Evaluation of Leaf extracts of Simarouba glauca on Experimentally Induced Inflammatory Bowel Diseases in Wistar rats

G. Ariharasivakumar*, T. Karthikaa, Ridahunlang Nongkhlaw, P. Packialakshmi

Department of Pharmacology, KMCH College of Pharmacy, Coimbatore-48, Tamil Nadu, India.

*Corresponding Author E-mail: karthikaathambidurai@gmail.com

ABSTRACT:

Aim: The aim of the study was to evaluate the Inflammatory Bowel Disorder activity of EASG and CHSG extracts of Simarouba glauca on Experimentally induced Inflammatory Bowel Disease in wistar rats. Methods: The levels of phytochemicals were quantified and HPTLC study was conducted using β-sitosterol as reference. In vitro antioxidant property of EASG and CHSG was evaluated using DPPH and ABTS assay. Evaluation of IBD was done in wistar rats. Rats were treated with indomethacin (7.5mg/kg, s.c) for 3days after 14 days pretreatment with extract and standard drug (prednisolone 10mg/kg). The MPO Level, macroscopic features scoring, body weight analysis and microscopic (histopathological study was carried out and evaluated. Antioxidant potential of intestinal tissue was determined by measuring activities of Superoxide Dismutase, Catalase, Glutathione peroxidase, Lipid per oxidation and reduced Glutathione. Results: EASG and CHSG possessed considerable amount of phytoconstituents such as flavonoid, phenols and β-sitosterol; and also significantly scavenged the free radicals making it a potent antioxidant. The results of macroscopic scoring and MPO level activity suggest that EASG and CHSG at 400mg/kg significantly (p < 0.001) reduced the inflammation in IBD induced animal models. In In vitro cytotoxicity study, a significant reduction in cell damage was observed when caco₂ cells were pretreated with EASG and CHSG extracts when compared to control. EASG and CHSG at 400mg/kg significantly reduced lipid per oxidation and increased the Superoxide Dismutase (SOD), Catalase (CAT) and Glutathione peroxidase (GPx). Reduced Glutathione (GSH) levels and produced significant antioxidant activity. Conclusion: Attributing to the potent antioxidant and anti-inflammatory activity, we could conclude that the EASG and CHSG of Simarouba glauca showed significant anti- inflammatory activity due to presence of β-sitosterol and antioxidants such as phenols and flavonoids.

KEYWORDS: Ethylacetate extract of Simarouba glauca (EASG) and Chloroform extract of Simarouba glauca (CHSG), Inflammatory Bowel Disease (IBD), Myeloperoxidase (MPO).

1. INTRODUCTION:

Inflammatory bowel disease is the general term used to describe two different chronic nonspecific disorders of the gastrointestinal tract i.e., ulcerative colitis (UC) and Crohn’s disease (CD) having many similarities but the conditions usually have distinctive morphological appearance. The disease can cause chronic inflammation of the intestinal tract, which leads to a variety of symptoms.

The inflammation can also cause inflammation to other organs other than the intestines. IBD is a lifelong disease with periods of active disease alternating with periods of disease control (remission). IBD is sometimes confused with but its different from that of irritable bowel disease.

Simarouba glauca bark, flower, stems, seeds, leaves, roots are used for various purpose. Constituents present in the plant are QUASSINOIDS namely, Ailanthinone, glaucarubinone, holocanthone, dehydroglaucarubinone, benzoquinone, canthin, glaucarubine, glaucarubolone, simarubin, melianone, simaroubidin, simarolide, sitosterol, tirucalla and flavonoids. An infusion of the leaves or bark is considered to be astringent, a digestion and menstrual stimulant and an antiparasitic remedy. It is taken internally for Cancer, diarrhoea, dysentery, malaria, colitis, Hemorrhage, Anemia, Rheumatoid arthritis, Hepatitis, Hyperacidity, Dyspepsia Fever, Ulcers and bleeding in alimentary system, it is used externally for wounds and sores. The present study is to investigate the effects of Mandarin oil on the experimental model of colitis induced by indomethacin in wistar rats, it is well established model of inflammatory bowel disease that has same histological and biochemical features of human disease.

2. MATERIALS AND METHODS:

Plant collection and authentication:

Simarouba glauca was collected from Alapuzha district, Kerela and authenticated from Botanical survey of India (BSI) Southern circle, Coimbatore Tamil Nadu. The authentication certificate number is No. BSI/SRC/5/23/2016/tech/604. Soon after collection, the leaves were dried in the shade and crushed to coarse powder for extraction.^(1,2).

Extraction of the plant material:

The leaves were first treated with petroleum ether, the defatted material was then extracted with ethyl acetate and chloroform using soxhlet apparatus. After extraction the solvent was evaporated by using rotary evaporator and dried at room temperature to give viscous mass. The crude extracts were weighed and stored at 4°C before analysis.

Phytochemical screening:

Preliminary qualitative Phytochemical analysis of the extract was carried out using various procedures to detect the presence of several phytochemical like Tannins, Terpenoids, Alkaloids, Flavonoids, Steroids and free Amino acid etc.

HPTLC analysis:

HPTLC analyses were carried out using CAMAG Linomat HPTLC instrument. Sample Preparation was done by taking weighed quantity of extract residue was dissolved in 1ml of chromatographic grade methanol which was used for sample application on pre-coated silica gel 60 F 254 aluminum sheets.

Procedure:

The samples; β-sitosterol 1μl, 3μl, 5μl, 7μl, 10μ, 12μl, 15μl and Chloroform extract of Simarouba glauca (CHSG) and Ethylacetate extract of Simarouba glauca (EASG) 10μl and 10μl were spotted in form of bands with a Camag microlite syringe on pre-coated Silica Gel glass plate 60F254 (10×10 cm with 0.2 mm thickness) using a Camag Linomat 5 applicator. The plates were pre-washed with methanol and activated at 60ºC for 10 min prior to chromatography. The sample loaded plate was kept in TLC twin trough developing chamber after chamber saturation with respective mobile phase. The optimized chamber saturation time for mobile phase was 5 min at room temperature. Linear ascending development was carried out and the plate was developed in the respective mobile phase up to 7cm. The developed plate was then dried by hot air oven for the development of bands. The dried plate was observed under UV light at 254nm and 366nm and photo documentation was done. Densitometric scanning was performed on Camag TLC scanner 3 in the absorbance mode at 366nm. The percentage of active ingredient (β-sitosterol) present in the SG leaf extract was compared with the standard^.(3-5)

In vitro antioxidant studies of EASG and CHSG:

DPPH free radical scavenging assay:

The antioxidant activity of the extract was measured in terms of hydrogen donating or radical scavenging ability using the stable DPPH radical (Blois method) as described⁽⁷⁾

ABTS radical cation assay:

ABTS radical scavenging activity of the extract was measured by Rice-Evans method as described⁽⁸⁾

Invitro cytotoxicity study of EASG and CHSG:

Determination of cell viability by MTT assay:

Cell viability assay (MTT assay) performed using the procedure as described⁽⁹⁾

Pharmacological study:

Animals:

Male wistar rats of 6-8 weeks old and 160-180 g body weight were obtained from the animal house, KMCH College of pharmacy, Coimbatore. All rats were kept at room temperature and allowed to accommodate in standard conditions at 12-hr light and 12-hr dark cycle in the animal house. Animals were fed with commercial pellet diet and water ad libitum freely throughout the study. The experimental procedure was reviewed and approved by IAEC (Institutional animal ethical committee) of KMCH College of Pharmacy, Coimbatore-48. Governed by CPCSEA Government of India. Proposal number: KMCRET/M. PHARM/03/2015-16).

Acute toxicity study:

Based on previously conducted study of Simarouba glauca, the dose was selected⁽¹⁰⁾

Indomethacin induced enterocolitis in rats:

The male wistar albino rats (160-180g) were selected and randomized into five groups of six animals in each group. Group 1 served as Normal or untreated animals, group 2 was positive control (induced) animals receive only Indomethacin (7.5mg/kg) s.c group 3 was treated group with Rats received prednisolone (10mg/kg, p.o), for 3days and indomethacin (7.5mg/kg, p.o) for 2days, group 4 was treated 14days pretreatment with CHSG and indomethacin (7.5mg/kg, s.c), for 3 days and group 5 was treated 14days pretreatment with EASG and indomethacin (7.5mg/kg, s.c), for 3 days.^{(11, 12)}

On the third day after 24 hours fasting the animals were sacrificed by cervical dislocation and dissected open to remove GIT (from stomach to anus). GIT s was flushed gently and cut open.

Duedenum, jejunum, ileum, caecum and colon were taken out to assess inflammation, based on physical parameters, macroscopy and microscopic features. Quantification of inflammation would be done using biochemical assay (MPO, lipid peroxides, GSH). Tissues were fixed in 10% formalin saline and examined histopathologically.^(12-14)

Macroscopic scoring:

Blood was collected through retro-orbital puncture. Following the blood collection, the animals were sacrificed and the small intestine (ileum segment) were excised and opened longitudinally. Gross findings were ranked using the following criteria, 0 - No visible change, 1-hyperemia at sites, 2-loss of mucosal integrity, 3- lesion having diameter 1mm or less, 4-lesions having diameter 2mm or less (no<5), 5- lesions having diameter 2mm or less (no 5-10), 6- lesions having diameter 2mm or less (no>10), 7- lesions having diameter more than 2 mm (no<5), 8- lesions having diameter more than 2mm (no5-10), 9- lesions having diameter more than 2mm (no>10)^(12,13)

Myeloperoxidase assay for quantification of inflammation:

MPO assay performed based on the procedure described^(15,16)

In vivo antioxidant activity:

Enzymatic anti-oxidant activiy:

Estimation of catalase (CAT), estimation of superoxide dismutase (SOD), estimation of glutathione peroxidase (GPX)

Enzymatic antioxidant studies were performed using procedure described^(18-21)

Non enzymatic antioxidant activity:

A. Estimation of reduced glutathione (GSH):

The protein estimation was performed by the procedure as described by Yerra Rajeshwar et al⁽¹⁷⁾

B. Determination of lipid peroxidation(LPO):

The LPO determination was performed by the procedure as described by M. Aslam et al⁽¹⁸⁾

Histopathological studies:

The tissues duodenum, jejunum, ileum, colon and caecum of all rats were isolated and were fixed in 10% formalin solution. The tissues were trimmed and prepare 5μm thick paraffin sections and stained in haematoxyline and eosin. The stained sections were examined for any inflammatory changes like infiltration of cells, necrotic foci, damage to tissue structures like payers patches, damage to nucleus, etc.⁽¹¹⁾

Statistical analysis:

The data’s of all the parameters were analysed using the software SPSS version 16 software The data’s of all parameters were analysed by means of one way ANOVA (Analysis of Variance) followed by Dunnet’s test. The results were expressed as mean ± SEM.

3. RESULTS:

Preliminary phytochemical analysis:

Table 1: Phytochemical Analysis of CHSG and EASG

Sl. No	Phytochemical constituents	CHSG and EASG
1	Carbohydrate	Positive
2	Alkaloids	Positive
3	Triterpenoid	Positive
4	Glycosides	Positive
5	Steroid and sterols	Positive
6	Phenols	Positive
7	Tannins	Positive
8	Saponins	Positive
9	Phenols	Positive
10	Proteins and Amino acids	Positive

Detection of bands:

3D display of β-sitosterol and EASG andCHSG:

Quantification of β-sitosterol in CHSG and EASG sample by HPTLC:

Table 2: quantification of β-sitosterol

Volume applied (μl)	Area	Amount of β- sitosterol present(µg)	% of β-sitosterol In 100 mg/g 0f extract
(EASG extract)10μl	16849.49	42.16	4.21
(CHSG extract)10μl	20538.19	51.39	5.13

In vitro antioxidant activity study in EASG and CHSG:

DPPH and ABTS radical scavenging activity:

Table 3: %Inhibition and IC50 values of DPPH radical by CHSG and EASG

S. No	Concentration (µg/ml)	% inhibition CHSG	% inhibition EASG	IC₅₀ (µg/ml) CHSG	IC₅₀ (µg/ml) EASG
1	10	48.29	42.67	11.94	15.97
2	15	50.23	46.45
3	20	56.20	55.31
4	25	62.04	59.32
5	50	68.28	65.36
6	100	73.19	76.12

Table 4: % inhibition and IC50 values of ABTS radical by CHSG and EASG

S. No	Concentration (µg/ml)	% inhibition CHSG	%inhibition EASG	IC₅₀ (µg/ml) CHSG	IC₅₀ (µg/ml) EASG
1	1	50.67	53.61	1.134	1.160
2	2	56.04	57.41
3	3	61.34	61.63
4	4	65.32	71.18
5	5	68.45	76.92
6	10	79.36	97.37

Invitro cytotoxicity study of EASG and CHSG:

MTT assay on CaCO2 with EASG and CHSG extract.

a) Representative photo micrograph shows morphological changes of Caco2 cell lines with EASG extract

Control CaCo2 EASG extract 500 (µg/ml) EASG extract 1000 (µg/ml)

b) Representative photo micrograph shows morphological changes of Caco2 cell lines with CHSG extract

Control CaCo2 CHSG extract 500 (µg/ml) CHSGextract1000 (µg/ml)

Figure: 2a, 2b

Evaluation of inflammatory bowel disease of CHSG and EASG.

Indomethacin-induced enterocolitis

Table 5: Evaluation based on bodyweight in indomethacin induced enterocolitis

Groups	1^st day body weight	14^th day body weight
Group-1normal	141.2±5.069	163.7±4.402
Group-II control	143±7.956	89.67±2.692***
GROUP-IIIStandard	149±16.06	119.7±8.086***
GROUP-IVEASG treated	138.0±7.412	111.7±4.201*
GROUP-VCHSG treated	139.0±4.211	121.3±3.836***

Statistical comparison: Each group (n=6), each value represents Mean ± SEM. One way Annova followed by Dunnett’s test was performed. ^aP<0.001 denotes comparison of Inflammatory bowel disease control with vehicle control and ns- non significant *P<0.05, **P<0.01, and ***P<0.001 denotes comparison of all groups with Inflammatory bowel disease control.

Table 6: Evaluation based on macroscopic feature and MPO activity in Indomethacin- induced enterocolitis.

Group	Treatment	Mean of macroscopic scores ± S.E.M	MPO activity (U/g)±S.E.M
Group I: normal	Only normal saline	0.0±0.0	6.733±0.1695
Group II: control	Indomethacin (7.5mg/kg, s.c)	5.667±0.1520***	24.90±0.2769***
Group III: Standard	Prednisolone (10mg/kg, orally) and indomethacin(7.5mg/kg))	1.000±0.2191***	8.473±0.1314***
Group IV: EASG (400mg/kg)	Ethyl acetate extract of simarouba glauca (400mg/kg) and indomethacin (7.5mg/kg)	1.133±0.2765***	14.77±0.1520***
Group V: CHSG (400mg/kg)	Chloroform extract of Simarouba glauca (400mg/kg, orally) and indomethacin (7.5mg/kg)	2.200±0.4099***	12.69±0.2143***

Estimation of In-vivo anti-oxidant:

Histopathological study:

Indomethacin-induced enterocolitis in rats:

5a) GROUP-I (normal group)

5b) GROUP-II (Indomethacin control group)

5c) GROUP-III (standard treated group)

5d) GROUP-IV (EASG Extract treated group)

5e) GROUP-V (CHSG Extract treated group)

4. DISCUSSION:

The present study was aimed to assess the efficacy of chloroform and ethylacetate leaves extract of Simarouba glauca for its anti-inflammatory activity in indomethacin induced enterocolitis in wistar rats. Indomethacin, a non-selective COX inhibitor produces enterocolitis. The mechanisms of indomethacin-induced enterocolitis have not been fully illustrated, but in previous reports it was suggested that, initial epithelial damage is mediated partly by synthesis inhibition of the protective prostaglandins PGE₁, PGE₂ and prostacylin. In addition, luminal bacterial and bacterial products also contribute to the inflammatory response in the indomethacin-induced colitis model. Indomethacin induced model affect the middle portion of small intestine (jejunum and proximal ileum) and most severely the caecum, the mechanism is due to decrease level of prostoglandin and generation of free radicals which damage the mucosal integrity

From the results obtained from HPTLC study, it was found that CHSG and EASG contains 51.39 and 42.16μg of β-sitosterol. From the DPPH results obtained, IC₅₀ value of ascorbic acid and CHSG and EASG was found to be 2.989μg/ml and 11.94μg/ml and 15.97μg/ml respectively. From the ABTS Assay IC₅₀ value of ascorbic acid was found to be 0.1142μg/ml whereas 1.134μg/ml and1.160 μg/ml for CHSG and EASG extracts. Thus from the result obtained it could be concluded that CHSG and EASG shows a good antioxidant activity which might be attributed to the presence of phytochemicals such as polyphenols.

The treatment with EASG and CHSG leaves extract of Simarouba glauca has shown a decrease in the macroscopic scores for the inflammation. A significant decrease in MPO activity was also observed. All these observations support the findings that the EASG and CHSG leaves extract of Simarouba glauca was able to offer significant protection in the models studied.

In IBD the activity of the SOD, CAT, GSH, and GPx were found to be increased in the ethylacetate and chloroform extract of Simarouba glauca in treated rats and decrease only in Indomethacin treated animals. From the In-vivo antioxidant studies it was found that CHSG extract has more In-vivo antioxidants activity when compared to EASG extract. The EASG and CHSG extracts treated group showed reduced intensity of lesions without any evidence of necrosis, regeneration or inflammatory reaction. Prednisolone treatment showed suppressed inflammatory reaction.

5. CONCLUSION:

Based on the results obtained, It can be concluded that the Ethylacetate and Chloroform extract of Simarouba glauca has a protective activity against experimentally induced IBD, due to its anti-inflammatory and antioxidant properties. The protective action of this plant may be due to the presence of active constituents β-sitosterol, flavanoids and polyphenols. All the Parameters of extract treated group animals have shown better results which are comparable with the standard prednisolone treated group. Possible mechanism of action may be due to decreasing number of neutrophils and reduction in the synthesis of inflammatory mediators like myeloperoxidase enzyme.

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Received on 07.08.2019 Modified on 19.09.2019

Research J. Pharm. and Tech. 2020; 13(4):1886-1892.

DOI: 10.5958/0974-360X.2020.00340.6