Original Research Article 1

Anti-inflammatory activity of the phenolic-rich 2

extract of Schotia brachypetalea Sond. Fam. 3

Fabaceae, Cultivated in Egypt 4

Esraa A. El-Hawary 1*, Ebrahim Abdel-Aziz 2, Rola M. Labib 1, Ahmed Esmat2, 5 Ashraf B. Abdel-Naim 2, Abdel Nasser B. Singab 1, Amani E. Khalifa2, Nahla 6 A. Ayoub 1 7 8

1 Department of Pharmacognosy, Faculty of Pharmacy, Ain Shams University, Abbasia 9 (11566), Cairo, Egypt 10

2 Department of Pharmacology, Faculty of Pharmacy, Ain Shams University, Abbasia 11 (11566), Cairo, Egypt 12

13 14

15 ABSTRACT 16

Aim: The current study evaluates the anti-inflammatory activity of the methanolic leaf extract of Schotia brachypetalea, isolate and identify the phenolic content and standardize the crude leaf extract using HPLC. Place and duration of study: This study was conducted in Department of Pharmacognosy and Department of Pharmacology, Faculty of Pharmacy, Ain Shams University, Cairo, Egypt during the period between July 2013 and December 2014. Methodology: Six groups of Adult male Sprague Dawley rats (weighing 180–200 g) were used to assess the anti-inflammatory activity in two models. Polyamide and sephadex column chromatography were used for fractionation, isolation and purification of compounds together with preparative paper chromatography. Isolated compounds were analyzed using UV and1H-NMR spectroscopy. HPLC analysis was used to standardize the extract. Folin-Ciocalteu and Aluminium chloride colorimetric assays were used for determination of total phenolic and total flavonoid contents, respectively. Results: Significant dose dependent edema inhibition was observed in the carrageenan at a dose 200 mg/kg (79.31%, 2hrs) and (85.19%, 3hrs). Also, levels of PGE2 had decreased significantly at this dose at two time intervals. In croton oil model, the methanolic leaf extract of S. brachypetalea (50 mg/kg) decreased the edema to about 39.56% while (100 and 200 mg/kg) doses decreased the edema to about 23 and 15.8%, respectively. Myloperoxidase (MPO) levels were reduced by 38, 45 and 63% in doses (50, 100 and 200 mg/kg), respectively. TNF-α level was reduced similarly. The anti-inflammatory activity was confirmed by the histopathological examination revealing that the 200 mg/kg dose of the methanolic leaf extract of S. brachypetalea had shown normal dermal structure with no histopathological changes. Total phenolic content of S. brachypetala leaves methanolic extract was calculated as 376 mg of caffeic acid equivalents (CAE) per one gram of the extract while the total flavonoid content was 67.87 mg in one gram extract calculated as quercetin equivalent (QE). Three phenolic constituents had been isolated namely 1 Gallic acid, 2 Myrecitin-3-O-α-L-1C4-rhamnoside and 3 Quercetin-3-O-α-L-1C4-rhamnoside for the first time from genus Schotia. The crude methanolic leaf extract was standardized using gallic acid as reference. Conclusion: The study proved potent anti-inflammatory activity of the methanolic leaf extract of S. brachypetalea, this might be due to its richness in phenolics.

17 Keywords: Schotia brachypetalea, Fabaceae, anti-inflammatory, HPLC, phenolic, PGE2, TNF, MPO. 18 19

1. INTRODUCTION 20 21

Inflammation is a complex biological response of vascular tissues to harmful stimuli, such as pathogens, damaged cells or 22

irritants. It is a protective attempt to remove injurious stimuli as well as initiate the healing process for the tissues [1]. 23

Prostaglandin E2 (PGE2) is generated from arachidonic acid by the enzyme cyclo-oxygenase (COX) at sites of inflammation 24

in substantial amounts and mediate many of the pathologic features of inflammation. One of the early cellular events of 25

inflammation is the migration of leukocytes, primarily neutrophils. This response can be measured by using the neutrophil 26

specific enzyme myeloperoxidase (MPO), an indicator of neutrophil accumulation [2]. 27

Although steroidal anti-inflammatory drugs and NSAIDS are currently used to treat inflammation, they show significant 28

side effects even with newer classes [2]. Therefore, there is an urgent need to find safer anti-inflammatory compounds [3]. 29

This demonstrates the need for natural products as safer choice [2]. Schotia brachypetala (S. brachypetalea) Sond. 30

(Fabaceae), commonly named weeping boer-bean and huilboerbean (Afrikaans), is a tree endemic to Southern Africa. 31

Traditionally, different organs of Schotia were used; Decoction of the bark was used to strengthen the body and treat 32

dysentery and diarrhea, nervous and heart conditions, flu symptoms and in excessive beer drinking as an emetic. The roots 33

were also used to treat diarrhea and heartburn. The seeds can be roasted and eaten [4]. 34

Many biological activities viz. antibacterial activity [4, 5], anti-diarrheal [6], anti-oxidant and anti-malarial [4] had been 35

reported from genus Schotia. The stalks showed activity against Alzheimer's disease, which was correlated to their anti-36

oxidant properties [7]. Reported secondary metabolites include: polyhydroxystilbenes which were isolated from the 37

heartwood of the tree [8]; two antibacterial fatty acids (9,12,15-octadecatrienoic (δ-linolenic acid) and methyl-5,11,14,17-38

eicosatetraenoate) were also isolated from the leaves [5]. Furthermore, Catechin and epicatechin were isolated from genus 39

Schotia [9]. Flavonol acylglucosides were recently reported from aerial parts of S. brachypetalea [4]. The stalks showed 40

presence of procyanidin isomers, procyanidin dimer gallate, quercetin 3-O-rhamnoside, quercetin-hexose-gallic acid, 41

quercetin-hexose-protocatechuic acid, quercetin 3-O-rhamnoside and ellagic acid [7]. A thorough survey had indicated the S. 42

brachypetalea wasn't extensively studied phytochemically and pharmacologically. The present work aimed at exploring the 43

potential anti-inflammatory activity of the methanolic leaf extract of S. brachypetala in two experimental models ; isolate and 44

identify the phenolic constituents of the methanolic leaf extract of S. brachypetalea as well as standardization of the 45

methanolic leaf extract and determining its total phenolic and total flavonoid content. 46

47

2. MATERIAL AND METHODS 48 49

2.1 Plant material 50

During spring season (April-May 2012), S. brachypetalea leaves were collected from trees grown in private garden, 51

Alexandia desert road. They were kindly authenticated by Prof. Dr. Mohamed El-Gabaly, Professor of Taxonomy, National 52

Research Center (NRC), Egypt. A voucher specimen (Sbf1513) was deposited at the Department of Pharmacognosy, 53

Faculty of Pharmacy, Ain Shams University. 54

2.2 Extraction and isolation 55 56

The intact air dried plant material (3kg) was comminuted to powder then boiled in distilled water for 2 hours then filtered while 57

hot. The filtrate was completely evaporated in vacuo at ≈ 70 ºC till dryness. The solid residue was then extracted with 58

methanol till exhaustion, then the combined methanol extract was evaporated in vacuo till dryness, then the residue obtained 59

(120g) was applied on top of polyamide 6 column. Gradient elution was done with water and polarity was decreased using 60

methanol till neat methanol to obtain four main fractions. Fractions II and III showed their richness in phenolics which were 61

detected with spray reagent (FeCl3). Fraction II (eluted with water) showed only one major spot to afford compound 1. 62

Fraction III (eluted with 10% to 60%MeOH) was applied on top of Sephadex-LH50 column using butanol saturated with water 63

as an eluent to afford compounds 2 and 3. After purification, compounds were identified using UV spectrophotometry and 1H-64

NMR spectrometry. For 1H-NMR experiments, samples were dissolved in deuterated DMSO-d6 solvent and measured in 5-65

mm tubes at 25 °C on Bruker 400 MHz NMR spectromete r (Faculty of Pharmacy Beni-Suef University, Beni-Suef, Egypt). 66

2.3 Solvents and chemicals 67

The solvents used were of HPLC grade: Methanol, water and acetic acid. All other laboratory chemicals used in current 68

study, in the isolation protocol, were of analytical grade and were purchased from Sigma-Aldrich Chemicals. Gallic acid 69

standard was purchased from Sigma-Aldrich Chemicals. PPC (preparative paper chromatography) was purchased from 70

Whatman Ltd. Maidstone, Kent, England. 71

2.4 Material for biological study 72 2.4.1 Experimental animals 73

Adult male Sprague–Dawley rats weighing 180–200 g were used. They were obtained from the animal facility, Misr 74

University for Science and Technology (MUST), Egypt. The study was conducted according to ethical guidelines (Ain 75

Shams University, Cairo, Egypt). Rats were housed at a temperature of 23 ± 2ºC with free access to water and standard 76

food pellets. Animals were acclimatized in our animal facility for at least one week prior to the experiment. They were then 77

equally divided into six groups (I-VI), six animals per each group. All animal tests were performed under laboratory 78

conditions. 79

2.4.2 Measurement of paw volume and PGE 2 in carrageenan-induced rat edema model [10] 80

Animals were fasted, with free access to water, 12 hours before the experiment. Using an intra-gastric tube, groups I and II 81

were given the vehicle (0.5% CMC-Na). Animals in Group III received Indomethacin (10 mg/kg) as a standard anti-82

inflammatory drug suspended in aqueous CMC-Na (0.5% w/v), whereas remaining groups were treated with methanolic leaf 83

extract of S. brachypetalea at 3 dose levels (50, 100 and 200 mg/kg, respectively). The dosing volume were kept constant 84

(10 ml/kg) for all the orally treated groups. One hour after oral treatment, group I received 0.05mL of saline, whereas groups 85

II-VI received 0.05 ml of carrageenan (1% solution in saline) subcutaneously on the plantar surface of the right hind paw. 86

The right hind paw volume was measured immediately after carrageenan injection by water displacement using a 87

plethysmometer (model 7140, Ugo Basile, Comerio, Italy). The paw volume was re-measured 1, 2, 3 and 4 hours after 88

carrageenan injection. Right hind paws were removed. A volume of 0.1 ml of saline containing 10µM indomethacin was 89

injected to aid removal of the eicosanoid-containing fluid and to stop further production of PGE2. Paws were incised with a 90

scalpel and suspended off the bottom of polypropylene tubes with Eppendorf pipette tips to facilitate drainage of the 91

inflammatory exudates. For the purpose of the removal of the inflammatory exudates, paws were centrifuged at 1,800 g for 92

15 minutes. PGE2 was quantified using enzyme-linked immunosorbent assay kit obtained from R&D Systems Inc. 93

(Minneapolis, MN, USA), according to the manufacturer instructions. This assay was based on the forward sequential 94

competitive binding technique in which PGE2 in a sample competes with horseradish peroxidase–labeled PGE2 for a limited 95

number of binding sites on monoclonal antibodies. PGE2 in the sample is allowed to bind to the antibody in the first 96

incubation. During the second incubation, horseradish peroxidase–labeled PGE2 binds to the remaining antibody sites. 97

Following a wash to remove unbound materials, a substrate solution is added to the wells to determine the bound enzyme 98

activity. The color development is stopped, and the absorbance is read at 450 nm. The intensity of the color is inversely 99

proportional to the concentration of PGE2 in the sample. 100

2.4.3 Assessment of ear edema, myloperoxidase (MPO ), and histopathology in croton oil–induced ear ede ma model 101

in rats [11] 102

An irritant solution was prepared by dissolving four parts croton oil (the irritant) in a solvent mixture of 10 parts ethanol, 20 103

parts pyridine, and 66 parts ethyl ether. The irritant solution was applied in a volume of 20 µl topically on both sides of the 104

right ear; the left ear was kept untreated to serve as a control. Group I served as the negative control and hence received 105

only the irritant-free solvent mixture, whereas group II received the croton oil solution. Animals in group III received 106

indomethacin (10 mg/kg) as a standard anti-inflammatory drug suspended in aqueous CMC-Na (0.5% w/v), whereas 107

remaining groups (IV-VI) were treated with the methanolic leaf extract of S. brachypetalea at 3 dose levels (50, 100 and 200 108

mg/kg, respectively). One hour later, the solvent mixtures were re-administered to the groups. After 4 hours, animals were 109

sacrificed. 8-mm cork borer was used to punch out discs from both the treated and the control ears. The two punches were 110

weighed immediately after decapitation, and the difference in weight was used to assess the inflammatory response. 111

The entire tissue of the right ear was homogenized for 10 minutes in an ice bath (10% w/v) in 50 mM phosphate buffer (pH 112

6.0) containing 0.5% hexa-decyl-tri-methylammonium bromide with a IKA homogenizer (Staufen, Germany). Tissue 113

suspensions were centrifuged at 40,000 g for 15 minutes. The supernatants were collected. An aliquot of 0.1mL of the 114

supernatant was added to 2.9mL of 50mM phosphate buffer (pH 6.0) containing 0.167mg/mL O-di-anisidine dihydrochloride, 115

serving as the MPO substrate, and 0.0005% hydrogen peroxide. The change in absorbance was measured at 460 nm with 116

a Shimadzu UV-1601 spectrophotometer at 25ºC. MPO activity was quantified kinetically; change in absorbance was 117

measured over a period of 2 minutes, sampled at intervals of 15 seconds. The maximal change in absorbency per minute 118

was used to calculate the units of MPO activity based on the molar absorbency index of oxidized O-di-anisidine 119

dihydrochloride, which equals 1.13X104 M-1 cm-1. One unit of MPO is defined as that degrading 1 µmol of peroxide/minute at 120

25 ºC. Results were expressed as units of activity per milligram of protein. 121

A representative ear tissue from each group was fixed in 10% formol saline for 72 hours. Well washing was done in tap 122

water, and then serial dilutions of alcohol (methyl, ethyl, and absolute ethyl) were used for dehydration. Specimens were 123

cleared in xylene and embedded in paraffin at 56 ºC in a hot air oven for 24 hours. Paraffin beeswax tissue blocks were 124

prepared for sectioning at 4 mm thickness with a sledge microtome. The tissue sections obtained were collected on glass 125

slides, deparaffinized, and stained by hematoxylin and eosin then examination was done using the light microscope. 126

127

2.4.4 Statistical analysis 128

Data are presented as mean ± S.D. Comparisons were carried out using one way analysis of variance (ANOVA) followed by 129

Tukey-Kramer’s test for post hoc analysis. Statistical significance was acceptable to a level of p< 0.05. All statistical 130

analyses were performed using GraphPad InStat software, version 3.05 (GraphPad Software, Inc. La Jolla, CA, USA). 131

Graphs were plotted using GraphPad Prism software, version 5.00 (GraphPad Software, Inc. La Jolla, CA, USA). 132

2.5 HPLC Standardization 133

The methanolic leaf extract of S. brachypetala was standardized using an Agilent 1200 series HPLC equipped with an 134

Agilent quaternary pump connected to a photodiode array detector with variable wavelengths, separation was carried out on 135

RP C18 column with dimensions (150 mm, 4.6 mm, 5 µm). Gallic acid was used as a reference standard. A stock solution 136

was prepared in a dilution 0.1296 g/10ml in HPLC grade methanol to give a stock solution from which serial dilutions were 137

prepared at different concentrations ranging from 0.02592 to 0.1036 mg/ml. All samples were filtered through PTFE 0.5 µm 138

syringe filter and injected in triplicates into the HPLC column using 4% acetic acid in water (solvent A) and methanol 139

(solvent B) in gradient elution. The gradient program was 0-4 minutes 100% A, 4-10 minutes 50% A in 50% B , 10-20 140

minutes 20% A in 80 % B, 20-22 minutes 50% A in 50% B, 22-26 minutes 100% B, with flow rate 0.6 ml/min and injection 141

volume 20µl, measurement was done at wavelength 280nm [12]. A standard calibration curve was designed by plotting the 142

different concentrations of gallic acid against peak area. Regression equation and co-efficient of correlation (R2) were 143

derived and concentration were calculated based on the regression equation. The assay was repeated five times in the 144

same day for intraday precision by the used method. Accuracy was measured by performing a recovery study of gallic acid 145

reference by adding (0.0732g) to the extract solution (0.0324g) and analyzed by the same method. 146

2.6 Determination of total phenolic content and tot al flavonoid content 147

The Folin-Ciocalteu method was used for the determination of the total phenolic content using caffeic acid as a reference 148

standard [13]. Total flavonoid content was determined by the Aluminum chloride colorimetric method using quercetin as the 149

reference standard [14]. 150

3. RESULTS AND DISCUSSION 151 152

3.1 Identification of the isolated compounds 153

Three phenolic compounds; one phenolic acid 1 (Gallic acid) and two flavonoid glycosides, 2 (Myrecitin-3- O-α-L-1C4-154

rhamnoside) and 3 (Quercetin-3- O-α-L-1C4-rhamnoside) were isolated and identified for the first time out of leaf extract of 155

S. brachypetalea. 156

Compound 1 showed one major violet spot on PC under UV and turned blue upon spraying with FeCl3. Thus, it was co-157

chromatographed with gallic acid standard. The spot showed same color under UV and same Rf value. 158

Compound 2 was obtained as yellow crystalline powder. It appeared on the chromatograms as dark purple spot on PC 159

under short UV light. Rf values: 24.5 (BAW) and 13.5 (6% AcOH). It gave dirty green colour with FeCl3, spray reagent 160

specific for phenolics [15]. Also, it exhibited UV spectrum which showed two bands at λmax MeOH (350nm band I and 161

258nm band II) which indicated the flavonol nucleus containing 3', 4', 5-trihydroxy substitution [16]. Upon addition of sodium 162

methoxide, bathochromic shift (11 nm) occurred in band II while upon using sodium acetate, bathochromic shift (14 nm) was 163

observed in band II and (20 nm) in band I, indicating free OH at positions 3', 4', 5' and 7. A bathochromic shift occured upon 164

addition of boric acid (61 nm) and (22 nm) in bands II and I, respectively. The 1H-NMR spectra showed the absence of 165

signal due to H-3, the presence of aromatic proton signals at δ=6.15ppm (1H, s, H-6) and 6.31ppm (1H, s, H-8), presence of 166

O-glycosidic anomeric signal at 5.2ppm (1H, s, H-1"), signal at δ=6.879ppm (2H, s, H-2', H-6') and signal for methyl of 167

rhamnose at 1.51ppm (3H, s, CH3 rhamnose). UV as well as 1H-NMR chemical shifts were found to be similar to those 168

reported for Myrecitin-3- O-α-L-1C4-rhamnoside [17]. 169

Compound 3 was obtained as yellow crystalline powder. It gave dark purple color on PC under short UV light. Rf values: 170

22.5 (BAW) and 7.5 (6% AcOH). Upon spraying with FeCl3, dirty green colour is developed. Also, it exhibited UV spectrum 171

which showed two bands at λmax MeOH (360nm band I and 264 nm band II) which indicated the flavonol nucleus [16]. It 172

showed a bathochromic shift (11 nm) on addition of sodium methoxide and (10nm) in band II with sodium acetate to prove 173

that the 3', 4' and 7 OH positions are free. No change in shift was observed with sodium acetate and boric acid. 1H-NMR 174

spectrum of compound 3 showed the absence of signal due to H-3, the presence of aromatic proton signals at δ =7.199 175

(1H, d, J=2.5 Hz, H-2'), δ=6.909 (1H, dd, J=2.5 Hz, 8 Hz, H-6'), δ =6.882 (1H, d, J=8 Hz, H-5'), presence of O-glycosidic 176

anomeric signal at δ=5.214ppm (1H, s, H-1"), signal for methyl of rhamnose at δ=1.5 ppm (3H, s, CH3 rhamnose) and 177

signals δ=6.22ppm (1H, s, H-6) and δ=6.43ppm (1H, s, H-8). UV as well as 1H-NMR chemical shifts were found to be similar 178

to those reported for Quercetin-3- O-α-L-1C4-rhamnoside [18]. 179

3.2 Biological Results 180 3.2.1 Carrageenan-induced rat paw edema model 181

The anti-inflammatory activity was expressed as percentage inhibition of edema volume in treated animals in comparison 182

with the control and carrageenan-treated groups. The percent edema inhibition was calculated as: 183

[(Vcar. – Vt) / (Vcar. – Vcon.) X 100] 184

where: Vcar., Vt and Vcon. are the paw volumes of carrageenan, extract treatment and control groups, respectively. 185

Intraplantar injection of carrageenan to rats resulted in severe discernible inflammation and significant increase in the mean 186

volume of the challenged paw compared with that of the untreated paws. As shown in Table [1], pretreatment with 187

methanolic leaf extract of S. brachypetalea (50 mg/kg) resulted in significant amelioration of carrageenan-induced paw 188

edema only after 3 and 4 hours. However, methanolic leaf extract of S. brachypetalea (100 mg/kg) significantly decreased 189

carrageenan-induced paw edema starting after 2 hours, while pretreatment with methanolic leaf extract of S. brachypetalea 190

(200 mg/kg) resulted in significant reduction of carrageenan-induced paw edema at all time intervals. In a similar pattern, 191

indomethacin caused significant inhibition of carrageenan-induced paw edema by about 57.14%, 75.86%, 92.59% and 90% 192

after 1, 2, 3 and 4 hrs, respectively. 193

Carrageenan challenge resulted in more than 10-fold increase in PGE2 concentration compared to animals in the control 194

group. Animals receiving methanolic leaf extract of S. brachypetalea (50, 100 & 200 mg/kg) or indomethacin (10 mg/kg) 195

showed significant reduction of carrageenan-induced PGE2 production in the inflamed skin tissues compared to 196

carrageenan-only group Figure [1]. It is worthy noted that pre-treatment with methanolic leaf extract of S. brachypetalea at 197

dose (200 mg/kg) restored normal PGE2 concentration with no significant difference from the control group. 198

Table 1. The effect of the methanolic leaf extra ct of S. brachypetalea on rat paw volume in carrageenan-induced 199 rat paw edema model 200

201

Group

1 hour 2 hours 3 hours 4 hours

paw volume

(ml)

% edema inhibition

paw volume

(ml)

% edema inhibition

paw volume

(ml)

% edema inhibition

paw volume

(ml)

% edema inhibition

Control (I) 0.61 ± 0.08

-- 0.58 ± 0.11

-- 0.57 ± 0.10

-- 0.53 ± 0.06

--

Carrageenin (II) 0.82 ± 0.10

-- 0.87 ± 0.04

-- 1.11 ± 0.17

-- 1.33± 0.16

--

Indomethacin (III) 10mg/kg

0.70b ± 0.11

57.14 % 0.65b ±

0.09 75.86%

0.61b ± 0.07

92.59% 0.61b ±

0.07 90%

S. brachypetalea leaf extract (IV) 50mg/kg

0.81a ± 0.18

4.76% 0.82a ±

0.08 17.24%

0.97a,b ± 0.10

25.93% 1.08 a,b±

0.09 31.25%

S. brachypetalea leaf extract (V) 100mg/kg

0.76a ± 0.09

28.57% 0.75 a,b±

0.05 41.37%

0.84 a,b± 0.13

50% 0.93 a,b±

0.07 50%

S. brachypetalea leaf extract (VI) 200mg/kg

0.68b ± 0.12

66.66% 0.64 b± 0.13

79.31% 0.65 b± 0.10

85.19% 0.72 b± 0.08

76.25%

202 *Data are presented as mean ± S.D. Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Tukey 203 post hoc test. 204 n=6 205 a statistically different from the corresponding control group at p < 0.05 206 b statistically different from the corresponding carrageenan-treated group at p < 0.05 207

0

200

400

600

800 ControlCarrageenanCar. + Indomethacin 10mg/kgCar. + SB extract 50 mg/kgCar. + SB extract 100 mg/kgCar. + SB extract 200 mg/kg

a

b b

a,b

a,b

PG

E2

(pg/

ml)

208

Fig.1. Effect of the methanolic leaf extract of S. brachypetalea on the level of PGE 2 in carrageenan-induced rat paw 209 edema model 210

211 *Data are presented as mean ± S.D. Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Tukey 212 post hoc test. 213 n=6 214 a statistically different from the control group at p < 0.05 215 b statistically different from the carrageenan-treated group at p < 0.05 216 3.2.2 Croton oil induced rat ear edema model 217

Application of croton oil to rat ears caused a significant increase in the weight of the ear punch from control animals by 218

53.95% as shown in Table [2] and Figure [2]. Indomethacin pretreatment caused insignificant increase of punch weight 219

from control values by about 10.07%. Furthermore, pretreatment of rats with the methanolic leaf extract of S. 220

brachypetalea (50, 100 & 200 mg/kg) significantly reduced the increase of punch weight from control ears to about 39.56%, 221

23.02% and 15.82%, respectively. It is worthy noted that pretreatment of rats with the methanolic leaf extract of S. 222

brachypetalea 200mg/kg did not show any significant increase in punch weight from the control group. 223

Assay of MPO activity in rat ears indicated that application of croton oil caused a significant (4-fold) increase in the activity 224

compared to the control group, as shown in Figure [3]. Indomethacin pretreatment resulted in significant protection against 225

croton oil–induced enhancement of MPO acivity by about 66% of the induced ear. In addition, pretreatment of rats with the 226

methanolic leaf extract of S. brachypetalea (50, 100 & 200 mg/kg) significantly reduced MPO activity by about 31.9%, 44%, 227

and 63% of the induced ear, respectively. 228

A similar pattern of effect was detected with TNF-α level, as shown in Figure [4]. Application of croton oil significantly 229

increased TNF-α level by about 20-folds, compared to control value. Indomethacin pretreatment resulted in significant 230

protection against croton oil–induced enhancement of TNF-α level by about 81.4% of the induced ear. Pretreatment of rats 231

with the methanolic leaf extract of S. brachypetalea (50, 100 & 200 mg/kg), however, significantly reduced TNF-α level by 232

about 31.4%, 43.1%, and 71.3% of the induced ear, respectively. 233

The histopathological examination of the ear tissue revealed: Figure [5] (A) shows normal histological structure of the 234

epidermal and dermal layers as well as underlying cartilage with no obvious neutrophil infiltration. Figure [5] (B) the croton 235

oil–treated rats shows congestion of dermal blood vessels accompanied with perivascular edema. Also, the blood vessels 236

shows large numbers of leukocytes mixed with RBCs, with the dermis shows large numbers of inflammatory cells mainly 237

macrophages and lymphocytes. Indomethacin pretreatment, Figure [5] (C) shows almost intact dermal and cartilaginous 238

structures with no histopathological changes. Figure [5] (D) which represents ear tissue treated with 50 mg/kg methanolic 239

leaf extract of S. brachypetalea, shows few inflammatory cells mainly macrophage and lymphocytes in the dermis. Edema 240

and congestion of dermal blood vessels is the most prominent lesion in the dermis of ear tissue treated with 100 mg/kg 241

methanolic leaf extract of S. brachypetalea, as shown in Figure [5] (E). However, ear tissues treated with 200 mg/kg of the 242

methanolic leaf extract of S. brachypetalea shows normal dermal structure with no histopathological changes, as shown in 243

Figure [5] (F). 244

Table 2. The effect of the methanolic leaf extract of S. brachypetalea on rat disc weight in croton oil-induced rat 245

ear edema model 246

Group Disc Weight (g) Percentage increase in punch weight from control

Control (I) 0.0139 ± 0.0011 --

Croton oil (Cro.) (II) 0.0214a ± 0.0023 53.95%

Cro. + Indomethacin (10mg/kg) (III)

0.0153b ± 0.001 10.07 %

Cro. + S.brachypetalea methanolic leaf extract (50mg/kg) (IV)

0.0194a,b ± 0.0024 39.56%

Cro. + S.brachypetalea methanolic leaf extract (100mg/kg) (V)

0.0171a,b ± 0.0015 23.02%

Cro. + S.brachypetalea methanolic leaf extract extract (200mg/kg) (VI)

0.0161b ± 0.0011 15.82%

*Data are presented as mean ± S.D. Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Tukey 247 post hoc test. 248 n=6 249 a statistically different from the corresponding control group at p < 0.05 250 b statistically different from the corresponding carrageenan-treated group at p < 0.05 251 252

0.00

0.01

0.02

0.03ControlCroton oil (Cro)Cro. + Indomethacin 10mg/kgCro. + SB extract 50 mg/kgCro. + SB extract 100 mg/kgCro. + SB extract 200 mg/kg

a

b b

a,ba,b

Dis

c w

eigh

t (gm

)

253 254

Fig.2. Effect of the methanolic leaf extract of S. brachypetalea on the ear disc weight in croton oil-induced rat e ar 255 edema model 256 *Data are presented as mean ± S.D. Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Tukey 257 post hoc test. 258 n=6 259 a statistically different from the control group at p < 0.05 260 b statistically different from the carrageenan-treated group at p < 0.05 261

0

5

10

15 ControlCroton Oil (Cro)Cro. + Indomethacin 10mg/kgCro. + SB extract 50 mg/kgCro. + SB extract 100 mg/kgCro. + SB extract 200 mg/kg

a

b b

a,ba,b

MP

O a

ctiv

ity (

U/m

g pr

otei

n

262 Fig.3. Effect of the methanolic leaf extract of S. brachypetalea on MPO activity in croton oil-induced rat ear edem a 263 model 264 *Data are presented as mean ± S.D. Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Tukey 265 post hoc test. 266 n=6 267 a statistically different from the control group at p < 0.05 268 b statistically different from the carrageenan-treated group at p < 0.05 269

0

50

100

150ControlCroton oil (Cro)Cro. + Indomethacin 10mg/kgCro. + SB extract 50 mg/kgCro. + SB extract 100 mg/kgCro. + SB extract 200 mg/kg

a

a,ba,b

a,ba,b

TNF-

αα αα le

vel (

ng/m

g pr

otei

n)

270

Fig.4. Effect of the methanolic leaf extract of S. brachypetalea on TNF-α level in croton oil-induced rat ear edema 271 model 272

*Data are presented as mean ± S.D. Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Tukey 273 post hoc test. 274 n=6 275 a statistically different from the control group at p < 0.05 276 b statistically different from the carrageenan-treated group at p < 0.05 277

A B

C D

FE

278 Fig.5. Effect of the methanolic leaf extract of S. brachypetalea extract on histopathological changes in the croton 279 oil–induced ear edema experiment 280 *Magnification X100. (A) Normal histological structure of the epidermal and dermal layers as well as underlying cartilage with no obvious 281 neutrophil infiltration. (B) Croton oil–treated rats with massive neutrophil infiltration in the dermal layer. (C) Ear tissue treated with 282

indomethacin shows almost intact structures (D, E, F) Ear tissues treated with 50, 100 & 200 mg/kg the methanolic leaf extract of S. 283 brachypetalea extract shows pathological lesions that differ in severity according to the dose used. 284 285

3.3 HPLC standardization of the methanolic leaf ext ract of S. brachypetalea 286 The methanolic leaf extract of S. brachypetalea showed an intense peak at Rt 3.815 minutes. Spiking with gallic acid 287

showed that this peak is identified as gallic acid. The standard solution of gallic acid was prepared and analyzed in the 288

concentration range of 0.02592 to 0.1036 mg/ml. The regression equation of the curve is Y=37172X+359.1 and the 289

coefficient of regression (R2) was 0.993 confirming the linearity. Repeatability was achieved by determining relative standard 290

deviation (RSD) which ranges from 1.9263 to 17.80518% for interday assay. Accuracy expressed as recovery showing 291

101%. Gallic acid content was calculated as 206 mg per one gram extract. 292

293 Fig.6. HPLC chromatogram for S. brachypetalea methanolic leaf extract 294 295

296 Fig.7. HPLC chromatogram for gallic acid standard 297 298

299 Fig.8. Calibration curve for gallic acid 300 301 302

303 304

3.4 Total phenolic content and total flavonoid cont ent 305

Phenolic compounds are regarded as the molecules with the highest potential to neutralize free radicals. Therefore, their 306

quantification is a common practice in different areas of food research. The phenolic compounds are the major contributors 307

to the antioxidant capacity of fruits, vegetables, and grains. Therefore, the quantification of such phenolic compounds is a 308

common practice when selecting genotypes, maturity stages, storage and processing conditions that allow the production of 309

fresh and processed food products with high potential to protect against free radicals [13]. Total phenolic content of S. 310

brachypetala leaves methanolic extract was 376 mg of caffeic acid equivalents (CAE) per one gram of the extract while the 311

total flavonoid content was 67.87 mg in one gram extract quercetin equivalent (QE). 312

313 4. CONCLUSION 314

Three phenolic compounds were isolated for the first time from genus Schotia. A validated HPLC method for the analysis of 315

the methanolic leaf extract using gallic acid as a reference proved to be accurate and reliable. 316

Lately, Extensive studies had been conducted to explore the anti-inflammatory activity of different plant extracts. In the 317

present study, S. brachypetalea methanolic leaf extract showed promising anti-inflammatory activity using indomethacin as 318

a reference drug. In two anti-inflammatory experimental models, the extract had decreased the inflammatory markers viz. 319

PGE2, TNF-α and MPO. It decreased also the paw volume and weight of ear disc. The anti-inflammatory activity was 320

confirmed by the histopathological examination of ear tissue. Both quercetin and myrecitin were proved to have anti-321

inflammatory activity by inhibiting MPO [19] so the activity of our extract may be attributed to its phenolic constituents. 322

323 ETHICAL APPROVAL 324 325 “All authors hereby declare that "Principles of laboratory animal care" (NIH publication No. 85-23, revised 1985) were 326 followed, as well as specific national laws where applicable. All experiments have been examined and approved by the 327 appropriate ethics committee” 328 329 330 REFERENCES 331 1. Rathee P. Mechanism of action of flavonoids as anti-inflammatory agents: A Review. Inflamm and Allergy, 2009. 332

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