Introduction
Many genera of the family Ranunculaceae have large and colorful flowers and are being
cultivated as ornamentals or for their pharmacological substances, which are commonly
used in traditional Asian herbal medicine [1]. Anemone coronaria L. belongs to the family Ranunculaceae and is a winter perennial, flowering in the
Mediterranean region from February until May [2]. A. coronaria was reported to contain anthocyanins (flowers), triterpene glycosides (tubers), and
alkaloids (herb) [3]
[4]
[5]
[6]. A. coronaria has been used as antineuralgic and antirheumatic in the folk medicine of Lebanon
[7]. Fresh A. coronaria flowers are reported to aid the healing of acute joint inflammation in traditional
Turkish medicine [1]. In traditional Chinese medicine, A. coronaria is used to substitute Pulsatilla chinensis (Bunge) Regel (Ranunculaceae) roots in the management of neuralgic toothache and
earache [8].
Currently, it is expensive for pharmaceutical companies to develop a single novel
drug. As reported by Forbes, it can cost from 1 up to 5 billion US dollars [9]. Thus, phytochemicals might be more economical alternatives, important especially
for low-income developing countries [10].
T1DM is a less-prevalent autoimmune disorder, comprising five percent of those diagnosed
with diabetes, and mostly affecting kids and young adults. However, it is just as
severe as type 2 diabetes [11].
T1DM is marked by demolished pancreatic beta cells resulting in insulin deficiency.
Mounting evidence assumes that hyperglycemia management alone might be inadequate
to prevent the progression of diabetic complications, thus, its early diagnosis and
management are crucial for better therapeutic outcomes. However, complications are
still the main cause of both mortality and morbidity in patients with T1DM [11].
DN is a common complication of T1DM and may cause serious pain. This pain might happen
spontaneously, as hyperalgesia associated with mild painful stimuli, or as allodynia
with non-noxious stimuli [12].
DN pain management substantiates an increasing therapeutic concern to clinicians,
given that 39% of DN patients were not properly treated [13]. Moreover, the rest of the patients have been treated with unspecific analgesics
like tramadol and non-steroidal anti-inflammatory drugs (NSAIDs), anticonvulsants
or antidepressants [14]. Thus, it is important to find alternative therapies that are more specific and
have fewer side-effects [15]
[16].
A literature review indicated that there are no detailed studies on the effect of
A. coronaria on diabetes-induced neuropathic pain. Consequently, the aim of the current work is
to probe A. coronaria and its possible active principles for antinociceptive potentials against diabetic-neuropathy
pain in a mouse model. This study also aimed to achieve a bio-guided fractionation,
and to isolate the most active principles of A. coronaria.
Results
To isolate the active principles of AC, a bio-guided fractionation was conducted.
Through RP-HPLC major peaks were identified as follows: I corrurine (12.6%); II catalin (9.3%); III thaliporphine (5.2%); IV glaucine (19.9%); V protopine (12.4%). A mobile phase of triethylamine (0.1%) and acetonitrile (60:40)
was used at a flow rate of 1 mL/min at 280 nm, using UV detection ([Fig. 1] and Fig. S1, Supporting Information). The A. coronaria ethanolic extract was fractionated using RP-column chromatography. Each fraction
was concentrated, dispersed in 0.9% cold sterile saline (vehicle), and examined for
hypoglycemic and antinociceptive effects. The main active fraction was directly injected
into the RP-HPLC apparatus to assess its patterns, and was found to be rich in alkaloids,
hence it was named isolated alkaloid rich fraction (SARF). Four peaks were obtained
from SARF, of which 3 peaks have been identified as catalin (21.1%); thaliporphine
(14.1%); glaucine (compound 3, 63.4%; [Fig. 1], [2]), using a previously reported method [17]. The peak concentrations were measured utilizing the area under-peaks, and the peak
areas were found to be directly proportional to the sample amount utilizing standard
calibration curves and steep-area method. A comparison of the 1H NMR and 13C NMR analysis to standards ascertained that SARF contained catalin, thaliporphine,
and glaucine (3; [Fig. 1] and Table. S1, Supporting Information).
Fig. 1 Isolated compounds from A. coronaria active fraction (SARF). a catalin; b thaliporphine; c glaucine.
Fig. 2 HPLC chromatogram of SARF. Peaks are as follows: 1 catalin (21.1%); 2 thaliporphine
(14.1%); 3 glaucine (63.4%) using mobile phase of triethylamine (0.1%) and acetonitrile
(60:40) at a flow rate of 1 mL/min at 280 nm using UV detector.
The experimental protocol of the in vivo tests is summarized in [Table 1]. The levels of HbA1c (%), measured at the end of eight weeks for different experimental
groups, are shown in [Fig. 3]. A significant elevation of the HbA1c level (p≤0.05) was detected in the vehicle
control mice (VEH; 8.30±0.10) when compared with normal control mice (NORM; 7.40±0.14).
After eight weeks of treatment with AC (200 mg/kg; 7.10±0.09), SARF (70 mg/kg; 7.41±0.08),
and compound 3 (100 mg/kg; 7.80±0.07) the HbA1c levels in alloxan-induced diabetic mice ameliorated
near to the normal-ranges, significantly (p≤0.05) relative to VEH mice (8.30±0.10)
and near to the normal ranges compared with NORM mice, indicating that AC, SARF, and
compound 3 worked as potential hypoglycemic agents.
Fig. 3 Effects of A. coronaria extract (AC), its alkaloid rich fraction (SARF) and glaucine (GLA) on HbA1c at pre-dose
and 8 weeks post-dose. Values represent the mean±SEM (n=7). (NORM) normal non-diabetic
untreated mice. *p<0.05 vs. diabetic vehicle control (VEH).
Table 1 Protocol of the experimental design.
|
Groups
|
n
|
Tested Substance(s)
|
Description
|
|
A Acute (0, 0.5, 2 and 6 h) and subchronic (1, 3, 5, and 8 days) effects of Anemone coronaria ethanolic extract (A. coronaria), its alkaloid rich fraction (SARF) and glaucine (GLA) on blood glucose, body weights,
and CAT serum levels:
|
|
I
|
7
|
Control
|
Normal mice: Vehicle [sterile cold saline (0.9%)]
|
|
II
|
7
|
Diabetic Control
|
Diabetic mice: Vehicle
|
|
III
|
7
|
GB
|
Diabetic mice: GB 5 mg/kg
|
|
IV
|
7
|
A. coronaria
|
Diabetic mice: A. coronaria 50 mg/kg
|
|
V
|
7
|
A. coronaria
|
Diabetic mice: A. coronaria 100 mg/kg
|
|
VI
|
7
|
A. coronaria
|
Diabetic mice: A. coronaria 200 mg/kg
|
|
VII
|
7
|
SARF
|
Diabetic mice: SARF 70 mg/kg
|
|
VIII
|
7
|
GLA
|
Diabetic mice: GLA 25 mg/kg
|
|
IX
|
7
|
GLA
|
Diabetic mice: GLA 50 mg/kg
|
|
X
|
7
|
GLA
|
Diabetic mice: GLA 100 mg/kg
|
|
B Subchronic (0, 2, 4, 6 and 8 weeks) effect of A. coronaria, SARF, and GLA on hot plate and tail withdrawal latencies, and von Frey paw withdrawal
thresholds:
|
|
XI
|
7
|
NORM
|
Normal mice: Vehicle [sterile cold saline (0.9%)]
|
|
XII
|
7
|
VEH
|
Diabetic mice: Vehicle
|
|
XIII
|
7
|
A. coronaria
|
Diabetic mice: A. coronaria 50 mg/kg
|
|
XIV
|
7
|
A. coronaria
|
Diabetic mice: A. coronaria 100 mg/kg
|
|
XV
|
7
|
A. coronaria
|
Diabetic mice: A. coronaria 200 mg/kg
|
|
XVI
|
7
|
SARF
|
Diabetic mice: SARF 70 mg/kg
|
|
XVII
|
7
|
GLA
|
Diabetic mice: GLA 25 mg/kg
|
|
XVIII
|
7
|
GLA
|
Diabetic mice: GLA 50 mg/kg
|
|
XIX
|
7
|
GLA
|
Diabetic mice: GLA 100 mg/kg
|
To assess the therapeutic effects of the tested compounds on diabetes, the acute anti-diabetic
activities of AC, SARF, and compound 3 were examined for 6 h ([Table 2]). Glibenclamide (5 mg/kg) was used as a positive control to validate the experimental
protocol and procedure, and showed a significant acute reduction in BGL [18]. Acute administration of AC to alloxan-induced diabetic mice at various doses (50,
100, and 200 mg/kg) induced a sustained decrease of 40.4, 43.7, and 53.1%, respectively,
in BGL over a 6-h period relative to the diabetic control-group. While SARF displayed
a 48.4% decrease in BGL relative to the diabetic control group after 6 h, compound
3 (25, 50, and 100 mg/kg) however, showed 36.8, 38.4, and 45.6%, respectively, reduction
in BGL after 6 h Consequently, the highest dose of AC showed a higher efficacy than
SARF and glaucine (3), acutely.
Table 2 Acute effect of A. coronaria hexane extract, its alkaloid rich fraction (SARF), and glaucine (GLA) on blood glucose.
|
Group
|
Dose (mg/kg)
|
Mean blood glucose concentration±S.E.M. (mg/dL)
|
|
0 h
|
0.5 h
|
2 h
|
6 h
|
|
Control
|
__
|
101.80±1.80
|
106.50±3.50
|
104.13±3.10
|
107.25±3.90
|
|
Diabetic control
|
__
|
204.79±5.60
|
214.63±4.50
|
216.91±9.70
|
212.25±7.30
|
|
GB
|
5
|
220.70±2.70
|
223.64±1.80
|
159.74±2.10
|
130.60±2.40**
|
|
A. coronaria
|
50
|
205.51±2.40
|
172.45±2.30
|
172.87±3.50
|
126.59±2.40*
|
|
A. coronaria
|
100
|
211.33±2.70
|
166.78±2.90
|
166.87±2.90
|
119.59±2.30*
|
|
A. coronaria
|
200
|
202.20±2.10
|
114.67±1.90
|
114.87±2.30
|
99.59±2.70*
|
|
SARF
|
70
|
211.90±2.80
|
124.83±1.90
|
126.81±1.80
|
109.58±1.30*
|
|
GLA
|
25
|
204.54±2.10
|
191.45±2.90
|
129.67±2.40
|
134.12±1.50*
|
|
GLA
|
50
|
212.90±2.70
|
180.36±2.10
|
147.33±2.40
|
130.76±1.70*
|
|
GLA
|
100
|
206.34±1.20
|
156.26±2.00
|
136.55±2.10
|
115.44±2.20*
|
S.E.M.: mean standard error
* p<0.05 significant from the control animals
** p<0.01 significant from the control animals
AC is commonly commercialized in the folk medicine of Lebanon for the management of
several disorders, including diabetes. This use has encouraged the performance of
this study to prove its preclinical efficacy as a potential hypoglycemic agent using
well-established mice models [19]. To verify this, subchronic administration of AC to alloxan-induced diabetic mice
at various doses (50, 100, and 200 mg/kg) induced a sustained decrease of 33.7, 45.2,
and 53.8%, respectively, in BGL for eight days as compared with the diabetic control
group ([Table 3]), while SARF had a 48.6% decrease in BGL relative to the diabetic control group
8 days post-administration. On the other hand, glaucine (3; 25, 50, and 100 mg/kg) showed 41.2, 42.8, and 45.4%, respectively, reduction in
BGL after 6 h post-administration relative to the diabetic control-group. Accordingly,
the highest dose of AC showed a higher efficacy than SARF and glaucine (3), subchronically.
Table 3 Subchronic effect of A. coronaria, SARF, and GLA on blood glucose.
|
Group
|
Dose (mg/kg)
|
Mean blood glucose concentration±S.E.M. (mg/dL)
|
|
1st day
|
3rd day
|
5th day
|
8th day
|
|
Control
|
__
|
105.10±2.50
|
107.10±3.60
|
106.10±2.20
|
110.40±3.70
|
|
Diabetic controla
|
__
|
202.79±5.60***
|
211.93±4.50***
|
215.21±9.70***
|
210.50±7.30***
|
|
GBb
|
5
|
184.20±3.70
|
177.03±2.90
|
159.04±2.40**
|
169.80±3.10
|
|
A. coronaria
a
|
50
|
127.59±2.40
|
150.67±2.30
|
144.33±2.30
|
139.63±2.40*
|
|
A. coronaria
a
|
100
|
120.41±2.30
|
122.69±2.10
|
120.45±1.90
|
115.33±2.10*
|
|
A. coronaria
a
|
200
|
100.59±2.70
|
105.88±2.70
|
105.73±2.40
|
97.34±2.90*
|
|
SARFa
|
70
|
110.58±1.30
|
109.89±3.10
|
109.22±2.50
|
108.13±1.30*
|
|
GLAa
|
25
|
135.12±1.50
|
139.76±2.10
|
124.76±2.10
|
123.76±2.50*
|
|
GLAa
|
50
|
131.66±1.70
|
129.48±2.40
|
125.76±2.00
|
120.55±2.20*
|
|
GLAa
|
100
|
120.44±2.20
|
120.26±2.20
|
119.26±2.10
|
114.98±2.10*
|
S.E.M.: mean standard error
* p<0.05 significant from the control animals
**p<0.01 significant from the control animals
*** p<0.001 significant from the control animals
a Compared to vehicle control
b Compared to diabetic control
The elevation in BW of the experimental-animals is an indicator for the amelioration
of hyperglycemia [20]
[21] ([Table 4]). Therefore, the effects of AC, SARF, and glaucine (3) on BW levels of the mice were monitored for eight days during the experimental period
([Fig. 4]). The BW levels of the control diabetic animals gradually decreased throughout the
experimental period, indicating an aggravation of diabetes. On the other hand, the
BW levels of the AC (50, 100 and, 200 mg/kg) treated group increased by 2.3, 6.3,
and 14.7%, respectively, 8 days post-administration as compared to the diabetic control
group. Eight days after SARF (70 mg/kg) administration, BW levels increased by 15.1%
relative to the diabetic control group. Compound 3 (25, 50, and 100 mg/kg) treated groups showed 9.0, 14.6, and 17.3%, respectively,
increase in BW levels when compared to the diabetic control group 8 days post-administration.
Accordingly, the diabetic mice treated with AC, SARF, and compound 3 had a significant elevation on BW gain relative to diabetic control-groups, indicating
the amelioration of hyperglycemia.
Fig. 4 Effect of A. coronaria extract (AC) and its alkaloid rich fraction (SARF), using tramadol (TRA) 10 mg/kg
as a positive standard, a on the hot plate latency and b on the tail flick latency, in alloxan treated mice (DIA) and untreated (NORM). “*”
means p<0.05 compared with control. Data (n=7) are expressed in mean±S.E.M.
Table 4 Subchronic effect of A. coronaria, SARF, and GLA on body weights in alloxan-induced diabetic mice.
|
Group
|
Dose (mg/kg)
|
Mean body weight±S.E.M. (gm) (% increase from the vehicle control)
|
|
1st day
|
3rd day
|
5th day
|
8th day
|
|
Control
|
__
|
26.40±0.50
|
26.50±0.60
|
26.66±0.97
|
26.72±0.70
|
|
Diabetic controla
|
__
|
27.18±0.70
|
27.60±0.20 (4.2%)
|
27.65±0.80 (3.7%)
|
27.00±0.50
(-0.4%)
|
|
GBa
|
5
|
25.40±0.70
|
30.67±1.70 (8.1%)
|
31.04±0.40 (8.8%)
|
32.87±1.10* (13.7%)
|
|
A. coronaria
a
|
50
|
28.00±1.80
|
30.10±1.30
(0.4%)
|
30.50±1.50
(1.8%)
|
30.63±1.40*
(2.25%)
|
|
A. coronaria
a
|
100
|
29.46±2.70
|
30.30±1.10
(3.0%)
|
30.90±1.30
(5.2%)
|
31.20±1.10*
(6.3%)
|
|
A. coronaria
a
|
200
|
28.46±2.10
|
30.88±1.70
(9.1%)
|
31.77±1.40
(12.5%)
|
32.34±1.90*
(14.7%)
|
|
SARFa
|
70
|
29.55±0.40
|
30.85±0.80
(4.7%)
|
32.61±1.20
(11.1%)
|
33.71±1.40*
(15.1%)
|
|
GLAa
|
25
|
29.56±0.80
|
28.66±0.30
(4.0%)
|
30.99±0.90
(5.2%)
|
32.05±0.40*
(9.0%)
|
|
GLAa
|
50
|
29.10±0.40
|
28.50±0.40
(5.2%)
|
31.50±0.50
(8.9%)
|
33.05±0.80*
(14.6%)
|
|
GLAa
|
100
|
28.50±0.20
|
27.90±0.70
(5.3%)
|
32.05±0.30
(13.4%)
|
33.09±0.20*
(17.3%)
|
S.E.M.: mean standard error
* p<0.05 significant from the control animals
a Compared to vehicle control
CAT enzyme activity, analyzed in alloxan-provoked diabetic mice, was significantly
(p<0.001) declined relative to normal controls ([Table 5]). After eight days of AC administration using 50, 100, and 200 mg/kg doses, the
antioxidant CAT levels were significantly (p<0.05) increased by 40.2, 40.7, and 59.0%,
respectively, as compared to the diabetic control group. The CAT levels were also
increased by 54.0% 8 days post-administration of the isolated SARF (70 mg/kg) as related
to the diabetic control group. Moreover, compound 3 at doses 25, 50 and 100 mg/kg improved CAT levels by 10.0, 15.5, and 29.8%, respectively,
after 8 days of administration as compared to the diabetic control group. The lipid
peroxidation, measured as TBARS levels, analyzed in alloxan-provoked diabetic mice,
was significantly (p<0.05) increased relative to normal control ([Table 6]). After 8 weeks of AC (200 mg/kg), SARF (70 mg/kg), and compound 3 (100 mg/kg) administration, the TBARS levels were significantly (p<0.05) decreased by 72.1, 63.8, and 46.6%, respectively, as compared to the diabetic
vehicle group. The glutathione levels, analyzed in alloxan-provoked diabetic mice,
were significantly (p<0.05) decreased relative to normal controls ([Table 6]). After 8 weeks of AC (200 mg/kg), SARF (70 mg/kg), and compound 3 (100 mg/kg) administration, the glutathione levels were significantly (p<0.05) elevated
by 66.6, 47.7 and 26.0%, respectively, as compared to the diabetic vehicle group.
As shown, the herbal extract showed a more powerful antioxidant effect than the isolated
active fraction and compound 3, which apparently was due to the additive properties of the active principles along
with other compounds in the plant extract.
Table 5 In vivo assessment of the antioxidant activity of A. coronaria, SARF, and GLA using CAT levels in serum of alloxan-induced diabetic mice.
|
Group
|
Dose (mg/kg)
|
Catalase level±S.E.M. (kU/I)
|
|
1st day
|
3rd day
|
5th day
|
8th day
|
|
Control
|
__
|
37.50±1.40
|
38.10±1.50
|
39.26±1.20
|
38.12±1.60
|
|
Diabetic controla
|
__
|
19.71±1.60***
|
19.63±1.30***
|
19.51±1.90***
|
18.75±1.40***
|
|
GBb
|
5
|
19.10±1.70
|
21.50±1.70
|
28.04±1.40
|
28.87±1.00**
|
|
A. coronaria
a
|
50
|
19.61±1.20
|
20.90±1.60
|
24.16±2.40
|
26.28±3.10*
|
|
A. coronaria
a
|
100
|
20.13±1.70
|
22.92±1.20
|
23.10±2.70
|
26.38±2.90*
|
|
A. coronaria
a
|
200
|
18.94±2.10
|
22.16±1.70
|
27.10±2.50
|
29.82±1.60*
|
|
SARFa
|
70
|
20.44±1.60
|
26.73±1.40
|
28.64±1.80
|
28.87±1.30**
|
|
GLAa
|
25
|
19.12±1.90
|
19.47±2.70
|
19.45±2.65
|
20.55±2.60**
|
|
GLAa
|
50
|
20.89±2.10
|
20.44±2.50
|
20.80±1.90
|
21.65±2.40**
|
|
GLAa
|
100
|
19.22±2.20
|
21.44±2.80
|
23.10±2.10
|
24.34±1.90**
|
S.E.M.: mean standard error
* p<0.05 significant from the control animals
** p<0.01 significant from the control animals
*** p<0.001 significant from the control animals
a Compared to vehicle control
b Compared to diabetic control
Table 6 Effect of A. coronaria, SARF and GLA on alloxan-induced alterations in TBARS and reduced GSH (mean±S.E.M.).
|
Group
|
Dose (mg/kg)
|
TBARS Level (nM/100 g) GSH (µg/mg)
|
|
Predose
|
8th Week
|
Predose
|
8th Week
|
|
NORM
|
__
|
0.75±0.10
|
0.78±0.20
|
72.70±2.50
|
73.80±2.5
|
|
Vehiclea
|
__
|
2.40±0.15*
|
2.90±0.10*
|
44.60±2.00*
|
41.90±2.40*
|
|
A.coronaria
b
|
200
|
2.45±0.18
|
0.81±0.12*
|
46.40±2.40
|
69.80±2.30*
|
|
SARFb
|
70
|
2.60±0.17
|
1.05±0.20*
|
45.80±2.30
|
61.90±2.20*
|
|
GLAb
|
100
|
2.50±0.20
|
1.55±0.25*
|
47.50±1.90
|
52.80±1.90*
|
S.E.M.: mean standard error
* p<0.05 significant from the control animals.
a Compared to normal control.
b Compared to vehicle control.
After 8 weeks of development of diabetes, diabetic mice developed thermal hyperalgesia
marked by a significant latency decrease to noxious heat (8.49±0.19 S at baseline
to 4.80±0.20 S in hot plate latency, and 5.29±0.02 S at baseline to 2.16±0.02 s in
tail flick latency; [Fig. 4]). To validate the procedure, tramadol (TRA; 10 mg/kg) was utilized as a positive
control and decreased alloxan-induced thermal hyperalgesia, elevating the hot plate
withdrawal latency by 85.2%, and that of tail-withdrawal latency by 173.1%, 8 weeks
post-administration ([Fig. 5]). Treatment of mice with various doses of AC (50, 100, and 200 mg/kg, orally) increased
the hot plate latency by 85.0, 87.5 and 95.6%, respectively, and that of tail-withdrawal
latency by 72.2, 89.9, 103.7%, respectively, 8 weeks post-administration as compared
to the diabetic control group ([Fig. 4]). In addition, SARF (70 mg/kg) also decreased alloxan-induced thermal hyperalgesia,
elevating the hot plate withdrawal latency by 54.0% and of that of tail-withdrawal
latency by 94.4%, 8 weeks post-administration ([Fig. 4]). Moreover, the oral administration of various doses of compound 3 (25, 50, 100 mg/kg) resulted in an increase of the hot-plate latency by 70.8, 74.8
and 79.2%, respectively, and that of tail-withdrawal latency by 62.0, 80.5, 94.4%,
respectively, 8 weeks post-administration as compared to the diabetic control group
([Fig. 5]).
Fig.5 Effect of glaucine (GLA) using tramadol (TRA) 10 mg/kg as a positive standard, a on the hot plate latency and b on the tail flick latency, in alloxan treated mice (DIA) and untreated (NORM). “*”
means p<0.05 compared with control. Data (n=7) are expressed in mean±S.E.M.
Mechanical allodynia was provoked in alloxan-induced diabetic mice after 8 weeks of
diabetes induction, which was marked by a significant decrease in paw-withdrawal threshold
upon application of Von-Frey filaments (9.10±0.20 g at baseline to 2.55±0.19 g after
8 weeks for vehicle-treated diabetic group; [Table 7]). The management of neuropathic animals with AC (200 mg/kg), SARF (70 mg/kg) and
compound 3 (100 mg/kg) significantly reduced the tactile allodynia, 8 weeks post-administration,
by 7.0, 6.0, and 5.3 folds, respectively ([Table 7]). To validate the procedure, treatment of neuropathic mice with TRA (10 mg/kg, utilized
as positive-control) also lowered the mechanical allodynia by 8.8 folds 8 weeks after
the treatment ([Table 7]).
Table 7 The effect of A. coronaria, SARF, GLA, and tramadol (TRA) 10 mg/kg on Von Frey filaments tactile allodynia thresholds
in neuropathic model in alloxan-induced diabetic mice.
|
Group
|
Dose (mg/kg)
|
Tactile allodynia thresholds±S.E.M. (gm)
|
|
Predose
|
2nd Week
|
4th Week
|
6th Week
|
8th Week
|
|
NORM
|
__
|
8.70±0.20
|
8.80±0.22
|
9.20±0.30
|
8.80±0.24
|
9.10±0.20
|
|
Vehiclea
|
__
|
2.50±0.21***
|
2.80±0.30***
|
2.45±0.27***
|
2.50±0.20***
|
2.55±0.19***
|
|
TRAb
|
10
|
3.00±0.10
|
24.00±1.00
|
24.50±1.10
|
24.60±1.30
|
25.10±1.00**
|
|
A.coronaria
b
|
200
|
3.20±2.20
|
7.50±0.12
|
8.70±0.20
|
17.00±0.40
|
20.50±0.20*
|
|
SARFb
|
70
|
3.00±0.10
|
8.00±0.20
|
8.60±0.40
|
16.80±0.30
|
18.00±0.50*
|
|
GLAb
|
100
|
3.10±0.20
|
7.50±0.30
|
7.80±0.50
|
13.00±1.20
|
16.00±1.20*
|
S.E.M.: mean standard error
* p<0.05 significant from the control animals
**p<0.01 significant from the control animals
*** p<0.001 significant from the control animals
a Compared to normal control
b Compared to vehicle control
The mortality rate in the untreated diabetic group was significantly increased (36.4%)
in comparison to the normal non-diabetic group. In contrast, animals treated with
AC showed a lower rate of mortality in correlation to the vehicle-control group (10.6%).
For SARF- and compound 3-treated groups, animals showed a lower mortality rate in comparison to the vehicle-control
group (11.4 and 13.7%).
Discussion
In folk medicine, A. coronaria is an essential medicinal plant used by the Lebanese population as antineuralgic
and antirheumatic [7]. Nevertheless, the exact neuroprotective activity of AC and its most active principles
were not studied thoroughly. Thus, this study was designed to assess AC and its possible
active principles for their phytochemical and pharmacological properties in experimental
models of hyperglycemia, thermal and mechanical nociception, as well as to investigate
mortality rates before and after administration. However, it is the first report demonstrating
a potential of A. coronaria for improvement in alloxan-induced neuropathy in mice. The bio-guided fractionation
and isolation of the active principles in AC showed that its most active fraction
is rich in alkaloids, hence it was named isolated alkaloid rich fraction (SARF). SARF
was found to be rich in the alkaloid glaucine (compound 3). Therefore, SARF and compound 3 were thoroughly studied to monitor their hypoglycemic and antinociceptive potentials.
In this study, chemical induction of T1DM was performed utilizing alloxan to provoke
and maintain hyperglycemia in mice during the experimental period. Alloxan-induced
persistent elevation in BGL resulted from an inadequate production and the release
of insulin attributed to pancreatic β-cells destruction by oxidative stress and superoxide radical formation provoked by
alloxan [22].
Data from the present study showed that the administration of AC, SARF and compound
3 for 6 h (acute) and 8 days (subchronic) ameliorated the elevated BGL of alloxan-induced
diabetic mice to near the BGL baseline range (107.25±7.30 mg/dL) in the non-diabetic
control group. Thus, this study has established the antidiabetic activity and a possible
future usage of AC for the amelioration of diabetes mellitus. Administration of various
doses of AC and compound 3 caused dose-dependent BGL lowering effects. However, the highest dose of AC showed
more powerful hypoglycemic activity than that of SARF and compound 3. Moreover, the highest doses of AC, SARF and compound 3 showed significant stabilizations of BGL for longer times (8 weeks) as indicated
by HbA1c-experiments. Furthermore, HbA1c levels showed more stabilization of BGL after
8 weeks of regular AC administration than in groups administered SARF and compound
3. Additionally, the administration of AC, SARF, and compound 3 caused a significant increase in mice BW. AC and SARF increased BW more significant
than compound 3. Nevertheless, to assess the activities of AC, SARF, and compound 3 to reverse alloxan-induced oxidative stress, CAT serum levels were monitored. Thus,
the administration of AC, SARF, and compound 3 showed significant elevations of CAT serum levels. However, AC showed more increase
in CAT serum levels than SARF and compound 3. In addition, the administration of AC, SARF, and compound 3 attenuated alloxan-induced raised levels of TBARS, a lipid peroxidation marker, and
significantly decreased levels of the endogenous antioxidant, glutathione. Thus, it
could be suggested that the antioxidant potential of AC, SARF, and compound 3 is taking a significant part in reversing alloxan-induced neuropathy, as indicated
before with similar compounds [23].
There is substantial evidence, that oxidative damage provoked by free radicals is
engaged in the pathogenesis of neurodegenerative diseases, like Parkinson and Alzheimer’s
disease [24]. Furthermore, ROS (reactive oxygen species) were reported to contribute to diabetes
and neuropathic-pain pathogenesis [25]
[26]
[27]. Previous reports utilizing other antinociceptive compounds with similar antioxidant
potentials strengthen the findings observed in mice receiving AC, SARF, and compound
3 [23]
[28].
Combined with the elevation in oxidative stress, alloxan showed a significant elevation
in BGL. Moreover, AC, SARF, and compound 3 showed powerful antidiabetic activity, reversing alloxan-induced hyperglycemia. However,
AC showed more powerful antidiabetic activity than its most active fraction (SARF)
and constituent (compound 3), indicating that there is an apparent additive effect between the AC constituents
potentiating its activity.
After 8 weeks of alloxan-induced diabetes, the mice showed evidence of thermal hyperalgesia
(8.49±0.19 S at baseline to 4.80±0.20 S in hot plate latency and 5.29±0.02 S at baseline
to 2.16±0.02 S in tail flick latency) and mechanical allodynia (9.10±0.20 g at baseline
to 2.55±0.19 g using Von Frey filaments), comparable to those illustrated before in
literature [12]
[20]
[29]
[30]. We investigated the antinociceptive potentials of AC, SARF, and compound 3 against hot plate and tail flick thermal hyperalgesia. Mice that were administered
AC, SARF, and compound 3 showed significant amelioration of thermal hyperalgesia. Moreover, the highest dose
of AC showed more hypoalgesic activity than that of SARF and compound 3. Furthermore, the antinociceptive activities of AC, compound 3, and SARF against mechanical allodynia were monitored utilizing Von Frey filaments.
Animals to which AC, SARF, and compound 3 were administered showed a significant amelioration of thermal hyperalgesia. However,
the highest dose of AC showed more amelioration of tactile allodynia than those of
SARF and compound 3. Thus, AC showed more powerful antinociceptive activity against thermal hyperalgesia
and mechanical allodynia than its most active fraction (SARF) and constituent glaucine
(3), showing that there is an apparent additive effect between AC constituents potentiating
its activity as concluded before in literature [12]
[20]
[29]
[30].
In conclusion, the findings of this work demonstrated that the ethanolic extract of
A. coronaria herb (AC) and its most active fraction (SARF) showed interesting antihyperglycemic
and antinociceptive activities and that its most active constituent was the alkaloid,
glaucine (compound 3). The results indicate a possible future use of AC in the management of diabetes
mellitus and DN. AC might have exerted the antinociceptive effects by the regulation
of blood glucose levels and reversing alloxan-induced oxidative stress. Comprehensively,
A. coronaria herb is a good candidate for further clinical investigation as a complementary therapy
with a satisfactory margin of safety for the management of diabetic neuropathy pain.
Materials and Methods
General
The protocols and experimental procedures ([Table 1]) used in this study were conducted abiding the standards of the local animal welfare
committee and were reviewed by the Beirut Arab University Institutional Review (IRB;
Register Number: 2016 A-0031-P-R-108).
A. coronaria herb
The A. coronaria herb was commercially purchased from Ibn El-Nafees herbalists (Beirut, Lebanon).
The herb was authenticated by Prof. J. Habib (LU, Lebanon) against an authentic sample,
and a reference sample was carefully preserved in the faculty’s herbarium (voucher
specimen number PS-15-14). The herb was dried on the ground in the shadow and stored
under refrigeration (4°C) until further processing.
A. coronaria extraction protocol
A. coronaria was extracted with 80% ethanol by sonication three consecutive times for 72 h at
normal pressure. The extracts were combined and dried utilizing a Buchi rotary evaporator
under reduced pressure at a temperature of 40°C. The dried extract was kept frozen
(−30°C) until the experiment time [20].
Bio-guided fractionation and isolation of A. coronaria active principles
The dried extract was dissolved in basified water (NH4OH, pH 9.5). The filtrate was
extracted with chloroform three consecutive times, and the extracts were combined
and concentrated. Then the sample was fractionated by analytical column chromatography.
RP-silica gel was used as stationary phase and a gradient mobile phase of a mixture
of (A) triethylamine (0.1%) and (B) acetonitrile was used (starting with 100% A, then
60% A, and finally 100% B). About 200 fractions were collected and every fraction
was examined for its hypoglycemic and antinociceptive effects. The 1H and 13C NMR spectra of the isolated compounds were measured in MeOH-d4 on a Bruker ARX-300
spectrometer. The main active fraction was injected directly into a JASCO RP-HPLC
apparatus, after dissolving in the mobile phase. The mobile-phase was formed of a
combination of 60% triethylamine (0.1%) and 40% acetonitrile at 1 mL/min flow rate,
utilizing a Merck C-18 endcapped-Lichrospher column (250.00×4.60 mm i.d.; 5.00 μM
particle size) as stationary-phase, at 40°C and 280 nm [17].
Statistical analysis
The results were measured in terms of the±standard error of the mean (S.E.M.). All
in vivo results were statistically assessed by one-way analysis of variance (ANOVA), then
the Dunnett test, using “OriginPro” statistics program, was done. The p-value≤0.05
was designated as a statistically significant result.
Supporting information
Details on chemicals, solvents and standards, animals, study design, assessment of
oxidative stress markers, and assessment of diabetic neuropathy methods are available
as Supporting Information.
