A new iridoid glycoside from Vitex negundo Linn (Verbenacea)

Article abstract of DOI:10.1080/14786410802696494

Five compounds have been isolated from the leaves of Vitex negundo Linn (Verbenacea) and their structures established by spectral analysis. Compound 1 has been identified and characterised as a new iridoid with a novel structure which has never before been reported in literature from Vitex or any other source. Its structure has been established as 1,4a,5,7a-tetrahydro-1 - β-D-glucosyl-7-(3′,4′-dihydroxybenzoyloxymethyl) -5-ketocyclopenta[c]pyran-4-carboxylic acid. Other compounds, 2, 3, 4 and 5, have been identified as luteolin-7-O -β- D glucoside, nishindaside, negundoside, and agnuside, respectively. Compound 2, a flavone-O-glycoside, is being reported from this plant for the first time, and compounds 3, 4 and 5 are structurally known iridoids.

Full text of DOI:10.1080/14786410802696494

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A new iridoid glycoside from Vitex
negundo Linn (Verbenacea)
a
b
b
Rattan L. Sharma , Anil Prabhakar , Kanaya L. Dhar & Anand
a
Sachar
a
Department of Chemistry , University of Jammu ,
Jammu-180004, India
b
Indian Institute of Integrative Medicine (IIIM) , Jammu
Tawi-180001, India
Published online: 05 Nov 2010.
To cite this article: Rattan L. Sharma , Anil Prabhakar , Kanaya L. Dhar & Anand Sachar (2009) A
new iridoid glycoside from Vitex negundo Linn (Verbenacea), Natural Product Research: Formerly
Natural Product Letters, 23:13, 1201-1209, DOI: 10.1080/14786410802696494
To link to this article: http://dx.doi.org/10.1080/14786410802696494
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Natural Product Research
Vol. 23, No. 13, 10 September 2009, 1201–1209
A new iridoid glycoside from Vitex negundo Linn (Verbenacea)
a
b
b
a
Rattan L. Sharma *, Anil Prabhakar , Kanaya L. Dhar and Anand Sachar
a
b
Department of Chemistry, University of Jammu, Jammu-180004, India; Indian Institute of
Integrative Medicine (IIIM), Jammu Tawi-180001, India
(
Received 6 June 2008; final version received 16 December 2008)
Five compounds have been isolated from the leaves of Vitex negundo Linn
(
Verbenacea) and their structures established by spectral analysis. Compound 1
has been identified and characterised as a new iridoid with a novel structure
which has never before been reported in literature from Vitex or any other source.
Its structure has been established as 1,4a,5,7a-tetrahydro-1-ꢀ-D-glucosyl-7-
0 0
(
3 ,4 -dihydroxybenzoyloxymethyl)-5-ketocyclopenta[c]pyran-4-carboxylic acid.
Other compounds, 2, 3, 4 and 5, have been identified as luteolin-7-O-ꢀ-D
glucoside, nishindaside, negundoside, and agnuside, respectively. Compound 2,
a flavone-O-glycoside, is being reported from this plant for the first time, and
compounds 3, 4 and 5 are structurally known iridoids.
Keywords: Vitex negundo; new iridoid glycoside
1
. Introduction
The genus Vitex belongs to the family Verbenacea, which includes 80 genera and
about 800 species. Vitex is a genus of trees and shrubs widely found in the tropic and warm
region. Vitex negundo Linn is a shrub which is quite prolific in India, occurring up to an
altitude of ꢀ1500 m. The leaf extract of V. negundo has been reported to exhibit a wide
range of pharmacological activities, including mosquito-repellant activity (D. Hebbalkar,
G. Hebbalkar, Sharma, Joshi, & Bhat, 1992), hepato protective action (Avadhoot & Rana,
1
991), anti-inflammatory activity (Chawala, Sharma, Handa, & Dhar, 1991), antigeno-
toxic effects (Balboa & Lim-Sylianco, 1993), antihistamine release properties (Rimando
et al., 1987), rehumatoid arthritis action (Kishor & Banerjee, 1988), activity of cattrachea
(
Dayrit, Lapid, Cagampang, & Lagurin, 1987), central nervous system (CNS) depressant
activity (Gupta, Mazumder, & Bhawal, 1999), and antifilarial activity (Bhargava, 1986)
in vitro. From the leaves, twigs, seeds and roots of V. negundo, a large number of
compounds with varied structural ranges have been reported, including glucononitol
(
1
(
Ghosh & Krishna, 1936), camphene, ꢁ-pinene, citral and ꢀ-caryophyllene (Masilungan,
955), casticin, orientin, isorientin, corymbosin and various new flavonoid glycosides
Achari, Chowdhury, Dutta, & Pakrashi, 1984; Banerji, Chadha, & Malshet 1969;
Banerji, Das, Chakrabarty, & Jha, 1988; Dayrit et al., 1987; Ferdous et al., 1984;
Hansal, Leuckert, Rimpler, & Schaaf, 1965; Sirait, Rimpler, & Hansal, 1962),
a diterpenoid, three triterpenoids, various long-chained unsaturated fatty acids
¨
¨
*
Corresponding author. Email: rlsharma_hod@rediffmail.com
ISSN 1478–6419 print/ISSN 1029–2349 online
ß 2009 Taylor & Francis
DOI: 10.1080/14786410802696494
http://www.informaworld.com

1
202
R.L. Sharma et al.
(
Vishnol, Shoeb, Kapil, & Popli, 1983), five iridoid glycosides, aucubin, agnuside,
nishindaside, negundoside and 6-p-hydroxybenzoyl mussaenosidic acid (Dayrit &
Lagurin, 1994), and a lignan (Chawala, Sharma, Handa, & Dhar, 1992). Now, we
report one new iridoid (1); a flavonoid glycoside, luteolin-7-O-ꢀ-D-glucoside (2); and three
known iridoids: nishindaside (3), negundoside (4) and agnuside (5) from the methanolic
extract of leaves of V. negundo. The methanolic extract has been found to exhibit very
potent antifeedant, anti-inflammatory, analgaesic and anti-convulsant activities.
2
2
. Experimental
.1. Plant material
Vitex negundo leaves were collected from Trivandrum (Kerala State in India) in the month
of August (1997) and authenticated by Dr B.K. Kapahi, Head of the Botany Division,
Regional Research Laboratory (CSIR), Jammu.
2
.2. Isolation
Air-dried leaves of V. negundo (2.25 kg) from Trivandrum were extracted thrice in a
ꢁ
percolator at room temperature with 18.0 L of petroleum ether (60–80 C), and kept for
over 24 h, affording a defatted mass which was dried under high vacuum and charged
in a percolator for extraction with 11.25 L of methanol again at room temperature
and kept for over 24 h to yield a dry brown solid (360 g). Si-gel CC of 50 g of this
residue over 300 g of gel gave, on elution with (EtOAc–MeOH 49 : 1), 0.22 g of 5, with
(
1
EtOAc–MeOH 19 : 1) 0.16 g of 3, with (EtOAc–MeOH 9 : 1) 0.11 g of 4 and 0.465 g of
and with (EtOAc–MeOH 17 : 3) 0.065 g of 2.
2
.3. Characterisation and spectral analysis of compounds 1–5
ꢁ
Compound
1:
White
needles,
m.p.
224 C,
Rf
0.65
solvent
system
(
C H O ; IR (KBr on a Shimadzu IR-435) bands: 1715, 1602 cm
EtOAc : MeOH : H O : : 100 : 17 : 13), [ꢁ]D ꢂ24 (MeOH ca. 0.121), analysed for
2
ꢂ1
1
;
H-NMR
2
3
24 14
(90 MHz, CDCl , TMS int. std.) ꢂ 3.25 to 4.85 (10H, methylene and methine protons
3
geminal to hydroxyl, C-1 methine proton and C-10 methylene protons of the iridoid
0
0
0
0
0
0
nucleus, i.e. CH-1, CH-1 , CH-2 , CH-3 , CH-4 , CH-5 , CH -6 , CH -10), 6.34 (1H, s, H-
2
2
2
0
0
00
00 13
), 6.45 (1H, s, H-7), 6.62 (1H, s, H-3), 6.86 (1H, d, H-5 ), 7.37 (1H, d, H-6 ); C-NMR –
þ
see Table 1; MS: m/z, 563 (M þ 39) on a FAB system confirming the molecular formula
þ
1
with M at m/z 524. The H-NMR data for the heptaacetate of 1 is explained in the
Results and discussion section.
ꢁ
Compound 2: Colourless crystals, m.p. 253ꢂ254 C, Rf 0.63 solvent system
(
CHCl : MeOH : H O : : 65 : 35 : 13), analysed for C H O ; UV
2
(MeOH) 254
max
3
21 20 11
and 348 nm, shifted to 274,389 and 393 nm after addition of AlCl þ HCl solution; IR
3
(
KBr on a Shimadzu IR-435) bands: 3435 (highly H-bonded hydroxyl bands spread
1
out beneath CH-stretching), 1686 (C¼O) and 1605 (Ar); H-NMR (90 MHz DMSO-d ,
6
TMS internal standard) did not provide much information and hence the compound was
1
subjected to acetylation. H-NMR of the heptaacetate in CDCl has been explained in the
Results and discussion section.
3

Natural Product Research
1203
ꢁ
Compound 3: Amorphous powder, m.p. 179 C (with decomposition), Rf 0.48
(
(
CHCl : MeOH : : 85 : 15), [ꢁ]D ꢂ83ꢁ (MeOH ca. 0.975) analysed for C H O ; IR
3
23 36 12
ꢂ1
1
KBr on a Shimadzu 1R-435) bands: 1740, 1710 cm ; H-NMR (90 MHz, CD OD,
3
0
TMS int. std.): ꢂ 3.41 (3H, s, OMe); 4.50 (1H, d, H-1 , 8 Hz), 4.56 (1H, d, 1-H, 8 Hz),
00 00
4
7
1
.95 (1H, d, 3-H, 6 Hz), 5.76 (1H, s, 7-H) 6.85 (2H, dd, 3 -H and 5 -H, 8 Hz),
þ
0
0
00
13
.85 (2H, dd, 2 -H and 6 -H, 8 Hz); C-NMR – see Table 2; MS: m/z 504 (M ),
38, 120, 94, 64.
1
3
6
Table 1. C NMR (DMSO-d ) of compound 1 and its acetate (1).
Carbon atom no.
Compound 1
Acetate of 1
Chemical shift
Chemical shift
1
3
4
5
6
7
8
9
94.01
157.16
114.10
104.19
182.71
91.90
157.59
112.11
107.03
176.31
109.45
143.12
30.08
109.47
144.72
Embedded in DMSO-d
6
1
1
0
1
1
2
3
4
5
6
1
2
3
4
5
6
21.75
164.63
98.25
79.65
82.16
73.81
168.32
95.82
76.88
74.81
72.75
0
0
0
0
0
0
0
0
0
0
0
0
73.84
71.62
70.02
68.78
0
0
0
0
0
0
119.92
122.12
146.52
156.54
116.95
122.26
129.72
123.22
153.81
157.59
122.04
124.83
O
C
1
64.10
168.10
OH
O
C
O
O
21.61, 21.71, 29.01
CH3
CH3
O
C
1
68.01, 169.12, 169.22, 178.11

1
204
R.L. Sharma et al.
1
3
3
Table 2. C NMR (CD OD) data of nishindaside (3).
Carbon
atom no.
Chemical
shift
Carbon
atom no.
Chemical
shift
0
1
3
4
5
6
7
8
9
98.5
100.0
30.1
44.5
80.6
131.3
149.3
142.3
63.0
1
2
3
4
5
6
100.0
74.9
77.8
71.2
77.9
0
0
0
0
0
62.5
0
0
1
121.7
132.6
116.1
163.3
56.1
0
0
00
00
2 , 6
3 , 5
00
1
0
0
0
4
OCH3
CO
167.6
1
3
Table 3. C NMR (CD
3
OD) data of negundoside (4).
Carbon
atom no.
Chemical
shift
Carbon
atom no.
Chemical
shift
0
1
3
4
5
6
7
8
9
94.8
151.0
113.3
30.7
30.0
41.1
79.5
52.1
24.2
169.7
1
2
3
4
5
6
1
97.5
75.5
78.0
71.6
78.1
62.5
122.0
132.6
116.0
163.3
167.0
0
0
0
0
0
00
00
00
00
2 , 6
00
3 , 5
1
1
0
1
00
4
COOH
–
ꢁ
Compound 4: Colourless crystals, m.p. 162–164 C, R 0.32 (CHCl : MeOH : : 85 : 15),
f
3
2
5
[
ꢁ] ꢂ130 (MeOH, ca. 0.120), analysed for C H O IR (KBr on a Shimadzu 1R-435)
D
23 28 11;
ꢂ1 13
bands: 3400, 1710, 1690, 1645, 1600, 1599, 1455 cm
4
;
C-NMR – see Table 3; MS: m/z
þ
96 (M ).
ꢁ
Compound 5: Colourless crystals, m.p. 147–151 C, R 0.68 (CHCl : MeOH : : 85 : 15),
f
3
analysed for C H O ; 1R (KBr on a Shimadzu 1R-435) bands: 3400, 1707, 1645, 1590,
26 11
2
2
ꢂ1 13 þ
1
455 cm
;
C-NMR – see Table 4; MS: m/z 466.44 (M ).
2
.3.1. Acid hydrolysis of 1
Acid hydrolysis was carried out with 45 mg of 1 with 7% H SO in MeOH (30 mL) by
2
4
refluxing for 12 h. A total of 20 mL of methanol was removed from the reaction mixture
by distillation, 20 mL of H O added, the reaction mixture extracted with 75 mL of CHCl ,
dried on anhydrous Na SO and the chloroform distilled off to yield the aglycon.
2
3
2
4

Natural Product Research
1205
13
3
Table 4. C NMR (CD OD) data of agnuside (5).
Carbon
atom no.
Chemical
shift
Carbon
atom no.
Chemical
shift
Carbon
atom no.
Chemical
shift
0
00
1
3
4
5
6
7
8
9
97.8
141.5
105.5
46.0
1
2
3
100.1
74.5
78.0
71.2
77.8
62.5
1
121.8
132.6
116.2
163.2
167.8
0
00
00
00
2 , 6
3 , 5
0
00
0
00
4
4
CO
0
82.5
5
0
132.2
142.6
48.5
6
1
0
63.5
The major moiety was identified as glucose on paper chromatogram by comparison with
standard sugars in solvent system (BuOH : AcOH : H O : : 4 : 1 : 5) and sprayed with
ninhydrin after drying.
2
2
.3.2. Alkaline hydrolysis of 1
A total of 50 mg of 1 was refluxed with 5% alcoholic KOH (10 mL) for 3 h, half of the
alcohol distilled off, 10 mL of H O added, the remaining quantity of alcohol again distilled
2
off, the aqueous solution acidified to pH2 and extracted with the benzene. After removal
of the solvent, the residue passed through silica gel and was eluted with ethyl acetate and
ꢁ
alcohol (1 : 1) to yield brownish powder, m.p. 199–200 C.
2
.3.3. Acetylation of 1
A total of 20 mg of 1 was dissolved in dry pyridine (2 mL); acetic anhydride (5 mL) was
added and the reaction mixture kept under anhydrous conditions at room temperature.
Progress of the reaction was monitored on TLC and further processed in the usual
manner. The picture became clear when 1 formed a heptaacetate, when all the sugar
protons were clearly visible at the appropriate position alongwith OCOCH protons at
3
ꢂ.182 (1 ꢃ CH ), 2.01 (3 ꢃ CH ), 2.32 (2 ꢃ CH ) and 2.42 (1 ꢃ CH ).
3
3
3
3
2
.3.4. Acid hydrolysis of 2
A total of 30 mg of 2 was refluxed with 7% H SO in MeOH (25 mL) for 14 h and
2
4
ꢁ
worked out, as in the case of the hydrolysis of 1. The aglycon obtained has m.p. ꢀ300 C
þ
(
dec) and was analysed as C H O ; MS: m/z 286 (M ). It was identified as luteolin,
1
5
10
6
ꢁ
m.p. 328–330 C (dec). The sugar was identified as glucose by comparison with the
standard sugar.
2
.3.5. Acetylation of 2
A total of 30 mg of 2 was dissolved in 3 mL of dry pyridine; 5 mL of Ac O was added and
2
refluxed for 1 h. After completion of the reaction, the product was worked up as usual to
ꢁ
give heptaacetate, m.p. 224–226 C, and analysed for C H O .
3
5
34 18

1
206
R.L. Sharma et al.
OH
O
COOH
OMe
O
HO
O
HO
O
O
HO
O
O
OH
HO
O
HO
O
O
(3)
O
OH
HO
COOH
CH OH
HO
1a)
OH
2
(
_
H
O
+
H
O
Me
OC
O
OH
O
O
CH OH
2
OH
O
O
HO
O
HO
O
(4)
HO
HO
HO
O
HO
O
O
H
H
HO
(1b)
OH
HO
O
O
O
OH
H
HO
O
(5)
O
COOH
O
OH
CH OH
2
O
HO
O
HO
HO
O
O
COOH
4
HO
O
O
6
5
′′
6′′
1′′
5
HO
7
8
3
OH
O
HO
11
2
4′′
O
(1c)
10
9
1
3
′′
2′′
HO
6′
O
OH
OH
HO
O
O
4
′
5′
1′
2
′
GO
O
HO
3
′
HO
OH
(Numbering of 1 according to iridoid system)
OH
O
(
2)
3
. Results and discussion
Compound 1: C H O , responds positively to Wieffering field test, giving blue
3
2
24 14
colouration, which indicates it as an iridoid. On acid hydrolysis, it gave a sugar and
an aglycon. The sugar moiety was identified as glucose on paper chromatogram
by comparison with standard sugars. Alcoholic KOH hydrolysis and repeated
chromatography of the acidified product yielded an acid which was identified as

Natural Product Research
1207
ꢁ
1
protocatechuic acid, m.p. 200 C. H-NMR of the acid showed two doublets (H-5) at ꢂ 6.88
1
and (H-6) at 7.37 and a singlet (H-2) at 6.40. H-NMR of 1 showed glucose methylene and
methine protons geminal to different hydroxyl groups between ꢂ 3.25 and 4.85. The
singlets at ꢂ 6.45 and 6.62 could be assigned to C-7 and C-3 olefinic protons of the iridoid
0
0
00
nucleus. Two doublets, one for each proton at ꢂ 6.86 and 7.37 were assigned to 5 and 6
0
0
protons of the aromatic nucleus alongwith one proton singlet at ꢂ 6.34 due to 2 proton.
The picture became clear when compound 1 formed a heptaacetate where all the sugar
protons were clearly visible at the appropriate positions, along with OCOCH protons at
3
ꢂ 1.82 (1 ꢃ CH ), 2.01 (3 ꢃ CH ), 2.32 (2 ꢃ CH ) and 2.42 (1 ꢃ CH ). Two downfield
3
3
3
3
signals of COCH at ꢂ 2.32 and 2.42 are due to two phenolic acetates and one due to an
3
enolic acetate of C-6 position of the iridoid nucleus, respectively. The unusual upfield
acetate proton at ꢂ 1.82 indicates the close proximity of shielding the aromatic ring as
0
0
00
benzoate at C-10 methylene. The two doublets due to 5 and 6 protons moved downfield
0
0
to ꢂ 7.41 and 7.73, as expected, and singlets due to C-7, C-3 and C-2 protons appeared at
ꢂ 6.61, 7.37 and 7.69, respectively. The methylene and methine protons attached to oxygen
1
3
function appeared between ꢂ 3.6 and 5.75 as expected. C NMR showed a signal at
ꢂ 182.7, which was assigned to C-6 carbon atom. This position is unique in having a partial
allylic carbonyl character and partially enolic ¼C(OH) – carbon function. The structure
with more enolic character due to strong hydrogen bonding with –COOH is borne by the
fact that C-9 has moved upfield in acetate to ꢂ 30.08 and C-10 metheylene carbon to 21.7
due to the allylic position. The carboxylic and ester carbonyl appeared as expected at ꢂ
1
structures 1 , 1 and 1 can be assigned to compound 1. Since the molecules have a partial
64.1 and 164.6 in compound 1. Keeping in view the above data and discussions,
a
b
c
keto carbonyl (182.7), the structure 1 is ruled out because its keto forms cannot show the
c
presence of one olefinic proton at ꢂ 6.45. Structure 1 is tautomeric to structure 1 , and
b
a
both appear to correctly represent the structure of compound 1. This structure is being
represented for the first time from a natural source and is new to the literature.
ꢁ
Compound 2: C H O m.p. 253–254 C responded to FeCl and Shinoda test for
20 11
21
3
ꢁ
flavanoids. On acid hydrolysis it gave an aglycon analysed for C H O , m.p. 279–278 C
10
1
5
6
and glucose. Compound 2 showed UVmax (CH OH) at 348 and 254 and shifted to 274, 389
3
and 393 nm after the addition of AlCl þ HCl solution, while sodium acetate solution
3
addition showed no significant shift, indicating that the 7-oxygen function is either absent
or bound with the carbon function, while 5-OH is present with a strong hydrogen bond with
1
C¼O function. H-NMR of 2 in DMSO-d did not provide much information, so the
6
1
compound was subjected to acetylation. The H-NMR of the heptaacetate in CDCl
3
showed four singlets (one for each proton) at ꢂ 7.37 (slightly meta coupled), 6.65, 6.59 (meta
0
coupled) and 6.46 (meta coupled) due to C-2 , C-3, C-6 and C-8 protons, respectively. Sugar
protons, as usual, centred at ꢂ 3.94, 4.23, 5.18 and 5.32. Acetate protons (each 3H) were
centred at ꢂ 2.43, 2.35 and 2.33 due to three phenolic OCOCH protons and ꢂ 2.07 due to
3
four OCOCH of the tetracetate sugar moiety. Anomeric protons of a sugar moiety at ꢂ 5.31
3
1
in H-NMR and ꢂ 98.2 in C NMR showed the glycosidic linkage to be ꢀ. The assignments
13
ꢁ
are in agreement with luteolin-7-O-glucoside, m.p. 254 C. This is the first report of this
compound from this plant.
ꢁ
Compound 3: C H O , m.p. 179 C gave a positive Wieffering field test, indicating
2
3
36 12
ꢂ1
it to be an iridoid. The absence of characteristic bands at 1645 and 1650 cm ruled out the
possibility of the O–C (3)¼C (4) moiety which is usually present in the iridoid. In
1
H-NMR, a singlet at ꢂ 3.41 integrating for three protons was assigned to a methoxy group

1
208
R.L. Sharma et al.
attached to the iridoid nucleus. Two doublets at ꢂ 4.95 (J ¼ 6 Hz) and 4.56 (J ¼ 8 Hz)
integrating for one proton each were assigned to acetate protons. The chemical shift and
coupling constant of a doublet at ꢂ 4.50 indicated it to be a ꢀ-glycoside. A broad singlet at
ꢂ 5.76 integrating for one proton indicated the presence of a trisubstituted double bond in
the cyclopentane ring. Two symmetrical double doublets at ꢂ 6.85 and 7.85 (J ¼ 8 Hz)
integrating for two protons each were assigned to aromatic protons. This indicated the
1
3
presence of a monohydroxy benzoyl group in the molecule. In C NMR spectrum, a total
of 21 signals were recorded in proton and noise decoupled mode. In single frequency off
resonance decoupled spectrum, one of the signals appeared as one quartet, 3 signals as
1
13
triplets, 13 as doublets and 4 as singlets. The IR, H-NMR, C NMR and mass spectral
data were observed to be in total conformity with nishindaside. So, compound 3 was
assigned the structure of nishindaside.
Compounds 4 and 5 were also found to be iridoids and their spectral data were in
conformity with negundoside and agnuside, respectively. Hence compound 4 was assigned
the structure of negundoside and 5 of agnuside.
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