Two new P-coumaroyl flavonoid glycosides having Cytokine increasing activity from Centaurium spicatum L

Article abstract of DOI:10.1016/j.phytol.2016.07.018

Two new P-coumaroyl flavonol glycosides, Kaempferol 3-O-[β-D-glucopyranosyl- (1?→?4)-α-L-rhamnopyranosyl-(1?→?4)-α-L-rhamnopyranosyl-(1?→?6)-(4 trans- P-coumaroyl)]-β-D- galactopyranoside (1) and Quercetin 3-O-[β-D-glucopyranosyl-(1?→?4)-α-L-rhamnopyranos

Full text of DOI:10.1016/j.phytol.2016.07.018

Phytochemistry Letters 17 (2016) 144–151
Contents lists available at ScienceDirect
Phytochemistry Letters
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Short communication
Two new P-coumaroyl flavonoid glycosides having Cytokine increasing
activity from Centaurium spicatum L
b
b
b
Ahmed E. Allama,b, , Alaa M. Nafady , Ahmed M.M. Hassanein , Mahmoud A.H. Mostafa ,
*
Mohamed A. El-Shanawanyc, Fumide Takanoa, Tomihisa Ohtaa
Pharmacognosy and Chemistry of Natural Products, School of Pharmaceutical Sciences, Kanazawa University, Kanazawa 920-1192, Japan, Japan
Department of Pharmacognosy, Faculty of Pharmacy, Al-Azhar University, Assiut 71524, Egypt, Egypt
Department of Pharmacognosy, Faculty of Pharmacy, Assiut University, Assiut 71526, Egypt, Egypt
a
b
c
A R T I C L E I N F O
A B S T R A C T
Article history:
Two new P-coumaroyl flavonol glycosides, Kaempferol 3-O-[
b
-D-glucopyranosyl- (1 !4)-
-D- galactopyranoside (1)
-rhamnopyranosyl-(1 !4)- -rhamnopyranosyl-
-D-galactopyranoside (2) were isolated from the aerial part of
a-L-rhamno-
Received 9 May 2016
Received in revised form 30 June 2016
Accepted 6 July 2016
Available online xxx
pyranosyl-(1 !4)-
and Quercetin 3-O-[
(1 !6)-(4 trans- P-coumaroyl)]-
a
-
L
-rhamnopyranosyl-(1 !6)-(4 trans- P-coumaroyl)]-
b
b
-D-glucopyranosyl-(1 !4)-
a
-L
a-L
b
Centaurium spicatum (L.) Fritsch (Gentianaceae). Structure elucidation of the new compounds was
established on the basis of 1D and 2D NMR analyses including 1H, 13C NMR, DEPT-135 NMR, HꢀꢀH COSY,
HMQC and HMBC experiments. In addition to HRFAB Mass Spectrometry. Also it is noted that these new
Keywords:
Centaurium spicatum
Gentianaceae
Flavonoid glycosides
P-coumaroyl
Cytokine increase activity
compounds significantly increase both TNF-a and IL-1b after 48 h of treatment (this level of increase of
cytokines (300/200) activates the cytokines like vaccines towards invasion of microorganisms).
ã 2016 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.
1. Introduction
assignments of the new flavonol glycosides in addition to their
effect on the cytokines activity are discussed in this paper.
Centaurium spicatum (L.) Fritsch (Gentianaceae) is an erect small
branched annual herb, widespread in Southern Europe and North
Africa. It is used together with other Centaurium species such as C.
pulchellum in traditional medicine for the treatment of abdominal
pain, hypertension, gallstones, kidney and ureter stones, renal
colic, wounds and diabetes (EL-Shanawany et al.,1989). It is known
in the arabic name as ‘Kantaryoun’. A survey of the current
literature revealed the isolation and identification of secoiridoids
(sweroside, swertiamarin, gentiopicrin and ester of swertiamarin
and secoxyloganic acid) and polyoxygenated xanthones from the
plant (EL-Shanawany et al., 1989, 2011; Bishay et al., 1979; Van der
Sluis and Labadie, 1981; Ahmed et al., 2011, 2012, 2015).
Alkaloids of the pyridine type (e.g. gentianine), spicatine and
the series of amides derived from the secoiridoid glucoside
swertiamarin, and kantaurin were also shown to be present
(Bishay et al., 1979). In the present work we reported the isolation
and structure elucidation of two new P-coumaroyl flavonol
glycosides 1 and 2. The structure elucidation and the spectral
2. Results and discussion
2.1. Identification of compound 1
Compound 1 was obtained as yellow amorphous powder
(40 mg), soluble in methanol with [
a]
22.3 – 18.3ꢁ (c = 0.6, MeOH).
D
Structure of compound 1was elucidated by 1D and 2D NMR
spectroscopy including 1H, 13C NMR, DEPT-135, HꢀꢀH COSY, HMQC
and HMBC experiments as well as HRFAB Mass Spectrometr. 1H
NMR spectrum of compound 1 indicated the presence of a
kaempferol moiety and four sugar moieties (two hexose and two
pentose moieties) in addition to the presence of p-coumaroyl
group attached to the terminal hexose moiety which were proved
from the presence of pair of doublets at dH 6.40 (H-6) and dH 6.70
(H-8) and two-spin system with the typical coupling pattern of 1,4-
disubstituted benzene ring [a pair of doublets each is equivalent to
two protons at dH 7.44 (H-20, H-60) and at dH 6.90 (H-30, H-50)]
which are two features characteristic of a flavonol with phenolic
groups at positions 5, 7 and 40. 1H NMR spectrum showed also
signals characteristic to p-coumaroyl moiety, where there is a
typical coupling pattern of 1,4-disubstituted benzene ring [a pair of
doublets each is equivalent to two protons at dH 8.10 (H-2,6
* Corresponding author at: Department of Pharmacognosy, Faculty of Pharmacy,
Al-Azhar University, Assiut 71524, Egypt.
E-mail address: aallam81@yahoo.co.uk (A.E. Allam).
http://dx.doi.org/10.1016/j.phytol.2016.07.018
1874-3900/ã 2016 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.

A.E. Allam et al. / Phytochemistry Letters 17 (2016) 144–151
145
p-coumaroyl) and at dH 6.80 (H-3,5 p-coumaroyl)] in addition to
the trans olefinic protons at dH 7.50 and 6.31, d, J = 16.0 (H-7,8) of
the p-coumaroyl moiety respectively (Table 1).
proton. In galactopyranose, which is the C-4 epimer of the
glucopyranose, all protons except H-4 are axial. Since H-4 in
galactose is equatorial, it has one Hax–Heq coupling with H-3. On
13C NMR and DEPT spectra were in agreement with 3-
substituted kaempferol moiety. Substitution of kaempferol in
position C-3 was evident from the chemical shift of C-2 (dC 159.6),
whereas in flavonols with an unsubstituted hydroxyl functionality
the other hand, H-2 has two
compound1 only H-3 of the galactoside residue could show
ax–Heq (with H-4) and Hax–Hax (with H-2) coupling and the
Hax–Hax couplings. Thus, in
H
doublet of doublets at dH 3.36 could be assigned to this proton
corresponding to13C NMR signal at dc 71.0 (C-3 of galactose) in
HMQC experiment and hence, the first hexose could be obviously
determined as galactose (Lambert et al., 1989). The anomeric
protons showed characteristic doublets in 1H NMR spectrum at dH
5.60 for galactose with a coupling constant of 7.5 Hz indicating the
at this position C-2 is expected around dc 147 (Markham et al.,
1978). The typical doublet of the C-6 secondary methyl group of
both rhamnose (a) and rhamnose (b) were found at dH 1.03, 0.99
(1H, d, J = 6.1 Hz) and dC 17.9, 17.6 in the 1H and 13C NMR spectra,
respectively.
1H NMR spectrum showed a signal at dH 3.36 which is a doublet
of doublets with coupling constants of 9.9 and 3.4 Hz, which was
obviously corresponded to an axially and equatorially coupled
presence of
rhamnose (a) and rhamnose (b) respectively with a broad singlet
splitting indicating their -configuration. (Markham et al., 1982).
b-configuration and at dH 4.41 and 5.19 for both
a
From HMBC spectrum, the proton at dH 5.33 had long-range
correlations with both C-3 of galactose (dH 73.8) and C-5 of
galactose (dH 77.7) which were also evidenced from HꢀꢀH COSY
this proton also correlated with the carbonyl carbon of the
coumaroyl group. This evidence showed that the coumaroyl group
connected with the galactose moiety at C-4. The downfield shift of
galactose C-4 (dC 71.9) of 0.3 ppm compared with the same carbon
in literature (Lambert et al., 1989) supported this conclusion.
A long-range correlation in HMBC was also observed between
the anomeric proton of glucose (dH 5.05) and C-4 of rhamnose (b)
Table 1
13C- and 1H NMR assignments for compounds 1 and 2 recorded in CD3OD.
Position Compound 1
13C NMR
Position Compound 2
13C NMR 1H NMR
1H NMR
, mult., J (Hz)]
(
d, mult.)
[
d
(
d
, mult.) , mult., J (Hz)]
[d
2
3
4
5
6
7
8
9
10
1”
2”0,60
4”0
3”0,50
159.6,s
134.3,s
179.1,s
162.5,s
100.6,d
164.2,s
95.8,d
2
3
4
5
6
7
8
159.6,s
134.5,s
179.3,s
162.6,s
100.7,d
164.3,s
95.7,d
–
–
–
–
–
(dC 74.0) suggesting that the glucose was connected at this site. The
6.40,d, 2.0
–
6.44,d, 2.0
–
fact that the signal of rhamnose (b) C-4 shifted downfield for
4.0 ppm compared with C-4 of rhamnose in literature (Markham,
1989) supported this conclusion. All these evidences demonstrated
that the glucose was attached to the rhamnose (b) at C-4.
In addition, optical rotation values of both D-galactopyranose
6.70,d, 2.0
6.72,d, 2.0
157.7,s
107.5,s
122.9,s
131.3, d
161.1,s
116.0,d
9
157.9,s
107.5,s
123.3,s
117.5
146.0,s
149.8,s
116.1,d
123.5,d
–
–
10
1”
2”0,60
4”0
3”0,50
–
–
–
7.44, 2H,d, 8.2
–
6.74,d, 2.0
–
and L-rhamnopyranose tetrabenzoate derivatives of the acid
31.5
6.90, 2H,d, 8.2
–
hydrolysis products of compound1 were [
CHCl3) for
(c 0.002, CHCl3) for
a]
+52.5 (c 0.019,
D
6.92,d,8.5
7.72,d, 8.5
31.8
b-D
-galactopyranose tetrabenzoate and [
a
]
+75.5
D
a-L
-rhamnopyranose tetrabezoate, which were
Galac.
1”
2”
3”
4”
Galac.
identical with the optical rotation values of the synthetic models of
both (Hasegawa et al., 2008a; Kasai et al., 2005). A long-range
correlation, observed in a HMBC experiment, between C-3 of
kaempferol (dC 134.3) and the anomeric proton of galactose (dH
5.60) confirmed that this was the site of glycosylation and that
galactose was the first sugar. Other long range correlations
observed in HMBC experiment between C-6 of the galactose (dC
66.6) and the anomeric proton of the rhamnose (a) (dH 5.19) from
one side, between C-4 of the rhamnose (a) (dC 71.0) and the
anomeric proton of rhamnose (b) (dH 4.41) from another side,
between C-4 of the rhamnose (b) (dC 74.0) and the anomeric proton
of glucose (dH 5.05) from the other side confirmed that the
attachment between rhamnose (a) and galactose is (1 !6),
between the rhamnose (b) and that of rhamnose (a) is (1 !4)
and between the terminal glucose and rhamnose (b) is (1 !4)
linkage.
100.4,d
71.02,da
73.8,db
71.9,da
77.7,d
5.60,d,7.5
3.36,dd, 9.9,7.5 2”
3.94,dd, 9.9,3.4 3”
5.33,d,3.4
3.94,m
1”
100.5,d
71.02,da
73.8,db
71.9,da
77.7,d
5.60,d,7.5
3.36,dd, 9.9,7.5
3.94,dd, 9.9,3.4
5.33,d,3.4
4”
5”
5”
6”
3.94,m
66.6,t
3.43,m, 3.20,m
6”
66.6,t
3.43,m, 3.20,m
Rham. (a)
Rham. (a)
1000
102.5,d 5.19,brs.
1000
102.1,d
71.9,da
73.7,db
71.0,da
69.8,d
17.9,q
5.19,brs.
3.71,m
3.74,m
3.35,m
3.30,m
2000
71.9,da 3.71,m
73.7,db 3.74,m
71.0,da 3.35,m
2000
3000
3000
4000
4000
5000
69.8,d
17.7,q
3.30,m
0.96,d, 6.1
5000
6000
6000
1.05,d, 6.1
Rham. (b)
1””
2””
3””
4””
5””
6””
Glu.
100000
200000
Rham. (b)
1””
3.88,dd, 3.4, 1.7 2””
102.7,d 4.41,brs.
102.8,d
72.1,da
73.7,db
74.0,d
69.8,d
17.5,q
4.41,brs.
3.88,dd, 3.4, 1.7
3.74,m
3.19,dd, 9.9, 9.6
4.12,dd,9.9,6.1
1.01,d, 6.1
72.1,da
73.7,db 3.74,m
3””
74.0,d
69.8,d
17.5,q
3.19,dd, 9.9, 9.6 4””
4.12,dd,9.9,6.1
0.85,d, 6.1
5””
6””
The molecular weight of compound 1 is 1048.91 which was
evident from an [M + H] + peak at m/z 1049 in the positive ion FAB
Mass spectrum and also from [HR-FAB] + at 1049 was in agreement
with the assigned structure. Hence, 1 could unequivocally be
Glu.
101.9,d 5.05,d,6.8
71.02,
100000
101.4,d
5.05,d,6.8
3.36,dd, 9.9,7.5
3.36,dd, 9.9,7.5 200000
71.02,da
da
300000
400000
500000
600000
74.5,d
71.1,da
78.1,d
62.3,t
3.49,dd, 9.9,3.4 300000
3.39,d,3.4
3.50,m
74.5,d
71.1,da
78.1,d
62.3,t
3.49,dd, 9.9,3.4
3.39,d,3.4
3.50,m
identified as; Kaempferol 3-O-[
rhamnopyranosyl-(1 !4)- -rhamnopyranosyl-(1 !6)-(4trans-
P-coumaroyl)]- –D-galactopyranoside and is a new compound.
b
ꢀD-glucopyranosyl-(1 !4)-
a-L-
400000
500000
600000
a-L
b
3.90,m, 3.68,m
3.90,m, 3.68,m
P-coumaroyl
P-coumaroyl
127.0,s
1
127.0,s
–
–
2.2. Identification of compound 2
2,6
4
3.5
7
132.3,d 8.10,2H,d,8.9
131.5,d
161.2,s
116.8,d
147.4,d
115.9,d
168.8,s
8.12,2H,d,8.5
–
161.4,s
–
Compound 2 was obtained as yellow amorphous powder
(34 mg), soluble in methanol with [
21.8 – 13.3ꢁ (c = 0.6, MeOH).
The structure of compound 2 was elucidated by 1D and 2D NMR
spectroscopy including 1H, 13C NMR, DEPT-135, HꢀꢀH COSY, HMQC
and HMBC experiments, as well as HRFAB Mass spectrometry.1H
116.8,d 6.80,2H,d,8.2
147.2,d 7.50,d,16.0
115.9,d 6.31,d,16.0
6.84,2H,d,8.5
7.62,d,15.8
6.41,d,15.8
–
a]
D
8
C
¼
O
168.6,s
–
a,bAssignments bearing the same superscript may be reversed.

146
A.E. Allam et al. / Phytochemistry Letters 17 (2016) 144–151
and 13C NMR spectra of compound 2 indicated the presence of a
quercetin moiety, four sugar moieties (two hexose and two
pentose moieties) in addition to the presence of p-coumaroyl
group attached to one of the two hexose moieties. 1H NMR
spectrum showed a pair of doublets at dH 6.44 (H-6) and at dH 6.72
(H-8) and a three-spin system with the typical coupling pattern of
10,30,40-trisubstituted benzene ring [dH 6.74 (H-20), 6.92 (H-50)
and 7.72 (H-60)] which are two features characteristic of a flavonol
with phenolic hydroxyl groups at positions 5, 7, 30 and 40. 13C NMR
and DEPT spectra were in agreement with 3-substituted quercetin
moiety. 1H NMR spectrum showed also signals characteristic to
p-coumaroyl moiety, where there is a typical coupling pattern of a
1,4-disubstituted benzene ring [a pair of doublets each is
equivalent to two protons at dH 8.12 (H-2,6 coumaroyl)] and at
dH 6.84 (H-3,5 p-coumaroyl)], in addition to the trans olefinic
protons at dH 7.62 and 6.41, d, J = 15.8 (H-7,8) of the p-coumaroyl
moiety respectively (Table 1). 13C NMR and DEPT spectra were in
agreement with 3-substituted quercetin moiety. The typical
doublet of the C-6 secondary methyl group of both rhamnose
(a) and rhamnose (b) were found at dH 1.05, 1.01 (1H, d, J = 6.1 Hz)
and dC 17.9, 17.5 in the 1H and 13C NMR spectra respectively.
R
OH
HO
O
O
O
OH
OH
O
OH
O
OH
O
H3C
O
OH
O
OH
O
HO
O
HO
OH
O
O
HO
OH
OH
HO
R
H
Compound 1
Compound 2
OH
Fig. 1. Structure of the new isolated compounds.

A.E. Allam et al. / Phytochemistry Letters 17 (2016) 144–151
147
1H NMR spectrum showed a signal at dH 3.36 which is a doublet
of doublets with coupling constants of 9.9 and 3.4 Hz which was
obviously corresponded to an axially and equatorially coupled
proton atom. The anomeric protons showed characteristic
doublets in 1H NMR spectrum at dH 5.60 for galactose, with a
the reaction mixture, stirred for 30 min, and then diluted with
EtOAc and aqueous Na2CO3, and the layers were separated. Each
organic layer was washed with brine and the combined aqueous
layers for each were extracted with EtOAc. Each combined organic
extracts were dried over MgSO4, and concentrated, was the
corresponding residual dark brown oil were individually purified
by silica gel cc (eluting with hexane/EtOAc 5:1) to give either D–
coupling constant of 7.5 Hz, indicating
b-configuration and at dH
4.41 and 5.19 for both rhamnose (a) and rhamnose (b) respectively
with a broad singlet splitting indicating their
(Markham, 1989).
a-configuration
galactopyranose tetrabenzoate (40 mg) [
a
]
31 + 53.5 (c = 1.2,
D
CHCl3) or L-rhamnopyranose tetrabenzoate (22 mg):
[a]
D
A
long-range correlation, observed in HMBC experiment,
29.6 + 75.0 (c = 1.6, CHCl3) as a colorless oil, respectively (Hasegawa
between C-3 of quarcitin (dC 134.5) and the anomeric proton of
galactose (dH 5.60) confirmed that this was the site of glycosylation
and that galactose was the first sugar. Other long range correlations
observed in HMBC experiment between C-6 of the galactose (dC
66.6) and the anomeric proton of the rhamnose-a (dH 5.19) from
one side, between C-4 of the rhamnose-a (dC 71.0) and the
anomeric proton of the rhamnose (b) (dH 4.41) from another side,
between C-4 of the rhamnose (b) (dC 74.0) and the anomeric proton
of glucose (dH 5.05) from another side, confirmed that the
attachment between the rhamnose (a) and galactose is (1 !6),
between the rhamnose (b) and rhamnose (a) is (1 !4) and that
between the terminal glucose and rhamnose (b) is (1 !4) (Fig. 1).
From HMBC spectrum, the proton at dH 5.33 had long-range
correlations with both C-3 of galactose (dH 73.8) and C-5 of
galactose (dH 77.7) which were also evidenced from HꢀꢀH COSY.
This proton also correlated with the carbonyl carbon of the P-
coumaroyl moiety. This evidence showed that the P-coumaroyl
moiety is connected to galactose at C-4. The downfield shift of
galactose C-4 (dH 71.9) by 0.3 ppm compared with the same carbon
in literature (Lambert et al., 1989) supported this conclusion.
The molecular weight of compound 2 is 1064.30 which was
evident from an [M+H] + peak at m/z 1065 in the positive ion FAB
Mass spectrum and also from [HR-FAB]+ at 1065 was in agreement
with the assigned structure and hence, compound 2 could
et al., 2008a,b; Kasai et al., 2005).
3.2. Plant material
Aerial parts of C. spicatum (L.) Fritsch (Gentianaceae) were
collected from New Valley, 200 km southwest of Assiut City, Egypt.
The plant was identified and authenticated by Prof. A. Fayed,
Professor of Plant Taxonomy, Faculty of Science, Assiut University.
A voucher specimen (CRs-3b = cpd. 1) was deposited at the
Department of Chemistry of Natural Products, School of Pharma-
ceutical Sciences, Kanazawa University, Kanazawa, Japan.
3.3. Extraction and isolation
Air-dried C. spicatum aerial parts (4 kg) was extracted thrice
with MeOH (5 L of each) at room temperature. The solvents were
combined and filtered through filter paper (Advantec MFS
Incorporated). The solvent was removed under reduced pressure
at 40 ꢁC to yield the methanol extract (550 g), which was
suspended in distilled water and partitioned between chloroform,
ethyl acetate and n-butanol (1 L each) to give the chloroform
fraction (163 g), ethyl acetate fraction (80 g), n-butanol fraction
(100 g) and the rest aqueous fraction (200 g). The ethyl acetate
fraction was in turn partitioned between (methanol-water 90%)
and n-hexane to give 90% methanol fraction (50 g) and n-hexane
fraction (20 g).
unequivocally be identified as Quarcitin 3-O-[
-rhamnopyranosyl-
-D- galactopyranoside which is a
b-D-glucopyrano-
syl-(1 !4)-
a
-
L
-rhamnopyranosyl-(1 !4)-
a-L
(1 !6)-(4 trans- P-coumaroyl)]-
b
The n-butanol fraction (100 g) was sub- fractionated on Diaion
HP-20 column using water (2 L) and methanol (25%, 50%, 75% and
100%) (2 L each). The fraction eluted by 75% methanol from the n-
butanol fraction (20.5 g) was further separated by chromatography
on ODS column (80 ꢂ 200 mm) (Cosmosil 140C18 PREP, Nacalai
Tesque, Tokyo, Japan) using six mobile phase systems of
CH3CNꢀꢀH2O (10, 25, 40, 50, 70 and 90% v/v; elution volume:
1.5 L of each) to give six corresponding fractions. The fraction
eluted with 40% CH3CN (1.9 g) was further chromatographed by
column chromatography on silica gel and eluted stepwise gradient
with CHCl3–MeOH (ratios of 9:1, 6:1, 4:1, 3:1and 1:1, v/v elution
volume: 200 mL each) to give five corresponding fractions. The
fraction eluted with 6:1 CHCl3 ꢀ MeOH was further separated by
preparative HPLC, ODS column: C30 UG-5 ODS (20 mm ꢂ 250 mm)
new compound.
After establishing the structures of the two new flavonoids, we
investigated their ability to regulate production of inflammatory
cytokines (TNF-
activated monocytes produce large amounts of inflammatory
cytokines such as TNF- , IL-1 , IL-6 and IL-12 and these cytokines
a and IL-1b) in THP-1 cells activity. Since fully
a
b
directly or indirectly cause tissue inflammation (Watson et al.,
1999).
After 24 and 48-h stimulation with lipopolysaccharide (LPS) at
1
m
M, an increase in production of TNF-
(Fig. 2) and it was noted that both compounds 1 and 2 significantly
increase both TNF- and IL-1 after 48 h of treatment by a level of
about 300/200 in a dose dependent manner 10, 25 and 50 M.
a and IL-1b was observed
a
b
m
This level of increase of cytokines which is not so high to attain
the septic shock but only to activates the cytokines like vaccines
towards invasion of microorganisms as this level is always released
from the local inflamed area and spread the request to other organs
to contribute to fighting the invader (Yinyao et al., 2010).
particle size: 5 mm, flow rate: 6 mL/min. (Develosil, Nacalai Tesque,
Tokyo, Japan) equipped with a UV detector (210 nm). The mobile
phase was 30% CH3CN in H2O. This resulted in elution of compound
(1).
3.4. Acid hydrolysis
3. Experimental
Acid hydrolysis of the glycosides was carried out by re- fluxing
5 mg of compound in 5 mL of 6% HCl in MeOH for 3 h. The reaction
mixture was partitioned against EtOAc (10 mL). The aglycone was
obtained from the EtOAc layer and identified as quercetin by co-
chromatography on silica gel with a reference sample (Sigma-
Aldrich, St. Louis, MO, USA). The aqueous layer was evaporated and
developed crystal needles with EtOAc–H2O–MeOHꢀꢀ HOAc
(13:3:3:4). Identification of galactose and rhamnose present in
the sugar fraction was carried out by comparison with authentic
3.1. General experimental procedures
3.1.1. Measurements of the optical rotation of D-galactopyranose and
L-rhamnopyranose tetrabenzoate derivatives
Benzoyl chloride (0.5 mL) was added to each ice-cooled solution
of either D–galactopyranose (18.0 mg) or L-rhamnopyranose or
(15.0 mg) in dry pyridine (1.0 mL), and each mixture was stirred at
room temperature for 15 h. MeOH (1.0 mL) was added dropwise to

148
A.E. Allam et al. / Phytochemistry Letters 17 (2016) 144–151
Fig. 2. Effects of the two new flavonoid glycosides on production of inflammatory cytokines in cultured THP-1 cells. Cells were directly treated with various concentrations
(10, 25 and 50 M) of compounds (CRs-15 = cpd1 and CRs–16 = cpd2) co-stimulated with 1 g/mL LPS. Culture supernatants were collected at 24 h after LPS stimulation and
cytokine levels such as TNF- and IL-1 production co-
were measured using ELISA. Data are means ꢃ SD of quadruplicate cultures. Correlation coefficients for TNF-
stimulated with LPS: r2 = 0.89629 (cpd1), r2 = 0.96711 (cpd2), Correlation coefficients for IL-1 production co-stimulated with LPS: r2 = 0.99760 (cpd 1), r2 = 0.99454 (cpd 2).
m
m
a
b
a
b

A.E. Allam et al. / Phytochemistry Letters 17 (2016) 144–151
149
Fig. 2. (Continued)

150
A.E. Allam et al. / Phytochemistry Letters 17 (2016) 144–151
samples, galactose (Rf 0.42), glucose (Rf 0.48) and rhamnose (Rf
0.64) (Sigma-Aldrich), in TLC over silica gel (CHCl3–MeOHꢀꢀH2O
8:5:1) using 5% H2SO4 in MeOH as spraying reagent followed by
heating the plates at 120 ꢁC for 15–20 min.
3.8. NMR data: compound 1 (40 mg), obtained as yellow amorphous
3
powder, soluble in methanol with [
a]
ꢀ18.3ꢁ (c = 6, MeOH)
D
1H NMR (600 MHz, CD3OD): [dH 6.40, 1H, d, J = 2.0, H-6], [dH
6.70,1H, d, J = 2.0, H-8], [dH 7.44, 2H, d, J = 8.2, H-20,60], [dH 6.90, 2H,
d, J = 8.2, H-30,50]
3.5. Evaluation of cytokine production in cultured THP-1 cells
Galac. [dH 5.60,1H, d, J = 7.5, H-100], [dH 3.36,1H, dd, J = 9.9,7.5, H-
200], [dH 3.94, 1H, dd, J = 9.9, 3.4, H-300], [dH 5.33, 1H, d, J = 3.4, H-400],
To determine the effects of the tested samples on the
production of inflammatory cytokines in monocytes, THP-1 cells
(Dainippon Pharmaceutical Company) suspended in RPMI-1640
medium (containing 5% fatal bovine serum) were seeded at
1.0 ꢂ105 cells/mL into 96-well tissue culture plates, and then
incubated at 37 ꢁC in a humidified CO2 incubator (Hasegawa et al.,
2008b). After 24-h incubation, the medium was replaced with
fresh medium. Cells were further incubated for 24 h with test
sample (purity >93%, dissolved in dimethyl sulfoxide/phosphate-
[
dH 3.94, 1H, m, H-500], [dH 3.43, 1H, m, H-6a00], [dH 3.20, 1H, m, H-
6b00]
Rham. (a) [dH 5.19, 1H, brs.H-1000], [dH 3.71, 1H, m, H-2000], [dH
3.74, 1H, m, H-3000v], [dH 3.35, 1H, m, H-4000], [dH 3.30, 1H, m, H-5000],
[
dH 0.96,1H, d, J = 6.1, H-6000]
Rham. (b) [dH 4.41,1H, brs.H-10000], [dH 3.88,1H, dd, J = 3.4,1.7, H-
20000], [dH 3.74, 1H, m, H-30000], [dH 3.19, 1H, dd, J = 9.9,9.6, H-40000], [dH
4.12, 1H, dd, J = 9.9,6.1, H-50000], [dH 0.85, 1H, d, J = 6.1, H-60000
]
buffered saline [DMSO/PBS] [1:1, v/v]) co-stimulated with 1
lipopolysaccharide (LPS) to increases the production of TNF-
m
and
M
Glu. [dH 5.05, 1H, d, J = 6.8, H-100000], [dH 3.36, 1H, dd, J = 9.9,7.5, H-
200000], [dH 3.49, 1H, dd, J = 9.9,3.4, H-300000], [dH 3.39, 1H, d, J = 3.4, H-
400000], [dH 3.5, 1H, m, H-500000], [dH 3.90, 1H, m, H-6a00000], [dH 3.68, 1H,
a
IL-1b. To measure cytokine production in cultured THP-1 cells,
culture supernatants were collected at 24 h and stored at ꢀ80 ꢁC
m, H-6b00000
]
until use. The levels of tumor necrosis factor (TNF)-
a
and
P-coumaroyl [dH 8.10, 2H, d, J = 8.9, H-2,6], [dH 6.80, 2H, d,
J = 8.2, H-3,5], [dH 7.50,1H, d, J = 16.0, H-7], [dH 6.31,1H, d, J = 16.08.2,
H-8]
interleukin (IL)-1b in supernatants were measured by enzyme-
linked immunosorbent assay (ELISA) using commercial kits
(Cytoscreen; Biosource) according to manufacturer’s instructions.
For 13C NMR, (150 MHz, CD3OD); [dc 159.6, s, C-2], [dc 134.3, s,
C-3], [dc 179.1, s, C-4], [dc 162.5, s, C-5], [dc 100.6, d, C-6], [dc 164.2,
s, C-7], [dc 95.8, d, C-8], [dc 157.7, s, C-9], [dc 107.5, s, C-10], [dc 122.9,
s, C-10], [dc 131.3, d, C-20,60], [dc 116.0, d, C-30,50], [dc 161.1, s, C-40]
Galac. [dc 100.4, d, C-100], [dc 71.02, d, C-200], [dc 73.8, d, C-300], [dc
71.9, d, C-400], [dc 77.7, d, C-500], [dc 66.6, t, C-600]
3.6. Cytotoxic assay
To determine the cytotoxic activity of the tested samples, THP-1
cells (180
m
L) were seeded in 96-well plates at 1.0 ꢂ105 cells per
well with tested samples (purity > 93%) (20
m
L in DMSO/PBS) at
Rham. (a) [dc 102.5, d, C-1000], [dc 71.9, d, C-2000], [dc 73.7, d, C-3000],
various concentrations. After 48-h cultivation, supernatants were
removed, non adherent cells (THP-1) incubated with 3-(4, 5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT;
[
dc 71.0, d, C-4000], [dc 69.8, d, C-5000], [dc 17.7, q, C-6000]
Rham. (b) [dc 102.7, d, C-10000], [dc 72.1, d, C-20000], [dc 73.7, d, C-
30000], [dc 74.0, d, C-40000], [dc 69.8, d, C-50000], [dc 17.5, q, C-60000
]
10
(w/v) sodium dodecyl sulphate (SDS; in 60% [v/v] dimethyl
formamide) solution (100 L) for 18 h. The absorbance was
measured at 570 nm using a microplate reader, and the cytotoxicity
calculated by comparing absorbance with that of the non treated
control culture. Cell growth curve was graphed using statistical
analysis software (Kaleida Graph version 4.00; Synergy Software),
and IC50 values calculated using simple linear regression.
m
L, 5 mg/mL in PBS) for 4 h, and then solubilized with 10%
Glu. [dc 101.9, d, C-100000], [dc 71.02, d, C-200000], [dc 74.5, d, C-300000],
dc 71.1, d, C-400000], [dc 78.1, d, C-100000], [dc 62.3, t, C-600000
P-coumaroyl [dc 127.0, d, C-1], [dc 132.3, d, C-2,6], [dc 11.6.8, d,
C-3,5], [dc 161.4, s, C-4], [dc 147.2, d, C-7], [dc 115.9, d, C-8], [dc
168.6, s, C O]
[
]
m
¼
Compound 2 (34 mg), obtained as yellow amorphous powder,
ꢁ
21.8
soluble in methanol with [
a
]
ꢀ13.3 (c = 0.6, MeOH).
D
1H NMR (600 MHz, CD3OD): [dH 6.44, 1H, d, J = 2.0, H-6], [dH
6.72,1H, d, J = 2.0, H-8], [dH 6.72,1H, d, J = 2.06, H-20], [dH 6.92,1H, d,
J = 8.5, H-50], [7.72, 1H, d, J = 8.5, H-60]
3.7. Spectroscopic methods
Galac. [dH 5.60,1H, d, J = 7.5, H-100], [dH 3.36,1H, dd, J = 9.9,7.5, H-
200], [dH 3.94, 1H, dd, J = 9.9, 3.4, H-300], [dH 5.33, 1H, d, J = 3.4, H-400],
NMR spectra were recorded on JOEL EC-600 spectrometer
operating at 600.17 MHz for 1H NMR and at 150.92 MHz for 13C
NMR at 20.1 ꢁC in CD3OD (99.5% D) in 5 mm sample tubes. The
solvent peaks at dH 3.30 in the 1H NMR spectra and at dC 49.00 in
13C NMR spectra respectively, were used as internal references
downfield of tetramethylsilane (TMS) at 0 ppm. Spectral widths
were 9008 Hz (26 K acquisition points) and 37878 Hz (26 K
acquisition points) for 1H- and 13C NMR, respectively. Chemical
shifts are presented in ppm downfield of TMS. For CHn groups, n
was determined in DEPT-135 yielding 180ꢁ phase difference
between ꢀCH2- signals on the one hand and ꢀCH- and ꢀCH3-
signals on the other. Proton-proton chemical shift correlations
were obtained in Hꢀꢀ H HOHAHA experiment. Proton-carbon
chemical shift correlations were obtained in inversely detected
HMQC and HMBC experiments. Fast atom bombardment (FAB)
Mass spectra were carried out on MStation instrument in the
positive ion mode, using glycerol as the liquid matrix.
[
dH 3.94, 1H, m, H-500], [dH 3.43, 1H, m, H-6a00], [dH 3.20, 1H, m, H-
6b00]
Rham. (a) [dH 5.19, 1H, brs.H-1000], [dH 3.71, 1H, m, H-2000], [dH
3.74, 1H, m, H-3000], [dH 3.35, 1H, m, H-4000], [dH 3.30, 1H, m, H-5000],
[
dH 1.05, 1H, d, J = 6.1, H-6000]
Rham. (b) [dH 4.41,1H, brs.H-10000], [dH 3.88,1H, dd, J = 3.4,1.7, H-
20000], [dH 3.74, 1H, m, H-30000], [dH 3.19, 1H, dd, J = 9.9,9.6, H-40000], [dH
4.12, 1H, dd, J = 9.9,6.1, H-50000], [dH 1.01, 1H, d, J = 6.1, H-60000
]
Glu. [dH 5.05, 1H, d, J = 6.8, H-100000], [dH 3.36, 1H, dd, J = 9.9,7.5, H-
200000], [dH 3.49, 1H, dd, J = 9.9,3.4, H-300000], [dH 3.39, 1H, d, J = 3.4, H-
400000], [dH 3.50,1H, m, H-500000], [dH 3.90,1H, m, H-6a00000], [dH 3.68,1H,
m, H-6b00000
]
P-coumaroyl [dH 8.12, 2H, d, J = 8.5, H-2,6], [dH 6.84, 2H, d,
J = 8.5, H-3,5], [dH 7.62, 1H, d, J = 15.8, H-7], [dH 6.41,1H, d, J = 15.8,
H-8]

A.E. Allam et al. / Phytochemistry Letters 17 (2016) 144–151
151
For 13Cꢀꢀ NMR, (150 MHz, CD3OD); [dc 159.6 s, C-2], [dc 134.5, s,
Appendix A. Supplementary data
C-3], [dc 179.3, s, C-4], [dc 162.6, s, C-5], [dc 100.7, d, C-6], [dc 164.3,
s, C-7], [dc 95.7, d, C-8], [dc 157.9, s, C-9], [dc 107.5, s, C-10], [dc 123.3,
s, C-10], [dc 117.5, d, C-20], [dc 146.0, s, C-30], [dc 149.8, s, C-40], [dc
116.1, d, C-50], [dc 123.5, d, C-60]
Spectroscopic data of compounds 1 and 2 are available as
Electronic Supplementary material. The online version of this
article contains Supplementary material, which is available to
authorized users.
Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/j.
phytol.2016.07.018.
Galac. [dc 100.5, d, C-100], [dc 71.02, d, C-200], [dc 73.8, d, C-300], [dc
71.9, d, C-400], [dc 77.7, d, C-500], [dc 66.6, t, C-600]
Rham. (a) [dc 102.1, d, C-1000], [dc 71.9, d, C-2000], [dc 73.7, d, C-3000],
[
dc 71.0, d, C-4000], [dc 69.8, d, C-5000], [dc 17.9, q, C-6000]
Rham. (b) [dc 102.8, d, C-10000], [dc 72.1, d, C-20000], [dc 73.7, d, C-
30000], [dc 74.0, d, C-40000], [dc 69.8, d, C-50000], [dc 17.5, q, C-60000
]
References
Glu. [dc 101.4, d, C-100000], [dc 71.02, d, C-200000], [dc 74.5, d, C-300000],
dc 71.1, d, C-400000], [dc 78.1, d, C-100000], [dc 62.3, t, C-600000
P-coumaroyl [dc 127.0, d, C-1], [dc 131.5, d, C-2,6], [dc 116.8, d,
C-3,5], [dc 161.2, s, C-4], [dc 147.4, d, C-7], [dc 115.9, d, C-8], [dc
168.8, s, C O]
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3.9. Statistical analyses
Mean cytokine production (pg/mL) and ratio (percentage of
control) of DPPH were determined. Data are mean ꢃ SD. Statistical
significance was determined by Dunnett’s multiple test after one-
way analysis of variance (ANOVA) with comparison to a control
group using statistical analysis software (Kaleida Graph ver 4.00).
The IC50 values of three experiments and correlation coefficient
values for the tested samples in each experiment were calculated
using the same software. Differences were considered significant
at p < 0.05.
Kasai, Y., Komatsu, K., Shigemori, H., Tsuda, M., Mikami, Y., Kobayashi, J., 2005.
Cladinol A, a polyketide glycoside from marine-derived fungus Gliocladium
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4. Conclusion
Lambert, J.F., Millman, W.S., Fripiat, J.J., 1989. Revisiting kaolinite dehydroxylation. J.
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Markham, K.R., Ternai, B., Stanley, R., Geiger, H., Mabry, T.J., 1978. Tetrahedron 34,
1389–1397.
The general methodology was used for the structure elucidation
and the spectral assignments of the two new flavonol glycosides
from C. spicatum in this paper.
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Flavonoids: Advances in Research, Chapt. 2. Chapman and Hall, London, pp. 23–
24.
It is noted that the new compound 1 significantly increased
both TNF-
a and IL-1b after 48 h of treatment with little increase
with compound 2 (this level of increase of cytokines (300/200)
activates the cytokines like vaccines towards invasion of micro-
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Van der Sluis, W.G., Labadie, R.P., 1981. Secoiridoids and xanthones in the genus
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Acknowledgements
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This work was supported in part by Pharmacognosy and
Chemistry of Natural Products, School of Pharmaceutical Sciences,
Kanazawa University, Kanazawa, Japan, for which we are thankful.