Phytomedicine Plus 2 (2022) 100172

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Phytomedicine Plus
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Anti-hemorrhoidal potential of standardized leaf extract of

Dolichandrone falcata
Suhas R. Dhaswadikar a, b, c, d, Komal M. Parmar a, Shantibhushan K. Kamble b, Ishita Kathuria c,
Mahaveer Dhobi c, Arunadevi Birajdar d, Satyendra K. Prasad a, *, Prakash R. Itankar a, *
a
Department of Pharmaceutical Sciences, Rashtrasant Tukadoji Maharaj Nagpur University, Nagpur, Maharashtra 440033, India
b
Covance, Inc., Qubix Business Park Pvt. Ltd., (SEZ) Blue Ridge, IT 6, Hinjewadi, Phase 1, Pune, Maharashtra 411057, India
c
Department of Pharmacognosy and Phytochemistry, Delhi Pharmaceutical Sciences and Research University, New Delhi, India
d
K. T. Patil College of Pharmacy, Barshi Road, Osmanabad, Maharashtra 413501, India

A R T I C L E I N F O A B S T R A C T

Keywords: Background and objective: Leaves of plant Dolichandrone falcata Seem. Are traditionally used by the healers of
Anti-hemorrhoidal activity “India” for the treatment of piles and diabetes. Therefore, the main objective of the present investigation was to
Antioxidant evaluate the anti-hemorrhoidal potential of ethanolic leaf extract from the plant D. falcata (EDF). Methods: D.
Croton oil
falcata leaf extract obtained after maceration process was initially subjected to phytochemical standardization
Cytokines
Evans blue dye
with the help of HPLC using rutin as a marker compound. Hemorrhoid was induced in the rats using croton oil rat
Rutin model, and the treatment was carried out with EDF suspension prepared using carboxymethyl cellulose for 5 days
using 100, 200 and 300 mg/kg p. o. The scoring of inflammation was calculated on the basis of the clinical
hemorrhoidal area and severity index, which was followed by estimating the level of Evans blue dye as an in­
flammatory marker. The hemorrhoidal tissues were also subjected to cytokine profiling, biochemical evaluation
and histopathology. Results: EDF demonstrated the presence of mainly polyphenols, tannins, flavonoids, alka­
loids, and saponins, while rutin was calculated to be 3.08% w/w in EDF following HPLC analysis. The results
depicted more potent anti-hemorrhoidal activity of EDF at 200 mg/kg orally which was evident through the
grading of inflammatory index. The result demonstrated significant decline in the levels of IL-1β, IL-6, TNF-α
expression and also showed restoration of altered antioxidants and liver enzymes. Histopathological study
showed minimal inflammation and reduced dilated blood vessels in treated animals, thereby confirming the
tissue recovery. Conclusion: Thus, we have successfully explored and substantiated the traditional use of the
leaves from the plant D. falcata for the first time in the treatment of hemorrhoids, which may be attributed to its
anti-inflammatory and its antioxidant potential.

1. Introduction aging, low-fiber diet, spicy diet and chronic straining. Major symptoms
associated with hemorrhoid complaints include inflammation, pain,
Hemorrhoids are anorectal disorders that affect about 4.4% of the bleeding and pruritus (Jebakumar et al., 2014). Treatment available for
world’s population and are more common in people between the age hemorrhoids mainly includes cryotherapy, laser treatment, sclerother­
group of 45–65 years (Sun and Migaly, 2016). Studies have revealed apy, hemorrhoidectomy, infrared photocoagulation and bipolar
that, around 5–10% affected patients from hemorrhoids hesitate to diathermy. Generally these therapies are very expensive and have
receive conventional treatments (Azeemuddin et al., 2014). The risk several side effects with no total recovery. Therefore, researchers are in
factors associated with the changes in pathophysiological condition of search of a treatment that may provide complete relief with less cost and
anal hemorrhoid includes constipation, diarrhea, alcohol consumption, low side effects (Lohsiriwat, 2012).

Abbreviations: ALP, Alkaline Phosphatase; ALT, Alanine Aminotransferase; AST, Aspartate Aminotransferase; CAT, Catalase; EB, Evans Blue Dye; EDF, Extract of
Dolichandrone falcata; LPO, Lipid peroxidation; MDA, Malonaldehyde; NO, Nitric Oxide; NIH, National Institute of Health; OECD, Organization for Economic Co-
operation and Development; RAC, Rectoanal Coefficient; SDPX, Standard Pilex; SOD, Superoxide Dismutase; TNF, Tumour Necrosis Factor.
* Corresponding author.
E-mail addresses: [email protected] (S.K. Prasad), [email protected] (P.R. Itankar).

https://doi.org/10.1016/j.phyplu.2021.100172
Received 17 May 2021; Received in revised form 12 November 2021; Accepted 20 November 2021
Available online 24 November 2021
2667-0313/© 2021 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
S.R. Dhaswadikar et al. Phytomedicine Plus 2 (2022) 100172

Due to their potential medicinal value, traditional medicines are Mo. U.S.A.) as a marker with the help of High Performance Liquid
emerging as an alternative source for therapy in various ailments, which Chromatography (HPLC, Shimadzu, Japan). For HPLC analysis, the
mainly involves medicinal plants and their bioactive molecule. Medic­ mobile phase used was acetonitrile: water (Merck Life Sciences Pvt. Ltd.,
inal plants are considered to be a conceivable source of phytocon­ Mumbai, India) in the ratio 95: 5 (Doshi and Une, 2016). HPLC grade
stituents with potential pharmacological activity (Kim et al., 2005). methanol (Merck Life Sciences Pvt. Ltd., Mumbai, India) was used for
Dolichandrone falcata Seem. is a deciduous plant belonging to the family the preparation of stock solutions of standard rutin (0.5 mg/ml) and EDF
Bignoniaceae and is commonly known by the name Medshingi in the (5 mg/ml), while flow rate for this analysis was fixed at 1.0 mL/min. The
regions of Marathwada and Vidarbha from Maharashtra State of ‘India’ volume of injection was fixed at 10 µL and detection wavelength was
(Badgujar and Surana, 2010; Patil, 2013). Traditionally, the leaves of kept at 256 nm.
plant D. falcata are consumed by the tribes of Melghat region from
Maharashtra as folk remedy in cases of food poisoning (mainly due to 2.3. Experimental animals
consumption of fish) and also to procure abortion (Kirtikar and Basu,
2005). The traditional healers of Osmanabad district of Maharashtra, Rats (Wistar strain) of either sex, having weight between 150 and
‘India’ use the leaf of the plant in treatment of piles and diabetes (Patil, 200 g, were obtained from animal house of the Department of Phar­
2013; Korpenwar and Borkar, 2011). The phytochemical investigations maceutical Sciences, R.T.M. Nagpur University (Registration number
of the plants D. falcata revealed the presence of dolichandrone A, 92/1999, dated 28/04/1999). Animals were grouped and housed at
β-tocopherol, vitamin E, n-hexadeconoic acid, chrysin, chrysin 7-rutino­ controlled environment (ambient temperature and standard relative
side. pinocembrin, cirsimaritin. α-lapachone, lapachol, verbascoside, humidity) and were fed with standard rat feed and water ad libitum.
dolichandroside A, sterol, coumarin derivative, monoterpine, phenol, Acclimatization of the animals to the experimental environment was
isobutaric acid, β-sitosterol, p-cresol, cyclohexen (Subramanian et al. done for one week before the start of the experiments. All the protocols
1972; Aparna et al. 2009; Ekade and Manik, 2013; Salve and Bhutkar under the experiments were approved by the Ethical Committee of the
2017). Pharmacological studies on the plant have reported University (No- IAEC/UDPS/2018/46. Dated 04/08/18) and were car­
anti-inflammatory, analgesic, anti-cancer, anxiolytic, antiestrogenic, ried out as per the standard NIH guidelines.
antioxidant (Jebakumar et al., 2014), antidiabetic (Mungle et al., 2012)
and abortifacient activities (Wikhe et al., 2012). 2.4. Acute oral toxicity study
Scientifically, the plant is reported to have potent anti-inflammatory
and analgesic activity while, traditionally the leaves of the plant are The OECD (Organization for Economic Cooperation and Develop­
used in treatment of hemorrhoids, which is still unexplored. Thus, taking ment) guideline 425 was refered for determination of acute oral toxicity
these evidences into consideration, the present investigation was un­ study of EDF. Overnight fasted Wistar female rats were orally adminis­
dertaken to scientifically validate the anti-hemorrhoidal potential of tered with EDF following the guidelines and were observed individually
leaves from the plant D. falcata using a suitable animal model. for about 48 h for any behavioral or neurological abnormalities viz.
diarrhoea, feeding behavior, convulsions, tremors, lacrimation, sleep,
2. Material and methods and salivation as a markers for toxicity. For confirming the mortality
rate if any, the rats were kept under observation for two weeks (OECD,
2.1. Plant material 2008).

The leaves of D. falcata were obtained from the local areas of Mar­ 2.5. Induction of hemorrhoids
athwada region of Maharashtra. The plant was authenticated by Dr.
Nitin Dongarwar, Department of Botany, R.T.M. Nagpur University, The overnight fasted rats were initially injected with Evans blue (EB)
Nagpur, Maharashtra, India and the plant (specimen No. 10,219) has dye (Sigma Aldrich, St, Louis Mo. U.S.A.) via tail veins, which act as a
been deposited in Department of Pharmaceutical Sciences, R.T.M. marker for judgement of inflammatory index. Half an hour after the
Nagpur University. The leaves were shade dried and were coarsely injection of EB dye, hemorrhoids were induced in rats by application of
powdered. Further, 500 g of powdered leaves were extracted using 1.5 L croton oil preparation, prepared by mixing deionized water, pyridin,
ethanol (S. D. Fine-Chem Pvt. Lid., Mumbai, India, analytical grade, diethyl ether (S D Fine-Chem Limited, Mumbai, India) and 6% croton oil
strength 99.9%) following a one-week maceration procedure. The (Sigma Aldrich, St, Louis Mo. U.S.A.) in diethyl ether in the ratio of 1: 4:
extract was then evaporated on a rotary evaporator (BUCHI India Pvt. 5: 10. Except normal group, the croton oil preparation was inserted in
Ltd, Mumbai, India).The yield of the extract was 5.6% w/w. It was the rectoanal portion of the rats, 20 mm deep into the anal opening of
stored in a desiccator until further use. rats, using sterile cotton swabs having diameter of 4 mm, soaked in 100
µl of prepared croton oil. After 7 to 8 hrs of croton oil application, the
2.2. Phytochemical standardization aninals were observed for any development of edema (Azeemuddin
et al., 2014; Saria and Lundberg, 1983).
Preliminary phytochemical screening of the ethanolic leaf extract of
D. falcata (EDF) was initially carried out to confirm the presence of 2.6. Grouping of animals
various primary and secondary metabolites (Trease and Evans, 2002).
Further, EDF was subjected to quantification of identified phytocon­ Hemorrhoid in rats was induced after 24 h of croton oil adminis­
stituents, where total alkaloid content was determined following the tration. This was followed by grouping of animals, which included six
gravimetric analysis as described by Wagner and blodt (1996), whereas groups i.e. Group 1 served as normal control and was given vehicle
total carbohydrate in EDF was quantified using the procedure adopted suspension of 0.5% carboxymethyl cellulose (CMC) (Merck Life Sciences
by Yemm and Willis (1954). Total phenolics and total tannin content in Pvt. Ltd., Mumbai, India) in normal saline. Group 2 was served as
EDF was quantified using Folin Ciocalteu reagent (S. D. Fine-Chem Pvt. hemorrhoid induced negative control group and was administered with
Lid., Mumbai, India). Aluminum chloride method was used for deter­ 0.5% CMC as vehicle in normal saline. Group 3 was served as standard
mining the total flavonoid content, while total saponin content in EDF treated group, which included hemorrhoid induced rats treated with
was determined by taking diosgenin (Sigma Aldrich, St, Louis Mo. U.S. Pilex granules at 400 mg/kg, p.o. (Himalaya Drug Company, Bengaluru,
A.) as standard following the procedure described earlier (Hirudkar India) suspended in 0.5% CMC (Azeemuddin et al., 2014), while Group
et al., 2020). 4, 5 and 6 included hemorrhoid induced rats treated with EDF at 100,
EDF was further standardized using rutin (Sigma Aldrich, St, Louis 200 and 300 mg/kg, p.o. suspended in 0.5% CMC. EDF was

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S.R. Dhaswadikar et al. Phytomedicine Plus 2 (2022) 100172

administered to the rats for five successive days, once in a 24 h period. instructions.

2.7. Inflammatory index 2.11. Histopathological studies

The Inflammatory index in the respective animals was calculated on Hemorrhoidal tissue samples obtained on the fifth day of treatment
the fifth day, one hour after treatment. For evaluating the severity of were also utilized for performing the histopathological examinations.
inflammation of the rectoanal portion, a scoring system was designed on The tissues were immediately blotted, dried and fixed in 10% formalin,
the basis of the clinical hemorrhoidal area and the severity index. which was followed by dehydration in acetone. The tissue samples were
Hemorrhoids were graded on a scale from 1 to 4 to measure severity i.e. then embedded in paraffin wax for microtome sectioning and 4–6 µm
Grade I: The anal cushions bleed but do not prolapse, Grade II: The anal thickness sections were obtained. After that, sections of the tissue were
cushions prolapse through the anus on straining but reduce spontane­ stained with hematoxylin and eosin and were subjected to photo
ously, Grade III: The anal cushions prolapse through the anus on microscopic evaluation.
straining or exertion and require manual replacement into the anal canal
and Grade IV: The prolapse stays out at all times and is irreducible
2.12. Statistical analysis
(Tjandra et al., 2007).
The results are expressed as mean ± SEM, with six rats in each group,
2.8. Estimation of Evans blue dye
and one-way analysis of variance was used to analyse the data. For
evaluating the statistical significance between different groups, the
The quantity of EB dye was estimated in the rectoanal tissue of rats
Newman–Kevis test for multiple comparisons was used. For all statistical
for determinning the extent of croton oil-induced plasma exudation
comparision, Graph Pad Prism, version 5 software (San Diego, CA, USA)
(Saria and Lundberg, 1983). As described earlier, EB dye (30 mg/kg; i.
was used and p values less than 0.05 were considered significant.
v.) was administered before 30 min. of croton oil administration, which
was followed by treatment for five days. Rats were euthanized on the
3. Results
fifth day, three hours post treatment by deep thiopental sodium anes­
thesia and about 20 mm rectoanal tissues of rats were dissected and
3.1. Phytochemical standardization
weighed. The tissues were further, subjected to extraction of EB dye by
using 2 mL of formalin (Merck Life Sciences Pvt. Ltd., Mumbai, India),
The preliminary phytochemical screening of ethanolic extract of
which was used for estimating the level of EB dye by recording the
D. falcata (EDF) revealed the presence of mainly polyphenols, tannins,
absorbance of sample at 620 nm using UV visible spectrophotometer
flavonoids, alkaloids and carbohydrates as major components (Table 1).
(Systronics India Pvt. Ltd., Ahmedabad, Gujrat, India) (Yasmina et al.
From the quantitative estimations the total alkaloid content in EDF was
2010).
estimated to be 0.62 ± 0.15% w/w, while total saponin and total car­
bohydrate content in EDF was observed to be 419.93 ± 11.95 mg/g
2.9. Estimation of rectoanal coefficient
diosgenin equivalent and 86.26 ± 0.26 mg/g dextrose equivalent
respectively. Total phenolic content in EDF was found to be 37.98 ±
For calculating rectoanal coefficient (RAC), the previously weighed
0.07 mg/g gallic acid equivalent, whereas total tannin content was
rectoanal tissues were compared with the body weight of the individual
estimated to be 55.77 ± 1.55 mg/g tannic acid equivalent. The total
rats and the obtained value represented the RAC, which helps in
flavonoid content quantified in the plant extract was found to be 86.82
judgement of severity of inflammation (Azeemuddin et al. 2014) using
± 0.54 mg/g rutin equivalent (Table 2). The presence of rutin in etha­
the formullae:
nolic extract of the plant was confirmed by HPLC analysis, which
Rectoanal coefficient=weight of rectoanal tissue (mg)/Body weight
revealed a well-resolved peak of rutin at Rt of 3.07 min. and from the
(g).
HPLC quantification, the content of rutin in EDF was calculated to be
3.08% w/w (Fig. 1).
2.10. Biochemical analysis and cytokine evaluations

Blood samples were obtained from the rats retroorbital sinus on the 3.2. Acute oral toxicity study
5th day, one hour after treatment (Faujdar et al., 2019) for determining
the level of liver enzymes ALP (Alkaline phosphatase), ALT (Alanine From the results of the acute oral toxicity trial, EDF was found to be
transaminase) and AST (Aspartate transaminase) in blood serum using safe up to 2000 mg/kg with no evidence of behavioural or neurological
the standard procedure (Sengar et al., 2015; Reitman and Frankel, 1957; toxicity.
Kind and King, 1954). The hemorrhoidal tissue of the rats were collected
on the fifth day of treatment after sacrificing them (three hours after 3.3. Anti-hemorrhoidal activity
treatment) and were rinsed vigorously with Tyrode solution for deter­
mining different biochemical parameters. A small portion of rectoanal Application of croton oil, significantly induced hemorrhoid in rats
tissue was weighed, dried and homogenised with phosphate buffer
before centrifugation, and the supernatant was used to determine nitric Table 1
oxide (NO) levels using the process described by Green et al. (1982), Phytochemical screening of ethanolic extract of D. falcata leaves.
whereas total protein content in the tissue samples were calculated using Plant phytochemicals Observations
the Lowry et al. (1951) method. In vivo antioxidants such as catalase Steroids +
(CAT), superoxide dismutase (SOD) and lipid peroxidation (LPO) in the Cardiac glycosides +
tissue samples were also evaluated using standard procedures as Cyanogenetic glycosides –
Alkaloids
described earlier (Sinha, 1972; Kakkar et al., 1984; Nehius and
+
Tannins +
Samuelson, 1968; Hirudkar et al., 2020). Flavonoids +
The pro-inflammatory cytokines IL-1ß, IL-6 and TNF-α in the tissue Carbohydrates +
homogenate were measured using commercially available enzyme Proteins +
Saponins
linked immunosorbent assay (ELISA) kits (Konabiotech, Korea). The +

ELISA protocol was followed according to the manufacturer’s In table + indicates presence and – indicates absence.

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S.R. Dhaswadikar et al. Phytomedicine Plus 2 (2022) 100172

Table 2 with EDF. The results depicted a more pronounced effect of EDF at 200
Quantitative estimations of ethanolic extract of D. falcata leaves. mg/kg, even when compared with EDF at 300 mg/kg (Fig. 3A), which
Quantitative Parameters Observations may be attributed to the ceiling effect of EDF at 200 mg/kg. The findings
Total alkaloid (% w/w) 0.62 ± 0.15 also demonstrated a higher concentration of Evans blue dye in the
Total saponin content (mg/g) equivalent to diosgenin 419.93 ± 11.95 negative control group as a marker of inflammation, which was found to
Total carbohydrate content (mg/g) equivalent to dextrose 86.26 ± 0.26
significantly (p<0.05) decrease after treatment with EDF (Fig. 3B). The
Total phenol content (mg/g) equivalent to gallic acid 37.98 ± 0.07
Total tannin content (mg/g) equivalent to tannic acid 55.77 ± 1.55 rectoanal coefficient was also calculated in the present investigation,
Total flavonoid content (mg/g) equivalent to rutin 86.82 ± 0.54 which was found to decrease significantly on treatment with EDF, where
more prominent effect was observed with EDF at 200 mg/kg, even when
compared with standard used. Thus, the findings confirmed the anti-
after 24 h of its application, which was clearly visible as blue color spots inflammatory potential of EDF (Fig. 3C). EDF at 100 mg/kg, p.o. was
on rectoanal portion, imparted through the Evans blue dye (Fig. 2). not found to be effective and thus, the dose of EDF at 100 mg/kg, p.o.
Further, the inflammatory scores calculated on fifth day of treatment, was not included in further studies.
showed significantly (p<0.05) higher score of hemorrhoid in negetive
control rats, which was found to significantly decrease after treatment

Fig. 1. HPLC Chromatogram of rutin in ethanolic extract of D. falcata. In figure, A indicates peak of standard rutin (Peak area 41,872 mAU*min) and B indicates peak
of rutin in ethanolic extract of D. falcata (Peak area 1,289,791 mAU*min).

Fig. 2. Images of anal hemorrhoids on treatment with ethanolic extract of D. falcata in croton oil-induced hemorrhoid rat model. In figure, G1 indicates normal
anorectal portion of normal control group rats, G2 indicates marked hemorrhoid development in hemorrhoid-induced negative control group rats, G3 indicates
recovery from hemorrhoid on treatment with standard pilex granules, G4 indicates the presence of hemorrhoid with very low recovery on treatment with ethanolic
extract of D. falcata at 100 mg/kg, p.o., G5 indicates marked recovery from hemorrhoid on treatment with ethanolic extract of D. falcata at 200 mg/kg, p.o. and G6
indicates slight recovery from hemorrhoids on treatment with ethanolic extract of D. falcata at 300 mg/kg, p.o.

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S.R. Dhaswadikar et al. Phytomedicine Plus 2 (2022) 100172

Fig. 3. Effect of treatment of ethanolic extract of D. falcata on different parameters in croton oil-induced hemorrhoid rat model. In figure A indicates inflammatory
score after treatment with extract, B indicates Evans blue dye extravasation results showing maximum protective nature of extract at 200 mg/kg and C indicates
effect of extract treatment on Rectoanal Coefficient (RAC) in the rectoanal tissue of rats confirming the anti-hemorrhoidal potential of extract at 200 mg/kg.

3.4. Biochemical estimations 3.5. Cytokine profiling

The results of the biochemical estimations carried out on the blood The impact of EDF treatment on various proinflammatory cytokines,
serum and anorectal tissues obtained from the croton oil-induced such as IL-1β, IL-6, and TNF-α, is represented in Fig. 4. The results from
hemorrhiodal rats is represented in Table 3. The findings showed a the negative control group rats demonstrated a significant increase in
significant (p<0.05) increase in the levels of all the liver enzymes ALT, the expression of all of above pro-inflammatory cytokines. However,
AST, and ALP in the blood serum of negative control hemorrhoid rat treatment with EDF resulted in a significant decrease in the elevated
group however, a significant (p<0.05) decrease in all these liver enzyme expression of these pro-inflammatory cytokines, where prominent effect
levels were observed after treatment with EDF. Furthermore, the anti­ was observed in case of EDF at 200 mg/kg, p.o. as compared to other
oxidant studies also revealed a significant increase in the levels of NO treated groups.
and LPO in the negative control group rats, while the levels of CAT and
SOD were found to decrease significantly. Nevertheless, on treatment
3.6. Histopathological studies
with EDF, a significant (p<0.05) restoration of all these altered enzymes
were observed, which was also supported with an concomitent increase
The histopathological examination of the anorectal tissues of nega­
in total protein content, suggesting the cellular proliferative potential of
tive control group rats showed degeneration, as indicated through the
EDF.
presence of inflammatory zones, hemorrhage and dilated blood vessels.

Table 3
Effect on various biochemical parameters in anorectal tissue of rats treated with ethanolic extract of D. falcata in croton oil-induced hemorrhoid rat model.
Biochemical Parameters Normal control Negative control Standard Pilex EDF (200 mg/kg) EDF (300 mg/kg)
AST (U/ml) 261.67±24.67 356.80±17.70a 313.34±26.81ab 301.33±18.37ab 329.83±24.87ab
ALT (U/ml) 71.83±16.88 115.67±14.98a 102.5 ± 18.46ab 93.83±14.68ab 107.33±19.61a
ALP (U/ml) 153.83±12.18 398.52±23.50a 222.66±19.5ab 159.5 ± 17.37ab 241.16±15.66ab
Protein (mg/100 mg) 10.32±1.08 7.92±1.09a 9.24±0.65b 9.21±0.45b 8.42±0.82 b
NO (units in mole/mg of protein) 0.43±0.03 1.03±0.01a 0.72±0.01ab 0.565±0.02ab 0.52±0.00ab
Catalase (U/min/mg of protein) 11.74±5.41 7.76±3.46a 8.95±3.99ab 9.76±4.42 ab 9.83±3.14ab
SOD (Units/mg of protein 2.68 ± 0.19 1.08 ± 0.12a 2.18 ± 0.20b 2.42± 0.34b 2.28 ± 0.15b
LPO (MDAnmol/ Umg of Protein) 6.41±1.09 12.43±1.28a 3.83±0.11ab 4.07±1.34ab 5.73±2.03b

Value are mean ± SEM (n = 6) where a: p<0.05 vs. Normal control group and b: p< 0.05 vs. negative control group (In Table, ALP: Alkaline phosphatase, ALT: Alanine
transaminase, AST: Aspartate transaminase, EDF: Ethanolic extract of D. falcata, LPO: Lipid peroxidation, NO: Nitric Oxide and SOD: Superoxide dismutase).

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Fig. 4. Effect on pro-inflammatory cytokines (IL-1β, IL-6 and TNF- α) on croton oil-induced hemorrhoidal tissues of rats after treatment with ethanolic extract of
D. falcata. The figure indicates that, treatment with ethanolic extract of D. falcata at EDF 200 mg/kg and 300 mg/kg caused significant decline in the levels of IL-1β,
IL-6, and TNF- α.

However, the images of rectoanal tissues of rats treated with EDF at 200 that occurs during the development of hemorrhoids after the application
mg/kg demonstrated a marked decline in the inflammatory zones and of croton oil, researchers used an EB dye extravasation test as a marker
also showed normal blood vessels with intact tissues (Fig. 5). of inflammation and RAC (Hamer et al., 2002; Azeemuddin et al., 2014).
The observations from our study showed promising effect of EDF in
4. Discussion reducing inflammatory score, EB dye extravasation and RAC on rats
treated with 200 mg/kg, especially when compared with standard Pilex
In the present investigation, croton oil was used to induce experi­ granules. However, EDF at 100 mg/kg, was found to be non-significant.
mental hemorrhoid in rats. Croton oil extracted from Croton tiglium L. As discussed earlier, croton oil causes massive increase in the level of
(Euphorbiaceae) is a phlogisitc oil that imparts irritant and vesiculant inflammatory mediators which damages the vessels by raising their
effect due to the presence of an phorbol esters, 12-O-tetradecanoyl­ permeability and inducing oxidative stress in the rectoanal tissues. A
phorbol-13-acetate, which promotes inflammation characterized by study performed on croton oil induced carcinoma in mouse skin
vasodilatation, polymorphonuclear leukocyte infiltration and leads to revealed that, croton oil causes the over expression of free radicals
edema formation in tissue (Cabrini et al., 2011). Studies have reported scavengers, leukocytes infiltration and increased production of ROS,
that, the phorbol esters from croton oil causes the release of soluble which hampers the antioxidant status by altering the levels of liver en­
factors such as inflammatory lipid metabolites like prostaglandins, leu­ zymes (ALP, AST and ALT) along with CAT and LPO (Majed et al., 2015).
kotrienes, kinins like bradykinins and chemokines, nitric oxide, and Similarly, our study also showed that, croton oil application hampered
cytokines which triggers inflammation. Individually or in combination, the antioxidant status, where a significant increase in the levels of NO,
these factors triggers the regulation of resident cells (endothelial cells, LPO and liver enzymes (ALT, AST, ALP) were observed, whereas a sig­
fibroblasts, mast cells, and macrophases) as well as newly assigned in­ nificant decrease in the levels of antioxidants SOD and CAT was noted in
flammatory cells like neutrophils, eosinophils, monocytes and lympho­ negative control group. The increased level of NO is due to a higher level
cytes that results in severe inflammation (Azeemuddin et al., 2014; of nitrotyrosine, which acts as a marker for NO-induced tissue damage
Tjandra et al., 2007). Application of croton oil in our study also triggered and is said to be an indicator of cellular damage following inflammation
the inflammatory response, which was confirmed through the inflam­ (Nemirovskiy et al., 2009; Esposito et al., 2011). Studies have also re­
matory scores. To better understand the series of inflammatory reactions ported that croton oil application contributes in over expression of iNOS,

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Fig. 5. Histopathological view of rectoanal tissue of rats treated with ethanolic extract of D. falcata in croton oil-induced hemorrhoidal rat model. In figure, G1
represents normal control group showing normal intact rectoanal tissue, G2 represents rectoanal tissue of negative control group rat showing hemorrhagic spot (#)
and dilatation of blood vessels (*), G3 represents rectoanal tissue of rat treated with standard Pilex showing mild hemorrhagic spot (#) and dilatation of blood vessels
(*), G4 represents rectoanal tissue of rat treated with ethanolic extract of D. falcata (200 mg/kg) showing very minimal dilatation of blood vessels (*) and G5
represents rectoanal tissue of rat treated with ethanolic extract of D. falcata (300 mg/kg) showing slight hemorrhagic spot (#) and dilatation of blood vessels (*).

that results in alleviation of NO level (Majed et al., 2015). LPO induces inflammatory agents has been well documented in the literature. (Sen­
lipid peroxidation, which causes oxidative damage to lipids and is gar et al. 2015; Yang et al. 2013). Rutin was selected as a marker for the
considered to be a major contributing factor in pathogenesis of hemor­ standardization of the EDF using HPLC, as its presence has already been
rhoidal disease (Ebru et al., 2013). However, after treatment with EDF at reported in the leaves of D. falcata (Joshi et al. 2016). Rutin is a naturally
200 mg/kg p.o, all of these altered enzyme levels (NO, LPO, CAT, SOD) occurring flavonoids responsible for its numerous pharmacological ac­
were significantly restored, indicating that EDF possessed a very high tions viz. anti-inflammatory, antioxidant and anti-hemorrhoidal activity
antioxidant capacity, which is known to be the most important prereq­ with their respective mechanisms (Faujdar et al. 2019). The role of rutin
uisite for anti-hemorrhoidal drugs (Nair et al., 1988). in inhibiting the expression of IL-1β, IL-6, TNF-α, and nitrite formation
Studies have reported that, the, 12-O-tetradecanoylphorbol-13-ace­ in a croton oil-induced hemorrhoidal model has already been reported
tate from croton oil activates the protein kinase C, which induces earlier. In an inflammation model, it has also been shown to restore the
inflammation following the release of inflammatory mediators like IL- alterations in LPO and CAT levels in stressful conditions (Jain and Par­
1β, IL-6, TNF-α along with other inflammatory mediators (Sangchart mar, 2011; (Romm et al., 2010). Thus, we may presume that, the pres­
et al., 2021; Azeemuddin et al., 2014; Tjandra et al., 2007). Therefore, in ence of rutin in EDF could be the contributing factor in its
our study also, we have evaluated the impact of croton oil application on anti-hemorrhoidal potential.
the expression of cytokines such as IL-1β, IL-6, and TNF-α and found that In conclusion, we have successfully justified the traditional claims of
croton oil caused a significant increase in the expression of these cyto­ the plant D. falcata in the treatment of hemorrhoids for the first time in
kines. However, the anti-inflammatory nature of EDF was confirmed as rats. The observed anti-hemorrhoidal potential of the leaves from the
it significantly reduced the levels of all these tested cytokines. plant was attributed to its anti-inflammatory and antioxidant potential,
Histopathologically, application of croton oil on rectoanal tissue where rutin could play a major contributing factor in conjugation with
causes an extreme vasodilation and filtration of inflammatory cells, other phytoconstituents. However, a detailed study on the isolated
along with hemorrhagic spots (Saria and Lundberg, 1983), which also phytochemicals from the plant is required to know the exact phyto­
corroborated with our results. Our findings from histopathological ex­ constituents responsible for the observed anti-hemorrhoidal potential of
amination revealed a marked to severe inflammation, hemorrhage and D. falcata with specific mode of action.
dilatation of blood vessels as observed in the rectoanal tissue of the rat in
negative control group. However, on treatment with EDF 200 mg/kg p. Declaration of Competing Interest
o, a marked recovery from inflammation was confirmed as the tissues
showed almost normal architecture with very minimal damage. The manuscript was submitted with the approval of all co-authors,
The phytochemical evaluations of EDF confirmed the presence of and we have no conflicts of interest to declare. The writers are solely
polyphenols, tannins, flavonoids and saponins as major phytocon­ responsible for the paper’s writing and content.
stituents. The importance of these phytoconstituents as anti-

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S.R. Dhaswadikar et al. Phytomedicine Plus 2 (2022) 100172

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