Background: The aim
of this study was to evaluate the therapeutic effect of pilescure in
hemorrhoids induced Sprague Dawley male rats.
Methods: Total
30 male Sprague Dawley rats were selected and randomized into three groups of
ten animals each. Group I was control, whereas group II and group III were
hemorrhoid and hemorrhoid plus pilescure treated groups. Hemorrhoid was induced
in group II and group III by application of 6.0% croton oil preparation. At the
end of experiment, rectoanal weight was recorded and blood sample was for
measurement of lipid peroxidation, white blood cells, nitric oxide and
C-reactive protein levels along with some antioxidants enzyme activities and
cytokine parameters in all groups. The histological analysis was performed in
recto-anal tissue of all groups.
Results: The
findings revealed that the LD50 of drug was found more than 2000
mg/kg in acute toxicity study. No clinical sign of toxicity, mortality were
found up to 2000 mg/kg body weight. In efficacy study, there was a
statistically significantly (p<0.0001) reduction in recto-anal weight, recto-anal
coefficient and the levels of WBC, neutrophil, CRP, NO, lipid peroxidation and
cytokines parameters along with increased stool frequency, level of hemoglobin,
platelet count and some antioxidant enzyme activities in pilescure treated
group as compared with hemorrhoids induced group and these levels were comes
back to control group after treatment with drug for three weeks. The
histopathological finding also clearly showed that inflammatory cells were
found low after treatment with drug as compared to hemorrhoid induced group.
Conclusion: The
study concluded that pilescure is most effective ayurvedic medicine for
reduction of inflammation along with increase antioxidant enzymes and
hematological parameters and decrease oxidative stress during hemorrhoids condition.
Keywords:
Pilescure, Hemorrhoids, Hematological and oxidative stress, Antioxidant and
anti-inflammatory, Recto-anal tissue
Abbreviations: RAC:
Recto-Anal Coefficient; CRP: C-Reactive Protein; WBC: White Blood Cells MDA:
Malonaldialdehyde; TNF-α: Tumor Necrosis Factor-α; IL-β: Interleukin-β; IL-6:
Interleukin-6; NO: Nitric Oxide; SOD: Superoxide Dismutase; GR: Glutathione
Reductase; GPx: Glutathione Peroxidase; GSH: Reduced Glutathione
The treatment of hemorrhoids in modern medicine
is still in infancy. There are many topical agents or suppositories are
available for treatment of hemorrhoids, there is little evidence to support and
their use [12,13]. Now a day's extensive research is going on in the field of
ayurveda for utilizing the natural resources for treating hemorrhoids. Various
medicinal plants are used in the treatment of hemorrhoids. Pilescure is one of
the ayurvedic medicines which consist of poly-ingredients. The ingredients of
pilescure are Abhaya (Terminalia chebula),
Nagkesar (Mesua ferrea), Arand mool (Ricinus communis) and Purified alum
(Sudhasphatika). These individual ingredients have been reported as an effective
for management of rectal pain, rectal bleeding, anti-inflammatory and
constipation, etc. [14-17]. Terminalia
chebula is one of them which is also cited in Charaka's Arshoghna
Mahakashaya [18]. This drug is manufacture under the GMP (Good Manufacturing
Practices) condition. Its botanical identification, quality parameters and
ayurvedic criteria are complied with pharmacopoeia standard. So the purpose of
this study was to investigate the protective effect of polyherbal drug
pilescure in experimental model of croton oil induced hemorrhoid in Sprague
Dawley rats.
MATERIALS
AND METHODS
Chemicals
All biochemical such as reduced glutathione,
NADPH, thiobarbutric acid, SDS, Croton oil, used in the present study were
procured from Sigma, St. Louis, MO, USA. Other biochemical and chemicals
(butanol, pyridine and diethyl ether, etc.) purchased locally were of HiMedia
and analytical grade.
Drug
Pilescure drug was obtained from sponsor
Lavanaya Pharmacy Pvt. Ltd., Lucknow, to conduct study on animals. Total 20
capsules were obtained from sponsor. The purity and chemical analysis is not
part of this study and it was responsibility of sponsor. The drug concentration
was 500 mg/capsules.
Acute
toxicity
An acute oral toxicity study was performed by
method of Parasuraman [19]. In acute oral toxicity, we followed the acute toxic
category method. Total three Sprague Dawley male rat (n=3) were selected at
different dose levels. The animals were kept overnight with access to water but
not food, after which the pilescure drug was administered orally at a different
dose level of 500, 1000 and 2000 mg/kg body weight and the animals were
monitored for clinical sign of toxicity, mortality for 24 h. If any mortality
was observed in 2 out of 3 animals, then the dose administered was identified
as a toxic dose. If mortality was observed in one animal, then the same dose
was repeated again to confirm the toxic dose.
Experimental
design
The study was carried out in animals after
getting ethical clearance from Institutional animal ethical Committee (IAEC).
The IAEC number for this study was IAEC-NIMSLAC/18-04. The study was conducted
under the Good Laboratory Practices (GLP) environment. The study was performed
on male SD rats weighing 200 ± 5 g, housed in polypropylene cages in an
air-conditioned room with temperature maintained at 25 ± 2°C and 12 h
alternating day and night cycles. The animals were allowed standard rat chow
diet and sterile distilled water. Total 30 male SD rats were selected and
divided into three groups of ten animals each. These groups are follows:
Group I (n=10): Control group
Group II (n=10): Hemorrhoids induced group
(6.0% Croton oil application)
Group III (n=10): Hemorrhoids plus Pilescure
treated group (206.66 mg/kg body weight)
Hemorrhoids were induced in the all groups,
except control group, by applying the croton oil preparation (pyridine, diethyl
ether, deionized water and 6% croton oil in diethyl ether in the ratio of
1:4:5:10 [8]. Followed by overnight fasting animals, sterile cotton swabs (4 mm
diameters) soaked with 200 µl of croton oil solution was inserted in to anus
for 20-25 s for 7 days. After 7 days, a linear development of edema was
observed after croton oil application and recto anal area was measured in all
groups by vernier caliper as evidence of inflammation. After changes in the
recto-anal area, stool frequency, hematological (WBC, Neutrophil) and
C-reactive protein parameters were measured in group II and group III animals,
further treatment was started with pilescure drug orally for 21 days only in
group III animals. After 21 days treatment, all animals were overnight fasted
and 2.0 ml blood samples were collected in heparin and non-heparin vials. The
1.5 ml blood sample was used for serum preparation and measured the oxidative
stress (lipid per-oxidation), inflammatory and antioxidant enzymatic parameters
and rest 0.5 ml was used for hematlogical examination. After collection of
blood samples, animals were euthanized exsanguinations under anesthesia and
recto-anal tissue were isolated and weight immediately and fixed in 10%
formalin solution for histological examination. The recto-anal coefficient was
calculated by using formula.
Recto-anal coefficient=Weight of recto-anal
tissue (mg)/Body weight (g)
Hematological
examination
Hemoglobin (Hb), White blood cell (WBC),
platelet count (PLT), neutrophil were examined through fully automatic cell
counter (Mindray Vector, Model No.BC-2300).
Oxidative
stress parameter
Free radical mediated damage was assessed by
the measurement of lipid peroxidation in the term of malon dialdehyde (MDA)
formed, essentially according to method of Ohkawa et al. [20]. It was
determined by thiobarbituric reaction. The reaction mixture consisted of 0.1 ml
serum samples, 0.20 ml of 8.1% sodium dodecyl sulphate (SDS), 1.5 ml of (20%,
pH 3.5) acetic acid, 1.5 ml of 0.8% thiobarbituric acid (TBA) and 0.7 ml
distilled water to see find volume of 4.0 ml. The tubes were kept in boiled
water at 95°C for 1 h and cooled immediately under running tap water. The
amount of 1.0 ml of water was added to and 5.0 ml of the mixture of n-butanol
and pyridine (15:1 v/v) and vortexed. The tubes were centrifuged at 3500 rpm
for 15-20 min. The upper layer was aspirated and optical density was measured
at 532 nm. The molar extension coefficient 1.56 × 105 was used for calculation.
Cytokines
assays
Cytokines parameters such as tumor necrosis
factor-α (TNF-α), Interlukin-β (IL-β) and Interlukin-6 (IL-6) were assayed in
the serum samples by ELISA Reader (Merck, Serial No. 21041098, MIOS-Jounior). The
assays were performed according to protocol recommended by the manufacturer’s
(Invitrogen San Jose CA, USA).
Nitric
oxide assay
The nitrite level was estimated in the serum
sample according to method of Tsai et al. [21]. 100 µl of serum sample was
mixed with 0.4 ml phosphate buffer saline (0.1 M, pH 7.2) and added 2.0 ml
Griess reagent. Then 2.0 ml of 5% TCA solution was added and mixed properly by
vortex shaker and kept for incubation for 15-20 min. After incubation, the
reaction mixture was centrifuged at 14000x g for 20 min and supernatant was
taken carefully in other clean tube and absorbance was recorded at 540 nm. The
concentration of nitrite was determined from stranded curve prepared with 0.1
ml of 100 µM sodium nitrite.
Anti-oxidant
parameters
We measured the some antioxidant enzymatic
parameters viz Catalase [22], reduced glutathione [23], glutathione peroxidase
[24] and glutathione reductase [25] by standard methods in the serum samples.
Histopathological
analysis
Recto-anal tissue was excised from all groups
of animals under anesthesia and fixed in 10% neutralized formalin solution for
24 h, dehydrated in gradual ethanol (50-100%), cleared in xylene and embedded
in paraffin wax. The sections, which were 5 mm thick, were then prepared using
rotary microtome (Leica RM 2125 RTS, Singapore) and stained with hematoxylin
and eosin dye for microscopic observation of histopathological changes using a
light microscope.
STATISTICAL
ANALYSIS
The results are expressed in mean ± SD.
Statistical significance was calculated by one way analysis of variance
(ANNOVA) followed by Turkey's t-test multiple comparison test using Graphpad
Prism (Version 5, software programe, San Diego, CA, USA). A P<0.05 was considered
to be statistically significant.
RESULTS
Acute
toxicity
In acute toxicity study, the animals did not
showed any clinical sign of toxicity and mortality after oral administration of
pilescure drug upto 2000 mg/kg dose level, so the LD50 of ayurvedic
drug Pilescure was found more than 2000 mg/kg. Hence the approximately 1/10th
of minimum dose was selected and administered for the present study.
Effect
on recto-anal weight, stool weight and recto-anal coefficient
In the present study, it was examined that a
significant (p<0.0001) elevated recto-anal weight and recto-anal coefficient
along with (p<0.001) decreased stool weight in hemorrhoid induced group
after application of croton oil preparation as compared to control group. After
treatment with pilescure drug for 21 days, the recto-anal weight and its
coefficient were found statistically significant (p<0.0001) decreased along
with improved stool weight in pilescure treated group as compared with
hemorrhoids induced group and come back to control group (Table 1).
Effect
on hematological and biochemical parameter
It was observed that a statistically
(p<0.0001) significant decreased hemoglobin and platelet count levels in the
hemorrhoid induced group as compared with control group. After treatment with
pilescure for 21 days, these levels were significant (p<0.0001) enhanced in
the treated group as compared to induced group and these levels were reached
almost near to control group. A white blood cell and C-reactive protein (CRP)
levels were also found significant (p<0.0001; p<0.001) increased the in
the hemorrhoid induced group as compared with control group. These parameters
were significant (p<0.001; p<0.0001, p<0.05) decreased in pilescure
treated group after treatment with drug for 21 days (Table 2).
Effect
on cytokines parameters
A significant (p<0.0001) enhancement was
found in the tumor necrosis factor-α (TNF-α), Interlukin-beta (IL-β) and
Interlukin-6 (IL-6) levels in the serum of hemorrhoid induced group after
administration of croton oil application. After treatment with pilescure drug
for 21 days, these inflammatory parameters were found significantly (p<0.0001)
reduced in the treated group and all levels were come back near to control
group (Figure 1).
Effect
on stress and inflammatory parameter
The results revealed that a significant
(p<0.0001) enhancement of malonaldialdehyde (MDA) and nitric oxide (NO)
levels in hemorrhoid induced group as compared with normal control group. These
levels were found statistically significant (p<0.0001) decreased in
hemorrhoid plus pilescure treated group after 21 days treatment and come back
to control group (Figures 2 and 3).
Effect
on antioxidant parameters
A significant (p<0.0001; p<0.001)
reduction of antioxidant enzymes viz. catalase, glutathione peroxidase,
glutathione reductase and reduced glutathione enzyme activities were found in
hemorrhoid induced group as compared with control group. After treatment with
pilescure drug in hemorrhoid plus pilescure treated group, these enzymes
activities were significantly (p<0.0001; p<0.001) increased when compared
with hemorrhoids induced group. These enzyme activities were normalized and
come back to control group (Figures 2
and 3).
Histopathological
examination
The histological examination of recto-anal
tissue showed that partly skin and anal mucosa with normal cytoarchitecture of
the recto-anal region found in control group (Figure 4A) whereas group II treated with croton oil application
developed the polypoid mass of smooth muscle layer by papillary mucosa and
significantly found higher inflammatory cells in recto-anal region (Figure 4B). After treatment with
pilescure drug in treated group, the section showed that the inflammatory cells
were seen less and tissue of rectum lined found by mucosa forming luminal folds
(Figure 4C).
normal
cytoarchitecture of the recto anal region in control group (4A), whereas,
slide B (hemorrhoid induced) showed that after application of croton oil in recto-anus
area, a developed the polypoid mass of smooth muscle layer by papillary mucosa
and found higher inflammatory cells (4B). After treatment with pilescure drug
for three weeks, the inflammatory cells were less seen and section showed the
normal tissue of rectum lined by mucosa forming luminal folds (4C).
DISCUSSION
Hemorrhoids are a pathological state that is
characterized by a severe vasodilatation at the region of recto-anal area that
causes the inflammation surrounding tissues, thus further leading to secondary
complications such as extravasation of fluid into fluid interstitial space
mainly due to increased vascular permeability and migration of large amount of
inflammatory cells [8]. So in this study croton oil used as inducer for
induction of hemorrhoid model in animals. Croton oil causes inflammation due to
release of soluble factors inflammatory lipid metabolites such as
prostaglandins [26], leukotrienes, nitric oxide (NO) [27] and cytokines (TNF-α,
IL-6 and IL-β) [28,29], etc. The present finding suggested that a significant
improvement in stool frequency on the basis of stool weight examination in
pilescure treated group when compared with hemorrhoid induced group. The result
clear indicated that drug suppresses the constipation symptom which is one of
the causative factors for hemorrhoid disease. Such action was seemed due to
presence of active ingredients Terminalia
chebula in a drug which act as laxative and vata detoxification and
digestive balancing. Recto-anal weight and recto-anal coefficients were found
significantly decreased along with increased the hematological parameters after
treatment with drug for 21 days in pilescure treated group as compared with hemorrhoid
induced group. After treatment with drug these parameters were come back to
control group. These findings clear suggesting that the active ingredients of
drug shows potent anti-inflammatory effect and improve the hematological
parameter due to homeostatic balance and its main action on the blood
capillaries, due to its KASHAYA RAS (Astringent) and SHEET VIRYA (cool nature).
The results also showed that there were significantly increased inflammatory
parameters C-reactive protein, neutrophil, cytokines (TNF-α, IL-6 and IL-β) and
nitric oxide in hemorrhoid induced group when compared with control group.
These results are indicating that croton oil leads to inflammation due to
release of soluble factors involving inflammatory lipid metabolites, kinins (chemokines)
and cytokines, etc. These factors alone or and/or in combination, regulate the
activation of resident cells (fibroblast, microphages, endothelial cells and
mast cells) and newly recruited inflammatory cells (lymphocytes, basophil and
neutrophil) causes the systemic response to inflammation [30]. After treatment
with drug for 21 days, these inflammatory parameters were significantly
decreased in pilescure treated group as compared with hemorrhoid induced group
and come near to control group. The inflammatory parameters were reduced in
pilescure treated group due to presence of anti-inflammatory property of Terminalia chebula, Mesua ferrea and Ricinus
communis herbs present in the drug. Various studies have been reported that
these herbs shows as anti-inflammatory effect [31-34].
Oxidative damage to biological compounds,
especially lipids through lipid peroxidation has been shown to play a significant
role in various pathophysiological states. Superoxide dismutase (SOD) is a
specific antioxidant enzyme that repairs the cells and reduced the damage
caused by superoxide anions (O-2), which is most common free radical
in the body. Free radical can lead to lipid peroxidation in organisms.
Malonaldialdehyde (MDA) is end product of poly unsaturated fatty acids (PUFAs)
in cells, and an increase in free radical causes overproduction of MDA. MDA is
biomarker of oxidative stress in various diseases [35]. So authors have
measured the malonaldialdehyde level as a biomarker of oxidative stress. In
normal cell, there is proper balance between oxidative stress and antioxidant
enzyme levels, any imbalance between these two ratios that lead to various
pathophysiological states. In our study, we found that there were statistically
(p<0.0001) significant decreased antioxidant enzymes activities (Catalase,
glutathione peroxidase, glutathione reductase and reduced glutathione) along
with increased MDA level in hemorrhoid induced group as compared with control
group. After treatment with pilescure drug for 21 days, these enzymatic
activities were significant improved along with lowered the MDA level in
pilescure treated group when compared with hemorrhoid induced group. These
results clear demonstrated that the drug consists of polyherbal has prominent
antioxidant effect. The similar result has been published by other investigator
in croton oil hemorrhoid induced animals [10]. Based on above findings it is
clear indicating that croton oil application causes severe inflammation that
altered recto-anal physiology, hematological, biochemical and cytokines
parameters along with enhancement of oxidative stress that leads to hemorrhoid.
After treatment with pilescure drug the above parameters were improved in
hemorrhoid condition. Base on above finding it is clear indicating that the
poly herbal drug pilescure is most therapeutic effects for reduction of
recto-anal pain, inflammation and inhibit the free radical generation and
enhance the antioxidant enzymes level. These anti-inflammatory and antioxidant
properties are present in a drug due to presence of active constituents of
tannin, alkaloids, and polyphenol ingredients in polyherbal ayurvedic
formulation.
CONCLUSION
So conclusion these finding suggesting that
pilescure is nontoxic with LD50 more than 2000 mg/kg body weight and
it is most effective ayurvedic polyherbal drug which reduces inflammation,
oxidative stress and antioxidants properties along with improvement of
haematological parameters during hemorrhoid condition. The drug can improve the
healthy and quality of life people who are suffering from hemorrhoid problem.
ACKNOWLEDGEMENT
The authors are thankful to Mr. Harish Yadav
(Technical officer) to provide the histopathological results. Authors are also
thankful to sponsor, formulation department of Lavanya Ayurvedic Pvt. Ltd.,
Lucknow to provide test item pilescure for carried out the study.
COMPETING
INTERESTS
None
SOURCE
OF SUPPORT
None
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