Non-cannabinoid constituents from a high potency Cannabis sativa variety

Article abstract of DOI:10.1016/j.phytochem.2008.07.010

Six new non-cannabinoid constituents were isolated from a high potency Cannabis sativa L. variety, namely 5-acetoxy-6-geranyl-3-n-pentyl-1,4-benzoquinone (1), 4,5-dihydroxy-2,3,6-trimethoxy-9,10-dihydrophenanthrene (2), 4-hydroxy-2,3,6,7-tetramethoxy-9,10-dihydrophenanthrene (3), 4,7-dimethoxy-1,2,5-trihydroxyphenanthrene (4), cannflavin C (5) and β-sitosteryl-3-O-β-d-glucopyranoside-2′-O-palmitate (6). In addition, five known compounds, α-cannabispiranol (7), chrysoeriol (8), 6-prenylapigenin (9), cannflavin A (10) and β-acetyl cannabispiranol (11) were identified, with 8 and 9 being reported for the first time from cannabis. Some isolates displayed weak to strong antimicrobial, antileishmanial, antimalarial and anti-oxidant activities. Compounds 2-4 were inactive as analgesics.

Full text of DOI:10.1016/j.phytochem.2008.07.010

Phytochemistry 69 (2008) 2627–2633
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Non-cannabinoid constituents from a high potency Cannabis sativa variety
a
a,b,*
a
a
c
Mohamed M. Radwan , Mahmoud A. ElSohly
, Desmond Slade , Safwat A. Ahmed , Lisa Wilson ,
c
a,d
a,d,
*
Abir T. El-Alfy , Ikhlas A. Khan , Samir A. Ross
a
National Center for Natural Products Research, School of Pharmacy, The University of Mississippi, University, MS 38677, USA
Department of Pharmaceutics, School of Pharmacy, The University of Mississippi, University, MS 38677, USA
Department of Pharmacology, School of Pharmacy, The University of Mississippi, University, MS 38677, USA
Department of Pharmacognosy, School of Pharmacy, The University of Mississippi, University, MS 38677, USA
b
c
d
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 2 May 2008
Received in revised form 3 June 2008
Available online 4 September 2008
Six new non-cannabinoid constituents were isolated from a high potency Cannabis sativa L. variety,
namely 5-acetoxy-6-geranyl-3-n-pentyl-1,4-benzoquinone (1), 4,5-dihydroxy-2,3,6-trimethoxy-9,10-
dihydrophenanthrene (2), 4-hydroxy-2,3,6,7-tetramethoxy-9,10-dihydrophenanthrene (3), 4,7-dime-
0
thoxy-1,2,5-trihydroxyphenanthrene (4), cannflavin C (5) and b-sitosteryl-3-O-b-
D
-glucopyranoside-2 -
O-palmitate (6). In addition, five known compounds,
a
-cannabispiranol (7), chrysoeriol (8), 6-prenylap-
Keywords:
igenin (9), cannflavin A (10) and b-acetyl cannabispiranol (11) were identified, with 8 and 9 being
reported for the first time from cannabis. Some isolates displayed weak to strong antimicrobial, antileish-
manial, antimalarial and anti-oxidant activities. Compounds 2–4 were inactive as analgesics.
Ó 2008 Elsevier Ltd. All rights reserved.
Cannabis sativa L.
Cannabaceae
High potency
Non-cannabinoid
Antimicrobial
Antileishmanial
Antimalarial
Anti-oxidant
1
. Introduction
Cannabinoids are phenolic compounds possessing a C21 ter-
2. Results and discussions
Compound 1 was obtained as an orange amorphous powder. Its
penophenolic structure uniquely found in Cannabis sativa L. (ElS-
ohly and Slade, 2005). Currently, 86 cannabinoids have been
isolated from cannabis (Ahmed et al., 2008; ElSohly and Slade,
positive mode HRESIMS exhibited a pseudomolecular ion at m/z
+
373.2425 [M+H] corresponding to
a
molecular formula of
,b-
max 1663 and
1780 cm , respectively. The H NMR spectrum of 1 displayed
C
23
32
H O
4
. The IR spectrum of 1 indicated the presence of an
a
2
005; Radwan et al., 2008). Non-cannabinoid constituents isolated
unsaturated ketone and ester carbonyl groups at
m
ꢀ1
1
from cannabis include flavonoids, spiroindans, dihydrostilbenes,
dihydrophenanthrenes, sterols and alkaloids, among others (Ross
and ElSohly, 1995; Turner et al., 1980). As part of our program to
study the constituents of high potency cannabis and their pharma-
cology (Ahmed et al., 2008; Radwan et al., 2008), we herein report
the isolation and structure elucidation of eleven non-cannabinoid
constituents including six new (1–6) and five known (7–11) com-
pounds as well as their antimicrobial, antileishmanial, antimalarial
and anti-oxidant activities. The analgesic activities of 2–4 are also
reported.
three olefinic methyl singlets (d
H
1.56, 1.63 and 1.69), one primary
2.32),
0
methyl triplet (d
H
3
0.87, H -5 ), one acetoxy methyl singlet (d
H
one aromatic singlet (d
H
6.53, H-2) and 7 methylene resonances (d
H
1
3
1.32–2.39). The C NMR, DEPT and HMQC spectra of 1 revealed 23
resonances including five methyl, seven methylene, one aromatic
methine, two olefinic methine and eight quaternary carbons. The
two carbonyl carbons resonating at d
teristic for a benzoquinone skeleton (Mossa et al., 1999), while
NMR spectroscopic analysis suggested an acetoxy (d 2.32, d
68.1, 20.5), a geranyl (Radwan et al., 2008) and an n-pentyl sub-
stituent (Ahmed et al., 2008; Radwan et al., 2008), indicating that
is a trisubstituted-1,4-benzoquinone derivative. HMBC analysis
C
187.1 and 180.8 are charac-
H
C
1
1
*
Corresponding authors. Address: National Center for Natural Products Research,
School of Pharmacy, The University of Mississippi, University, MS 38677, USA. Tel.:
1 662 915 1031; fax: +1 662 915 7989 (S.A. Ross), tel.: +1 662 915 5928; fax: +1
(Fig. 1) placed the geranyl substituent at C-6 due to the correlation
00
of H
2
-1 (d
H
3.10) with C-6 (d
C
135.6), C-1 (d
C
187.1) and C-5 (d
6.53) with C-
C
+
1
6
49.1). The three bond HMBC correlation of H-2 (d
H
6
62 915 5587 (M.A. ElSohly).
0
(d
C
135.6) and C-4 (d
C
180.8), and the correlation of H
2
-1 (d
H
E-mail addresses: melsohly@olemiss.edu (M.A. ElSohly), sross@olemiss.edu (S.A.
Ross).
2.39) with C-4 (d
C
180.8) and C-2 (d
C
132.7) placed the n-pentyl
0
031-9422/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2008.07.010

2
628
M.M. Radwan et al. / Phytochemistry 69 (2008) 2627–2633
moiety at C-3. The location of the acetoxy group at C-5 was
determined by the four bond HMBC correlation between the acet-
d
C
30.6 (C-9) and 31.8 (C-10) in the HMQC spectrum], a pair of sig-
nals for an ortho-coupled AB spin system [d 6.74 (1H, d, J = 8.0 Hz,
H-7) and 6.79 (1H, d, J = 8.0 Hz, H-8) correlated to d 109.2 (C-7)
and 119.1 (C-8), respectively, in the HMQC spectrum], and an iso-
lated aromatic proton [d 6.50 (s, H-1)] suggesting that 2 is a
H
oxy methyl (d
tyl and geranyl groups were confirmed by COSY correlations
Fig. 1), establishing 1 as 5-acetoxy-6-geranyl-3-n-pentyl-1,4-
H
2.32) and C-5 (d
C
149.1). The presence of the n-pen-
C
(
H
benzoquinone.
2,3,4,5,6-pentasubstituted 9,10-dihydrophenanthrene (Crombie
Compound 2 was isolated as a brown amorphous powder. Its
et al., 1979; Leong et al., 1997; Stermitz et al., 1983). The 13C
NMR (Table 1) and APT spectra indicated the presence of five oxy-
genated quaternary aromatic carbons. Three of these carbons have
molecular formula was determined as C17
18 5
H O from the positive
+
13
mode HRESIMS ion at m/z 325.1100 [M+Na] and C NMR spec-
1
13
trum. The H and C NMR spectra of 2 (Table 1) displayed two sets
of methylene protons [d 2.64 (4H, s, H -9 and H -10) correlated to
methoxyl substituents as established by three sharp singlets in the
1
H
2
2
H
H NMR [d 3.85, 3.86 and 3.89 (3H each)], while the remaining

M.M. Radwan et al. / Phytochemistry 69 (2008) 2627–2633
2629
Table 1
1
H (400 MHz) and 13C (100 MHz) NMR spectroscopic data of 2–4 (CDCl
3
, d in ppm, J in
a
Hz)
Position
2
3
4
HMBC
COSY
d
H
d
C
d
H
d
C
d
H
d
C
1
2
3
4
4a
6.50 s
–
–
–
–
–
–
–
6.74 d (8.0)
6.79 d (8.0)
–
2.64 s
2.64 s
–
3.89 s
3.86 s
–
104.4
152.0
136.4
147.2
114.9
121.1
141.7
147.6
109.2
119.1
132.6
30.6
6.39 s
–
–
–
–
–
8.02 s
–
–
6.79 s
–
2.74 s
2.74 s
–
3.88 s
3.89 s
–
3.92 s
3.92 s
103.9
150.3
134.5
146.8
114.7
107.8
111.5
147.2
147.1
111.1
125.4
29.5
–
–
140.2
140.2
107.6
161.5
124.2
117.4
156.6
108.9
161.0
102.0
129.0
137.7
122.9
132.7
–
O
O
6.15 s
–
–
–
–
O
4
b
5
6
7
8
6.93 d (2.0)
–
6.82 d (2.0)
–
8.06 d (8.4)
8.12 d (8.4)
–
–
O
8a
1
9
0
1
31.8
30.9
Fig. 1. HMBC and COSY correlations of 1.
1
0a
136.4
56.3
130.1
56.2
OMe-2
OMe-3
OMe-4
OMe-6
OMe-7
61.0
61.3
–
–
two carbons have hydroxyl substituents as was confirmed by the
–
–
3.96 s
–
3.90 s
57.2
ꢀ
1
presence of a characteristic absorption band at
m
max 3420 cm
in the IR spectrum. The J HMBC correlations between H-8 (d
.79) and C-6 (d 147.6), C-4b (d 121.1) and C-9 (d
H-7 (d 6.74) and C-8a (d 132.6) and C-5 (d
the methoxyl group at d 3.85 and C-6 (d
3.85 s
–
56.1
56.0
56.1
–
3
–
55.7
H
6
C
C
C
30.6); between
a
Assignments confirmed by APT, gHMQC, gCOSY and gHMBC experiments.
H
C
C
141.7) and between
C
147.6), located this
H
methoxyl group at C-6, as was confirmed by ROESY correlation
3
Compound 3 was isolated as a pale brownish, amorphous pow-
(
Fig. 2). The J HMBC correlation between the isolated aromatic
proton at d 6.50 and C-10 (d 31.8) and C-4a (d 114.9) deter-
mined its location at C-1. The ROESY correlation between the
remaining two methoxyl groups at d 3.86 and 3.89 in conjunction
with the ROESY correlation between the methoxyl group at d 3.89
and H-1 (d 6.50) fixed their positions at C-3 and C-2, respectively,
der. The molecular formula of 3 was determined as C18
H
20
O
5
from
H
C
C
+
the positive mode HRESIMS at m/z 339.1279 [M+Na] , a mass dif-
1
13
ference of 14 amu compared to 2. The H and C NMR spectra of
(Table 1) were similar to 2 except for the replacement of one hy-
droxyl by a methoxyl group (d 3.92, d 56.1). Therefore, 3 exhib-
ited four methoxyl groups [d 3.88 (3H, s), 3.89 (3H, s) and 3.92
H
3
H
H
C
H
which was confirmed by HMBC correlations (Fig. 2), establishing 2
as 4,5-dihydroxy-2,3,6-trimethoxy-9,10-dihydrophenanthrene.
H
(
6H, s); d
C
56.0, 56.1, 56.2 and 61.3] and one hydroxyl group. Their
OMe
MeO
OH HO
OMe
H
OH
MeO
H
HO
O
O
H
H
2
OH
5
OH
3
HMBC
O
O
H
HO
2'
ROESY
3
'
H
1
'
O
OH
O
(
CH2)14CH3
6
Fig. 2. HMBC and ROESY correlations of 2, 5 and 6.

2
630
M.M. Radwan et al. / Phytochemistry 69 (2008) 2627–2633
locations on the dihydrophenanthrene skeleton were determined
the location of the geranyl group at C-8 instead of C-6, establishing
5 as 8-geranyl-5,7,4 -trihydroxy-3 -methoxyflavone (cannflavin C).
Compound 6 was obtained as an optically active white amor-
1
13
0
0
by comparison to the H and C NMR data of 2 (Table 1) and con-
firmed by HMBC and ROESY correlations, placing two methoxyl
groups at C-2 and C-3 and the hydroxyl at C-4. The remaining
methoxyl groups are therefore attached to ring B. The presence
phous powder. Its molecular formula was deduced from the positive
+
. The 1
mode HRESIMS [M+Na] ion at m/z 837.6621 as C51
90
H O
7
H
of a pair of isolated aromatic singlets [d
H
6.79 (H-8) and 8.02 (H-
)] in the H NMR assigned the two methoxyl groups to C-6 (d
47.2) and C-7 (d 147.1) (Leong et al., 1997), which was confirmed
3.92 (OMe-6 and OMe-7)/H-5 and H-8; H-8/H -9]
NMR spectrum displayed two tertiary [d
(Me-19)], three secondary [d 0.63 (Me-26 and Me-27) and 0.90
(Me-21)] and one primary [d 0.63 (Me-29)] methyl groups in addi-
tion to an olefinic proton at d 5.33 (bs, H-6), indicating a sitosterol
H
0.75 (Me-18) and 0.99
1
5
1
C
H
C
H
by ROESY [d
H
2
H
and HMBC (H-5/C-7, C-4a; H-8/C-6, C-8a, C-9) correlations. Thus,
the structure of 3 was established as 4-hydroxy-2,3,6,7-tetrameth-
oxy-9,10-dihydrophenanthrene.
skeleton (Kovganko et al., 2000; Takemoto et al., 1967). The pres-
0
ence of an anomeric proton [d
H
4.33 (d, J = 7.6 Hz, H-1 )] and carbon
0
[d
C
101.6 (C-1 )] in the HMQC spectrum indicated monoglycosyla-
Compound 4 was isolated as a reddish brown powder. Its
tion at C-3 (d
C
80.1) (Ishii et al., 1977) that was confirmed by HMBC
0
0
0
molecular formula was determined as C16
mode HRESIMS (m/z 571.1630 [2M-H] ) and C NMR data. The
UV spectrum of 4 (kmax 258, 282 and 303 nm) is characteristic for
H
14
13
O
5
from negative
(H-3/C-1 ; H-1 /C-3) and ROESY (H-1 /H-3) correlations (Fig. 2). The
sugar moiety was identified as b-D-glucopyranose by acid hydroly-
ꢀ
1
sis of 6 and TLC comparison with authentic sugar samples. The H
1
13
a phenanthrene skeleton (Leong et al., 1997). The H NMR (Table
and C NMR spectroscopic data of 6 were similar to those reported
1
) displayed a pair of signals for an ortho-coupled AB spin system
8.06 (1H, d, J = 8.4 Hz, H-9), 8.12 (1H, d, J = 8.4 Hz, H-10)], two
meta-coupled protons [d 6.82 (1H, d, J = 2.0 Hz, H-8), 6.93 (1H, d,
J = 2.0 Hz, H-6)] and an isolated aromatic singlet [d 6.15 (1H, s,
H-3)] indicative of a pentasubstituted phenanthrene (Réthy et al.,
for b-sitosterol-3-O-b-D-glucopyranoside (Chang et al., 1981) with
[d
H
the addition of 16 resonances characteristic for a palmitate moiety
(Segre and Mannina, 1997), which was confirmed by methylation
of the alkaline hydrolysis product of 6 followed by GCMS analysis.
H
H
0
The downfield esterification shift of C-2 (+2 ppm), upfield shifts
1
13
0
0
2
006; Leong et al., 1997). The H and C NMR spectra of 4 showed
of C-1 (ꢀ4 ppm) and C-3 (ꢀ3 ppm) and the four bond HMBC corre-
0
00
two aromatic methoxyl groups [d 3.90 (3H, s) and 3.96 (3H, s)]
and five oxygenated quaternary carbons, indicating that 4 has
H
lation of H-1 (d
H
4.33) and C-1 (d
C
174.6) (Fig. 2) placed the palmi-
0
tate moiety at C-2 (Terui et al., 1976; Yamasaki et al., 1977). Thus,
three hydroxyl groups. The ROESY correlation of the methoxyl
the structure of 6 was established as b-sitosteryl-3-O-b-
ranoside-2 -O-palmitate.
D
-glucopy-
0
group at d
established its location at C-7 (d 161.0), which was confirmed by
HMBC [d 3.90 (OMe-7)/C-7; H-8/C-7, C-6, C-8a, C-9; H-6/C-7, C-
] correlations. The ROESY and HMBC correlations of the methoxyl
group at d 3.96 with H-3 (d 6.15) and C-4 (d 161.5), respectively,
H
3.90 with the protons at d
H
6.93 (H-6) and 6.82 (H-8)
Compound 7 was obtained as colorless prisms. Its positive
+
H
mode HRESIMS gave an [M+H] ion at m/z 249.2393 corresponding
8
20 3
to a molecular formula of C15H O . The GCMS showed a molecular
H
H
C
ion at m/z 248 (33%) and two characteristic ions at m/z 189 (100%)
and 176 (65%) suggesting that 7 is a spiroindan derivative (El-
assigned its location at C-4. Therefore, the structure of 4 was deter-
mined as 4,7-dimethoxy-1,2,5-trihydroxyphenanthrene.
1
3
Feraly et al., 1986). The C NMR, DEPT and HMQC spectra of 7 dis-
Compound 5 was obtained as a yellow amorphous powder. Its
positive mode HRESIMS displayed an [M+Na] ion at m/z
played 15 resonances including one methoxyl, six methylene, two
aromatic methine, one sp oxymethine and five quaternary car-
+
3
4
59.1766 suggesting C26
H
28
O
6
as the molecular formula and 13 de-
bons. The spectroscopic data of 7 are similar to those reported
for b-cannabispiranol (Boeren et al., 1977; Shoyama and Nishioka,
grees of unsaturation. The IR spectrum showed absorption bands at
ꢀ1
mmax 3421 and 1662 cm due to hydroxyl and carbonyl groups,
1978; Radwan et al., 2008) except for the downfield shift of the
0
respectively, while the UV absorption maxima at kmax 275 (band
oxymethine carbon (+5.6 ppm), indicating a 4
a
-configuration.
I) and 340 (band II) nm were indicative of a flavone skeleton
(
Although 7 is a known cannabis constituent (Crombie and Crom-
bie, 1982), this is the first report of the full NMR assignments.
The flavones 8 and 9 were isolated as yellow amorphous pow-
ders. Their molecular formulae were determined from the HRESIMS
Mabry et al., 1970). The 1H NMR spectrum of 5 established a
hydrogen-bonded hydroxyl proton [d
was confirmed by the bathochromic UV shift (+25 nm) of band II
upon the addition of AlCl to a methanolic solution of 5. Bathochro-
H
13.05 (s, HO-5)] which
3
12 6 18
as C16H O and C20H O5, respectively. Their spectroscopic data
mic UV shifts upon the addition of NaOMe (+61 nm) and NaOAc
(UV and NMR) were in agreement with reported values for chrysoe-
riol (8) (Toth et al., 1980; Agrawal, 1989) and 6-prenylapigenin (9)
(Abegaz et al., 1998). This is the first report of their isolation from
cannabis. The NMR spectra of 10 and 11 were identical with those
of cannflavin A (Agrawal, 1989; Choi et al., 2004) and b-acetyl cann-
abispiranol (Shoyama and Nishioka, 1978), respectively.
0
(
(
+5 nm) suggested hydroxylation at C-4 and C-7, respectively
Mabry et al., 1970). The 1H NMR spectrum displayed two sharp
singlets at d
H
6.66 (1H, H-3) and 6.36 (1H, H-6), one methoxyl
7.01 (1H, d,
group (d 3.98) and an ABX spin system of ring B [d
H
H
0
0
J = 8.0 Hz, H-5 ), 7.58 (1H, d, J = 2.0 Hz, H-2 ), 7.64 (1H, dd, J = 2.0,
0
8
.0 Hz, H-6 )]. The presence of a geranyl group was deduced from
The antimicrobial, antileishmanial, antimalarial and anti-oxi-
dant activities of the isolated compounds were tested. Compound
the three methyl singlets at d
H
1.48, 1.53 and 1.82 and two olefinic
1
proton triplets at d 5.02 and 5.35 in the H NMR spectrum (Ahmed
H
1 displayed weak anti-MRSA (IC50 15.0
ishmanial (IC50 13.0 g/mL) and mild antimalarial activity against
Plasmodium falciparum (D6 clone) and P. falciparum (W2 clone)
with IC50 values of 2.8 and 2.6 g/mL, respectively. Compound 5
had moderate antileishmanial activity (IC50 17.0 g/mL). Com-
pound 9 showed moderate anti-MRSA (IC50 6.5 g/mL), weak anti-
candidal (IC50 20.0 g/mL) and mild antimalarial activity against P.
falciparum (D6 clone) and P. falciparum (W2 clone) with IC50 values
of 2.8 and 2.0 g/mL, respectively. Compound 10 exhibited strong
antileishmanial activity (IC50 4.5 g/mL). Compound 11 displayed
weak antileishmanial activity (IC50 31.0 g/mL).
lg/mL), moderate antile-
13
et al., 2008; Radwan et al., 2008). The C NMR, DEPT-135 and
HMQC spectra displayed 26 resonances including three methyl,
one methoxyl, three methylene, seven methine and 12 quaternary
carbons. The location of the methoxyl group was determined to be
l
l
l
0
0
0
0
0
0
0
0
at C-3 from HMBC (OMe-3 /C-3 ; H-5 /C-3 ; H-2 /C-3 , C-4 ) and
l
0
0
ROESY (OMe-3 /H-2 ) correlations (Fig. 2). The carbon resonance
at d 98.9 corresponding to a proton singlet at d 6.36 in the HMQC
l
C
H
spectrum indicated an unsubstituted C-6 position (Agrawal, 1989).
The location of the geranyl group at C-8 was confirmed by the
l
l
HMBC correlation of the benzylic protons [d
H
3.57 (2H, d, J = 6.8
162.4) and C-9 (d 155.4)
Fig. 2). The spectroscopic data of 5 are similar to those reported
for cannflavin A (10) (Agrawal, 1989; Choi et al., 2004) except for
l
00
Hz, H
2
-1 )] with C-8 (d
C
106.5), C-7 (d
C
C
Compounds 2, 5 and 11 displayed strong, 1, 7 and 10 moderate
and 8 and 9 weak anti-oxidant activities in the DPPH assay, with 6
being inactive.
(

M.M. Radwan et al. / Phytochemistry 69 (2008) 2627–2633
2631
Compounds 2, 3 and 4 exhibited no antinociceptive action in
both tail-flick and hot-plate assays up to 120 min following injec-
tion (Supplementary data).
silica gel column (EtOAc/petroleum ether, 90:10) to give 16 sub-
fractions (B1–16). Subfraction B (265 mg) was purified by Si HPLC
(EtOAc/n-hexane, 5:95, 25 mL/min, UV 270 nm) to afford
12.3 mg, rt. = 4.0 min). Subfraction B (236.0 mg) was purified by
Si-SPE column eluting with MeOH/CH Cl (1:19) yielding
21.6 mg). Fraction D (14.3 g) was subjected to silica CC (EtOAc/
petroleum ether, 5:95 to 20:80) followed by C18 flash chromatog-
raphy (MeOH/H O, 8:2) and C18 HPLC (MeCN/H O, 50:50, 25 mL/
min, UV 270 nm) to afford 9 (5.0 mg, rt. = 3.8 min), 10 (1.2 mg,
rt. = 4.6 min), (15.0 mg, rt. = 6.7 min) and (11.6 mg,
rt. = 8.7 min). Fraction F (28.5 g) was applied to a silica gel column
EtOAc/n-hexane, 10:90 to 60:40), yielding 42 fractions (F1–42,
00 mL each). Fraction F32–35 (5.5 g) was subjected to silica gel
CC (MeOH/CH Cl , 3:97) to yield 24 subfractions (SF1–24). SF2–3
210 mg) was applied to Si-SPE column (EtOAc/n-hexane,
0:90) followed by C18 HPLC purification (MeOH/H O, 85:15,
5 mL/min, UV 279 nm) to yield 3 (2.3 mg, rt. = 6.3 min) and 4
1
1
(
3
3
. Conclusion
2
2
6
(
In previous studies (Ahmed et al., 2008; Radwan et al., 2008) we
reported the isolation, identification and biological activities of
seventeen new cannabinoids from high potency C. sativa. In a con-
tinuation of this work, the isolation and identification of eleven
non-cannabinoid constituents is reported. Among them, 1–6 are
new metabolites. Flavones 8 and 9 were identified for the first time
from this plant. Compounds 1, 5, 10 and 11 displayed antileishma-
nial activity, while 1 and 9 showed anti-MRSA activity. Compounds
2
2
7
2
(
2
2
2
(
1
2
a
2, 5, 7, 10 and 11 exhibited anti-oxidant activity.
2
4
. Experimental
(3.9 mg, rt. = 8.8 min). SF3–6 (1.9 g) was purified by Sephadex LH-
0 CC (MeOH) followed by C18-SPE purification (MeOH/H O,
2
2
4.1. General
75:25) to give 5 (12.9 mg), 8 (284.5 mg) and 11 (15.0 mg).
1H NMR (400 MHz), 13C NMR (100 MHz), DEPT-135, APT and 2D
4.3.1. 5-Acetoxy-6-geranyl-3-n-pentyl-1,4-benzoquinone (1)
NMR spectra were recorded using the residual solvent signal as
internal standard on a Varian AS 400. IR spectra were measured
on a Bruker Tensor 27. UV spectra were obtained on a Varian Cary
Orange amorphous powder; UV kmax (MeOH): 205, 270, 384 nm;
mmax (neat): 1663 (C@O, ketone), 1780 (C@O, ester), 1610 (C@C)
IR
cm 1; 1H NMR (CDCl , 400 MHz): d 0.87 (3H, t, J = 6.4 Hz, H -5 ),
ꢀ
0
3
H
3
0
0
00
5
0 Bio UV–visible spectrophotometer. Optical rotation was mea-
1.32 (4H, m, H
2
-3 and H
2
-4 ), 1.56 (3H, s, H
3
-8 ), 1.57 (2H, m, H
2
00
-
0
00
00
sured on an Autoplot IV automatic polarimeter. High resolution
mass spectra were measured using a Bruker BioApex. HPLC was
performed on a Waters Delta Prep 4000 Preparative Chromatogra-
phy System connected to a Waters 486 Tunable UV Absorbance
detector using Phenomenex Luna C18 and Si columns
2 ), 1.63 (3H, s, H -9 ), 1.69 (3H, s, H -10 ), 1.94 (2H, m, H -4 ),
3
3
2
0
0
2.05 (2H, m, H
2
-5 ), 2.32 (3H, s, OCOCH
H -1 ), 3.10 (2H, d, J = 7.2 Hz, H -1 ), 4.99 (1H, t, J = 7.2 Hz, H-2 ),
5.02 (1H, t, J = 7.2 Hz, H-6 ), 6.53 (1H, s, H-2); C NMR (CDCl
3
), 2.39 (2H, t, J = 7.6 Hz,
0
00
00
2
2
0
0
13
3
,
100 MHz): dC 187.1 (C-1), 132.7 (C-2), 148.3 (C-3), 180.8 (C-4),
0
0
0
(
250 ꢁ 21.2 mm, 5
l
m, 100 Å). GCMS analysis was carried out on
149.1 (C-5), 135.6 (C-6), 28.9 (C-1 ), 27.6 (C-2 ), 31.6 (C-3 ), 22.6
0
0
00
00
00
a HP 6890 series GC, equipped with a split/splitless capillary injec-
tor, a HP 6890 series injector autosampler and an Agilent DB-5 ms
(C-4 ), 14.1 (C-5 ), 23.0 (C-1 ), 118.4 (C-2 ), 138.4 (C-3 ), 39.8 (C-
4 ), 26.7 (C-5 ), 124.1 (C-6 ), 131.8 (C-7 ), 17.9 (C-8 ), 25.9 (C-9 ),
16.4 (C-10 ), 20.5 (OCOCH ), 168.1 (OCOCH ); HRESIMS m/z
3 3
33 4
373.2425 [M+H] (Calc. for C23H O , 373.2379).
0
0
00
00
00
00
00
0
0
column (30 m ꢁ 0.25 mm ꢁ 0.25
mass selective detector (MSD). The injector temperature was
50 °C and 1 L injections were performed in the splitless mode,
lm), interfaced to a HP 5973
+
2
l
with the splitless time set at 60 s, the split flow set at 50 mL/min
and the septum purge valve set to close 60 s after the injection oc-
curred. The oven temperature was raised from 70 to 270 °C (hold
4.3.2. 4,5-Dihydroxy-2,3,6-trimethoxy-9,10-dihydrophenanthrene (2)
Brown amorphous powder; UV kmax (MeOH): 220, 267, 310 nm;
ꢀ1
IR
mmax (neat): 3420 (OH), 1610, 1537, 1462 (benzene ring) cm ;
1
13
2
0 min) at a rate of 5 °C/min, for a total run time of 60 min; the
For H and C NMR spectroscopic data, see Table 1; HRESIMS
+
transfer line temperature was 280 °C.
18 5
m/z 325.1100 [M+Na] (Calc. for C17H O Na, 325.1052).
4
.2. Plant material
4.3.3. 4-Hydroxy-2,3,6,7-tetramethoxy-9,10-dihydrophenanthrene (3)
Pale brownish amorphous powder; UV kmax (MeOH): 220, 267,
ꢀ1
C. sativa plants were grown from high potency Mexican seeds.
310 nm; IR
m
max (neat): 3420 (OH), 1610, 1537, 1462 cm : For
1
13
The seeds and plants were authenticated by Dr. Suman Chandra,
The University of Mississippi, and a specimen (S1310V1) was
deposited at the Coy Waller Complex, The University of Missis-
sippi. Whole buds of mature female plants were harvested, air-
dried, packed in barrels and stored at low temperature (ꢀ24 °C).
H and C NMR spectroscopic data, see Table 1; HRESIMS m/z
339.1279 [M+Na] (Calc. for C18H O Na, 339.1208).
20 5
+
4.3.4. 4,7-Dimethoxy-1,2,5-trihydroxyphenanthrene (4)
Reddish brown amorphous powder; UV kmax (MeOH): 258, 282,
ꢀ
1
1
3
and
03 nm; IR mmax (neat): 3413 (OH), 1610, 1533, 1462 cm : For H
1
3
4.3. Extraction, isolation and characterization
C NMR spectroscopic data, see Table 1; HRESIMS m/z
ꢀ
5
71.1630 [2M-H] (Calc. for C32
H
27
O10, 571.1604).
The plant material (9.0 kg) was sequentially extracted with hex-
0
0
anes (48 L), CH
36 L, 1:1) and H
evaporated under reduced pressure at 40 °C to afford hexanes
1.48 kg), CH Cl (0.15 kg), EtOAc (0.13 kg), EtOH (0.09 kg), EtOH/
O (0.77 kg) and H O (0.54 kg) extracts for a total extract of
.16 kg (35.1%, w/w). Portions of the CH Cl , EtOAc and EtOH ex-
2
Cl
2
(40 L), EtOAc (40 L), EtOH (40 L), EtOH/H
2
O
4.3.5. 8-Geranyl-5,7,4 -trihydroxy-3 -methoxyflavone (Cannflavin C)
(5)
(
2
O (40 L) at room temperature. The extracts were
Yellow amorphous powder; UV kmax (MeOH): 275, 340,
(+NaOMe) 280, 342, 401, (+NaOAc) 280, 340, (+AlCl
(
H
3
2
2
3
) 300, 346,
2
2
360, (+AlCl +HCl) 300, 346, 360 nm; IR mmax (neat): 3421 (OH),
3
ꢀ1
1
2
2
1662 (C@O) cm
;
H NMR (acetone-d
6
, 400 MHz): d
H
1.48 (3H,
0
0
00
00
tracts were combined (191.0 g) since they showed similar TLC pro-
files (EtOAc/n-hexane, 4:6) and were subjected to silica gel VLC,
eluting with EtOAc/n-hexane [0:100, 10:90, 20:80, 30:70, 40:60,
0:50, 75:25, 100:0 (2 L of each mixture)] followed by EtOH (4 L),
yielding 9 fractions (A-I). Fraction B (10 g) was fractionated on a
s, H -9 ), 1.53 (3H, s, H -8 ), 1.82 (3H, s, H -10 ), 1.96 (2H, m, H -
-5 ), 3.57 (2H, d, J = 6.8 Hz, H
OMe-3 ), 5.02 (1H, t, J = 6.8 Hz, H-6 ), 5.35 (1H, t, J = 6.8 Hz, H-2 ),
6.36 (1H, s, H-6), 6.66 (1H, s, H-3), 7.01 (1H, d, J = 8.0 Hz, H-5 ),
7.58 (1H, d, J = 2.0 Hz, H-2 ), 7.64 (1H, dd, J = 2.0, 8.0 Hz, H-6 ),
3
3
3
2
0
0
00
00
4 ), 2.05 (2H, m, H
2
2
-1 ), 3.98 (3H, s,
0
00
00
0
5
0
0

2
632
M.M. Radwan et al. / Phytochemistry 69 (2008) 2627–2633
1
2
1
1
1
1
3.05 (1H, s, HO-5); 13C NMR (acetone-d
), 103.3 (C-3), 182.7 (C-4), 160.5 (C-5), 98.9 (C-6), 162.4 (C-7),
6
, 100 MHz): d
C
164.1 (C-
nas aeruginosa ATCC 27853, Mycobacterium intracellulare ATCC
23068, Aspergillus fumigat ATCC 90906, Methicillin Resistant Staph-
ylococcus aureus ATCC 43300) (Bharate et al., 2007; Babu et al.,
2006), antileishmanial (Radwan et al., 2008) and antimalarial
activity [P. falciparum (D6 clone) and P. falciparum (W2 clone)]
(Bharate et al., 2007).
0
0
06.5 (C-8), 155.4 (C-9), 104.5 (C-10), 124.4 (C-1 ), 109.7 (C-2 ),
0
0
0
0
00
48.3 (C-3 ), 151.1 (C-4 ), 115.8 (C-5 ), 120.7 (C-6 ), 21.7 (C-1 ),
00
00
00
00
00
23.0 (C-2 ), 135.1 (C-3 ), 39.7 (C-4 ), 26.7 (C-5 ), 124.4 (C-6 ),
00
00
00
00
0
31.0 (C-7 ), 17.9 (C-8 ), 25.1 (C-9 ), 16.0 (C-10 ), 55.8 (OMe-3 );
+
28 6
HRESIMS m/z 459.1766 [M+Na] (Calc. for C26H O Na, 459.1784).
4.7. Anti-oxidant activity
0
4
.3.6. b-Sitosteryl-3-O-b-
D
-glucopyranoside-2 -O-palmitate (6)
White amorphous powder; [
a
]
D
: + 91.3 (c 0.05, CHCl
3
); IR
, 400 MHz): d
-18), 0.83 (3H, t,
m
max
A
TLC autographic assay for DPPH (1,1-diphenyl-2-pic-
ꢀ1
1
(
0
neat): 1736 (OH), 1610 cm
;
H NMR (CDCl
3
H
rylhydrazyl) radical-scavenging effect was used to determine
anti-oxidant activity (Takamatsu et al., 2003). The isolated com-
pounds were dissolved in DMF (2 mg/mL) and applied in the form
.63 (9H, m, H
3
-26, 27, 29), 0.75 (3H, s, H
3
00
3
J = 7.2 Hz, H -16 ), 0.90 (3H, d, 6.8 Hz, H
3
-21), 0.99 (3H, s, H
3
-19),
-4 , 15 ),
0
0
00
00
00
1
2
5
.24 (18H, bs, H
2
0
-5 to H
2
-13 ), 1.28–1.30 (4H, m, H
2
of a spot (4 lL, 4–5 mm in diameter) on silica gel GF plates. The
0
0
2
.34 (2H, m, H -2 ), 3.49 (1H, m, H-3), 3.35-3.82 (4H, H-2 to H-
residual DMF was removed under vacuum (15–20 min). A similar
amount of vitamin E in DMF was used as positive anti-oxidant con-
trol. The radical-scavenging effects of the compounds were de-
tected on the TLC plate using DPPH spray reagent (0.2% w/v in
MeOH). The plate was observed 30 min after spraying. Active
compounds are observed as yellow spots against a purple back-
ground. Relative radical-scavenging activity was assigned as
‘strong” (compounds that produce an intense bright yellow spot),
‘medium” (compounds that produce a clear yellow spot), ‘‘weak”
compounds that produce a weakly visible yellow spot), or ‘‘not
0
0
0
), 4.33 (1H, d, J = 7.6 Hz, H-1 ), 4.1-4.2 (2H, m, H
2
-6 ) 5.33 (1H,
37.6 (C-1), 29.6 (C-2),
0.1 (C-3), 39.2 (C-4), 140.6 (C-5), 122.2 (C-6), 32.2 (C-7), 32.1
C-8), 50.3 (C-9), 36.9 (C-10), 21.3 (C-11), 40.0 (C-12), 42.5 (C-
1
3
3 C
bs, H-6); C NMR (CDCl , 100 MHz): d
8
(
1
1
4
1
3), 57.0 (C-14), 24.5 (C-15), 28.3 (C-16), 56.4 (C-17), 12.1 (C-18),
9.6 (C-19), 36.4 (C-20), 19.0 (C-21), 34.2 (C-22), 26.4 (C-23),
6.0 (C-24), 29.4 (C-25), 19.2 (C-26), 20.0 (C-27), 23.3 (C-28),
‘
‘
(
0
0
0
0
2.2 (C-29), 101.6 (C-1 ), 73.5 (C-2 ), 76.6 (C-3 ), 70.8 (C-4 ), 73.8
0
0
00
00
00
00
(
C-5 ), 64.1 (C-6 ), 174.6 (C-1 ), 34.5 (C-2 ), 25.2 (C-3 ), 29.6 (C-4
0
0
00
00
00
to C-13 ), 30.0 (C-14 ), 22.9 (C-15 ), 14.3 (C-16 ); HRESIMS m/z
37.6621 [M+Na] (calc. for C51H O Na, 837.6584).
90 7
active” (compounds that produce no sign of any yellow spot).
Vitamin E produced an intense bright yellow spot.
+
8
4
.3.7.
Colorless prisms (MeOH/H
10, 222 nm; IR max (neat): 3410, 3180 (OH), 1610, 1596 cm
a-Cannabispiranol (7)
Acknowledgments
2
O); mp 182 °C; UV kmax (MeOH):
ꢀ1
2
m
;
1
0
0
The project described was supported in part by Grant No.
5P20RR021929-02 from the National Center for Research Re-
sources and in part by the National Institute on Drug Abuse,
Contract # N01DA-5-7746. The content is solely the responsibility
of the authors and does not necessarily represent the official views
of the National Center for Research Resources or the National
Institutes of Health. We are grateful to Dr. Bharathi Avula for assis-
tance with the HRESIMS, and to Dr. Melissa Jacob, Ms. Marsha
Wright, Dr. Babu Tekwani and Dr. Shabana Khan for conducting
the antimicrobial and antiprotozoal testing.
H NMR (C
5
D
5
N, 400 MHz): d
H 2
1.75 (4H, m, H -2 , 6 ), 2.12 (2H, t,
0
0
J = 7.2 Hz, H
3
2
-2), 2.25 (4H, m, H
2
-3 , 5 ), 2.90 (2H, t, J = 7.2 Hz, H
2
-
0
), 3.70 (3H, s, OMe-5), 4.10 (1H, m, H-4 ), 6.52 (1H, bs, H-4), 6.67
1
3
(
1H, bs, H-6); C NMR (C
D
5 5
N, 100 MHz): d
C
49.2 (C-1), 36.4 (C-
2
1
5
2
), 31.9 (C-3), 102.0 (C-4), 161.1 (C-5), 101.3 (C-6), 156.7 (C-7),
0
0
0
0
0
29.9 (C-8), 146.9 (C-9), 34.6 (C-2 , 6 ), 34.3 (C-3 , 5 ), 70.8 (C-4 ),
+
21 3
5.6 (OMe-5); HRESIMS m/z 249.2393 [M+H] (Calc. for C15H O ,
49.1491); GCMS m/z (rel. int.): 248 (M , 33%), 230 (13%), 215
+
(
12%), 201 (12%), 189 (100%), 176 (65%), 161 (18%).
4.4. Acid hydrolysis of 6
Appendix A. Supplementary material
Compound 6 (5 mg) was refluxed with 3% H
for 8 h. The reaction mixture was neutralized with Na
2
SO
4
in MeOH (4 mL)
CO and ex-
tracted with EtOAc (3 ꢁ 10 mL). TLC comparison of the aqueous
layer with authentic sugar samples (CH Cl /MeOH/H O, 6:4:1; n-
BuOH/AcOH/H O, 5:5:1) identified the glycone as b- -glucose.
2
3
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.phytochem.2008.07.010.
2
2
2
2
D
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