# Chloroformic and Methanolic Extracts ofOlea europaea L. Leaves Present Anti-Inflammatory and Analgesic Activities

Authors: R Chebbi Mahjoub; M. Khemiss; M. Dhidah; A. Dellaï; A. Bouraoui; F. Khemiss
Journal: ISRN Pharmacology
 (2011)

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## Abstract

Olea europaea L. is used in traditional medicine in the Mediterranean areas. Its natural products are used in the treatment of different disorders, like fighting fever and some infectious diseases such as malaria, the treatment of arrhythmia, and relief of intestinal spasms.
The aim of the current study is to investigate the possible anti-inflammatory and anatinociceptive effects of methanol and chloroformic extracts prepared from leaves of Olea europaea L. The anti-inflammatory and antinociceptive effects of the different extracts of Olea europaea leaves were assessed after intraperitoneal administration into rats and mice, using the carrageenan-induced paw edema model in rats to test the anti-inflammatory effect and the acetic acid-induced writhing in mice to test the analgesic effect. The chloroformic and methanolic leaves extracts, studied at the doses of 50, 100, and 200 mg/kg (Body Weight: BW), exhibited significant dose-dependent anti-inflammatory and analgesic activities.
Based on the results obtained, it can be concluded that Olea europaea leaves extracts
have anti-inflammatory and antinociceptive effects.


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## Body

## 1. Introduction

The medicinal uses of different parts  ofOlea europaea  L. have been  known  for  a long time  in  all  the  countries  of  the  Mediterranean  basin.  Olive  oil  was  recognized  as  an important component of a healthy diet. Several epidemiological studies have shown that the incidence of coronary heart disease and certain cancers is low in the Mediterranean basin where the diet is rich in olive products [1, 2]. Historically, olive leaves have been used as a folk remedy for combating fevers and other diseases such as malaria. Previous studies showed that some extracts of this product decreased blood pressure in animals and  increased  blood  flow  in  coronary  arteries [3], relieved  arrhythmia,  and  prevented intestinal muscle spasms [4].A phytochemical investigation reported that oleuropein was  isolated  from the leaves ofOlea europaea [5].  This compound is known  for  possessing  a  wide  range  of pharmacologic and health promoting properties including anti-arrhythmic, spasmolytic, immuno-stimulant, cardioprotective, hypotensive, antihyperglycemic, antimicrobial, and anti-inflammatory effects [6–8]. Olea europaea is also rich in other bioactive phenolic compounds that are reported to have anti-inflammatory activities [9] However, there is no sufficient scientific studies reporting the anti-inflammatory and analgesic activities of Olea europaea leaves.As a contribution  to  the  study  of  the  pharmacological  activities  of  the  different components of olive leaves, we searched the possible anti-inflammatory and antinociceptive effects  of  the  chloroformic  and methanolic olive leaves extracts using an in vivo experimental models, that is, the carrageenan-induced hindpaw edema for  the  anti-inflammatory  activity and the acetic acid-induced abdominal writhing test for the analgesic activity.


## 2. Materials and Methods

### 2.1. Plant Material and Extraction Procedure

Leaves  ofOlea  europaea  L.  were  collected  from  the  region  of  Sfax  in  the  south  of Tunisia. A voucher specimen (No. 0506) was deposited in the herbarium of the botanic laboratory (Faculty of Pharmacy, Monastir, Tunisia).The olive leaves were dried at room temperature and then powdered. The powder was extracted  successively  with chloroform  and  methanol  by  maceration  at  room temperature  for  24 hours, and then  it  was  filtered.  The solvent was removed from resulting solution under vacuum in a rotary evaporator.


### 2.2. Phytochemical Screening

The  different  extracts  of  olive  leaves  were  screened  for  iridoids,  flavonoids, tannins, saponosides, anthracene heterosides, cardiotone heterosides and alkaloids.


### 2.3. Chemical Study by Nuclear Magnetic Resonance (NMR)

The  analysis  of  our  extract  was  carried  out  on  a  spectrometer  “Bruker  Avance-300 (300 MHZ).” The dimethylsulfoxyde (DMSO-d6) and the deuterium Chloroform (CDCl3) were used as solvents. The chemical movements (δ) are expressed in part per million (ppm).


### 2.4. Chemical Study by Infra-Red (IR)

IR spectrums were recorded on a spectrophotometer “Philips Anlytical PU 9800” in liquid film on pastills of sodium chloride. The frequencies  of  the  absorption  bands are expressed in cm−1.


### 2.5. Animals

Wistar rats (140–180 g)  and Swiss  albino mice  (18–25 g) were  obtained from Pasteur Institute  (Tunis, Tunisia). They were all kept in polypropylene cages in a room under controlled condition. All animals were fed according to a standard diet ad libitum and had free access to drinking water.


### 2.6. Determination of Anti-Inflammatory Activity of Olea Leaves Extract

The carrageenan-induced hind paw edema model according to the method of Winter [10] was used to test the anti-inflammatory activity. The chloroformic extract was dissolved in  Ethanol : Tween 80 : distilled water  (0.5 : 0.25 : 4.25),  and  the  methanolic  extract  was dissolved in the distilled water. The wistar rats were divided into groups of six animals each. The control group received 2.5 mL/kg (BW) of vehicle (saline solution or Ethanol-Tween80-distilled water 0.5 : 0.25 : 4.25).  The standard group received acetyl salicylic acid (ASA) at a dose of 300 mg/kg (BW) or dexamethasone at a dose of 1 mg/kg (BW). The test groups received, intraperitoneally, the chloroformic and methanolic extracts at doses of 50, 100, and 200 mg/kg (BW). After thirty minutes, each rat was injected with 0.05 mL of 1% carrageenan suspension as a phlogistic agent into the subplantar tissue of the left hind paw.  The  paw  volume,  up  to  the  tibiotarsal  articulation,  was  measured using a plethysmometer (model 7140, Ugo Basile, Italy). The measures were taken at 0, 1, 2, 3, 4, and 5 hours after the carrageenan injection. The degree of inflammation was determined by the difference between the left and the right paw volumes. The percent of the edema’s inhibition was calculated in comparison to the control groups.


### 2.7. Determination of Antinociceptive Activity of Olea Leaves Extract

The writhing test, a chemical visceral pain model, was chosen in this study to evaluate the  antinociceptive  effect  and  was  based  on  the  procedure  which  was  described by Koster  et  al. [11].  Nociception was induced by an intraperitoneal injection of acetic acid to the mice. The animals were divided into groups of six mice each. The first group, a control group, was treated with 10 mL/kg (BW) of vehicle (saline solution or Ethanol : Tween 80 : distilled water 0.5 : 0.25 : 4.25) intraperitoneally. The second group, the standard group, was treated with ASA (200 mg/kg, BW), as a reference drug. The last group was treated intraperitoneally with the olive leaves extracts at different doses (50, 100, and 200 mg/kg, BW).  30 minutes   after the administration of these different substances, all the animals received 10 mL/kg of 1% acetic acid intra-peritoneally. The number of abdominal writhes as a pain indicator was counted for 30 min, five minutes after the acetic acid injection.  The antinociceptive activity was expressed as a percentage of inhibition of abdominal writhes.


### 2.8. Statistical Analysis

Data were expressed as mean ± S.E.M. Results were statistically evaluated using a one-way analysis of variance (ANOVA), followed by Dunnett’s test. Results were considered significant whenP<.05 (*P<.05; **P<.01; ***P<.001).


## 2.1. Plant Material and Extraction Procedure

Leaves  ofOlea  europaea  L.  were  collected  from  the  region  of  Sfax  in  the  south  of Tunisia. A voucher specimen (No. 0506) was deposited in the herbarium of the botanic laboratory (Faculty of Pharmacy, Monastir, Tunisia).The olive leaves were dried at room temperature and then powdered. The powder was extracted  successively  with chloroform  and  methanol  by  maceration  at  room temperature  for  24 hours, and then  it  was  filtered.  The solvent was removed from resulting solution under vacuum in a rotary evaporator.


## 2.2. Phytochemical Screening

The  different  extracts  of  olive  leaves  were  screened  for  iridoids,  flavonoids, tannins, saponosides, anthracene heterosides, cardiotone heterosides and alkaloids.


## 2.3. Chemical Study by Nuclear Magnetic Resonance (NMR)

The  analysis  of  our  extract  was  carried  out  on  a  spectrometer  “Bruker  Avance-300 (300 MHZ).” The dimethylsulfoxyde (DMSO-d6) and the deuterium Chloroform (CDCl3) were used as solvents. The chemical movements (δ) are expressed in part per million (ppm).


## 2.4. Chemical Study by Infra-Red (IR)

IR spectrums were recorded on a spectrophotometer “Philips Anlytical PU 9800” in liquid film on pastills of sodium chloride. The frequencies  of  the  absorption  bands are expressed in cm−1.


## 2.5. Animals

Wistar rats (140–180 g)  and Swiss  albino mice  (18–25 g) were  obtained from Pasteur Institute  (Tunis, Tunisia). They were all kept in polypropylene cages in a room under controlled condition. All animals were fed according to a standard diet ad libitum and had free access to drinking water.


## 2.6. Determination of Anti-Inflammatory Activity of Olea Leaves Extract

The carrageenan-induced hind paw edema model according to the method of Winter [10] was used to test the anti-inflammatory activity. The chloroformic extract was dissolved in  Ethanol : Tween 80 : distilled water  (0.5 : 0.25 : 4.25),  and  the  methanolic  extract  was dissolved in the distilled water. The wistar rats were divided into groups of six animals each. The control group received 2.5 mL/kg (BW) of vehicle (saline solution or Ethanol-Tween80-distilled water 0.5 : 0.25 : 4.25).  The standard group received acetyl salicylic acid (ASA) at a dose of 300 mg/kg (BW) or dexamethasone at a dose of 1 mg/kg (BW). The test groups received, intraperitoneally, the chloroformic and methanolic extracts at doses of 50, 100, and 200 mg/kg (BW). After thirty minutes, each rat was injected with 0.05 mL of 1% carrageenan suspension as a phlogistic agent into the subplantar tissue of the left hind paw.  The  paw  volume,  up  to  the  tibiotarsal  articulation,  was  measured using a plethysmometer (model 7140, Ugo Basile, Italy). The measures were taken at 0, 1, 2, 3, 4, and 5 hours after the carrageenan injection. The degree of inflammation was determined by the difference between the left and the right paw volumes. The percent of the edema’s inhibition was calculated in comparison to the control groups.


## 2.7. Determination of Antinociceptive Activity of Olea Leaves Extract

The writhing test, a chemical visceral pain model, was chosen in this study to evaluate the  antinociceptive  effect  and  was  based  on  the  procedure  which  was  described by Koster  et  al. [11].  Nociception was induced by an intraperitoneal injection of acetic acid to the mice. The animals were divided into groups of six mice each. The first group, a control group, was treated with 10 mL/kg (BW) of vehicle (saline solution or Ethanol : Tween 80 : distilled water 0.5 : 0.25 : 4.25) intraperitoneally. The second group, the standard group, was treated with ASA (200 mg/kg, BW), as a reference drug. The last group was treated intraperitoneally with the olive leaves extracts at different doses (50, 100, and 200 mg/kg, BW).  30 minutes   after the administration of these different substances, all the animals received 10 mL/kg of 1% acetic acid intra-peritoneally. The number of abdominal writhes as a pain indicator was counted for 30 min, five minutes after the acetic acid injection.  The antinociceptive activity was expressed as a percentage of inhibition of abdominal writhes.


## 2.8. Statistical Analysis

Data were expressed as mean ± S.E.M. Results were statistically evaluated using a one-way analysis of variance (ANOVA), followed by Dunnett’s test. Results were considered significant whenP<.05 (*P<.05; **P<.01; ***P<.001).


## 3. Results

### 3.1. Extracts Yield

The results of yield extracts are shown in Table1.Table 1
Yield of different extracts.
ExtractsResultsChloroformic extractMethanolic extractMass (g)3160Yield (%)10,320


### 3.2. Chemical Composition of Olea Leaves Extract

The preliminary phytochemical qualitative screening of the chloroformic and methnolic extracts of the  leaves  ofOlea europaea  revealed the  presence of iridoids, flavonoids, and tannins as shown in Table 2.Table 2
Phytochemical screening of olive leaves extracts.
FlavonoidsIridoidsTanninsSaponosidsAnthracenic heterosidesCardiotonic heterosidesAlkaloïdsChE+++++−−−−ME++++++++−−−−(+): detectable and (−): not detectable. Abbreviations: Ch E: chloroformic extract; ME: methanolic extract.


### 3.3. NMR Analysis

The  spectrums  determined  by  NMR  of  our  extracts  allow  us  to  detect  peaks corresponding to different protons in Table3.Table 3
Different functions ofOlea europaea determined by NMR analysis.
Methanolic extractChloroformic extractType of protonδ  in ppmδ  in ppmRadical phenyl6.2 and 7.66 and 7.5Alcohol3.63.5Amine4.14.2Hydrocarbon2.41.3 and 1.5


### 3.4. IR Analysis

The spectrums determined by IR appear in the form of bands relative to the different functions in Table4.Table 4
Different functions ofOlea europaea determined by IR analysis.
Methanolic extractChloroformic extractFunctionsFrequencies of bands in cm−1Frequencies of bands in cm−1-C=C-16001600-C=O-17501700Amine3100–32502900–3250Alcohol3100–32502900–3250


### 3.5. Anti-Inflammatory  Effects of Olea  Leaves Extract

The results reported in Figures1, 2, and 3  showed  the  effect  of  the  chloroformic and methanolic  extracts  of  olive  leaves  (50,  100,  and  200 mg/kg)  administered  intraperitoneally. These results revealed significant dose-dependent inhibitory effects of the carrageenan-induced  paw  edema  at  1,  2,  3, 4, and  5 hours  after  injection.  The effect appeared 1 hour after the injection and persisted all along the duration of the experiment (5 hours).  The peak anti-inflammatory  effect  of  the  leaves  extracts  recorded  with  the dose of 200 mg/kg (BW) at the fifth hour was77.8±2% for the chloroformic extract and 68.1±3.8%  for  the  methanolic  extract  compared  to  the  standard  drugs  (ASA  and Dexamethasone)  which  decreased  the  paw  edema  by  62.9±3.1%  and  78.9±3.3%, respectively, at the fifth hour.Figure 1
Effect of intraperitoneal administration of chloroformic (CHCl3) olive leaves extract on the carrageenan-induced rat hind paw edema. Values are mean ± SEM. *P<.05, **P<.01, and ***P<.001 versus control group, (n=6).Figure 2
Effect of intraperitoneal administration of methanolic (MeOH) olive leaves extract on the carrageenan-induced rat hind paw edema. Values are mean ± SEM. *P<.05, **P<.01, and ***P<.001 versus control group, (n=6).Figure 3
Effect of intraperitoneal administration of drug references (ASA and Dexamethasone) on the carrageenan-induced rat hind paw edema. Values are mean ± SEM. **P<.01, ***P<.001 versus control group, (n=6).


### 3.6. Antinociceptive  Effects of Olea Leaves Extract

The effects of the used extracts on writhing response in mice are reported in Figures4 and 5. The intraperitoneal administration of the chloroformic and methanolic extracts at doses of 50, 100, and 200 mg/kg (BW) caused a significant reduction in the number of the writhing episodes induced by acetic acid compared to the control. The percentage of inhibition  of  the  constrictions  was  calculated  as  37.7±6.8%  (chloroformic  extract  50 mg/kg), 60±4.5%  (chloroformic extract 100 mg/kg), 71.5±3.5% (chloroformic extract 200 mg/kg),  31.4±4%  (methanolic  extract  50 mg/kg),  44.9±4.5%  (methanolic  extract 100 mg/kg),  57.3±1.3%  (methanolic  extract  200 mg/kg), and  64.2±2.1% (ASA). The 200 mg/kg dose of chloroformic extract was statistically highest than that of the reference drug ASA (P<.05).Figure 4
Effect of intraperitoneal administration of chloroformic (CHCl3) olive leaves extract on acetic acid-induced writhing in mice. Values are mean ± SEM. **P<.01, ***P<.001 versus control group, (n=6).Figure 5
Effect of intraperitoneal administration of methanolic (MeOH)  olive leaves extract on acetic acid-induced writhing in mice. Values are mean ± SEM. **P<.01, ***P<.001 versus control group, (n=6).


## 3.1. Extracts Yield

The results of yield extracts are shown in Table1.Table 1
Yield of different extracts.
ExtractsResultsChloroformic extractMethanolic extractMass (g)3160Yield (%)10,320


## 3.2. Chemical Composition of Olea Leaves Extract

The preliminary phytochemical qualitative screening of the chloroformic and methnolic extracts of the  leaves  ofOlea europaea  revealed the  presence of iridoids, flavonoids, and tannins as shown in Table 2.Table 2
Phytochemical screening of olive leaves extracts.
FlavonoidsIridoidsTanninsSaponosidsAnthracenic heterosidesCardiotonic heterosidesAlkaloïdsChE+++++−−−−ME++++++++−−−−(+): detectable and (−): not detectable. Abbreviations: Ch E: chloroformic extract; ME: methanolic extract.


## 3.3. NMR Analysis

The  spectrums  determined  by  NMR  of  our  extracts  allow  us  to  detect  peaks corresponding to different protons in Table3.Table 3
Different functions ofOlea europaea determined by NMR analysis.
Methanolic extractChloroformic extractType of protonδ  in ppmδ  in ppmRadical phenyl6.2 and 7.66 and 7.5Alcohol3.63.5Amine4.14.2Hydrocarbon2.41.3 and 1.5


## 3.4. IR Analysis

The spectrums determined by IR appear in the form of bands relative to the different functions in Table4.Table 4
Different functions ofOlea europaea determined by IR analysis.
Methanolic extractChloroformic extractFunctionsFrequencies of bands in cm−1Frequencies of bands in cm−1-C=C-16001600-C=O-17501700Amine3100–32502900–3250Alcohol3100–32502900–3250


## 3.5. Anti-Inflammatory  Effects of Olea  Leaves Extract

The results reported in Figures1, 2, and 3  showed  the  effect  of  the  chloroformic and methanolic  extracts  of  olive  leaves  (50,  100,  and  200 mg/kg)  administered  intraperitoneally. These results revealed significant dose-dependent inhibitory effects of the carrageenan-induced  paw  edema  at  1,  2,  3, 4, and  5 hours  after  injection.  The effect appeared 1 hour after the injection and persisted all along the duration of the experiment (5 hours).  The peak anti-inflammatory  effect  of  the  leaves  extracts  recorded  with  the dose of 200 mg/kg (BW) at the fifth hour was77.8±2% for the chloroformic extract and 68.1±3.8%  for  the  methanolic  extract  compared  to  the  standard  drugs  (ASA  and Dexamethasone)  which  decreased  the  paw  edema  by  62.9±3.1%  and  78.9±3.3%, respectively, at the fifth hour.Figure 1
Effect of intraperitoneal administration of chloroformic (CHCl3) olive leaves extract on the carrageenan-induced rat hind paw edema. Values are mean ± SEM. *P<.05, **P<.01, and ***P<.001 versus control group, (n=6).Figure 2
Effect of intraperitoneal administration of methanolic (MeOH) olive leaves extract on the carrageenan-induced rat hind paw edema. Values are mean ± SEM. *P<.05, **P<.01, and ***P<.001 versus control group, (n=6).Figure 3
Effect of intraperitoneal administration of drug references (ASA and Dexamethasone) on the carrageenan-induced rat hind paw edema. Values are mean ± SEM. **P<.01, ***P<.001 versus control group, (n=6).


## 3.6. Antinociceptive  Effects of Olea Leaves Extract

The effects of the used extracts on writhing response in mice are reported in Figures4 and 5. The intraperitoneal administration of the chloroformic and methanolic extracts at doses of 50, 100, and 200 mg/kg (BW) caused a significant reduction in the number of the writhing episodes induced by acetic acid compared to the control. The percentage of inhibition  of  the  constrictions  was  calculated  as  37.7±6.8%  (chloroformic  extract  50 mg/kg), 60±4.5%  (chloroformic extract 100 mg/kg), 71.5±3.5% (chloroformic extract 200 mg/kg),  31.4±4%  (methanolic  extract  50 mg/kg),  44.9±4.5%  (methanolic  extract 100 mg/kg),  57.3±1.3%  (methanolic  extract  200 mg/kg), and  64.2±2.1% (ASA). The 200 mg/kg dose of chloroformic extract was statistically highest than that of the reference drug ASA (P<.05).Figure 4
Effect of intraperitoneal administration of chloroformic (CHCl3) olive leaves extract on acetic acid-induced writhing in mice. Values are mean ± SEM. **P<.01, ***P<.001 versus control group, (n=6).Figure 5
Effect of intraperitoneal administration of methanolic (MeOH)  olive leaves extract on acetic acid-induced writhing in mice. Values are mean ± SEM. **P<.01, ***P<.001 versus control group, (n=6).


## 4. Discussion

Both the chloroformic and methanolic extracts of olive leaves showed a significant anti-inflammatory effect in the acute phase of the inflammation process when compared with the standard anti-inflammatory drugs. The effect was observed from the first hour. The results  showed that the anti-inflammatory activity of the chloroformic extract was more pronounced than the methanolic one.The  in  vivo  acute  inflammation  model  (Carrageenan-induced  paw  edema)  has  been frequently used to assess the anti-inflammatory effect of natural products [10, 12]. Several hypotheses  have been advanced  to explain the carrageenan mechanism  action. In fact, various  mediators  are  released  by  carrageenan in  the  rat paw. Thus,  while  the  initial phase is mainly mediated by the release of histamine and serotonin, the second phase is due  to  the  release  of  prostaglandins,  protease,  and  lysosome  [13–15].  Our  results indicate  that  the  olive  leaves  extracts  significantly  inhibit  the  development  of carrageenan-induced  rat  paw  edema.  Similar results were noted in the presence of aqueous extract of citrillus colocynthis [16]. In addition, the mechanism of action may involve  inhibition of production  of proinflammatory  cytokine (IL-6; IL-1β)  and of the expression  of  COX-2  and at stimulation of the  anti-inflammatory  cytokine (IL-4)  [17].Interestingly, the anti-inflammatory effect of both tested extracts appears to  be similar to  that  of dexamethasone,  a  steroidal  anti-inflammatory drug,  since in both  cases the effect  becomes more marked with  time.  In our experiment, we were limited to five hours after the injection of the phlogistic agent.  It would be interesting to extend the time of our experimentation (24 h) to observe the effects of the extracts.For the antinociceptive activity, different tests were widely employed in mice such as p-benzoquinone and acetic-acid-induced writing tests and hot plate test [18, 19].The olive leaves extract seems to exhibit a notable antinociceptive effects in the acetic acid-induced writhing test in the mouse. It is reported that acetic acid causes an increase in the  peritoneal  fluid  levels  of  prostaglandins  (PGE2 and  PGF2α),  involving  in  part peritoneal receptors [20]. Prostaglandins may be involved in the antinociceptive action of the olive leaves extracts.The presence of iridoids and flavonoids in these organic extracts could be responsible for the anti-inflammatory and antinociceptive effects recorded in this study as it was reported earlier  [16, 18, 21].  However,  other  results  [22]  suggest  that  the  anti-inflammatory mechanism of ethanol extract of Mahonia oiwakensis may be related to the decrease of neutrophil  infiltration, nitric  oxide  synthetase  (iNOS), COX-2, protein  expression, NO release, and TNFα  level serum.Further studies are needed to isolate the different fractions in our global extract and to isolate and purify the active ingredients in the different fractions responsible for the anti-inflammatory and antinociceptive effects ofOlea europaea.


## 5. Conclusion

Our current study is a first attempt to evaluate and validate  the anti-inflammatory and analgesic activities ofOlea europaea  leaves  confirming the  traditional use of this plant in treating ailments associated with inflammation and pain. We suggest that the iridoids and flavonoids contents in olive leaves are major contributors to the anti-inflammatory and antinociceptive effects of olive leaves. A chemical study by NMR and IR of our extracts showed the presence of different compounds like hydrocarbon phenyl and ketone amine. These  natural  products  are  considered  as  active  analgesic  and  anti-inflammatory ingredients [23, 24].However,  further  detailed  studies  are  required  to  determine  the  active  ingredients responsible  for  these  effects  and  to  determine  the  mechanism  of  action  of  these compounds in the anti-inflammatory and analgesic processes.



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*Source: 564972-2011-05-26.xml*