Author's Accepted Manuscript

Attenuation of inflammatory mediators, oxi-dative stress and toxic risk evaluation ofAporosa lindleyana Baill bark extract

Yakub Ali, Mohammad Sarwar Alam, HinnaHamid, Asif Hussain, Chetna kharbanda, Sa-meena Bano, Syed Nazreen, Saqlain Haider

PII: S0378-8741(14)00554-6DOI: http://dx.doi.org/10.1016/j.jep.2014.07.035Reference: JEP8937

To appear in: Journal of Ethnopharmacology

Received date: 7 December 2013Revised date: 11 June 2014Accepted date: 16 July 2014

Cite this article as: Yakub Ali, Mohammad Sarwar Alam, Hinna Hamid, AsifHussain, Chetna kharbanda, Sameena Bano, Syed Nazreen, Saqlain Haider,Attenuation of inflammatory mediators, oxidative stress and toxic riskevaluation of Aporosa lindleyana Baill bark extract, Journal of Ethnopharmacol-ogy, http://dx.doi.org/10.1016/j.jep.2014.07.035

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Attenuation of inflammatory mediators, oxidative stress and toxic risk evaluation of

Aporosa lindleyana Baill bark extract

Yakub Alia, Mohammad Sarwar Alama, Hinna Hamida, Asif Hussainb, Chetna kharbandaa,

Sameena Banoa, Syed Nazreena, Saqlain Haidera

*aDepartment of Chemistry, Faculty of Science, Jamia Hamdard (Hamdard University), New

Delhi-110062, India.

bDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Jamia Hamdard (Hamdard

University), New Delhi-110062, India.

Correspondence to Prof. Mohammad Sarwar Alam, Jamia Hamdard, New Delhi-110062, Email

ID. msalam@jamiahamdard.ac.in msalam5555@gmail.com Phone: +91 11 26059688(5555),

Fax: +91 11 26059663

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Abstract

Ethnopharmacological relevance: Traditionally, Aporosa lindleyana Baill. has been used

against various ailments viz. jaundice, fever, headache, seminal loss and insanity. The present

study aims to evaluate the anti-inflammatory and anti-oxidant activity of the ethanolic extract of

Aporosa lindleyana Baill. bark and its fractions.

Method: The anti-inflammatory activity of ethanolic extract of Aporosa lindleyana Baill. bark

and its various fractions at doses of 200 mg/kg and 300 mg/kg b.w. has been carried out by

carrageenan induced hind paw edema method. To establish the probable mechanism of action,

TNF-α and NO levels have been estimated by ELISA method and the effect of active fraction on

COX-2 and NF-κB expressions has been evaluated. The effect on the levels of anti-oxidative

enzymes (CAT, SOD & GPX) by the ethanolic extract and its fractions has also been

investigated. Furthermore, peptic ulcer and hepatotoxic risk evaluation has also been carried out

at three times higher dose than that used in inflammatory in vivo model.

Results: Among the extract and its various fractions tested for anti-inflammatory activity, the

methanolic fraction at a dose of 300 mg/kg showed significant inhibition in paw edema by 73%

as compared to Indomethacin which showed 77% inhibition after 5h. The same dose of

methanolic fraction also caused significant reduction in TNF-α (59.27%) and NO concentration

(57.12%) while Indomethacin showed inhibition of 63.91% and 60.12%, respectively. The active

methanolic fraction was also found to inhibit the expression of NF-κB and COX-2 induced by

carrageenan. Histological studies showed that the ethanolic extract and its fractions did not cause

any damage to the stomach as well as to liver. Moreover, the active fractions also decreased lipid

peroxidation levels and increased the antioxidant enzyme activities (SOD, CAT, GPX).

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Conclusion: The results of present study demonstrated that significant anti-inflammatory

activity of methanolic fraction of A. lindleyana may be attributed to the modulation of pro-

inflammatory mediators. Same fraction was also found to be effective against oxidative stress as

it was found to elevate the levels of anti-oxidative enzymes. It can therefore be concluded that

the methanolic fraction could be explored as a disease modifying agent against inflammation and

oxidative stress.

Keywords: Anti-inflammatory, Anti-oxidant, Aporosa lindleyana, TNF-α, NO, ulcerogenic

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1. Introduction

Inflammation plays a decisive role in immune surveillance and responses to therapy. It has also

been evident during last decade that the chronic inflammatory microenvironment is a substantive

requirement for the development and progression of chronic diseases like tumor, rheumatoid

arthritis, asthma, psoriasis and dermatitis (Karin, 2006). Since the oxidative stress plays a pivotal

role in pathogenesis of inflammation, prevention against oxidative stress might also result in

preventing the progression of related diseases. Oxidative stress is a consequence of discrepancy

between the production of reactive oxidative species (ROS) and antioxidants in a system.

Antioxidants have the ability to alleviate disease by scavenging ROS and by reducing resultant

oxidative stress (Oday et al., 2012). From ancient times, several medicinal plants and their

products are being used for treatment of various ailments. Aporosa lindleyana Baill, commonly

called kotili in Kerala and kodali in Tamil, belongs to the family Euphorbiaceae (Kirtikar and

Basu, 1993). This plant is traditionally being used against various ailments viz. jaundice, fever,

headache, seminal loss, insanity and excessive thirst (Chopra et al., 1992; Kirtikar and Basu,

1987, Anonymous, 1985). It is a branched, evergreen, glabrous tree, grown in southern part of

India and Sri Lanka. The antimicrobial and analgesic activity of Aporosa lindleyana bark has

been well reported (Lingadahalli et al., 2008). The root of this plant has been reported to exhibit

hypoglycemic (Jayakar and Suresh, 2003), anti-oxidant (Shrishailappa et al., 2005), antiviral and

diuretic (Venkataraman et al., 2010) activities. Keeping in view the ethnopharmacological use of

this plant, the present study has been carried out. The aim of this study is to evaluate the anti-

inflammatory and anti-oxidant potential of the ethanolic extract of bark of A. lindleyana and its

various fractions.

2. Materials and Methods

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2.1. Collection and identification of plant material

The bark of A. lindleyana was collected from district Thiruvananthapuram of Kerala during the

month of May and was identified by Dr. Sunita Garg, taxonomist, NISCAIR, CSIR, New Delhi

(Voucher Number: 2013/2223/04).

2.2. Plant preparation and ethanolic extract

The bark of plant was air dried under shade (25-35°C with 45-60% relative humidity) and

powdered. The powdered plant material was then extracted using ethanol (95% v/v) in a Soxhlet

apparatus and concentrated under vacuum. The yield of the ethanolic extract was 9.42% (w/w).

The ethanolic extract was fractionated by different solvents of increasing polarity using

petroleum ether, chloroform and methanol. The yields of petroleum ether, chloroform and

methanolic fractions were 1.1% w/w, 4.2% w/w and 6.5% w/w, respectively.

2.3. Animals

Albino Wistar rats of either sex (130-160 g) were obtained from Central Animal House,

Hamdard University, New Delhi. The animals were kept in cages at the room temperature and

fed with food and water ad libitum. Before starting the experiment, the animals were fasted

overnight. The experiments were performed in accordance with the rules of Institutional Animals

Ethics Committee (registration number- CPCSEA 863)

2.4. Drugs and Chemicals

Indomethacin, carrageenan, carboxymethylcellulose, Griess reagent, acetonitrile (HPLC grade),

trichloroacetic acid and thiobarbituric acid were purchased from Sigma-Aldrich Chemicals Pvt.

Limited, Bangalore, India.

2.5. Phytochemical screening

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The ethanolic extract of A. lindleyana was subjected to phytochemical analysis for determining

the presence of alkaloids, phenols, lipids, flavonoids, saponins, sterols, tannins, carbohydrates

and terpenoids (Wagner et al., 1984).

2.6. RP-HPLC profiling

Reverse Phase High Performance Liquid Chromatography (RP-HPLC) of ethanolic extract of A.

lindleyana was carried out using C-18 reverse phase HPLC column (250 x 4.6mm) with low

pressure gradient. The sample (0.1g) was dissolved in 10ml methanol/water (1:1) and filtered.

The column was eluted using acetonitrile (A) and 1% orthophosphoric acid (B) for 40min after

loading 10μL of the sample. Gradient elution was carried out by changing concentration of A

from 30% to 90% at a flow rate of 1.0 ml/min and the chromatograph was recorded at 254nm.

Toxicity Study

The selected albino Wistar rats of either sex were used to determine the dose of the 95%

ethanolic extract of A. lindleyana. The animals were fasted overnight before the start of the

experiment and were divided into six groups containing five rats in each. The Karbers method

(Kharbanda et al., 2014) was used to determine the dose. Carboxymethylcellulose (1%w/v) was

used as vehicle to suspend the extract and administered orally. The other groups received the

extract in one of the following doses– 100, 200, 300, 400, 500, 600, 700, 800, 900 & 1000

mg/kg. The animals were observed continuously for first four hours for behavioral changes after

dosing and for mortality at the end of 24, 48 and 72h. No mortality was seen even after 72 h.

This experiment indicated that the ethanolic extract is safe up to a single dose of 1000 mg/kg

b.w.

2.7. Anti-inflammatory activity

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Anti-inflammatory activity was carried out by Carrageenan-induced hind paw edema method

(Winter et al., 1962; Kumar et al., 2010; Haider et al., 2011, 2012). Two groups of five rats each

were given 0.5% carboxymethylcellulose solution and Indomethacin (20mg/kg/b.w.) which

served as control and standard, respectively. Rest of the groups was administered with ethanolic

extract and its various fractions orally at doses of 200 and 300mg/kg b. w. A freshly prepared

solution of Carrageenan (1.0% in sterile 0.9% NaCl solution) in a volume of 0.1 ml was injected

subcutaneously into the subplantar region of the right hind paw of rats after 1 h of administration

of the test extract and fractions. Right hind paw volume was measured at 3 h and 5 h after

Carrageenan injection with the help of a digital plethysmometer. The percent anti-inflammatory

activity was calculated according to the formula given below:

% Anti-inflammatory Activity = [VC -Vt /VC] x 100

where, Vt represents the mean increase in paw volume in rats treated with test extracts and VC

represents the mean increase in paw volume in control group of rats.

2.8. TNF-α Assay

Overnight fasted rats were divided into 11 groups of 6 rats in each group. Standard

(Indomethacin, 20mg/kg b.w.) and test samples of extract and its fractions (200 & 300 mg/kg

b.w.) suspended in vehicle (1% CMC in water in volume 10ml/kg) were administered orally to

respective groups. Control (Carrageenan control), standard and test groups received a subplantar

injection of Carrageenan (0.1ml of 1% suspension in normal saline) in the right hind paw. The

right hind paw of each rat was cut at the level of the calcaneous bone after 5 h of dosing. Paws

were washed in saline and gently centrifuged at 4000 rpm for 30 min in order to recover

oedemantous fluid. The fluid recovered was then filtered through Millipore cut-off filter (10,000

mol. wt.) to remove traces of blood cells if any (Millipore, Bedford, MA, U.S.A.). The level of

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TNF-α was determined using a commercially available ELISA kit (e-Bioscience, San Diego,

CA.) according to the manufacturer’s instruction (Syed et al., 2013).

2.9. NO assay

Nitrite assay was done using Griess reagent by the reported method of Green et al., 1982 with

some modifications (Rehman et al, 2013). In brief, 100 μl of Griess reagent (1:1 solution of 1%

sulfanilamide in 5% phosphoric acid and 0.1% naphthylethylenediaminedihydrochloride in

water) was added to 100 μl of PMS incubated for 5-10 min at room temperature protected from

light. Purple/magenta color began to form immediately. Absorbance was measured at 546 nm,

nitrite concentration was calculated using a standard curve for sodium nitrite, and nitrite levels

were expressed as l mol/mg protein.

2.10. Determination of antioxidant enzyme activity in paw tissue

For all antioxidant enzyme assays, dosing pattern was kept same as that for anti-inflammatory

activity. After 5 h, the treated animals were sacrificed and carrageenan induced paw tissue has

been taken for performing biochemical assay. 10% homogenate of paw tissue was centrifuged at

10,000 rpm for 10 min at 4oC to obtain post mitochondrial solution (PMS).

2.10.1. Catalase (CAT) activity

Catalase (CAT) activity was carried out by previously reported method (Claiborne, 1985). The

reaction mixture consisted of 1.95 ml phosphate buffer (0.1 M, pH 7.4), 1.0 ml hydrogen

peroxide (0.019 M) and 0.05 ml of tissue post mitochondrial solution (PMS, 10 %) in a final

volume of 3 ml. Absorbance was recorded at 240 nm. Catalase activity was calculated as nmol

H2O2 consumed per min per mg protein.

2.10.2 Superoxide dismutase (SOD) activity

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The Superoxide dismutase (SOD) activity was measured by the reported method (Marklund &

Marklund, 1974). The reaction mixture consisted of 2.87 ml Tris–HCl buffer (50 mM, pH 8.5),

pyrogallol (24 mM in 10 mM HCl) and 100 µl of tissue 10% PMS making a total volume of 3

ml. The absorbance of SOD was measured at 420 nm and was expressed as units/mg protein.

One unit is defined as the enzyme activity that inhibits auto-oxidation of pyrogallol by 50%.

2.10.3 Glutathione peroxidase activity

The Glutathione peroxidase (GPX) activity was measured by previously reported the method

(Jollow et al., 1974). In this method, 1.44 ml phosphate buffer (0.1 M, pH 7.4), 0.1 ml EDTA (1

mM), 0.1 ml sodium azide (1.0 mM), 0.05 ml GR (1 eu/ml), 0.05 ml GSH (1.0 mM), 0.1 ml

NADPH (0.2 mM), 0.01 ml H2O2 (0.25 mM) and 0.1 ml PMS (10%) was mixed in a total

volume of 2.0 ml. The absorbance was read at 340 nm. The enzyme activity was calculated as

nmol NADPH oxidized/min/mg protein with the help of the molar extinction coefficient of

6.22x103 M-1 cm-1.

2.11. Ulcerogenic risk evaluation

The ethanolic extract and its various fractions were further evaluated for their ulcerogenic

effects. The ulcerogenic studies (Vogel and Vogel, 1997) were carried out after oral

administration of test extracts and standard drug at a dose of 900 & 60 mg/kg b.w., respectively

which is three times of the dose used for anti-inflammatory activity. Control rats were orally

administered suspension of 1% carboxymethylcellulose only. Animals were sacrificed 5 h after

administration of standard and test samples.

2.12. Lipid peroxidation content

The reported method was followed for evaluating lipid peroxidation (LPO) content (Ohkawa et

al., 1979). The gastric mucosa was scraped with two glass slides and weighed (100 mg) and

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homogenized in 1.8 ml of 1.15% ice cold KCl solution. One milliliter of suspension medium was

taken from the supernatant, 0.5 ml of 30% trichloroacetic acid followed by 0.5 ml of 0.8%

thiobarbituric acid reagent were added to it. The tubes were covered with aluminum foil and kept

in a shaking water bath for 30 min at 80°C. After 30 min, tubes were taken out and kept in ice

cold water for 10 min. These were then centrifuged at 3000 rpm for 15 min. The absorbance of

supernatant was read at 540 nm at room temperature against the blank on UV.

2.13. Hepatotoxicity studies

For this study, a dose of 900 mg/kg b.w. of the ethanolic extract and its fractions were

administered orally (three times the dose used for anti-inflammatory activity). The standard drug

indomethacin was administered at 60 mg/kg b.w. dose. A group of healthy rats served as control

and was administered with1% carboxymethylcellulose. The rats were sacrificed after 5h of the

administration of the test sample and standard drug and their liver specimens were removed. The

specimens were stored in 10 % formalin solution. (Lambert et al., 2010)

2.14. Immunohistochemistry

The liver tissues were fixed in formalin and embedded in paraffin. Sections of 5 µm thickness

were cut onto poly-lysine coated glass slides. Sections were deparaffinized three times (5 min) in

xylene followed by dehydration in graded ethanol and finally rehydrated in running tap water.

For antigen retrieval, sections were boiled in 10mM citrate buffer (pH 6.0) for 5-7 min. Sections

were incubated with hydrogen peroxide for 15 min to minimize non-specific staining and then

rinsed three times (5 min each) with 1X PBST (0.05% Tween-20). Blocking solution was applied

for 10 min, then sections were incubated with diluted (1:100) primary anti-bodies, purified rabbit

polyclonal anti-NF-κB antibody (BioLegend) and rabbit polyclonal anti-COX-2 antibody (Bio

Vision), overnight at 4oC in humid chamber. Further processing was done according to the

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instructions of Ultra Vision plus Detection System Anti-Polyvalent, HRP/DAB (Ready-To-Use)

staining kit (Thermo scientific system). The peroxidase complex was visualized with 3,3’-

diaminobenzidine (DAB). Lastly the slides were counterstained with haematoxylin, cleaned in

xylene, dehydrated with ethanol and after that DPX mounting microscopic (BX 51 Olympus)

analysis was done at 40x magnification.

2.15. Statistical analysis

Data was analyzed by one way ANOVA followed by Dunnett’s ‘t’ test (n = 5), *p < 0.05, **p <

0.01 & ns p > 0.05 significant in comparison to control.

3. Results

3.1. Phytochemical screening and RP-HPLC profiling

Preliminary chemical tests of ethanolic extract confirmed the presence of alkaloids, flavonoids

and triterpenoids. RP-HPLC performed on the ethanolic extract of A. lindleyana showed

numerous peaks in the chromatogram with the retention time ranging between 0 to 40 min at the

wavelength of 254 nm. Two major peaks appearing at retention time 25.69 and 27.11 min were

identified as stigmasterol and β-sitosterol, respectively. The chromatogram also displayed many

peaks before the retention time of 12.00 min (Fig.1) which suggested that highly polar

phytoconstituents are present in the ethanolic extract. RP-HPLC profile thus generated can be

used as a fingerprint to authenticate the identity of the plant material.

3.2. Anti-inflammatory Activity

The results of anti-inflammatory activity of the ethanolic extract and its various fractions of A.

lindleyana bark are shown in table 1. Among the tested fractions, at a dose of 300mg/kg, the

methanolic fraction showed comparable activity (73% inhibition) to that of Indomethacin (77%

inhibition). It was observed that chloroform fraction and ethanolic extract showed moderate

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activity whereas the petroleum ether fraction did not show any significant anti-inflammatory

activity as compared to standard drug.

3.3. TNF-α Level

The effect of ethanolic extract and its various fractions on the level of TNF-α are shown in Fig.

2. The amount of TNF-α in the serum of the rats treated with methanolic fraction at a dose of

300mg/kg b.w. was reduced by 59.27% as compared to that of the control group. The percentage

reduction seen in case of methanolic fraction treated group (300mg/kg b.w.) was comparable to

that of Indomethacin (63.91%). The rest of the fractions also exhibited inhibitory activity in

TNF-α production. Inhibition was significant with chloroform fraction and ethanol extract at

dose of 300 mg/kg b.w. However, the petroleum ether fraction was not seen to be much effective

in inhibiting TNF-α level.

3.4. Nitrite level

Effect of ethanolic extract of bark of A. lindleyana and its various fractions on nitrite levels are

shown in Fig. 3. The level of nitrite in the serum of the rats treated with methanolic fraction at a

dose of 300mg/kg b.w. was reduced by 57.12% as compared to that of the control group. The

percentage reduction seen in case of methanolic fraction treated group (300mg/kg b.w.) was

comparable to Indomethacin (60.12%). The rest of the fractions also exhibited inhibitory activity

in nitrite levels. Inhibition was significant with chloroform fraction and ethanol extract at dose of

300 mg/kg b.w. However, the petroleum ether fraction was not seen to be much effective in

inhibiting the level of nitrites.

3.5. Anti-oxidant enzyme activity

The results of CAT, SOD, and GPX activities in rat paw edema are shown in Table 2. It can be

deduced from the results that carrageenan decreased the activities of CAT, SOD and GPX in the

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rat paw by 39.49%, 35.68% and 43.58%, respectively, as compared to control group. The

ethanolic extract of A. lindleyana and its fractions increased the activities of CAT, SOD and

GPX as compared to carrageenan group. The results indicated that among all fractions and

ethanolic extract, the methanolic fraction increases the activities of CAT, SOD and GPX by

73.30%, 75.28% and 74.14% at a dose of 300mg/kg when compared to standard drug ascorbic

acid which increase the activities by 80.23%, 79.32% and 82.38%, respectively.

3.6. Ulcerogenic activity

The results of the histopathological studies are shown in Fig. S1. When compared with

Indomethacin, ethanolic extract and its fractions did not cause any gastric ulceration and

disruption of epithelial cells at the given oral dose. Stomach wall of indomethacin treated group

at low power (10x) photomicrograph showed damage of the mucosa and sub mucosa. High

power (40x) photomicrograph of same section showed some loss of epithelial cells from the

superficial and deep layer of the mucosa whereas test group animals revealed no surface

epithelial damage and sub mucosal damage.

3.7. Lipid peroxidation

The results of lipid peroxidation activity were measured as nmol of MDA per 100 mg of gastric

mucosa tissue and are shown in fig 4. The level of lipid peroxidation in the carrageenan induced

animals was significantly increased. The elevation in lipid peroxidation was also seen in groups

treated with indomethacin and petroleum ether fraction but not as pronounced as in case of

carrageenan treated group. However, the level of lipid oxidation was greatly reduced in the

presence of ethanolic extract and its various fractions. Again, the methanolic fraction caused

significant reduction in lipid peroxidation in comparison to standard group.

3.8. Hepatotoxicity study

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From the photomicrograph obtained from hepatotoxicity study, it was observed that the

chloroform fraction, methanol fraction and ethanolic extract did not cause any damage to the

liver as compared to indomethacin which caused significant inflammation to the portal and

centrizonal area and sinusoidal dilation. However, the petroleum ether fraction exhibited slight

inflammation in the regions of portal and sinusoidal vein (Fig S2).

3.9. Immunohistochemistry

Hepatic expressions of COX-2 and NF-κB have been shown in the figures 5a and 5b,

respectively. The intensity of brown colour in the animals treated with carrageenan only (group

II) clearly indicated more number of cells having COX-2 and NF-κB expression as compared to

group I containing healthy rats. Treatment with standard drug Indomethacin in the group III and

active methanolic fraction in the group IV reduced the number of cells showing expression of

these proteins as compared to group II. However the active methanolic fraction reduced slightly

more number of cells as compared to group III standard.

4. Discussion

Carrageenan induced rat paw edema has been a well established biphasic inflammatory model to

investigate the anti-inflammatory potential of a drug. The first phase involves the release of

histamine, serotonin and kinins whereas the second phase is related to the production of

prostaglandins and slow reacting substances which peak at > 4h (Manfred F. et al., 1985). In the

present study, the methanolic fraction has shown comparable inhibition of inflammation to the

standard drug Indomethacin whereas the other fractions have shown moderate activity at 5 h.

Therefore it could be expected that the ethanolic extract as well as fractions of A. lindleyana

might be inhibiting the production of several mediators of inflammation like TNF-α, NF-κB,

COX-2 etc. TNF-α is a proinflammatory mediators and its inhibition may prove effective in

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healing inflammation (Schimmer et. al., 2001). From the results of TNF-α assay, it was found

that the ethanolic extract of A. lindleyana and its various fractions decreased the level of TNF-α.

The methanolic fraction showed prominent attenuation in TNF-α level, which was comparable to

the standard drug Indomethacin. The evaluation has also been carried out on the production of

nitrite oxide (NO), another important mediator in the pathogenesis of various inflammatory

diseases (Rehman et al., 2013). The results showed that carrageenan induced group markedly

increased the nitrite levels indicating that the induction with carrageenan clearly facilitated the

process of inflammation while this effect was quite controlled by the ethanolic extract of A.

lindleyana and its various fractions. Among all the fractions, the methanolic fraction was

observed to be the most effective in inhibiting the production of nitrite levels. A pro-

inflammatory enzyme cyclooxygenase (COX) is involved in the process of inflammation

(Dubois et. al 1998). Its two major forms, COX-1 and COX-2 are mechanistically similar but

different in their expression in genes. The expression of COX-1 involves normal homeostasis of

prostaglandins while the expression of COX-2 can be induced by various stimuli like growth

factors, cytokines and oncogenes (Smith et al., 2000). Therefore, the suppression in the activity

of COX-2 enzyme would inhibit the development of inflammation. The findings of the current

study demonstrated that the methanolic fraction which exhibited significant anti-inflammatory

activity also inhibited carrageenan induced hepatic expression of COX-2. NF-κB also plays a

pivotal role as a redox sensitive transcription factor for inflammation. It is a constituent in the

cytoplasm of resting cell. On exposure to different stimuli, it gets translocated into the nucleus

where it mediates the transcription of different proinflammatory mediators, cytokines,

chemokines, and adhesion molecules (Baeuerle and Baltimore, 1996; Chao et al., 2010).  The

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present study showed that the active methanolic fraction strongly suppressed the activation of

NF-κB in rats by carrageenan which in turn interrupted the various transcriptional processes.

Since oxidative stress is involved in triggering proinflammatory cytokines and mediators (TNF-

α, NO, COX-2 and NF-κB), the reduction in the levels of TNF-α, NO and in the expression of

COX-2, NF-κB can also be taken as the consequence of diminishing effect on oxidative stress

(Alessandro et al., 2007). Several assays have been carried out to study the effect of the ethanolic

extract and its various fractions on the activities of various antioxidative enzymes such as SOD,

CAT and GPX. These enzymes are directly involved in free radical scavenging and therefore in

attenuating oxidative stress. From the results, it could be implied that the protective effects of

methanolic fraction of A. lindleyana might be attributed to its potential to elevate the activity of

antioxidant enzymes during inflammation.

Major side effects associated with currently available non steroidal anti-inflammatory drugs

(NSAIDs) are gastric ulceration and hepatotoxicity (Gil, 2002). Therefore, there is a need to

evaluate the efficacy of any new anti-inflammatory treatment with respect to their peptic and

hepatic tolerance. In the present work, the ethanolic extract of A. lindleyana and its various

fractions were evaluated for their ulceration and hepatotoxic risk at three times higher dose than

that used in the evaluation of the in vivo anti-inflammatory activity. Again, it has been found that

the active methanolic fraction did not show any gastric ulceration as well as hepatotoxicity.

Gastric ulceration is also associated with cellular membrane disruption and destabilizing via lipid

peroxidation. It has already been reported that the process of lipid peroxidation proceeds by free

radical chain reaction which is scavenged through enzymatic and non enzymatic antioxidants.

Our results are in complete agreement with the earlier observations indicating that the

methanolic fraction significantly brought down the level of LPO as compared to carrageenan

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group. Furthermore, the decreased expression of COX-2 and NF-κB in treated groups suggested

that the hepatic tolerance has increased after treatment than in carrageenan induced control

group.

Preliminary phytochemical screening of the ethanolic extract of A. lindleyana has shown the

presence of alkaloids, flavonoids and triterprnoids. These phytoconstitutions have already been

reported for several biological activities (Huss et al., 2002) and might be responsible for

biological activity of ethanolic extract of A. lindleyana bark and its fractions.

5. Conclusion

It could be concluded that the methanolic fraction of ethanolic extract of A. lindleyana bark

exhibited significant anti-inflammatory and anti-oxidant activity. The methanolic fraction

attenuated the level of proinflammatory mediators like TNF-α, NO, NF-κB, COX-2 and elevated

the activity of anti-oxidative enzymes (SOD, CAT & GPX). Therefore, the bark of A. lindleyana

might provide a potent agent against inflammation and oxidative stress.

Acknowledgements

The authors are thankful to Dr. G. N. Qazi, Vice Chancellor, Jamia Hamdard for providing the

necessary facilities to the Department of Chemistry. Yakub Ali is also thankful to Hamdard

National Foundation for providing the financial assistance.

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Table I. In-vivo anti-inflammatory activity of ethanolic extract of bark of A. lindleyana and its various fractions.

Group Dose (mg/kg po)

Change in paw volume(ml) Mean + SEM

% inhibition

3hr 5hr 3hr 5hr

Carr Control 2ml/kg 1.45±0.024 1.48±0.066 - -

Indomethacin 20 0.94±0.012** 0.90±0.023** 70 77

Petroleum ether fraction

200 1.33±0.012ns 1.35±0.030ns 14.89 15.79

300 1.29±0.016ns 1.31±0.030ns 20.66 18.73

Chloroform fraction

200 1.03±0.016* 1.04±0.029* 55.30 56.47

300 1.03±0.026* 1.02±0.012* 56.44 58.67

Methanol fraction

200 0.98±0.012* 0.96±0.026** 62 66.94

300 0.97±0.015** 0.93±0.026** 66.18 73.00

Ethanolic Extract

200 1.08 ±0.011* 1.08±0.012* 53.89 52.34

300 1.07±0.019* 1.05±0.020* 48.59 56.47 Data is analyzed by one way ANOVA followed by Dunnett’s ‘t’ test and expressed as % inhibition and mean ± SEM from five observations

where * indicates p < 0.05, ** indicates p < 0.01 & ns indicates p > 0.05.

 

 

 

 

 

 

 

23 

 

Table.II Effects of the ethanolic extract and its various fractions of A. lindleyana and

indomethacin on catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase

(GPX) activities in carrageenan (Carr) induced edema paw.

Group Dose (mg/kg po)

CAT(U/mg protein) % SOD(U/mg

protein) % GPX(U/mg protein) %

Control 2ml/kg 8.59±1.02 - 7.96±0.79 - 20.61±0.61 - Carrageenan (Carr) 0.1mL 3.43±0.89ns 39.49 2.84±0.63ns 35.68 8.98±1.40ns 43.58

Carr+ Ascorbic acid

20mg/kg 6.89±1.29** 80.23 6.32±0.82** 79.32 16.97±0.61** 82.38

Carr+Petroleum ether fraction

200 3.62±0.64ns 42.22 3.08±0.42 ns 38.70 8.76±0.88 * 42.59

300 3.89±0.62 ns 45.28 3.14±0.35 ns 39.49 9.44±0.98* 45.81

Carr+Chloroform fraction

200 5.10±0.20 * 59.38 4.94±0.23 * 62.07 12.37±0.53** 60.02

300 5.52±0.38** 64.27 5.20±0.42 * 65.33 13.26±0.86** 63.80

Carr+Methanol fraction

200 5.83 ±0.76** 67.84 5.73±0.42 * 71.99 14.50±0.69** 70.36

300 6.35±0.48** 73.30 5.99±0.31** 75.28 15.58±0.61** 74.14 Carr+Ethanolic extract 200 4.95 ±0.38 ns 51.03 4.53±0.23 ns 56.91 10.74±0.96** 51.91

300 5.19±0.28 ns 53.50 4.58±0.25 ns 57.54 11.44±0.39** 55.51 Data is analyzed by one way ANOVA followed by Dunnett’s ‘t’ test and expressed as mean ± SEM from five observations where * indicates p <

0.05, ** indicates p < 0.01 & ns indicates p > 0.05 and % change in activities are also given.

24 

 

Figure Captions Fig.1 (A) RP-HPLC profiling of ethanolic extract of A. lindleyana Baill. (B) RP-HPLC profiling of pure β-sitosterol. (C) RP-HPLC profiling of pure sigmasterol Fig.2. In-vivo TNF-α activity of ethanolic extract of barks of A. lindleyana Baill. and its various fractions. Fig. 3. In-vivo NO activity of ethanolic extract of barks of A. lindleyana Baill. and its various fractions. Fig. 4. In-vivo LPO activity of ethanolic extract of barks of A. lindleyana Baill. and its various fractions. Fig.5a Hepatic expressions of COX-2 activation Representative photomicrographs (magnification 40x) Group I (only control), Group II (only carrageenan), Group III ( carrageenan + standard drug Indomethacein), Group IV ( carrageenan + methanol fraction).

Fig.5b Hepatic expressions of NF-κB activation. Representative photomicrographs (magnification 40x). Group I (only control), Group II (only carrageenan), Group III( carrageenan + standard drug Indomethacein), Group IV ( carrageenan + methanol fraction).

25 

 

Fig.1 (A) RP-HPLC profiling of ethanolic extract of A. lindleyana Baill. (B) RP-HPLC profiling of pure β-sitosterol. (C) RP-HPLC profiling of pure sigmasterol

 

26 

27 

 

Fig.2. In-vivo TNF-α activity of ethanolic extract of barks of A. lindleyana Baill. and its various fractions.

Data is analyzed by one way ANOVA followed by Dunnett’s ´t´ test and expressed as percentage inhibition ± SEM from three observations; ** indicates p < 0.01, *

indicates p < 0.05 & ns indicates p > 0.05.

28 

 

Fig. 3. In-vivo NO activity of ethanolic extract of barks of A. lindleyana Baill. and its various fractions.

Data is analyzed by one way ANOVA followed by Dunnett’s ´t´ test and expressed as percentage inhibition ± SEM from three observations; ** indicates p < 0.01, *

indicates p < 0.05 & ns indicates p > 0.05. 

29 

 

Fig. 4. In-vivo LPO activity of ethanolic extract of barks of A. lindleyana Baill. and its various fractions.

Data is analyzed by one way ANOVA followed by Dunnett’s ´t´ test and expressed as mean ± SEM from three observations; ** indicates p < 0.01, * indicates p <

0.05 & ns indicates p > 0.05.

 

Fig. 5a(magnific+ standar

Fig. 5b(magnific+ standar

a Hepatic cation 40x) rd drug Indo

b Hepatic cation 40x). rd drug Indo

expressionsGroup I (on

omethacein),

expressionsGroup I (on

omethacein),

s of COXnly control), Group IV (c

s of NF-κnly control), Group IV (c

30 

X-2 activatGroup II (oncarrageenan

κB activatiGroup II (oncarrageenan

tion Represnly carragee+ methanol

ion. Represnly carragee+ methanol

sentative penan), Group fraction).

sentative penan), Group fraction).

photomicrogrp III (carrage

photomicrogrp III (carrage

raphs eenan

raphs eenan