Ž .Fitoterapia 72 2001 255�262

Antinociceptive effect of Elaeagnusangustifolia fruit seeds in mice

Mohammad Ramezani�, Hossein Hosseinzadeh,Nosratollah Daneshmand

Faculty of Pharmacy, Mashhad Uni�ersity of Medical Sciences, P.O. Box 91775-1365, Mashhad,I.R. Iran

Received 13 May 2000; accepted in revised form 16 October 2000

Abstract

The antinociceptive effect of different Elaeagnus angustifolia fruit seed extractives wasstudied in mice using hot-plate and writhing tests. Following intraperitoneal injection, the

Ž . Ž . Ždecoction EaDE , the ethanol extract EaEE , the aqueous and n-butanol fractions EaAF,.and EaBF, respectively of a polyphenolic fraction, and two flavonoid-enriched fractions of

Ž .EaBF EaBCF1 and EaBCF2 showed significant antinociceptive activity in both tests,markedly and dose-dependently increasing the pain threshold. � 2001 Elsevier Science B.V.All rights reserved.

Keywords: Elaeagnus angustifolia; Antinociceptive activity

1. Introduction

Ž .The fruit of Elaeagnus angustifolia L. Elaeagnaceae is used in Iranian traditio-nal medicine as an analgesic agent for the alleviation of pain in rheumatoidarthritis patients. Species of this family have a variety of medicinal uses. The ripe

� �fruits of E. philippinsis have been used to treat amoebic dysentery 1 . Antibacterial� �agents such as epigallocatechin from the bark of E. glabra were identified 2 .

� Corresponding author. Fax: �98-51-837075.Ž .E-mail address: mapharm@sinasoft.net M. Ramezani .

0367-326X�01�$ - see front matter � 2001 Elsevier Science B.V. All rights reserved.Ž .PII: S 0 3 6 7 - 3 2 6 X 0 0 0 0 2 9 0 - 2

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Phytochemical investigations have indicated the presence of flavonoids in E.� �angustifolia 3,4 . It has been reported that some flavonoids have antinociceptive

� �and anti-inflammatory activities 5�7 .On the basis of the above information, the present study was undertaken to

investigate the antinociceptive activity of E. angustifolia seed and the possible roleof therein contained flavonoids in exerting this activity.

2. Experimental

2.1. Plant material

ŽFruits of E. angustifolia were collected from Nishabour region Khorassan.province , north-east of Iran in November of 1997, and dried at room temperature.

Seeds were separated from pericarp and powdered. The plant was authenticated byFerdowsi University and voucher samples were preserved for reference in theherbarium of Department of Pharmacognosy, School of Pharmacy, MashhadŽ .Taheri, 97-0501-1 .

2.2. Preparation of extracts

2.2.1. DecoctionŽ .The seed powder 400 g was added to boiling distilled water and boiled for 15

min. The mixture was filtered and the filtrate was evaporated to dryness underŽ .reduced pressure to afford a viscous residue EaDE; 2 g . Phytochemical screening

� �8�10 gave positive tests for anthocyanins, flavonoids and saponins.

2.2.2. Ethanol extractŽ .Powdered seeds 400 g were macerated for 3 days in diethyl ether, chloroform

Ž .and EtOH 96%, v�v , successively. The EtOH extract was filtered and the filtrateŽ .was dried under reduced pressure to afford a brown residue EaEE; 3.2 g giving

� �positive tests 8�10 for alkaloids, anthocyanins, flavonoids and saponins.

2.2.3. Preparation of polyphenolic fractionsŽ .The seed powder 400 g was extracted first with 10% MeOH and then with 50%

MeOH. In each step, sufficient solvent was added to make a liquid slurry whichwas left for 24 h at room temperature with occasional stirring. The two filtrateswere mixed and evaporated to dryness under reduce pressure to give brown residueŽ .7 g . The residue was suspended in water and extracted with EtOAc. The EtOAclayer was decanted and the aqueous layer was further extracted with n-BuOHsaturated with water. Aqueous and n-BuOH layers were separated and evaporated

Ž . Ž . Ž .to dryness to give EaAF 5.0 g and EaBF 2.1 g , respectively. EaBF 1.5 g wasŽ .Si-gel CC eluting with n-BuOH�water�AcOH 1:5:4 upper layer to give

Ž . Ž . � �flavonoid-enriched fractions EaBCF 0.55 g and EaBCF 0.63 g 11 .1 2

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2.3. Animals

Male and female Swiss mice of either sex, weighing 23�28 g, were obtained fromŽa random bred colony and maintained on standard rodent diet Khorassan Javane

.Co, Mashhad, I.R. Iran in the animal house of Mashhad University of MedicalSciences. Animals were housed in colony room on 12�12 h light�dark cycle at21 � 2�C, and had free access to water and food.

2.4. Maximum tolerated doses

Extractives were preliminarily injected in grade doses to groups of five miceeach. After 72 h, the highest dose that did not induce any mortality was taken as

Ž .the maximum tolerated dose MTD . Doses in the range from 10 to 90% of MTDwere chosen for activity tests.

2.5. Hot-plate test

� �Hot-plate test according to Koster et al. 12 was performed on groups of sixŽ .mice 3 male � 3 female each. The temperature of metal surface was maintained

Ž .at 55 � 0.2�C. Latency to a discomfort reaction licking paws or jumping wasŽ .determined at the time of intraperitoneal treatment time 0 and after 30, 60, 90

and 120 min. The cut-off time was 40 s. Administered extracts and doses areŽ . Ždetailed in Tables 1�3; controls received saline 10 ml�kg ; indomethacin 1

. Ž .mg�kg and morphine 5 mg�kg were used as reference drugs.

2.6. Writhing test

� �The test was performed according to Vogel and Vogel 13 on groups of six miceŽ .3 male � 3 female each. Briefly, 1 h after the intraperitoneal treatment, mice

Žwere given an intraperitoneal injection of 0.5% v�v acetic acid solution 0.1 ml�10.g . Five minutes after treatment the number of writhings induced by acetic acid

injection was counted for 10 min. Administered extracts and doses are detailed inŽ . Ž .Table 4; controls received saline 10 ml�kg , indomethacin 1 mg�kg and mor-

Ž .phine 5 mg�kg were used as reference drugs.

2.7. Statistical analysis

The data, expressed as mean values � S.E.M., were tested with analysis ofŽ .variance ANOVA followed by the multiple comparison test of Tukey�Kramer.

Differences with P � 0.05 were considered statistically significant.

3. Results

Preliminary phytochemical screening of the E. angustifolia seed ethanol extract

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Table 1aŽ . Ž . Ž .Antinociceptive activity of decoction EaDE and ethanol extract EaEE of E. angustifolia fruit seeds hot-plate test in mice

Ž .Treatment Dose Reaction time s at min after treatmentŽ .g�kg, i.p. 0 min 30 min 60 min 90 min 120 min

Ž .Control saline, 10 ml�kg, i.p. � 5.96 � 0.28 6.05 � 0.32 6.13 � 0.20 7.12 � 0.14 6.13 � 0.40EaDE 0.134 6.35 � 0.30 7.51 � 0.30 8.20 � 0.37 7.97 � 0.32 7.73 � 0.33

�EaDE 0.268 6.08 � 0.34 7.43 � 0.29 8.08 � 0.37 9.38 � 0.31 7.91 � 0.39��� ��� ���EaDE 0.536 6.16 � 0.19 7.95 � 0.28 9.63 � 0.65 10.11 � 0.61 9.10 � 0.35��� ��� ���EaDE 1.07 6.71 � 0.35 7.97 � 0.25 11.73 � 0.84 11.02 � 0.31 8.85 � 0.34

Ž .Control saline, 10 ml�kg, i.p. � 5.97 � 0.33 6.03 � 0.27 6.25 � 0.14 6.45 � 0.18 6.18 � 0.26EaEE 0.117 5.81 � 0.36 7.58 � 0.28 7.90 � 0.40 8.10 � 0.19 7.70 � 0.29EaEE 0.234 6.43 � 0.34 7.80 � 0.31 8.40 � 0.37 7.93 � 0.28 7.40 � 0.19

��� ���EaEE 0.468 6.18 � 0.41 7.75 � 0.46 9.42 � 0.51 9.60 � 0.31 8.10 � 0.34� ��� ��� �EaEE 0.936 6.30 � 0.32 8.03 � 0.36 11.38 � 0.78 10.43 � 0.65 8.40 � 0.23

a Ž . � ���Values are mean � S.E.M. n � 6 ; P � 0.05, P � 0.001 vs. control, Tukey�Kramer test.

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Table 2Ž . Ž . ŽAntinociceptive activity of aqueous EaAF and n-butanol EaBF soluble fractions of polyphenolic extract of E. angustifolia fruit seeds hot-plate test in

a.mice

Ž .Treatment Dose Reaction time s at min after treatmentŽ .g�kg, i.p. 0 min 30 min 60 min 90 min 120 min

Ž .Control saline, 10 ml�kg, i.p. � 5.28 � 0.35 6.68 � 0.32 6.77 � 0.25 6.45 � 0.34 6.80 � 0.50EaAF 0.201 6.03 � 0.27 7.43 � 0.27 8.02 � 0.34 8.12 � 0.28 7.87 � 0.38

�� ���EaAF 0.402 6.57 � 0.29 8.08 � 0.20 9.25 � 0.53 9.97 � 0.46 9.13 � 0.47��� ��� �EaAF 0.804 6.65 � 0.22 8.02 � 0.28 11.00 � 0.75 11.02 � 0.37 9.40 � 0.45

EaBF 0.083 6.65 � 0.34 7.48 � 0.19 8.62 � 0.36 8.07 � 0.25 8.31 � 0.48��� ��� ��EaBF 0.332 6.65 � 0.28 7.57 � 0.39 11.13 � 0.74 11.17 � 0.73 9.65 � 0.56

a Ž . � �� ���Values are mean � S.E.M. n � 6 ; P � 0.05, P � 0.01, P � 0.001 vs. control, Tukey�Kramer test.

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Table 3Ž .Antinociceptive activity of flavonoid-enriched fractions EaBCF and EaBCF of n-butanol soluble fraction of polyphenolic extract from E. angustifolia1 2

aŽ .fruit seeds hot-plate test in mice

Ž .Treatment Dose Reaction time s at min after treatmentŽ .mg�kg, i.p. 0 min 30 min 60 min 90 min 120 min

Ž .Control saline, 10 ml�kg, i.p. � 6.55 � 0.24 6.43 � 0.37 6.67 � 0.19 6.65 � 0.39 6.52 � 0.22�EaBCF 67 6.82 � 0.41 6.82 � 0.47 9.37 � 0.63 8.75 � 0.37 8.33 � 0.231� ���EaBCF 134 7.40 � 0.53 7.48 � 0.40 10.75 � 1.00 10.88 � 0.87 9.50 � 0.421�EaBCF 67 6.60 � 0.27 7.89 � 0.35 9.15 � 0.33 8.88 � 0.55 7.98 � 0.292��� ���EaBCF 134 7.32 � 0.40 7.82 � 0.43 10.12 � 0.74 10.03 � 0.65 8.72 � 0.502

Indomethacin 1 6.67 � 0.38 7.68 � 0.20 8.78 � 0.60 8.03 � 0.35 8.03 � 0.48��� ��� ���Morphine sulfate 5 7.07 � 0.47 8.27 � 0.46 12.13 � 0.72 13.10 � 1.24 12.20 � 0.70

a Ž . � ���Values are mean � S.E.M. n � 6 ; P � 0.05, P � 0.001 vs. control, Tukey�Kramer test.

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Table 4Ž .Antinociceptive activity of extractives from E. angustifolia seeds writhing test in mice

a b Ž .Treatment Doses Writhing count Protection %Ž .mg�kg, i.p.

Ž .Control saline, 10 ml�kg, i.p. � 26.6 � 1.7 ����EaDE 201 12.8 � 1.9 52���402 7.7 � 1.8 71���804 2.2 � 1.1 92

Ž .Control saline, 10 ml�kg, i.p. � 26.3 � 2.0 ����EaEE 201 12.8 � 1.8 51���402 9.5 � 1.1 64���804 4.8 � 1.4 82

Ž .Control saline, 10 ml�kg, i.p. � 27.0 � 2.0 ����EaAF 201 13.7 � 1.5 49���402 9.2 � 0.8 66���804 2.5 � 1.0 91���EaBF 83 14.0 � 1.1 48���332 2.3 � 1.2 92

Ž .Control saline, 10 ml�kg, i.p. � 27.8 � 2.0 ����EaBCF 67 14.5 � 1.1 481���134 4.2 � 1.4 85�EaBCF 67 20.7 � 1.3 262���134 12.1 � 1.4 57���Indomethacin 1 16.0 � 1.7 42���Morphine sulfate 5 2.8 � 1.2 90

a EaDE, decoction; EaEE, ethanol extract; EaAF and EaBF, aqueous and n-butanol solublefractions of polyphenolic extract; EaBCF and EaBCF , flavonoid-enriched fractions from EaBF.1 2

b Ž . � ���Values are mean � S.E.M. n � 6 ; P � 0.05, P � 0.001 vs. control, Tukey�Kramer test.

Ž .EaEE indicated the presence of flavonoids, alkaloids, anthocyanins and saponins;Ž .the decoction EaDE showed only the presence of flavonoids, anthocyanins and

saponins.Following intraperitoneal administration in mice, both EaDE and EaEE demon-

strated a significant and dose-dependent antinociceptive activity in hot-plate testŽ .after 60�90 min of treatment Table 1 . The number of writhings was also

Ž .decreased significantly compared to control Table 4 .� �Polyphenolic fraction 11 was partitioned between water and n-butanol to give

Ž .EaAF and EaBF, respectively. The results reported in Table 2 hot-plate test showthat EaAF significantly increased the reaction time of mice at doses of 0.4 and 0.8g�kg, again after 60 min of treatment, EaBF being as effective as EaAF but at a

Ž .lower dose 0.33 g�kg . In the writhing test, both EaAF and EaBF reduced theŽ .number of writhings significantly, being comparable to morphine sulfate 5 mg�kg

Ž .at doses of 800 and 330 mg�kg, respectively Table 4 .The flavonoid-enriched fractions EaBCF and EaBCF , obtained by column1 2

chromatography of EaBF, were tested at doses of 67 and 134 mg�kg. In hot-platetest, maximum activity was observed between 60 and 90 min after administration,EaBCF being the more active fraction; indomethacin did not show significant1

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Ž .activity Table 3 . In writhing test, EaBCF and EaBCF significantly decreased the1 2number of writhings in mice in a dose-dependent manner. Again, EaBCF ap-1peared to be more active, the protection reaching 85% at the dose of 134 mg�kg,compared to 90% for morphine sulfate at 5 mg�kg and 42% for indomethacin at 1

Ž .mg�kg Table 4 .

4. Discussion

The present results indicate that the various extractives of E. angustifolia seedsexhibit antinociceptive activity, both in the hot-plate and writhing tests.

� �As the hot-plate test is a specific central antinociceptive test 14 , it is possibleŽ .that E. angustifolia extractives exert their effect through central mechanism s . On

the other hand, since it is known that the peripheral antinociceptive activity of� �non-steroidal anti-inflammatory agents can be determined by writhing test 13 , the

efficacy of E. angustifolia extracts and fractions in this test suggests that aperipheral mechanism may also be involved.

The polyphenolic fraction of E. angustifolia seeds was further fractionated usingactivity-directed approach. By transfer of active components into n-butanol fol-lowed by fractionation on silica gel, a high antinociceptive activity was observed in

Ž .two flavonoid-enriched fractions EaBCF and EaBCF , suggesting flavonoids as1 2the active constituents of the seeds.

References

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