INTRODUCTION:
Popular as piles, Hemorrhoids are abnormal, dilated, soft veins that can be on the outside of the anus or inside the anus. Occur in both the sexes tend to run in families. Hemorrhoids do not bother unless they are damaged or diseased. Based on their location, two types of hemorrhoids occur internal and external. Internal piles completely prolapsed. The stool rubs against hemorrhoids, causing them to bleed. External piles forms thrombus, are painful often ruptures while walking and bleeds. Hemorrhoids are a genetic predisposition; the other factors such as pregnancy, smoking, aging, chronic constipation, and too much spice in the diet is also responsible in its pathogenesis1.
Euphorbia hirta Linn. is a small herb from the family Euphorbiaceae, found abundant in waste grounds, in the hotter parts of India and occurring in all tropical countries. In Hindi, it is called Dudhi. Traditionally the plant is used as a medicine in treatment of various alignments, different parts of the plant are, used for different types of disease, the leaves along with the areal parts are, used as decoction for the treatment of gastrointestinal disorders, diarrhea, it is also very much useful in asthma as a result it is known as asthma weed. The paste is applied topically to destroy warts, wounds, ulcers, oedemas, and phlegmons, the juice of the root given in case of vomiting and snake bite, and its prescribed in gonorrhea. The whole plant is given in worms, bowel complaints, cough, and breastfeeding women when their supply of milk is deficient or fails2.
Several studies revealed that Euphorbia hirta Linn. possess anti-inflammatory activity ( Meher B, et al. 2012)3, anti-oxidant activity (Showkat A.G et al. 2018)4, anti-oxidant activity (Teeli R.A, et al. 2018)5, antimalarial activity (Pereran SD, et al. 2018)6, anti-Cancer activity (Kwan YP, et al. 2015)7, anti-anaphylactic activity (Parmar G, et al. 2018)8, anti-venom activity (Kadiyala G, et al. 2015)9, anti-stress activity (Tiwari N, et al. 2015)10, anti-bacterial / anti-fungal activity (Jakhar S, et al. 2017)11, anti-tumour activity (Patil BS, et al. 2011)12, antidiabetic activity (Uppal G, et al. 2012)13, burn wound healing activity (Tuhin RH, et al. 2017 )14, anti-ulcer activity (Rathnakumar K, et al. 2013)15.
The present study aims to evaluate the anti hemorrhoid activity of the ethanolic extract, isolated rutin, and a rutin derivative semi synthesized drug from Euphorbia hirta Linn, by inducing hemorrhoid using croton oil preparation in Wistar Albino rats16.
MATERIAL AND METHODS:
Collection and Authentification of plant material:
Mature fresh, disease-free whole plants of Euphorbia hirta Linn. was, collected from several habitats of Sultanpur district, a region of Uttar Pradesh. The collected samples were stored in sterile containers for the present study.
The plant Euphorbia hirta Linn. was identified and authenticated by Dr. Narendra Kumar scientist at CSIR-CIMAP, Lucknow. A voucher specimen no (CIMAP/ Bot-Pharm./2018/07) was deposited at Department of CSIR- CIMAP.
Drugs and Chemicals:
The chemicals used for the present study were bromobenzene, sodium methoxide, isopropanol, Petroleum ether, Chloroform, Ethyl acetate, Ethanol and methanol, Pyridine, diethyl ether, These chemicals were purchased from (CDH, Central drug house (P) Ltd, and Avantor, Avantor Performance Materials India Limited). Croton oil (Sigma Aldrich, St, Louis, USA) Pilex ointment (The Himalayan drug company, Bangalore, India)
Sample preparation:
The mature fresh, disease-free, specimen of plants were washed thoroughly 2-3 times with running tap water, ones with 70% ethanol and ones with sterile water, shade-dried without any contamination. The dried plant material was, then powdered by an electric mill and stored in dry bottles for further use
Extraction of plant material:
50 g of dried powdered plant material was extracted by Continuous hot percolation with 250ml of ethanol extract was concentrated by distilling off the solvent and then evaporated to dryness on the water bath17.
Isolation of rutin:
50 g powdered material was, extracted with 500 ml of 80% ethanol. Extract was, evaporated on the water bath, 25 ml water was added, again Extracted with Petroleum ether (50ml×3) and Chloroform (50×3) successively, aqueous layer was collected, Precipitate was filtered and washed with (Chloroform: Ethyl acetate: Ethanol) Undissolved part was dissolved in hot methanol and filtered The filtrate was, evaporated 100 mg Yellow color powder was obtained18.
Semi-synthesis of rutin derivative:
6.64 g of rutin (0.01mol) was dissolved in 180 ml sodium methoxide (containing 0.23g sodium), Refluxed heating for 30 minutes, and subsequently was treated with 0.01 mol of bromobenzene. We obtain crude derivatives through precipitate with isopropanol filtration and ambient temperature drying19.
A Rutin derivative was, synthesized according to the scheme.
Croton oil preparation:
Deionized water, pyridine, diethyl ether, and 6% croton oil in diethyl ether in the ratio of 1:4:5:10.
Anti hemorrhoid studies:
Experimental Animals:
Male and female Wistar Albino rats weighing 200-250g were, used for anti- hemorrhoid activity. Animals were, given a standard diet and distilled water. They were housed in standard environmental condition like- ambient temperature (250C±10C), relative humidity (550C±5%), and 12/12 hour light-dark cycle. Committee for the Purpose of Control and Supervision on Experiments on Animals (CPCSEA) ethical committee of the institution (Hygia institute of pharmaceutical education and research, Lucknow) had given the approval (HIPER/IAEC/34/18/10) to perform the animal experiments on animals20,21.
Acute toxicity studies:
Acute toxicity of the corton oil was, performed as per the OECD 420 guidelines, animals (200-250g) were, divided into different groups having 5 animals each, different doses 10, 100, 1000, 2000 were given to each group and observed for the first 4 hours for behavioral changes, thereafter animals kept under observation for 2 weeks22,23.
Experimental protocol:
Male and female Wistar rats (200–250g) were randomized based on their body weights and were, divided into six groups (G-1–G-6), with each group consisting of 6 animals (𝑛 = 6). G-1 animals received Normal saline 0.9% NaCl (10ml/kg) and served as normal controls. G-2 animals served as disease control they also received Normal saline 0.9% NaCl received (10mg/kg) G-3 animals served as standard control received standard drug (10mg/kg) G4 animals received plant extract (400mg/kg ) G5 animal received isolated rutin (100mg/kg) G6 animals received synthesized drug (200mg/kg) respectively. By applying croton oil preparation hemorrhoid induced to all the groups. Ones all the animals were conformed to be produced with hemorrhoid, all the groups (G-1–G-6) were treated once daily for five days to their respective treatment. On the fifth day, 1 hour after the procedure, all the animals were euthanized by exsanguinations under deep chloroform anesthesia, and rectoanal tissue (20mm in length) were, isolated. They were evaluated for the severity score, weighed, and fixed in 10% neutral buffered formalin solution for histological examination. The RAC was calculated using the formula.
Histological observation of the rectoanal tissue was made to note the appearance of inflammatory cells, congestion, hemorrhage, vasodilatation, and medium to high degrees of necrosis24.
RESULTS AND DISCUSSION:
Identification of isolated compound:
IUPAC: 2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-3-[(-3,4,5-trihydroxy-6-{[(3,4,5-trihydroxy-6-methyloxan-2-yl]oxymethyl]oxan-2-yl]oxychromen-4-one
Molecular formula: C27H30O16, Yield: 70%, Mol. Wt.: 610.52, Colour: Yellow, Appearance: Solid powder Melting point: 194-196◦C, Solubility: Slightly soluble in ethanol and water, Soluble in methanol and ethyl acetate, Freely soluble in DMF, Insoluble in chloroform and ether, TLC Analysis: Solvent: Dichloromethane – Methanol (2:1 v/v) Rf : 0.85
FTIR (KBr)cm-1
The infrared spectra for the isolated compound were recorded using PerkinElmer FTIR spectrometer using KBr
769.88(Ali C-C), 1223.23(C-O), 1546.09( Ar C-C), 1648.99(C=O), 2921.84(Ali C-H), 3296.3(Ar C-O-H), 3698.86(Ali C-O-H)
1H NMR (300 MHz, DMSO-d6
The Nuclear magnetic resonance spectroscopy for the isolated compound was recorded using Bruker DRX-300 NMR Spectrometer using DMSO –d6
0.981-1.001 (d, 3H,-CH3), 3.041-3.209 (m,6H,Ali-OH), 3.268-3.444 (m,8H,rutinoside protons and CH2O), 3.691(d,1H,CH-CH3 ramnose), 3.723 (d,1H,CH-CH2O glucose), 4.382 (d,1H,OCHO ramnose), 4.385 (d, 1H, OCHO glucose) 5091-5.356 (m, 4H,Ar-OH), 6.189-6.195 (d,1H, Ar-H), 6.380-6.406 (d,1H,Ar-H), 6.826-6.856 (d,1H,Ar-H), 7.529-7.559 (m,2H,Ar-H).
Identification of semi-synthesized compound:
IUPAC: 5, 7-dihydroxy-2-(3-hydroxy-4-phenoxyphenyl)-3-[(3, 4, 5-trihydroxy-6-{[(3, 4, 5- trihydroxy-6-methyloxan-2-yl)oxy]methyl}oxan-2-yl)oxy]-4H-chromen-4-one
Molecular formula: C33H34O15, Yield: 64%, Mol. Wt: 692.66, Colour: Brown, Appearance: Solid powder Melting point: 198-200◦C, Solubility:Slightly soluble in ethanol, Soluble in Water, Freely soluble in DMF, Insoluble in chloroform and ether, TLC Analysis Solvent: Dichloromethane – Methanol (2:1 v/v) Rf: 0.85
FTIR (KBr) cm-1
The infrared spectra for the synthesized compound were recorded using PerkinElmer FTIR spectrometer using KBr
794.11(Ali C-C), 1235.29(C-O), 1558.82(Ar C-C), 1676.47(C=O), 2852.94(Ali C-H), 3411.76(Ar C-O-H), 3666.47(Ali C-O-H).
1H NMR (300 MHz, DMSO-d6
The Nuclear magnetic resonance spectroscopy for the synthesized compound was recorded using Bruker DRX-300 NMR Spectrometer using DMSO –d6
0.979-0.999 (d, 3H,-CH3), 3.218-3.269 (m, 6H, Ali –OH) 3.832-4.253 (m,8H, rutinoside protons and CH2O), 3.396 (d,1H,CH-CH3 ramnose), 3.688 (d, 1H, CH-CH2O glucose), 4.268 (d,1H,OCHO ramnose), 4.378-4.382 (d, 1H, OCHO glucose) 5.330-5.355 (m, 3H, Ar-OH) 6.179-6.195 (d, 1H, Ar-H), 6.380-6.387 (d, 1H,Ar-H), 6.824-6.853 (d,4H,Ar-H), 7.526-7.570 (m,4H,Ar-H).
Anti- Hemorrhoid activity:
Table 1: LD50 Determination of corton oil
|
Dosage (mg/kg) |
Animal died/Animal survived |
|
10 |
0/5 |
|
100 |
0/5 |
|
1000* |
1/4 |
|
2000 |
ND |
Effect of corton oil on the body weights of rats before and after induction:
In this case, the loss in weight of the animals after induction is a symptom of hemorrhoids due to loss of blood and severe inflammation in the recto-anal region of the rats coupled with anal itching was, seen in the animal groups.
Table 2: Effect of corton oil on the body weights of rats before and after induction
|
Test groups |
Weight before (g) 1-Day |
Weight after (g) 5 -Day |
|
G1 Normal control |
234.46 ± 2.57 |
229.41 ± 2.53 |
|
G2 Disease control |
233.83 ± 9.56 |
157.53 ± 6.70 |
|
G3 Standard drug |
234.93 ± 6.76 |
224.36 ± 6.45 |
|
G4 Isolated rutin |
234.33 ± 10.39 |
216.45 ± 10.81 |
|
G5 Plant extract |
233.83 ± 23.75 |
221.45 ± 24.18 |
|
G6 Test drug |
234.10 ± 10.18 |
220.49 ± 11.63 |
Fig 1: Effect of corton oil on body weights of rats before and after induction.
Effect of drugs on the rectum after treatment:
The normal control group showed normal cell architecture of the rectoanal region; however, test drug and plant extract showed remarkable vasoconstriction of the rectum. The most significant healing of the rectum was shown by standard drug and isolated rutin, which was evident in the reduction of tissue weight after treatment.
Table 3: Effect of drugs on the rectum after treatment
|
Drugs, Dose (mg/kg) |
Rectoanal coefficient |
Inference |
|
G1 Normal control No treatment |
0.014 ± 0.0016 |
- |
|
G2 Disease control No treatment |
0.042 ± 0.0024 *** |
- |
|
G3 Standard drug (10 mg) |
0.014 ± 0.0017 |
Strong healing |
|
G4 Plant extract (400 mg) |
0.020 ± 0.0039 ** |
Moderate healing |
|
G5 Isolated rutin (100 mg) |
0.015 ± 0.0023 |
Strong healing |
|
G6 Synthesized drug (200 mg) |
0.020 ± 0.0035 * |
Moderate healing |
Effect of the drug's on rectum after treatment values are, given as Mean ± SD of experimental animals (n=6) ***P < 0.05 represents high lesions on rectum compared to normal control. (***P<0.05) Represents, high inflammation in rectum compared to normal control. In the case of treatment group, standard drug and isolated rutin show remarkable treatment having minimum soreness when compared to normal. In the case of Plant extract and synthesized drug, there is very little inflammation in the rectum (* P < 0.05) as compared to normal control.
Fig 2: Effect of drugs on rectum after administration.
Effect of the drug's on body weight before and after treatment.
Table 4: Effect of drugs on body weight before and after treatment
|
Test group |
The difference in weight among 1-5 day |
|
G1 Normal control |
5.05 ± 0.13 |
|
G2 Disease control |
76.30 ± 13.03 *** |
|
G3 Standard drug |
10.56 ± 1.10 |
|
G4 Plant extract |
17.88 ± 1.48 ** |
|
G5 Isolated rutin |
12.35 ± 1.78 |
|
G6 Synthesized drug |
13.60 ± 2.52 * |
Effect of the drug's on body weight after treatment. Values are, given as Mean±SD of experimental animals (n=6) (*P<0.05) represents statistical significance against normal control. (***P<0.05) Represents, a high loss in body weight compared to normal control. In case of treatment group standard drug and isolated rutin show remarkable treatment minimum loss in weight when compared to normal. Plant extract represents the loss in body weight (** P < 0.05) whereas in the case of synthesized drug, there is very little loss in body weight (* P < 0.05).
Fig 3: Effect of drugs on body weight after treatment