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May 2013, Vol.11, No.3 206 Journal of Integrative Medicine

1 Introduction

Ulcerative colitis (UC) is a subcategory of inflammatory bowel disease (IBD). Colitis affects the inner most lining

or mucosa of the colon and rectum where a continuous area of inflammation and ulceration with no segments of normal tissue is observed[1]. The two primary types of IBD are Crohn’s disease and UC. In IBD, the intestine (bowel) becomes inflamed, often causing recurring abdominal

● Research Article

Amaranthus roxburghianus root extract in combination with piperine as a potential treatment of ulcerative colitis in miceSunil A. Nirmal1, Jayashri M. Ingale1, Shashikant R. Pattan2, Sanjay B. Bhawar3

1. Department of Pharmacognosy, Pravara Rural College of Pharmacy, Loni 413736, Maharashtra, India2. Department of Pharmaceutical Chemistry, Pravara Rural College of Pharmacy, Loni 413736,

Maharashtra, India3. Department of Pharmacology, Pravara Rural College of Pharmacy, Loni 413736, Maharashtra, India

OBJECTIVE: The present work was undertaken to determine the effects of Amaranthus roxburghianus Nevski. (Amaranthaceae) root alone and in combination with piperine in treating ulcerative colitis (UC) in mice.METHODS: Swiss albino mice were divided into seven groups (n = 6). Standard group received prednisolone(5mg/kg, intraperitoneally).Treatmentgroupsreceivedhydroalcoholicextractofroots of A. roxburghianus (50 and 100 mg/kg, per oral) and a combination of hydroalcoholic extractof rootsofA. roxburghianus (50 and 100 mg/kg, per oral) and piperine (5 mg/kg, per oral).Ulcer index,colitisseverity,myeloperoxidase(MPO),malondialdehydeandglutathionewereestimatedfrombloodandtissue.Columnchromatographyof theextractwasdoneandpurifiedfractionswereanalyzedbygaschromatography-massspectroscopy(GC-MS).RESULTS: Treatmentwith thecombinationofhydroalcoholicextractofA. roxburghianus and piperine showed minimal ulceration, hemorrhage, necrosis and leucocyte infiltration by histopathological observation. Acetic acid increased MPO levels in blood and colon tissue to355U/mLand385U/mg, respectively.Thecombinationof hydroalcoholic extract ofA. roxburghianus (100 mg/kg) and piperine (5 mg/kg) significantly decreased MPO in blood and tissue to 182 U/mL and 193 U/mg, respectively (P < 0.05). Similarly, this combination significantlyreducedmalondialdehydelevelsandincreasedglutathionelevelsinbloodandtissue.VariousphytoconstituentsweredetectedbyGC-MS.CONCLUSION: The combination of hydroalcoholicextractofA. roxburghianus and piperine is effective in the treatment of UC and the effects are comparable with the standard drug prednisolone.4H-pyran-4-one,2,3-dihydro-3,5-dihydroxy-6-methyl,eugenolandbenzene,and1-(1,5-dimethyl-4-hexenyl)-4-methylarereportedhavinganalgesic,anti-inflammatory,andantioxidantproperties;theymayplayaroleinthebiologicalactivityofA. roxburghianus root.KEYWORDS: Amaranthus;plantextracts;ulcerativecolitis;myeloperoxidase;malondialdehyde;glutathione; mice

DOI: 10.3736/jintegrmed2013022Nirmal SA, Ingale JM, Pattan SR, Bhawar SB. Amaranthus roxburghianusrootextractincombinationwithpiperine as a potential treatment of ulcerative colitis in mice. J Integr Med. 2013; 11(3): 206-212.Received September 17, 2012; accepted January 24, 2013.Open-access article copyright © 2013 Sunil A. Nirmal et al. Correspondence: Sunil A. Nirmal, PhD, Professor; Tel: +91-9766126772; E-mail: [email protected]

May 2013, Vol.11, No.3207Journal of Integrative Medicine


cramps and diarrhea. Although the exact cause of UC remains undetermined, the condition appears to be related to a combination of genetic and environmental factors. Among the pathological findings associated with IBD are increases in certain inflammatory mediators, signs of oxidative stress, a deranged colonic milieu, abnormal glycosaminoglycan content of the mucosa, decreased oxidation of short-chain fatty acids, increased intestinal permeability, increased sulfide production, and decreased methylation. However, no one factor has been identified as the initial trigger for IBD[2]. Hypochlorous acid, produced by the action of myeloperoxidase (MPO) on hydrogen peroxide in the presence of chloride ions, is involved in the inflammatory reaction in colitis[3]. One of the most frequently used biomarkers that provides an indication of the overall lipid peroxidation level is the plasma concentration of malondialdehyde (P-MDA); it is used to measure the level of oxidative stress in humans[4]. The role of prostaglandins in the course of IBD and their possible usefulness as predictive indicators of inflammation remain largely speculative. Plasma and mucosal prostaglandin E2 (PGE2) levels have been found to be directly correlated with degree of colonic injury[5]. Because there is moderate correlation between mucosal injury and PGE2 content, measurement of plasma PGE2 is a potential surrogate marker of bowel inflammation[5].

Amaranthus roxburgianus Nevski. (Amaranthaceae) is a small-sized tree, 30-65 cm in height, native to China and India. It is commonly known as “tandulja” in Marathi. The effect of varying pH on protein composition and yield of Amaranth blitum has been studied[6]. The roots of A. roxburghianus are traditionally reported to be used for the treatment of colic pains. Piperine has been shown to improve drug bioactivity[7]; thus, this study examines the potential of combining piperine with plant extracts for the treatment of UC.

2 Materials and methods

2.1 Plant materialsThe roots of A. roxburghianus were collected from

Ahmednagar district (Maharashtra State, India) in December 2011 and authenticated by Dr. J. Jayanthi, Joint Director, Botanical Survey of India, Pune, India (Voucher specimen – Ingale-1). Berries of Piper nigrum were collected from Ahmednagar district in December 2012. Authentication of plant material was done by comparison with authentic specimen stored. 2.2 Extraction and isolation 2.2.1 Extraction

A. roxburghianus roots were shade dried and powdered. Extraction of the dried powdered material (160 g) with 70% ethanol was carried out in a reflux condenser.

Extract was vacuum dried to yield 3.75% (weight ratio) of hydroalcoholic extract. Powder of Piper nigrum berries (250 g) was extracted using ethanol (analytical grade reagent) in reflux condenser to yield 6.4% (weight ratio) of ethanol extract. 2.2.2 Isolation of piperine

Twenty milliliters of 10% alcoholic potassium hydroxide was added with constant stirring to the concentrated extract and filtered. The alcoholic solution was allowed to stand overnight, where upon crystals of piperine settled out of solution (2.5%, weight ratio)[8]. 2.2.3 Column chromatography

Hydroalcoholic extract of A. roxburghianus roots (5 g) was subjected to column chromatography over silica gel column by using a step gradient of benzene/methanol (9:1, 1L, F1), benzene/methanol (1:1, 1L, F2), benzene/methanol (3:7, 1L, F3), benzene/methanol (1:9, 1L, F4) and methanol (1L, F5). Significant fractions F3 and F5 were purified and analyzed by gas chromatography-mass spectroscopy and Fourier transform infrared spectroscopy. The compound detection employed the NIST Ver. 2.0 - Year 2005 library. The biological activities were based on Dr. Duke’s Phytochemical and Ethnobotanical Databases by Dr. Jim Duke of the Agricultural Research Service/USDA. 2.3 Animals

Male Swiss mice (20-25 g) were housed under standard laboratory conditions and fed with standard rodent diet and water ad libitum. Rodent diet was composed of crude protein 16%, crude fat 3.8%, crude fiber 2%, amino acids, vitamins, and minerals. Animals were kept in constant temperature ((22 ± 2) °C), humidity (55%) and light-dark condition (12/12 h light/dark). Experimental protocol was approved by the Institutional Animal Ethical Committee (Approval No. PRCOP/IAEC/2011-12/10 dated 2/03/12). 2.4 Pharmacological screening2.4.1 Induction of experimental colitis

Animals were divided into seven groups with six in each group. Control group mice received vehicle (0.2 mL of 5% Tween 80 in distilled water). Standard group received prednisolone (5 mg/kg, intraperitoneally). Animals from groups III and IV received hydroalcoholic extract of roots of A. roxburghianus at doses of 50 and 100 mg/kg, orally, respectively suspended in vehicle for 7 d. Animals from group V received a combination of hydroalcoholic extract of roots of A. roxburghianus (50 mg/kg, per oral) and piperine (5 mg/kg, per oral). Animals from group VI received a combination of hydroalcoholic extract of roots of A. roxburghianus (100 mg/kg, per oral) and piperine (5 mg/kg, per oral). Animals from group VII received only piperine (5 mg/kg, per oral). On the 8th day colitis was induced by intrarectal administration of 150 µL of 5% acetic acid (pH 2.5), 3 cm from the anal margin.



Treatment was continued up to the 10th day[9,10]. 2.4.2 Determination of ulcer index

The entire colon was isolated, opened longitudinally and rinsed with phosphate buffer saline (PBS) (freshly prepared). The ulceration of the opened colon was measured with help of magnifying glass and the ulcer index was calculated by following formula[11]:

Ulcer index (%) = (UPC – UT) ×100% / (UT – UNC)Where, UPC = grade of ulcer in positive control; UT

= grade of ulcer in test; UNC = grade of ulcer in normal control.2.4.3 Assessment of colitis severity

After 48 h of colitis induction, mice were sacrificed by cervical dislocation and dissected for colon removal. Colons were isolated in their entirety, opened longitudinally and rinsed with PBS. Histological scoring of the colon damage was performed. For each mouse, the ulcer area was determined by adding together the sizes of lesions measured microscopically. The total area of damage was expressed as the relative percentage of the total surface area of the colon[12].2.4.4 Determination of MPO activity in colon and blood

After the microscopic measurements, the excised colons (100-150 mg) were homogenized with 200 mL of PBS (pH 7.4) and centrifuged at 78 × g for 20 min at 4 °C. MPO activity of supernatant fluid was then assayed by mixing the supernatant with citric phosphate buffer (pH 5.0) containing 0.4 mg/mL O-phenylene diamine and 0.015% hydrogen peroxide. The change in absorbance at 492 nm was measured spectrophotometrically and compared with the standard dilution with horseradish peroxidase[13].2.4.5 Determination of MDA levels in colon and blood

The reaction mixture contained 0.1 mL tissue sample, 0.2 mL 8.1% sodium dodecyl sulphate, 1.5 mL 2% acetic acid and 1.5 mL 0.8% aqueous solution of thiobarbituric acid. The mixture pH was adjusted to 3.5 and final volume was made of 4 mL distilled water, and 5 mL n-butanol and pyridine (15%) mixture. The mixture was shaken vigorously. After centrifugation at 1 252 × g for 10 min, the absorbance of organic layer was measured at 532 nm. MDA was expressed as nmol/mg of protein[14].2.4.6 Estimation of reduced glutathione

The estimation of the reduced glutathione (GSH) in the different isolated organs was carried out using the method of Jollow et al[15]. In most instances, the reduced form of GSH is mainly comprised of a cellular non-protein sulfhydryl group. This method is therefore based upon the development of a relatively stable yellow color when 5,5′-dithiobis- (2-nitrobenzoic acid), Ellman reagent is added to sulfhydryl compound. The absorbance was read at 412 nm and the equivalent GSH was estimated from the standard GSH curve supplied in the kits.

2.5 Histopathological study Colon tissue was fixed with 10% formalin for 24-36 h,

trimmed at the appropriate site, washed under running tap water for 2 h, then dehydrated with increasing grades of alcohol (50% alcohol overnight, 70% alcohol for 2 h, 80% alcohol for 2 h, 90% alcohol for 2 h and absolute alcohol for 2 h). The tissue was then cleaned with xylene for 1 h and embedded with paraffin wax at 60 ℃. Blocks were prepared and stored in a freezer for 45 d. Slices of tissue were cut at 5-µm thickness. Slices were taken on clean grease-free glass slides smeared with egg albumin in water bath at 60 ℃. Tissue was deparaffinized partially with heat, followed by immersing in xylene for 3 min intervals (3 changes of 3 min each). Sections were rehydrated with decreasing grades of the alcohol (100%, 90%, 80%, and 50%; 3 min in each). Slides were kept in distilled water (5 min) and in hematoxylin (10 min). One dip was given in 1% ammonia water and immediately washed under running tap water (5 min). Alcoholic eosin (2-3 drops) was given and the tissue was again dehydrated with increasing grades of alcohol (70%, 80%, 90%, and 100%; 3 min in each). Slices were cleaned with xylene (3 changes of 3 min each), mounted with DPX mountant and viewed under suitable magnification[16].2.6 Statistical analysis

All results are expressed as mean ± standard error of mean (n = 6). The statistical significance was calculated by the software Prismprimer. The statistical significance (P < 0.05) of differences between mean was assessed by analysis of variance followed by Tukey-Kramer multiple comparison test.

3 Results

3.1 Histopathological studyHistopathological observation showed ulceration,

hemorrhages, necrosis and leucocyte infiltration in the colon of mice treated with acetic acid (Figure 1). Treatment with the combination of hydroalcoholic extract of A. roxburghianus and piperine showed minimal ulceration, hemorrhage, necrosis and leucocyte infiltration (Table 1, Figure 1). 3.2 Determination of ulcer index and MPO, MDA and GSH levels

Results from the ulcer index showed better protective effect with the combination of hydroalcoholic extract of A. roxburghianus and piperine, as compared to individual extracts. Acetic acid caused increases in MPO levels in blood and tissue, up to 355 U/mL and 385 U/mg, respectively. After treatment with the combination of hydroalcoholic extract of A. roxburghianus (100 mg/kg) and piperine (5 mg/kg), MPO levels in blood and tissue decreased significantly to 182 U/mL and 193 U/mg,



Table 1 Histopathological observations after treatment with Amaranthus roxburghianus root extract alone and in combination with piperine

Group nDegree of histopathological changes in colon

Ulceration Hemorrhages Necrosis Leucocytic infiltration

Control (5% acetic acid) 6 ++++ +++ +++ +++

Prednisolone (5 mg/kg, i.p.) 6 ++ ++ + +

Hydroalcoholic extract (50 mg/kg, p.o.) 6 +++ ++ ++ ++

Hydroalcoholic extract (100 mg/kg, p.o.) 6 ++ ++ ++ ++

Hydroalcoholic extract (50 mg/kg, p.o.) and piperine (5 mg/kg, p.o.) 6 + ++ + +

Hydroalcoholic extract (100 mg/kg, p.o.) and piperine (5 mg/kg, p.o.) 6 + + — +

Piperine (5 mg/kg, p.o.) 6 +++ +++ +++ ++

—: no abnormality detected; +: damage/active changes up to less than 25%; ++: damage/active changes up to less than 50%; +++: damage/active changes up to less than 75%; ++++: damage/active changes more than 75%. i.p.: intraperitoneally; p.o.: per oral.

respectively (P<0.05). A significant dose-dependant reduction was observed after treatment with individual extract also but reduction after treatment with the combinaion was found to be more prominent. Similar results were obtained with MDA. Acetic acid caused increases in MDA levels in blood and tissue up to 9.40 nmol/mL and 9.38 nmol/mg, respectively. After treatment with combination of hydroalcoholic extract of

A. roxburghianus (100 mg/kg) and piperine, the MDA levels in blood and tissue decreased significantly to 5.88 nmol/mL and 5.71 nmol/mg, respectively (P<0.05). A significant dose-dependent reduction in MDA levels was also observed after treatment with individual extracts, but reduction after treatment with the combination was found to be more prominent. Regarding the redox state, acetic acid caused a significant decrease in colonic non-

Figure 1 Histopathological observations of colon tissue after the treatment with Amaranthus roxburghianus root extract alone and in combination with piperine (Light microscopy, ×400) A: Model control (5% acetic acid); B: Standard (prednisolone, 5 mg/kg, i.p.); C: Hydroalcoholic extract (50 mg/kg, p.o.); D: Hydroalcoholic extract (100 mg/kg, p.o.); E: Hydroalcoholic extract (50 mg/kg, p.o.) and piperine (5 mg/kg, p.o.); F: Hydroalcoholic extract (100 mg/kg, p.o.) and piperine (5 mg/kg, p.o.). i.p.: intraperitoneally; p.o.: per oral.



Table 2 Effects of Amaranthus roxburghianus root extract alone and in combination with piperine on ulcer and MPO, MDA and GSH levels

(Mean ± standard error of mean)

Group n Microscopic ulcer index (%)

MPO MDA Reduced GSH

Blood (U/mL)

Tissue (U/mg)

Blood (nmol/mg)

Tissue (nmol/mg)

Blood (nmol/mg)

Tissue (nmol/mg)

Control (5% acetic acid) 6 0 355.00±0.31 385.00±0.32 9.40±0.21 9.38±0.11 1.25±0.12 1.21 ± 0.12

Prednisolone (5 mg/kg, i.p.) 6 10 215.00±0.12* 225.00±0.41* 6.33±0.31* 6.26±0.17* 3.25±0.13* 3.21±0.13*

Hydroalcoholic extract (50 mg/kg, p.o.) 6 30 240.00±0.14 252.00±0.22 7.31±0.41 7.12±0.09 2.10±0.14 1.75±0.27

Hydroalcoholic extract (100 mg/kg, p.o.) 6 20 207.00±0.17* 212.00±0.17* 6.72±0.47* 6.62±0.32* 2.37±0.12 2.17±0.35

Hydroalcoholic extract (50 mg/kg, p.o.) and piperine (5 mg/kg, p.o.)

6 10 191.00±0.21* 201.00±0.29* 6.12±0.36* 6.02±0.31* 3.01±0.15* 2.91±0.12*

Hydroalcoholic extract (100 mg/kg, p.o.) and piperine (5 mg/kg, p.o.)

6 10 182.00±0.27* 193.00±0.31* 5.88±0.27* 5.71±0.17* 3.17±0.11* 3.15±0.15*

Piperine (5 mg/kg, p.o.) 6 0 342.00±0.21 343.00±0.12 9.25±0.25 9.32±0.31 1.27±0.22 1.29±0.17*P<0.05, vs control group. MPO: myeloperoxidase; MDA: malondialdehyde; GSH: glutathione.

Table 3 Phytoconstituents identified in Amaranthus roxburghianus root extract using gas chromatography-mass spectroscopy

Fraction Compound identified ActivityF3 4H-Pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl Anti-inflammatoryF3 2-Furancarboxaldehyde, 5-(hydroxymethyl) —F3 Eugenol Analgesic, anti-inflammatory, antioxidant F3 Benzene, 1,2-dimethoxy-4-(2-propenyl) —F3 Diethyl N-benzylanilinophoshonate —F5 Benzaldehyde, 4-methoxy —F5 Benzene, 1-(1,5-dimethyl-4-hexenyl)-4-methyl Antioxidant, anti-inflammatoryF5 1-Eicosene —

F5 3-[3-Acetyl-4,10a,10b-trimethyl-7-(4-methyl pentyl)-5,8-dioxotetradecahydro-9-oxapentalenol [2,1-a] naphthalene —

enzymatic defense systems; however, this effect was normalized by the treatment with the combination of hydroalcoholic extract of A. roxburghianus (100 mg/kg) and piperine in mice. GSH level increased after treatment to 3.17 nmol/mL and 3.15 nmol/mg from 1.25 nmol/mL and 1.21 nmol/mg in blood and tissue, respectively (P<0.05). Animals treated with only piperine did not show any significant improvement in their ulcer conditions. MPO, MDA and GSH levels were almost similar to those of the control group (Table 2). 3.3 Column chromatography and structure elucidation

Various phytoconstituents isolated from the fractions III and V are listed in Table 3 along with their activities.

4 Discussion

Hydroalcoholic extract of A. roxburghianus root along with piperine reduced MPO and MDA levels significantly. This combination potentially plays an anti-inflammatory role in the treatment of colitis, because MPO has been shown to be involved in the inflammatory reaction in colitis[3]. Additionally, increased level of MDA signals oxidative stress in organs, and thus causes inflammation[4]. Cytokines are responsible for modulating intestinal inflammation and injury[17,18]. Since the intestines are in a constant state of controlled inflammation, amplification



of the inflammatory response activates infiltration of inflammatory cells that trigger pathological responses and symptoms of IBD[19]. Our study showed that acetic acid elevates the levels of colonic MPO, indicating infiltration of neutrophils and perturbation of the inflammatory system[20]. This fact is documented in both animal models[3,21], and patients with IBD[22]. In IBD, oxidative stress plays a role in disease initiation and progression[23]. Reactive oxygen species (ROS) attack the cellular macromolecules, disrupting epithelial cell integrity and hindering mucosal recovery, which is especially the case in the presence of impaired endogenous defense systems[24]. In this study, acetic acid-induced ROS formation is inhibited by hydroalcoholic extract of A. roxburghianus root along with piperine. The hydroalcoholic extract of A. roxburghianus inhibited free radical generation, shown here when it restored the redox state of the colonic mucosa. This offers another explanation of the anti-ulcerogenic activity of A. roxburghianus root.

Results showed that the combination of hydroalcoholic extract of A. roxburghianus and piperine is effective in the treatment of UC and results are comparable with the standard drug prednisolone. Piperine itself has no effect on the treatment of UC, which leads us to postulate that piperine increases bioactivity, and improves the action of the herbal extract. 4H-Pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl, eugenol, benzene, and 1-(1,5-dimethyl-4-hexenyl)-4-methyl are reported having analgesic, anti-inflammatory, and antioxidant properties, and they may be important constituents for the biological actions of A. roxburghianus root. This study lends pharmacological support to folkloric, ethno-medical uses of the plant in the management of inflammatory gastrointestinal tract disorders.

5 Competing interests

The authors declare that they have no competing interests.

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