Antiulcer
Activity of Colebrookea oppositifolia Sm
MM Ghaisas1*, Surbhi Sharma2, GP
Ganu2 and RP Limaye3
1Indira
College of Pharmacy, Tathwade, Pune
411 033
2Padmashree
Dr. D.Y. Patil Institute of Pharmaceutical Sciences and Research, Dept. of
Pharmacology, Pimpri, Pune-18 (Maharashtra), India.
3BVDU Medical College and Hospital, Sangli.
ABSTRACT:
In the
present study, the antiulcer activity of hydroalcoholic extract of roots of
Colebrookea oppositifolia Sm. (CO) was evaluated in ethanol and swim
stress induced peptic ulcer models. In both the models, various parameters like
ulcer index, percentage protection, gastric wall mucus, catalase, reduced
glutathione (GSH), lipid peroxidation (LPO) and superoxide dismutase (SOD) were
estimated. CO
significantly reduced the ulcer index and increased the gastric wall mucus in
both the models. CO showed significant antioxidant activity as indicated by
significant decrease in LPO and increase in catalase, GSH and SOD levels in
stomach tissue homogenate. The results suggest that hydroalcoholic extract of roots of
Colebrookea oppositifolia possess antiulcer activity due to its
muco-protective action and antioxidant potential.
KEY WORDS: Antiulcer, Colebrookea
oppositifolia, Antioxidant.
INTRODUCTION:
Among the
different disorders of gastrointestinal system, peptic ulcer is more prevalent
and has greatest clinical impact1. It occurs due to imbalance
between offensive factors (acid-pepsin secretion, H. pylori, bile,
increased free radicals and decreased antioxidants) versus impaired mucosal
resistance (mucus, bicarbonate secretion, prostaglandins, blood flow and the
process of restitution and regeneration after cellular injury)2.
Numerous
plants and herbs are used to treat gastrointestinal disorders in traditional
medicine. Colebrookea oppositifolia
Sm. (Lamiaceae; Labiatae)3 is a woolly shrub4. It is a
subtropical plant mostly distributed in
the hilly valleys of India,
in subtropical Himalayas5. It is commonly known as binda, bindu and
pansra in Hindi. In folk medicine, stem is used to extract worms from bad sores
in leg6. In Nepal,
infusion of root is used to relieve peptic ulcers7. Leaves are used
to reduce sneezing in cold8 cataract, cough9, dysentery,
headache, conjunctivitis, sinusitis, gastric troubles10, mouth
ulcer, wound and bruises11. Plant is used in fractures, traumatic
injuries, rheumatoid arthritis12, asthma and tumor13. Colebrookea oppositifolia possesses CNS
depressant5, larvicidal14, antifertility5,
hepatoprotective15, wound healing activity13 and has
shown increase in bioavailability of amoxycilline16. Roots contain
5,6,7-trimethoxy flavones, 4',5,6,7-tetramethoxy flavone17, acylated
flavone glycoside, echioidinin 2'-O-β-D-(2''-O-acetyl) glucopyranoside,
5,6,7,8,5'-pentamethoxy-3',4'-methylenedioxyflavone,
5,2',6'-trihydroxy-7-methoxyflavone, kaempferol 7,4'-dimethyl ether 3-O-b-D
glucopyranoside18, triacontane, triacontanol, β-sitosterol,
palmitic acid, stearic acid, oleic acid19 and amino acid20. Present study was
aimed to explore the antiulcer and antioxidant activity of CO.
MATERIALS AND
METHODS:
MATERIALS:
Animals:
Swiss albino mice (25-30 gm) and Wistar rats
(180-250 gm) were purchased from National Toxicology Centre (NTC), Pune. The
animals had free access to feed pellets (Amrut Laboratory Animal Feed,
manufactured by Pranav Agro Industries Ltd., Sangli, Maharashtra)
and water ad libitum.
The Institutional Animal Ethics Committee approved the
experimental protocol (IAEC registration no. 198/ 99/ CPCSEA).
Chemicals:
Alcian blue,
carboxy methyl cellulose sodium salt, absolute alcohol, 5, 5– dithio bis (2- nitro
benzoic acid), EDTA, n-hexane, magnesium chloride hexahydrate crystals,
sucrose, tris buffer (Research Lab Fine Chem. Industries, Mumbai),
thiobarbituric acid (Spectrochem Pvt. Ltd, Mumbai), trichloroacetic acid,
(Universal Laboratories Pvt. Ltd, Mumbai) and tyrosine (Central Drug House Pvt.
Ltd, Mumbai).
Drugs:
Adrenaline bitartarate Injection I.P.
(Helichem Lab Pvt. Ltd., Palghar) and pantaprazole sodium sesquihydrate (Matrix
Laboratories Ltd., Secunderabad).
Methods:
Roots of Colebrookea oppositifolia Sm. were collected from Ranital area in
District Kangra, Himachal Pradesh in February, 2009. After collection material
was authenticated by Dr. Brij Lal (Scientist, Biodiversity Division) at
Institute of Himalayan Bioresource Technology (I.H.B.T.) Palampur (Himachal Pradesh, India) and preserved a specimen
(Collection # PLP 12576) at their herbarium.
Extraction of Colebrookea
oppositifolia Sm.:
The collected material was shade dried and powdered.
Powder was defatted using n- Hexane. Extraction was done using continuous hot
extraction method with ethanol and water (70:30) at 60 şC for 24 hrs. The
extract was vacuum dried, solvent recovered and stored in refrigerator till
further use. The hydroalcoholic extract
was dark brown colour (yield 5.6% w/w, pH in between 7-8).
Phytochemical investigation of the extract21:
Various phytochemical tests
were performed included Biuret test, Dragendroff’s test, Foam test, Ninhydrin test and Shinoda test
for detecting the presence of proteins, alkaloids, saponins, amino acids and
flavonoids.
Acute Toxicity Study22:
Mice were fasted
overnight prior to drug administration. Each animal received a single dose of
hydroalcoholic extract of Colebrookea
oppositifolia (2,000 mg/ kg, p.o). Food was withheld for further 3–4 h. Animals
were observed individually at least once for first 30 min after dosing,
periodically during the first 24 h (with special attention given during the
first 4 hours) and daily thereafter for a period of 14 days. The animals were
observed for a period of 2 weeks for mortality.
Selection of dose of the
extract:
No signs of acute
toxicity were observed at 2000 mg/ kg, p.o. Hence further study was carried out
at doses of 100, 200 and 400 mg/kg, p.o. in mice.
Pharmacological Screening:
Ethanol induced peptic ulcers in rats23:
Animals were divided into six groups (n=6). Group I and II received 1 % w/v NaCMC. Group III received pantaprazole orally
(20 mg/ kg) twice daily for 5 days. Group IV, V and VI received CO 70, 140 and
280 mg/ kg, p.o., respectively, twice daily for 5 days. On 6th day
gastric ulcers were induced in group II to VI by administering ethanol (1 ml/
200 g). The animals were sacrificed 1h after ethanol administration by cervical
dislocation. The stomachs were removed and analyzed for the following
parameters.
Ulcer score24 and percentage protection25:
The number of
ulcers was counted under a magnifying glass. Each ulcer was then measured with
a verniar calliper to assess the diameter and the percent protection was
calculated. The ulcer index was expressed as the sum of scores given to
ulcerative lesions as described below
Score 1: maximal
diameter of 1 mm.
Score 2: maximal
diameter of 1-2 mm.
Score 3: maximal
diameter of 2-3 mm.
Score 4: maximal
diameter of 3-4 mm.
Score 5: maximal
diameter of 4-5 mm.
Score 10: an
ulcer over 5 mm in diameter.
Score 25: a
perforated ulcer.
% Protection = (UI control − UI treated) X 100
(UI
control)
UI - ulcer index
Gastric wall mucus (barrier mucus) determination26:
A segment from
stomach was weighed. Each segment was transferred immediately to 10 ml of 0.1%,
w/ v alcian blue solution (in 0.16 M sucrose solution, buffered with 0.05 M
sodium acetate adjusted to pH 5.8 with HCl). After immersion for 2 h, excess
dye was removed by two successive rinses with 10 ml of 0.25 M sucrose, first
for 15 min and then for 45 min. Dye complexed with gastric wall mucus was
extracted with 10 ml of 0.5 M magnesium chloride (MgCl2) by shaking
intermittently for 1 min at 30 minutes interval for 2 h. The resulting blue
solution was shaken vigorously with an equal volume of diethyl ether and then
centrifuged at 3000 rpm for 10 min and the absorbance of the aqueous layer
against blank standard MgCl2 solution was recorded at 580 nm. The
quantity of alcian blue recovered per gram of tissue was then calculated.
In vivo antioxidant activity28:
Stomachs were
homogenized in tris buffer (10 mM, pH 7.4) at a concentration of 10% (w/v). The
homogenates were centrifuged at 10,000 rpm for 20 min. The clear supernatant was used for the
estimation of lipid peroxidation and endogenous antioxidant enzymes.
Determination of lipid
peroxidation:
The tissue
homogenate (supernatant) (2.0 ml) was added to 2.0 ml of freshly prepared 10%
(w/v) trichloroacetic acid (TCA) and the mixture was allowed to stand in an ice
bath for 15 min. After 15 min, the precipitate was separated by centrifugation
and 2.0 ml of clear supernatant solution was mixed with 2.0 ml of freshly
prepared 0.67% thiobarbituric acid (TBA). The resulting solution was heated in
a boiling water bath for 10 min. It was then immediately cooled in an ice bath
for 5 min. The color developed was measured at 532 nm against reagent blank.
The values are expressed as nmoles of malonaldehyde/ g tissue.
Determination of superoxide
dismutase (SOD):
Tissue homogenate
(0.5 ml) was diluted with 0.5 ml distilled water, to which 0.25 ml of ice-cold
ethanol and 0.15 ml of ice-cold chloroform was added. The mixture was mixed
well for 5 min and centrifuged at 2500 rpm. To 0.5 ml of supernatant, 1.5 ml of
carbonate buffer (0.05 M, pH 10.2) and 0.5 ml of EDTA solution (0.49 M) were added.
The reaction was initiated by the addition of 0.4 ml of epinephrine (3 mM) and
the change in optical density/minute was measured at 480 nm against reagent
blank. SOD activity was expressed as units/ g tissue.
Determination of catalase:
Hydrogen peroxide
(30 mmol/ l) was added to 2 ml of diluted sample. The blank was prepared by
mixing 2ml of diluted sample with 1ml of phosphate buffer (50 mmol/ l, pH 7.0).
The decrease in absorbance was measured at 240 nm. Catalase activity was
expressed as µmoles of H2O2 consumed/ min/ g tissue.
Determination of reduced
glutathione (GSH):
Supernatant (2ml)
and 20% TCA (2 ml) were mixed. The precipitated fraction was centrifuged and to
0.25 ml of supernatant, 2 ml of 0.6mM 5, 5-Dithiobis (2-nitro benzoic acid)
reagent was added. The final volume was made up to 3 ml with phosphate buffer
(0.2 M, pH 8.0). The colour developed was read at 412 nm against reagent blank.
The amount of reduced GSH was expressed as µmoles of GSH/ g tissue.
Swimming stress induced peptic
ulcers in mice29, 23.
Animals were
divided into six groups (n=6). Group I
and II received 1 % w/v
NaCMC. Group III received pantaprazole (28 mg/ kg, p.o.) twice daily for 5
days. Groups IV, V and VI received CO 100, 200 and 400 mg/kg, p.o.,
respectively, twice daily for 5 days. On 6th day, thirty minutes
after respective treatment, the animals from group II to VI were placed
individually inside a vertical cylinder filled with water maintained at 20–25
°C up to a height of 10 cm. Mice were removed from the cylinder after 3 h and
sacrificed. The stomachs were removed and analyzed for the following
parameters:
Ulcer score24,
percentage protection25, gastric wall mucus (barrier
mucus) content26 and in vivo antioxidant activity28.
Statistical evaluation:
Data was expressed
as mean ± SEM. Statistical analysis was done using either unpaired t-test
or one way analysis of variance (ANOVA) followed by Dunnett’s multiple
comparison test. Probability of less than 0.05 was considered statistically
significant.
RESULTS:
Phytochemical screening:
The phytochemical screening indicated the presence of
flavonoids, tannins, saponins, alkaloids, amino acids and proteins in the
hydroalcoholic extract of Colebrookea
oppositifolia.
Acute toxicity studies:
No signs of mortality were observed on oral
administration of hydroalcoholic extract of Colebrookea
oppositifolia up to the dose of 2000 mg/ kg in mice. Hence the doses of
100, 200 and 400 mg/ kg, p.o. were selected for further study.
In ethanol
induced ulcer model CO 140 and 280 treated groups showed significant (p<
0.01) decrease in ulcer index in comparison
to ulcerated control. CO 140 and 280 treated groups showed significant (p<
0.01) increase in gastric wall mucus in comparison to ulcerated control.
Pantaprazole and CO 280 treated groups showed significant (p< 0.05) and
(p< 0.01) increase respectively in reduced glutathione levels in comparison to
ulcerated control. CO 280 group showed significant (p< 0.01) decrease in lipid peroxidation in comparison to ulcerated control. CO
140 and 280 treated groups showed significant (p< 0.001) increase in superoxide dismutase levels in
comparison to ulcerated control (Table 1 and 2).
In swim stress induced peptic ulcer model, CO 200 and
400 treated groups showed significant (p< 0.01) decrease in ulcer index in comparison to ulcerated control. CO
200 and 400 treated groups showed significant (p< 0.05) increase in gastric
wall mucus in comparison to ulcerated control. CO 400 treated group showed
(p< 0.05) significant increase in catalase
levels in comparison to ulcerated control. CO 200 and 400 treated groups
showed significant increase (p< 0.05) in
reduced glutathione levels in
comparison to ulcerated control. CO 200 and 400 treated groups showed
significant (p< 0.01) decrease in lipid
peroxidation in comparison to
ulcerated control. CO 400 treated group showed significant (p< 0.01) in
increase in superoxide dismutase
levels in comparison to ulcerated control (Table 3 and 4).
DISCUSSION:
Peptic ulcer is a
common cause of discomfort. The gastric mucosal protection can be mediated
through a number of mechanisms that include enhancement of the gastric mucosal
defense through increase in mucus and /or bicarbonate production, reducing the
volume of gastric acid secretion or by simply neutralizing the gastric acidity30.
In the present study, pH of CO was found to be in between 7-8. The weak basic
pH of CO may contribute to antiulcer activity by neutralizing the gastric acid.
Platelet thrombi, damage to capillary endothelium and
release of arachidonate metabolites, especially LTC4/ D4 (metabolites
of lipooxygenase pathway) and PAF play a key role in the development of ulcers,
induced by irritant agents such as ethanol31. Oral administration of
absolute ethanol produces a decrease in gastric blood flow and induces
solubilization of mucus constituents in the stomach. The mucus contributes to
defense by providing a physical barrier to aggressive factors and acting as a
lubricant to reduce physical abrasion of the mucosa32. The reactive
oxygen species (ROS) generated by the metabolism of arachidonic acid,
platelets, macropages and smooth muscle cells may contribute to gastric mucosal
damage33. Lesions caused by ethanol have been attributed to free
radical formation and subsequent formation of lipid peroxidation products34.
Tissue damage begins with the formation of lipid radicals in cell membranes,
continues with the conversion of these radicals to lipid hydro peroxides, and
finally to products such as aldehyde, alkene sand monoaldehyde 35.
Ethanol after metabolization in the body releases superoxide anion25,
these free radicals cause breaking of DNA strands and protein denaturation36.
GSH may protect mucus, since mucus subunits are joined by disulfide bridges
that, if reduced, render mucus water-soluble 32.
In the present study, reduction of ulcer index was
reflected by increase in % protection with increase in dose of CO in
experimental model of ethanol induced ulcers. CO prevented ethanol induced gastric damage by increasing the
gastric wall mucus significantly. This may be explained with a correlation to
strengthening of the defense factors of gastric mucosa.
RESULTS:
Ethanol induced peptic ulcers:
Table 1: Effect of
hydroalcoholic extract of Colebrookea
oppositifolia on ulcer index, percentage protection and gastric wall mucus
in ethanol induced peptic ulcers.
|
Groups (n=6)
|
Ulcer index
|
Protection (%)
|
Gastric wall mucus (µg
Alcian blue/ gm tissue)
|
|
UC
|
93.00 ± 2.25
|
-
|
68.50 ± 2.59
|
|
Panta
|
11.33 ± 2.23**
|
87.81
|
96.50 ± 2.52**
|
|
CO70
|
87.66 ± 1.99
|
6.74
|
75.00 ± 2.00
|
|
CO140
|
56.66 ± 2.31**
|
39.07
|
85.66 ± 3.04**
|
|
CO280
|
47.33 ± 1.94**
|
49.10
|
92.00 ± 1.59**
|
UC- ulcerated control; Panta- pantaprazole (20 mg/ kg,
p.o.); CO 70, 140, 280- hydroalcoholic
extract of Colebrookea oppositifolia 70, 140 and 280 mg/ kg, p.o., respectively.
Values are expressed as mean ± SEM, compared with ulcerated control by one-way
ANOVA followed by Dunnett’s multiple comparisons test (** p< 0.01).
Table 2: Antioxidant effect of hydroalcoholic extract of Colebrookea oppositifolia in stomach in
ethanol induced peptic ulcers.
|
Groups (n=6)
|
Catalase (µmoles H2O2 consumed/
g tissue)
|
GSH (µmoles/ g tissue)
|
LPO (nmoles
MDA/ g tissue)
|
SOD (Units/ g tissue)
|
|
NC
|
9.26 ± 0.92
|
6.76 ± 1.12
|
6.66 ± 1.81
|
10.02 ± 0.82
|
|
UC
|
3.30 ± 0.76#
|
1.96 ± 0.76#
|
20.63 ± 0.64#
|
3.32 ± 0.15#
|
|
Panta
|
8.09 ± 1.00**
|
6.73 ± 0.63**
|
9.93 ± 1.30**
|
8.98 ± 0.42**
|
|
CO70
|
4.51 ± 0.63
|
4.60 ± 0.40
|
17.16 ± 0.71
|
3.40 ± 0.35
|
|
CO140
|
5.46 ± 0.62
|
5.90± 0.66*
|
15.30 ± 1.44*
|
5.98 ±0.18**
|
|
CO280
|
7.28 ± 1.06*
|
6.26 ± 0.95**
|
7.66 ± 1.42**
|
9.10 ± 0.64**
|
NC- normal control; UC- ulcerated control; Panta-
pantaprazole (20 mg/ kg,p.o.); CO 70, 140 and 280- hydroalcoholic extract of Colebrookea oppositifolia 70, 140 and
280 mg/ kg, p.o., respectively; GSH-reduced glutathione; LPO- lipid
peroxidation; MDA- malonaldehyde; SOD- superoxide dismutase. Values are
expressed as mean ± SEM, compared with normal control by unpaired t-test (#p< 0.05) and with
ulcerated control by one-way ANOVA followed by Dunnett’s multiple comparisons
test (*p< 0.05, **p< 0.01).
Swim stress induced peptic
ulcers:
Table 3: Effect of
hydroalcoholic extract of Colebrookea
oppositifolia on ulcer index, percentage protection and gastric wall mucus
in swim stress induced peptic ulcers.
|
Groups (n=6)
|
Ulcer Index
|
Protection (%)
|
Gastric wall mucus (µg Alcian
blue/ gm tissue)
|
|
UC
|
32.66 ± 1.64
|
-
|
67.50 ± 2.18
|
|
Panta
|
12.00 ± 1.06**
|
63.25
|
98.83 ± 2.24**
|
|
CO100
|
28.83 ± 1.38
|
11.72
|
68.00 ± 2.35
|
|
CO200
|
23.33 ± 1.14**
|
28.56
|
76.16 ± 2.83*
|
|
CO400
|
18.33 ± 0.88**
|
43.87
|
77.66 ± 1.76*
|
UC- ulcerated control; Panta- pantaprazole (28 mg/ kg,
p.o.); CO 100, 200 and 400- hydroalcoholic extract of Colebrookea oppositifolia 100, 200 and 400 mg/ kg, p.o.,
respectively. Values are expressed as mean ± SEM, compared with ulcerated
control by one-way ANOVA followed by Dunnett’s multiple comparisons test (*p< 0.05, **p< 0.01).
Table 4: Antioxidant effect of hydroalcoholic extract of Colebrookea oppositifolia in stomach in
swim stress induced peptic ulcers.
|
Groups
(n=6)
|
Catalase (µmoles H2O2
consumed/ g tissue)
|
GSH
(µmoles/ g tissue)
|
LPO (nmoles
MDA/g tissue)
|
SOD
(Units/ g tissue)
|
|
NC
|
9.26 ± 0.27
|
6.93 ± 0.90
|
7.06 ± 1.63
|
11.56 ± 0.40
|
|
UC
|
6.37 ± 0.54#
|
3.12 ± 0.24#
|
15.92 ± 1.47#
|
7.83 ± 0.30##
|
|
Panta
|
9.22 ± 0.76**
|
6.70 ± 0.52**
|
8.73 ± 0.61**
|
10.66 ± 0.43**
|
|
CO100
|
6.66 ± 0.40
|
3.86 ± 0.60
|
14.36 ± 0.58
|
8.30 ± 0.43
|
|
CO200
|
7.95 ± 0.17
|
5.12 ± 0.42*
|
10.60 ± 1.26*
|
9.66 ± 0.83
|
|
CO400
|
8.35 ± 0.18*
|
5.43 ± 0.53*
|
9.49 ± 1.98*
|
10.21 ± 0.34*
|
NC- normal control; UC- ulcerated
control; Panta-pantaprazole (28 mg/ kg, p.o.);
CO 100, 200 and 400 - hydroalcoholic
extract of Colebrookea oppositifolia 100,
200 and 400 mg/ kg, p.o., respectively; GSH-
reduced glutathione ; LPO- lipid peroxidation; MDA- malonaldehyde; SOD-
superoxide dismutase. Values are expressed as mean ± SEM, compared with normal
control by unpaired t-test (#p<
0.01, ##p< 0.001) and with ulcerated control by one-way ANOVA
followed by Dunnett’s multiple comparisons test (*p< 0.05, **p<
0.01).
It was observed
that MDA, a lipid peroxidation product increased significantly in ulcerated
control which was decreased significantly by CO in gastric tissue. In the
present study, exposure to ethanol led to a decrease in the levels of GSH in
gastric mucosa. Pretreatment with CO significantly increased the GSH content in
the gastric tissue. The SOD and catalase activity was decreased significantly
in stomach tissue on administration of ethanol when compared to the normal
control. CO significantly increased catalase and SOD levels in stomach
homogenate.
Ulcers due to
stress are both due to physiological and psychological factors37.
Stress hormones and vagal over-activity, increase the glandular secretion38
and histamine release29 respectively, which increase the secretion
of gastric acid. The highly concentrated hydrochloric acid (0.1 M) secreted by
the parietal cells of the gastric mucosa in the fundus denatures proteins in
plasma membranes and catalyses the hydrolysis of polysaccharide moieties of
proteoglycans in the protective mucous coat covering the luminal surface of the
stomach to a perilous extent38.
Stress induced
ulcers are due to increase in free radical generation apart from acid pepsin
factors23. SOD converts the reactive superoxide radicals to H2O2,
which if not scavenged by the catalase can by itself cause lipid peroxidation
by increase in the generation of hydroxyl radicals23. A significant
decrease in catalase levels might lead to increase in accumulation of reactive
products and thus, cause increased lipid peroxidation and tissue damage. H2O2
can stimulate acid secretion to aggravate mucosal damage and inhibits
prostaglandin (PG) production to inhibit gastroprotection offered by PGs39.
Oral
administration of CO was effective in lowering ulcer index significantly and
increasing gastric wall mucus significantly in swim stress induced peptic ulcer
model. In the present study, treatment with CO reversed the oxidative changes
induced by stress in stomach. The ulcer protective effects of CO could be due
to its antioxidant activity leading to restoration of GSH, SOD and catalase
levels.
The flavonoids, tannins, saponins and alkaloids present CO may be
responsible for the gastroprotection. Flavonoids, by virtue of their high
chemical reactivity with membrane phospholipid, have been reported to affect
enzymes altering endogenous phospholipid metabolism leading to inhibition of
synthesis of leukotriens and PAF31 which are released by irritant
action of ethanol. It is also known that flavonoids offer gastroprotection via
decreased histamine secretion from mast cells39. The antioxidant
activity of flavonoids is efficient in trapping superoxide anion, hydroxyl,
peroxyl and alcohoxyl radicals33.
Thus, protective efficacy of CO against peptic ulcers can be attributed
to antisecretory, gastroprotective and antioxidant activities, which may be due
to the presence of flavonoids, tannins and saponins. However, further studies
are required for elucidating the exact mechanism of action.
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