and anti-proliferative effects apart from alleviating pain.
The
assigned as C21
m/z 331.2272 [M + H] (calcd for C21
H
30
O based on the protonated molecular ion at
3
+
current study was initiated to examine the cellular effects of
naturally occurring cannabinoids on regulating the Wnt/β-catenin
signaling pathway by analysing the impact of these cannabinoids
on TCF-dependent β-catenin mediated transcription regulation.
H
31
O , m/z 331.2273) in its
3
positive-ion HR-ESI-MS, confirmed seven indices of hydrogen
deficiency. Detailed analyses of 2D NMR spectroscopic data
(Table S1) provided structure of 2a as shown in Figure 2. The
relative configuration of 2a was determined by analyzing
NOESY NMR correlations (Fig. 2). The NOESY correlations
In this study, we isolated six known compounds from
Cannabis sativa, and an epoxy derivative 2a was synthesized
from 2. We characterized these cannabinoids by 1D and 2D
NMR spectral data. We then assessed the anti-proliferative
activity of these cannabinoids on a panel of cancer cell lines.
Since cannabinoids are known to alleviate pain, we examined the
impact of these cannabinoids on modulating Wnt/β-catenin
signalling pathway, one of the most critical pathways implicated
in regulating neuropathic pain.
between H
-7, H-2, H-1, H-8 were on same side (α oriented), and epoxy
was β oriented (Fig. 2).
3
-7/H-2, H-2/H-1 and H-1/ H-8 (Fig. 2) suggested that
H
3
We extracted air dried and powdered leaves of Cannabis
sativa with hexane and fractionated it in an open column using
silica gel (100-200 mesh) column which resulted in 10 fractions
1
1
1
1
Figure 2. Key H– H COSY (
), HMBC (
) and H– H
NOESY ( ) correlations of 2, 3-epoxy derivative (2a).
(
Fr.1-Fr.10). Fr.3 (10 g) had major cannabinoids. Upon further
passing through Sephadex LH20, we collected ten fractions
Fr.3-1 to Fr.3-10). Fr.3-5 (450 mg) was filtered through a 0.45
Determining effect of cannabinoids on cell proliferation by
MTT assay. Since the major drawback of many chemotherapeutic
agents is their cellular toxicity. Thus, these six cannabinoids were
evaluated for their cytotoxicity by MTT assay on a panel of
cancer cell lines that include HCT-116, OVCAR, A549, MCF7,
PC-3, HepG2 and SH-SY5Y cells spanning different tumour
types (colon, ovary, lungs, breast, prostate, liver and
neuroblastoma). The addition of CBDs to the culture medium of
these cell lines for 24, 48 and 72 h resulted in a concentration-
dependent inhibition of the cell growth as determined by MTT
assay. The range of concentrations tested was from 1 μM to 100
μM. The results shown in Table-1 indicate that cannabinoid
treatment for 72 hours caused a significant decrease in the
viability of these cell lines when compared to DMSO control.
These cannabinoids showed varying degrees of growth inhibition
across different cell lines. Since HepG2 cells are considered a
standard cell line for screening small molecule inhibitors of
Wnt/β-catenin signalling due to the presence of an interstitial
deletion at the N-terminal domain of β-catenin protein, we
calculated IC50 values for these compounds in HepG2 cells.
Compounds 3, 4, 5 and 6 were relatively less effective than 1, 2
and 2a, and we compared the effect of 1, 2 and 2a in HepG2 cells
with that of salinomycin, a natural product-based inhibitor of
Wnt/β-catenin signalling pathway. Compounds 1, 2 and 2a are
seen to be less toxic than salinomycin in these cells under similar
conditions.
(
µm filter, and the MeOH soluble fraction was analysed by semi-
preparative HPLC. Its chromatogram showed six peaks in major
quantity, and all the six compounds were isolated with > 95%
purity: 1 (6 mg, t
2
R
14.7 min), 2 (42 mg, t
1.4 min), 4 (30 mg, t 24.9 min), 5 (48 mg, t
34.7 min). The structures of the compounds were
R
18.3 min), 3 (15 mg, t
R
R
R
30.5 min), 6 (27
mg, t
R
18
19
1
characterized as cannabidivarin (1), cannabidiol (2), Δ -
18
19
9
tetrahydrocannabidivarol
(3),
cannabinol
(4),
Δ -
tetrahydrocannabinol (5)19 and cannabichromene (6)
20
by
HRESIMS, NMR (1D and 2D spectroscopy analysis, and
compared with those reported in literature (Fig. 1).
Figure 1. Structures of compounds isolated from cannabis
sativa.
Further, 2, 3-epoxy derivative (2a) was synthesized by
treatment of CBD-5 in acetone with oxone at room temperature
2
1
for overnight which afforded 95% yield (Scheme 1).
Scheme 1. Reagents and conditions: (a) Oxone (3.0 equiv.),
acetone, rt, overnight
Indeed Yamamoto et al synthesized 2a from 2 as colourless
needles with mp 65 °C in two steps via CBD-2'-6'-diacetate and
1
22
characterized it by only H NMR (CDCl ). However, the
3
Figure 3. Comparison of dose-response curve of selected
cannabinoids in HepG2 cells. The experiments performed in
triplicate; 50% inhibition of cell growth (IC50) used as the
analysis parameter calculated by Prism GraphPad (version 5.0)
software. The One-Way ANOVA analysis was used to compare
the mean values (p < 0.05).
compound 2a that we synthesized here is assumed to be its
stereoisomer as it is a yellow colour liquid, and most of its H
NMR chemical shifts mismatched with reported data. Thus, we
characterized the structure of 2a by HRESIMS, and 1D- and 2D-
NMR spectroscopic data analyses. The molecular formula was
1
22
Nalli, Yedukondalu
