d
11.5 (2H), 12.6 (1H) and 13.2 (1H) and two pairs of
AA0 BB0 -type signals; one pair at d 7.95 (d, J = 8.0 Hz)
and 6.90 (d, J = 8.0 Hz) belonged to the protons at the 20 ,
60 and 30 ,50 protons of the flavonoid moiety and was partially overlapped with the alkene protons of the pcoumaroyl system. The other pair at d 8.18 (d, J = 8 Hz)
and 7.00 (d, J = 8 Hz) belongs to the 200 ,600 and 300 ,500
positions of the p-coumaroyl system. Due to the vicinal
coupling constants in the signals at d 7.95(d, J = 8.0 Hz,
H-700 ) and d 6.90 (d, J = 8.0 Hz, H-800 ) being 8.00 Hz,
they were assigned to a cis-configured double bond. For a
trans-configured double bond the coupling constant value
will be on the order J = 15.0 Hz [14, 15]. A cross peak
was observed in the NOSEY spectrum between the above
two signals, supporting the cis configuration. The two
doublets at d 6.40 and 6.52 (each 1H, J = 2 Hz) were
assigned to H-8 and H-6, respectively, of the flavonoid
nucleus. The carbon resonance signals at d 170.6, 161.6,
148.4, 132.6, 133.1, 115.7, 115.6 and 115.4 in the 13C
NMR spectrum supported the presence of p-coumaroyl

123

dC

170.6

170.5

residue. A cleavage at the carbonyl carbon of the

p-coumaroyl moiety was observed in the mass spectrum by
exhibiting a signal at m/z 286 of the aglycone moiety. The
HMBC NMR spectrum showed key correlations between
H-700 (d 7.95) and C-200 , C-600 and C-900 (d 132.6 and 131.6,
170.6, respectively), H-200 , H-600 (d 8.18) and C-400
(d 161.6), H-20 , H-60 (d 7.95) and C-40 (d 148.8), and
between H-30 , H-50 (d 6.90) and C-10 (d 125.6). Without the
coumaroyl moiety, it was similar to that of kaempferol. The
structure of castilliferol 1 was therefore elucidated as
kaempferol-3-p-coumarate.
Castillicetin 2 was isolated as a yellow powder which
decomposes at 310 C. Elemental analysis and ESIMS
negative mode indicated the molecular formula to be
C24H16O10. The UV spectrum has adsorptions at 253, 329
and 368 nm, showing that compound 2 was a flavone
derivative. The band at 368 nm was shifted by +46 nm
upon the addition of NaOMe and by +31 nm when NaOAc
was added, indicative of a free hydroxyl group at the C-40
position. A bathochromic shift of 32 nm in the band at
253 nm with AlCl3 and AlCl3/HCl revealed the presence of
a chelated hydroxyl in C-5. The 13C NMR spectrum of 2
displayed signals for 24 carbons, which were edited by

J Nat Med (2008) 62:369373

HSQC. SEFT NMR spectra of 2 showed the presence of 10

methine and 14 quaternary carbons, which includes two
carbonyls at d 171.7 and 173.4 and nine oxygenated carbons. The 1H NMR spectrum (500 MHz, CH3COCH3d6)
showed resonances due to six hydroxyl groups at d 12.4
(4H) and 11.8 (2H) and a pair of ABX proton spin systems
was observed at d 7.12 (d, J = 2.1 Hz), 7.00 (dd, J = 8.0,
2.1 Hz), 7.75 (dd, J = 8.0, 2.1 Hz) and 7.00 (dd, J = 8.0,
2.1 Hz), 6.80 (d, J = 8.0 Hz), and 7.85 (dd, J = 8.0,
2.1 Hz), characteristic of two 1,3,4-trisubstituted benzene
units. In addition, two doublet signals at d 6.32 and 6.50
(each 1H, J = 2 Hz), assignable to H-8 and H-6, respectively, of the flavonoid nucleus, were also observed. Two
doublets at d7.65 and 6.98 (each 1H, J = 8.3 Hz) appeared
as an AB system. Due to the vicinal coupling constant
being 8.3 Hz, they were assigned to a cis-configured double bond. A cross peak was observed in the NOSEY
spectrum between the above two signals supporting the cis
configuration.
The carbon signals at 170.5, 159.3, 152.6, 148.6, 126.4,
119.5, 117.4, 117.1 and 116.6 in the 13C NMR spectrum
indicated the presence of a caffeoyl moiety. Without the
caffeoyl moiety signals, it was similar to that of Quercetin.
The HMBC NMR spectrum showed key correlations
between H-200 (d 7.00) and C-700 (d 148.6), H-200 (d 7.00)
and C-400 (d 152.3), H-600 (d 7.85) and C-700 (d 148.6), H-600
(d 7.85) and C-400 (d 152.3), H-700 (d 7.65) and C-200
(d 117.4), H-700 (d 7.65) and C-600 (d 117.1), H-500 (d 6.80)
and C-300 (d 148.62), H-700 (d 7.65) and C-900 (d 170.52),
H-20 (d 7.12) and C-40 (d 148.50), H-20 (d 7.12) and C-2
(d 159.2), H-60 (d 7.75) and C-2 (d 159.2). Detailed analysis of the 1-D, 2-D NMR data enabled the full assignment
of all protons and carbons of this compound (Table 1). The
structure of castillicetin 2 was therefore elucidated as
Quercetin-3-caffeate.
Compound 3 was identified as isochlorogenic acid by
comparing its spectral data with those in the literature
[16, 17].
Antioxidant activity was measured by a decrease in the
absorbance at 516 nm of methanolic solution of colored
2,2-diphenyl-1-picryl hydrazyl radical solution (DPPH)
brought about by the sample. A stock solution of DPPH
(1.3 mg/ml methanol) was prepared such that 75 ll of it in
3 ml methanol gave an initial absorbance of 0.9. This stock
solution was used to measure the antioxidant activity.
Decreases in the absorbance in the presence of compound 1
and 2 at different concentrations were noted after 20 min.
IC50 was calculated from percentage of inhibition
(Table 2). Gallic acid was used as positive control. Castilliferol 1 and castillicetin 2 exhibited good antioxidant
activity with IC50 values of 23.10 and 13.31 lg/ml,
respectively, when compared to the control (IC50 value of
14.58 lg/ml) (Fig. 1).

371
Table 2 Antioxidant activity of compounds 1 and 2
Sample

Concentration
(lg/ml)

Percentage of
inhibition

9.04

25

57.08

50

84.74

18.10

25

75.44

50

85.24

IC50
(lg/ml)
23.10

13.31

Gallic
acid

14.58

3'

2'

HO

4'

1'

8
9

OH

2
5'

6'

2''
6

10
5

4''
9''

OH

3''

CH

CH

8''

OH

1''

7''

5''

6''

OH

OH

HO

OH

O
OH

HO
O
HO

2''

5''

6''

C
a

HO

7''
COOH

1''

CH

OH

CH

3''

4''

2'

a'
O

OH

C
O

OH

1'
3'

'
6'

4'
5'

OH

Fig. 1 Structures of compounds 1, 2 and 3

Materials and methods

General experimental procedures
Melting points were determined on a Veego (Mumbai,
India) melting point apparatus and are uncorrected. IR
spectra were recorded as KBr pellets on a PerkinElmer
(Waltham, MA, USA) 2000 FTIR spectrophotometer. UV
spectra were measured on a Shimadzu (Tokyo, Japan) UV2101 PC UVVis scanning spectrophotometer. 1H NMR
(500 MHz) and 13C NMR (150.9 MHz) spectra were
measured on a JEOL (Tokyo, Japan) JNM LA-500
instrument using acetone-d6 solvent. ESI-MS were determined on an Agilent (Palo Alto, CA, USA) MSD 1100
single-quadrapole spectrometer. Elemental analysis was
carried out on a PerkinElmer 240C instrument. For TLC,
precoated Si gel 60 F254 plates were used and spots were

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372

detected under UV light and further visualized by spraying

with vanillin-sulfuric acid. Column chromatography was
carried out on Si gel (60120 mesh, Merck, Darmstadt,
Germany). DPPH was purchased from Sigma Chemical
Co. (St. Louis, MO, USA). HPLC purification was performed on a LC-8A, SPD with FRC-10A Shimadzu
apparatus.

Plant material
The whole plant material of C. asiatica was collected from
the Nilgris district, Tamilnadu, India in August 2005. The
Botany Department of Government Arts College,
Udhagamandalam, The Nilgiris identified the plant
material and a voucher specimen has been deposited at the
college.

Extraction and isolation

The air-dried material (2 kg, whole plant) was ground and
extracted exhaustively with MeOHH2O (6:4, 3 9 5.0 l) for
2 h. The extracts were combined and freed of solvent to give
160 g of residue. The extract was dissolved in a mixture of
MeOHH2O (1:1, 500 ml) and extracted with n-butanol
(3 9 300 ml) to give, after concentration in vacuo, 102 g of
a n-butanol soluble extract. The residue was subjected to
column chromatography on 200 g of Si gel and eluted with
EtOAc (500 ml) followed with EtOAc containing increasing
amounts of MeOH. Eighty fractions (25 ml each) were
collected and pooled according to their similiarity on
analytical TLC plates and dried. Combined fractions 538
(CAI, 10.7 g), which were eluted with EtOAc, and 5279
(CAII, 9 g), eluted with EtOAcMeOH (9:1), were obtained.
Fraction CAI was subjected to chromatography on 140 g of
Si gel and eluted with pet. ether to start with and further
fractions were obtained by increasing the polarity of the
solvent by suitable additions of EtOAc and MeOH. Sixty
fractions (25 ml each) were collected and pooled according
to their similarity on analytical TLC plates and concentrated
in vacuo. This resulted in fractions 2239 (A, 14 g)
and fractions 4260 (B, 220 mg) when eluted with
EtOAc-pet.ether (3:7). A was rechromatographed in another
Si gel column, eluted with CH3COCH3-pet. ether (3:7) to
yield 1 (500 mg). B was rechromatographed in another Si gel
column, eluted with CH3COCH3-pet. ether (3:7) to yield 2
(109 mg). Compounds 1 and 2 were purified by RP-HPLC
(Inertsil C-18 column, 2.50 9 4.6 mm, acetonitrile and
orthophosphoric acid buffer, 1.5 ml/min, photodiode array
detection monitored at 254 nm). CAII was subjected to
column chromatography on 55 g diaion and eluted with
MeOHH2O (9:1) to yield 3 (41 mg).

123

J Nat Med (2008) 62:369373

Kaempferol-3-p-coumarate 1: yellow powder; mp 274

C (dec.); UV (MeOH) kmax (log e) 366 (3.51), 260 (2.52)
nm; (MeOH + NaOMe) kmax (log e) 410 (3.48), 320
(1.90), 272 (2.41) nm; (MeOH + NaOAc) kmax (log e) 399
(3.49), 320 (1.87), 271 (2.43) nm; (MeOH + AlCl3) kmax
(log e) 411 (3.29), 320 (1.98), 302 (1.69), 272 (2.51) nm;
(MeOH + AlCl3 + HCl) kmax (log e) 411 (3.31), 3.20
(1.89), 301 (1.68), 271 (2.48) nm; IR (KBr) mmax 3,344,
1,694, 1,664, 1,517, 1,267 cm-1; 1H NMR (CH3COCH3
d6, 500 MHz) and 13C NMR , see Table 1; elemental
analysis, found: C 66.6532, H 3.7327 (calc. C 66.6536, H
3.7319 for C24H16O8 ); ESI-MS negative mode m/z 431
[M-H]- ; ESI-MS positive mode m/z 433 [M+H]+.
Quercetin-3-caffeate 2: yellow powder; mp 310 C
(dec.); UV (MeOH) kmax (log e) 253 (2.46), 329 (1.87), 363
(3.21) nm; (MeOH + NaOCH3) kmax (log e) 269 (2.36),
320 (1.71), 344 (1.98), 409 (3.42); (MeOH + NaOAc)
kmax (log e) 273 (2.35), 320 (1.92), 394 (3.12);
(MeOH + AlCl3) kmax (log e) 273 (2.49), 308 (1.67), 332
(1.89), 433 (3.22); (MeOH + AlCl3 + HCl) 273 (2.49),
307 (1.59), 340 (1.91), 401 (3.17); IR (KBr).
1
H NMR (CH3COCH3d6, 500 MHz) and 13C NMR,
see Table 1; elemental analysis, found: C 62.0578, H
3.4753 (calc. C 62.0590, H 3.4747, for C24H16O10); ESIMS negative mode m/z 463 [M-H]-; ESI-MS positive
mode m/z 465 [M+H]+.

Antioxidant activity
Different concentrations (5, 25, 50 lg/ml) of test solution
and reference standard were placed in different microtitre
plates; 10 ll DPPH (1.3 mg/ml in methanol) solution was
added to each well. Finally, sufficient methanol was added
to make the volume up to 250 ll in each well. The reaction
mixture was mixed and incubated at room temperature for
20 min; the absorbance was measured at 516 nm. Gallic
acid was used as positive control.

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