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Analgesic effect of Allium ampeloprasum: Evidence for the involvement of beta-adrenergic system
Journal of Functional Foods 57 (2019) 1–6
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Analgesic effect of Allium ampeloprasum: Evidence for the involvement of beta-adrenergic system
T
Manal A. Abbas Faculty of Pharmacy and Medical Sciences, Al-Ahliyya Amman University, 19328 Amman, Jordan
A R T I C LE I N FO
A B S T R A C T
Keywords: Allium ampeloprasum Analgesic Antinociceptive Beta-adrenergic receptor Antidepressant Anxiolytic
Allium ampeloprasum (leek) is a bulbous perennial edible vegetable closely related to garlic (A. sativum L.). In this study, the antinociceptive, antidepressant and anxiolytic effects of A. ampeloprasum were studied. A. ampeloprasum inhibited abdominal cramps in writhing test and increased latency time in hot-plate and tail-flick tests. In formalin test, A. ampeloprasum inhibited paw licking in both early and late phases. Propranolol, but not naloxone or yohimbine, reversed this effect. A. ampeloprasum decreased the number of lines crossed in open field test but has no effect on the time spent in open arm in elevated plus maze or the immobility time in forced swimming test. GC–MS analysis resulted in the identification of 18 compounds with phytol acetate, linoleic acid and tricosane as major constituents. In conclusion, the analgesic action of A. ampeloprasum was mediated by interaction with β-adrenergic receptor. No antidepressant or anxiolytic effects were exerted by A. ampeloprasum.
1. Introduction Allium genus has over 500 species (Mohammadi, Zarei, Mahmoodi, Zarei, & Nematian, 2015) which differ in maturing, color and taste. However, they are similar in biochemical content (Dey & Khaled, 2015). The presence of organosulfur compounds is responsible for the characteristic taste, aroma, and lachrymatory effects of the Allium species (Najda, Błaszczyk, Winiarczyk, Dyduch, & Tchórzewska, 2016). Allium ampeloprasum (leek) is a bulbous perennial plant belonging to the family Amaryllidaceae (Acharya, Choubey, & Acharya, 2013). The native range of this edible plant is the Mediterranean region (south Europe, northern Africa to west Asia) (Dey & Khaled, 2015). However, it is cultivated in many countries (Rahimi-Madiseh, Heidarian, Kheiri, & Rafieian-Kopaei, 2017). Leek is closely related to garlic (A. sativum L.) (Najda et al., 2016). It is rich in organosulphur compounds especially γglutamyl peptides. Also, it contains alliin and allicin but at lower levels compared to garlic (Kim et al., 2018). In addition to the presence of saponins (Adão, Da Silva, & Parente, 2011), A. ampeloprasum leaves and bulbs contain higher levels of polyphenolics, phenolic acid, flavonoids, and tannins than garlic (Najda et al., 2016). In addition to culinary purposes, A. ampeloprasum is used for several medical purposes (Mohammadi et al., 2015). It has been reported that A. ampeloprasum has antibacterial (Bareemizadeh et al., 2014), anticancer (An, Zhang, Yao, Li, & Ren, 2015), anti-hemorrhoidal (Mosavat, Ghahramani, Sobhani, Haghighi, & Heydari, 2015) and antiulcer effects (Adão et al., 2011). Also, it exerted hypoglycemic, hypolipidemic, and
anti-oxidative effects in alloxan-induced diabetic model in rats (RahimiMadiseh et al., 2017). In addition, steroidal saponins in the bulbs exerted anti-inflammatory effects similar to dexamethasone (Adão et al., 2011). Antinociceptive action was reported in many Allium species like garlic (A. sativum L.) (Dange, Mathew, Datta, Tilak, & Jadhav, 2016), A. hirtifolium (Mohammadi et al., 2015), A. paradoxum (Maghsoodia, Afsharb, Rabania, Mahmoodic, & Ebrahimzadeh, 2018), A. humile (Singh, Gangwar, Singh, Jadon, & Ranjan, 2014), A. stracheyi (Ranjan et al., 2010) and A. jesdianum (Khaksarian, Meshkat Alsadat, Farazifard, & Safarpour, 2008). According to Acharya et al., A. ampeloprasum has the same medicinal virtues as garlic, but in a much milder and less effective form (Acharya et al., 2013). Since garlic has well documented analgesic effects in different animal models of pain, this study was conducted to investigate the antinociceptive action of A. ampeloprasum in mice. In addition, antidepressant and anxiolytic effects were investigated in different behavioral models. 2. Materials and methods 2.1. Plant collection, extraction and chemical identification of constituents A. ampeloprasum was collected from Subaihi, Jordan in May 2015. The plant was authenticated by Prof. Barakat Abu-Irmaileh/ The University of Jordan. A voucher specimen was deposited in Graduate Studies Laboratory at Al-Ahliyya Amman University (Voucher number
E-mail address: [email protected]. https://doi.org/10.1016/j.jff.2019.03.046 Received 6 December 2018; Received in revised form 2 March 2019; Accepted 25 March 2019 1756-4646/ © 2019 Published by Elsevier Ltd.
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2.6. Hot-plate test
Alliaceae#5-2015). Purple inflorescences of A. ampeloprasum growing in the wild were immediately dipped and extracted in acetone for 3 days at room temperature. The choice of acetone as a solvent was due to its water miscibility since flowers were extracted while they were fresh and because it has low boiling point so that the specimen was exposed to temperatures not exceeding 40 ˚C, and because acetone is useful in extracting biologically active compounds like flavonoids (Chebil et al., 2007). After evaporation of the solvent, the extract was stored at −20 °C. A. ampeloprasum extract was dissolved in sterile normal saline immediately before use and administered intraperitoneally (i.p). Gas chromatography–mass spectrometric (GC–MS) analysis was performed utilizing a Varian CP-3800 GC/MS/MS-220 (Saturn, the Netherlands) system, equipped with a DB-5 GC capillary column (95% dimethyl polysiloxane, 5% diphenyl, 30 m × 0.25 mm i.d., 0.25 μm film 1 thicknesses). The analysis method was as in Jaffal and Abbas (2018).
The hot-plate test was used to determine the latencies in pain reaction as in Dange et al. (2016). The temperature of the beaker on a hotplate was adjusted at 55 ± 1 °C. The test was done only once for each mouse. The time between the animal’s placement and first jumping was recorded as an indication for the latency of pain reaction. Latency time was measured after 45 min and 90 min. A cutoff time of 60 sec was determined to avoid tissue damage. 2.7. Paw licking test Paw licking test was carried out by injecting twenty microliters of formalin (2.5%) intraplantarly to the right hind paw of the animal as in Jaffal and Abbas (2018). In all tests, animals were pretreated i.p with vehicle (sterile distilled water, negative control), 150 mg/kg, 300 mg/ kg A. ampeloprasum flower extract or 30 mg/kg diclofenac sodium (positive control) 30 min prior to formalin injection. Antagonists used were naloxone (5 mg/kg, injected 30 min before extract), propranolol (10 mg/kg, given 20 min before extract) and yohimbine (1 mg/kg, given in 15 min before extract). Choice of antagonist dose and administration time was according to previous studies (Afify & Andijani, 2017; Janssen, Maiello, Wright, Kracinovsky, & Newsome, 2017; Saldanha et al., 2017). Nociception was determined by counting the time of licking the injected paw, moving while lifting the leg or exhibiting flinching behavior. Counting was performed during the early phase (the first 5 min) and during the late phase (25–30 min after formalin injection). The percentage inhibition was calculated using this formula:
2.2. Drugs and chemicals Clomipramin hydrochloride was purchased from Novartis, Switzerland. Diazepam was purchased from Medochemie LTD, Cyprus. Naloxone hydrochloride and Yohimbine were purchased from Tocris Bioscience (UK), Diclofenac sodium was from Novartis, Switzerland. Propranolol hydrochloride was from APM, Jordan. All drugs were dissolved in sterile normal saline and administered i.p. 2.3. Experimental animals All experiments were conducted in agreement with the Jordanian Animal Welfare By-Law No. (11) of the year 2010 and approved by the ethical committee at Al-Ahliyya Amman University (ethical approval number AAU-2/4/2018). Male BALB/c mice (weight: 20–25 g) were used in all experiments. Mice were brought from the animal house at Al-Ahliyya Amman University, Jordan. Animals were kept at 23 ± 2 °C with 12 h lightness and 12 h darkness. Water and pellets of food were available ad libitum. Before tests, the animals were acclimatized to the experimental room for at least 2 h. In each of the following experiments, 9 mice per group were used.
average time of paw licking in control − average time of paw licking in extract % inhibition = − treated animals × 100% average number of paw licking in control 2.8. Open field test (OFT) The open field was performed as in Mousavi, Fashi, Jahromy, and Rasooli (2017). Briefly, a 30 × 40 × 20 cm high arena divided into 20 equal squares was used. The ambulatory locomotion of each mouse was recorded for 3 min by video camera. The number of squares crossed by each animal and the number of rearing (i.e. the number of times the animal stood on the hind paws) was counted. A square crossing is considered when the animal totally crosses from one square to the next. The arena was cleaned with ethanol and dried after each experiment. The experimental animals were i.p treated with vehicle, A. ampeloprasum extract (150, and 300 mg/kg) or diazepam (2 mg/kg) 30 min before evaluation.
2.4. Writhing test The writhing test was conducted by injecting acetic acid solution (1%, 10 ml/kg) i.p, 30 min after receiving the vehicle, A. ampeloprasum flower extract (150 mg/kg or 300 mg/kg) or 30 mg/kg diclofenac sodium as in Jaffal and Abbas (2018). Ten minutes after acetic acid injection, the number of writhes was counted for 20 min. A contraction of the abdominal muscles with elongation of the body and extension of the forelimbs is considered a writhe. The percentage inhibition of abdominal cramps was calculated using this formula:
2.9. Elevated plus maze (EPM)
% inhibition
The EPM was performed as in Pitchaiah, Kumar, and Kumar (2015). Briefly, a maze consisted of two open (10 × 5 cm) and two closed (10 × 5) arms, extending from a center platform (5 × 5 cm) and elevated to a height of 50 cm above the floor was used. Mice were individually placed on the center of the maze facing a closed arm, and the time spent in closed and open arms were recorded during a 5 min by video camera. The maze was wiped clean with ethanol and dried after each trial. The experimental animals were i.p treated with vehicle, A. ampeloprasum extract (150, and 300 mg/kg) or diazepam (4 mg/kg) 30 min before evaluation in the maze.
average number of writhes in control =
− average number of writhes in extract − treated animals average number of writhes in control × 100%
2.5. Tail-flick test The tail-flick test was performed as in Dange et al. (2016) by immersing the tail in water at 55 ± 1 °C. Latency time was measured after 45 min, 90 min and 180 min. Different animals were used for measuring tail flick for each time. A cutoff time of 10 sec was determined.
2.10. Forced swim test (FST) FST was performed as in Mousavi et al. (2017) with minor 2
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modifications. The mouse was placed into a glass container (25 cm in height and 10 cm in diameter) containing 19 cm-deep water at 24–25 °C and left there for 5 min. Video recording started two minutes from the placement of mouse in water and lasted for 3 min to record immobility time. The mice were considered immobile when they remained floating in the water, without struggling, making only very slight movements necessary to keep the head above water. Mice were divided into 4 groups: one group (the control group) was treated with normal saline, 2 groups received i.p injection of A. ampeloprasum flower extract (300 or 150 mg/kg) and the forth group received clomipramin hydrochloride (40 mg/kg); used as a reference drug. Treatments were given 30 min before video recording. 2.11. Statistical analysis For all measured parameters, one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test was conducted using GraphPad Prism version 6. 3. Results
Fig. 1. Writhing test. *Significant difference compared with the vehicle-treated control, P < 0.05.
3.1. GC–MS analysis GC–MS analysis of the acetone extract of A. ampeloprasum inflorescence resulted in the identification of 18 compounds. The major constituents were found to be phytol acetate (33.94%), linoleic acid (11.96%) and tricosane (11.40%) (Table 1). 3.2. Writhing test Both doses of A. ampeloprasum (150 and 300 mg/kg) decreased the number of writhings in a significant manner compared to non-treated group (Fig. 1). Abdominal cramps produced by acetic acid were inhibited by 78.6% and 93.5% using 150 mg/kg and 300 mg/kg of A. ampeloprasum flower extract, respectively compared to 43.9% inhibition produced by diclofenac sodium (30 mg/kg). 3.3. Hot-plate test and tail-flick tests A significant increase in latency time in hot-plate was observed after 45 min using 150 and 300 mg/kg A. ampeloprasum flower extract (Fig. 2). In tail-flick test, 300 mg/kg A. ampeloprasum increased latency time after 45 min as well as after 90 min and 180 min (Fig. 3). Propranolol reversed the effect of A. ampeloprasum extract in hot plate test (Fig. 2). In tail flick test, propranolol antagonized the action of A. Table 1 Results of GC–MS analysis of A. ampeloprasum. No
Retention index
Compound
%
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
867 889 904 1057 1164 1211 1344 1346 1364 1567 1833 1878 1960 2041 2100 2133 2218 2300
Allyl propyl sulfide Allyl (E)-1-propenyl sulfide 3,4-Dimethylthiophene Thiophene (2-butyl) Methyl (E)-1-propenyltrisulfide Dimethyltetrasulfide 5-methyl-1,2,3,4-tetrathianene Methyl methylthiomethyl trisulfide Allyl methyl tetrasulfide Ionone < Dimethyl > Cyclopentadecanolide Cubitene Hexadecanoic acid Cyclic octaatomic sulfur Heneicosane < n- > Linoleic acid Phytol acetate < (E)- > Tricosane < n- >
2.88 2.65 4.29 1.41 1.80 1.30 1.82 1.24 2.17 4.05 6.49 3.39 5.40 2.11 1.63 11.96 33.94 11.40
Fig. 2. Hot plate test. * Significant difference compared with the vehicle-treated control, P < 0.05. **Significant difference between A. ampeloprasum (150 mg/ kg) and propranolol with A. ampeloprasum (150 mg/kg), P < 0.05.
ampeloprasum extract partially. However, the effect was not statistically significant (Fig. 3). Diclofenac sodium increased latency in both hotplate and tail-flick tests significantly.
3.4. Paw licking test (Formalin test) Both doses of A. ampeloprasum (300 and 150 mg/kg) inhibited significantly paw licking in the early phase of formalin test (Fig. 4). Percentage inhibition in the early phase was 90.3%, 70.7.8% and 52.7% for high dose (300 mg/kg), low dose (150 mg/kg) of A. ampeloprasum and diclofenac sodium (30 mg/kg), respectively. In late phase, both doses of A. ampeloprasum produced significant inhibition (Fig. 5). The percentage inhibition was 82.9%, 85.5% and 64.0% for high dose of A. ampeloprasum (300 mg/kg), low dose (150 mg/kg) and diclofenac 3
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Fig. 3. Tail flick test. *Significant difference compared with the vehicle-treated control, P < 0.05.
Fig. 5. Formalin test (late phase). *Significant difference compared with the vehicle-treated control, P < 0.05. **Significant difference between A. ampeloprasum (150 mg/kg) and propranolol with A. ampeloprasum (150 mg/kg), P < 0.05.
clomipramin hydrochloride (standard drug) decreased it significantly (Table 2). In OFT, both doses of A. ampeloprasum decreased the number of lines crossed and rearing significantly (Table 2). Similarly, both doses of A. ampeloprasum decreased the total time spent in open arm while diazepam increased it significantly (Table 2).
4. Discussion GC–MS analysis of the acetone extract of A. ampeloprasum inflorescence resulted in the identification of 18 compounds. The major constituents were found to be phytol acetate (33.94%), linoleic acid (11.96%) and tricosane (11.40%). It should be noted that the studied plant in this work was collected from the wild and not cultivated. Similar to our results, García-Herrera et al. (2014) found a predominance of polyunsaturated fatty acids, mainly linoleic acid, in wild leek (A. ampeloprasum). Also according to Morales et al. (2012), linoleic acid constituted 53.45% of the A. ampeloprasum bulbs and bottom of the leaves. Phytol and ethyl linoleate were previously isolated from leek bulbs (Mnayer et al., 2014) and tricosanoic acid was isolated from the bulb and bottom of the leaves (Morales et al., 2012). On the other hand, the essential oil of the aerial parts of A. ampeloprasum L. var, atroviolaceum contained D-limonene (26.9%), beta-pinene (25.3%), 9-octadecanoic acid (17.3%) and hexadecanoic acid (14.9%) as major constituents (Bareemizadeh et al., 2014). The difference in the findings of this study and our study can be explained by the difference in plant part analyzed, differences in geographical region and time of plant collection as well as differences between wild and cultivated plants. In the present study, A. ampeloprasum extract was effective in alleviating pain in chemical and thermal models. Up to our best knowledge, this study represents the first investigation of analgesic effect of A. ampeloprasum flower inflorescence in normal laboratory animals. Both the lower dose of A. ampeloprasum used in this study (150 mg/ kg) and the higher dose (300 mg/kg) were more effective than 30 mg/ kg diclofenac sodium in reducing abdominal cramps. Similar results were obtained by other Allium species like A. stracheyi (Ranjan et al., 2010), A. hirtifolium (Mohammadi et al., 2015), A. paradoxum
Fig. 4. Formalin test (early phase). *Significant difference compared with the vehicle-treated control, P < 0.05. **Significant difference between A. ampeloprasum (150 mg/kg) and propranolol with A. ampeloprasum (150 mg/kg), P < 0.05.
sodium (30 mg/kg), respectively. Involvement of opioid receptor was not evident since the opioid antagonist naloxone didn’t abolish the effect of A. ampeloprasum extract. Similarly, pretreatment with yohimbine, an alpha-adrenergic receptor antagonist didn’t reverse A. ampeloprasum action. On the other hand, propranolol reversed A. ampeloprasum action in both early and late phases (Figs. 4 and 5). 3.5. OFT, EPM and FST In FST, A. ampeloprasum has no effect on immobility time while 4
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Table 2 Results of FST, OFT and EPM (values are represented as mean ± SE). Test
Control (vehicle only) A. ampeloprasum (150 mg/kg) A. ampeloprasum (300 mg/kg) Standard drug◊
OFT
OFT
EPM
FST
Number of lines crossed
Number of rearing
Time spent in open arm (sec)
Immobility time (sec)
66.62 ± 6.71 9.00* ± 2.88 3.37* ± 0.73 99.5* ± 5.98
22.88 ± 2.00 3.83* ± 0.90 2.50* ± 0.64 22.0 ± 1.11
5.57 ± 2.51 0.0 ± 0.00 0.0 ± 0.00 33.2* ± 2.69
161.75 ± 4.02 146.85 ± 9.31 151.14 ± 5.27 109.57* ± 9.91
◊
Clomipramin hydrochloride (40 mg/kg) in FST, diazepam (2 mg/kg) in OFT and diazepam (4 mg/kg) in EPM. * Significant difference compared with the vehicle-treated control, P < 0.05.
could be one of the active constituents mediating the effect of this plant. Also, it is possible that more than one active constituent act synergistically to produce the analgesic effect. In a recent clinical study, A. ampeloprasum cream was applied topically to treat hemorrhoids. A significant decrease was achieved in the grade of bleeding severity and defecation discomfort. However, no significant change was observed in pain scores (Mosavat et al., 2015). This cream contained fresh aqueous liquid obtained from cutting A. ampeloprasum subsp iranicum. Our study demonstrated the effectiveness of flower acetone extract in reducing pain in different pain models. Therefore, the use of A. ampeloprasum flower extract to treat hemorrhoids is suggested to alleviate pain as well as discomfort in patients. In OFT, A. ampeloprasum decreased the number of lines crossed and rearing significantly. This may indicate the presence of sedative action of A. ampeloprasum. Sedative properties have been documented in several Allium species such as onion (A. cepa) (Janapati, 2012) and A. jesdianum extract that decreased the number of lines crossed in OFT (Mousavi et al., 2017). In our investigation, no anxiolytic action of A. ampeloprasum was observed as both doses of A. ampeloprasum had no effect on the total time spent in open arm in EPM while diazepam increased it significantly. This indicates that A. ampeloprasum differs from onion (A. cepa) (Pitchaiah et al., 2015) and garlic (Gilhotra & Dhingra, 2016) that exerted anxiolytic activity in EPM model. Unlike garlic (A. sativum), A. jesdianum, and onion (A. cepa), A. ampeloprasum exerted no antidepressant-like activities in FST (Dhingra & Kumar, 2008; Mousavi et al., 2017; Sakakibara, Yoshino, Kawai, & Terao, 2008). The Forced swimming test is the most widely used model for assessing antidepressant activity. Drugs with antidepressant activity reduce that parameter (immobility time), while depressant drugs exert the opposite effect (Mousavi et al., 2017).
(Maghsoodia et al., 2018) and A. humile (Singh et al., 2014) in acetic acid-induce writhing model. Also, A. sativum (garlic) shoot extract reduced writhing induced by 4% NaCl (Dange et al., 2016). In thermal pain models, A. ampeloprasum increased the latency time in hot-plate and tail-flick tests. Similarly, garlic shoot extract exerted antinociceptive action in tail flick and hot plate models of experimental pain (Dange et al., 2016). Also, A. jesdianum leaf-extract increased tail flick and hot plate latencies (Khaksarian et al., 2008). In Hot plate test, A. paradoxum extract increased pain threshold compared to control specifically in 30th minute of the test (Maghsoodia et al., 2018). Also, A. hirtifolium had antinociceptive effect in tail-flick test (Mohammadi et al., 2015). In formalin test, both doses of A. ampeloprasum flower extract inhibited significantly paw licking in the early and late phases. Interestingly, A. sativum bulb powder completely abolished the early phase using 300 mg/kg dose and was also effective in the late phase (Jayanthi & Jyoti, 2012). In our study, the same dose of A. ampeloprasum extract produced 90.3% and 85.5% inhibition in the early and late phases of formalin test, respectively. Similarly, A. hirtifolium decreased pain score in both phases of formalin test (Mohammadi et al., 2015). On the other hand, treatment of diabetic rats for one month with A. ampeloprasum extract attenuated hyperalgesia in chronic phase of the formalin test only (Roghani & Aghaie, 2007). The presence of phytol in A. ampeloprasum extract may explain, at least partially, its antinociceptive activity since the analgesic effects of phytol were reported earlier (Santos, 2013). Concerning the mechanism of action of A. ampeloprasum extract, the results of our study exclude the possibility of involvement of opioid receptor but suggests an interaction with beta; but not alpha, adrenergic receptors. It is well known that adrenergic systems modulate nociceptive transmission which is mediated through their respective receptors (Pertovaara, 2006). Alpha-adrenergic receptors were implicated in the mediation of noradrenergic analgesia (Carroll, Mackey, & Gaeta, 2007). However, this was not the case in A. ampeloprasum analgesic action since the alpha-adrenergic antagonist yohimbine failed to antagonize it. Up to our best knowledge, no previous report exists concerning the involvement of alpha-adrenergic receptors in A. ampeloprasum pharmacological activities. On the other hand, beta-adrenergic receptors mediated the analgesic effect of A. ampeloprasum in this study and in the action of this plant in decreasing ileum spasms in vitro (Masoumeh, Mosayyeb, Nasri, Hadi, & Mahmoud, 2014). Several lines of evidence exist suggesting that beta-adrenergic agonists exert central effects in mice when given i.p producing analgesic effects in hot plate test (Brochet, Micó, Martin, & Simon, 1986) and abdominal constriction test (Bentley & Starr, 1986). The interaction of A. ampeloprasum with beta adrenergic receptor sub-types remains to be investigated in the future since in vivo and in vitro studies are lacking. Other members of the garlic family like A. neapolitanum and A. subhirsutum interacted with beta-2 receptors in vitro (Nencini, Franchi, & Micheli, 2010). According to Loesberg, Spence, Nijkamp, and Houslay (1994), linoleic acid, one of the major constituents of A. ampeloprasum, induced changes in respiratory beta-adrenergic receptor function. Therefore, it
5. Conclusions Wild A. ampeloprasum inflorescence extract has significant analgesic effects in chemical and thermal acute pain models. Its mechanism of action involves interaction with beta-adrenergic receptor. On the other hand, it is devoid of anxiolytic and antidepressant actions.
Ethical statement All experiments were conducted in agreement with the NIH guiding principles in the care and use of animals and the Jordanian Animal Welfare By-Law No. (11) of the year 2010.
Acknowledgments The authors thank Prof. Barakat E. Abu-Irmaileh at the University of Jordan for plant authentication. This work was published with the support of Al-Ahliyya Amman University. 5
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Conflict of interest
(Allium sativum L.), elephant garlic (Allium ampeloprasum L.) and onion (Allium cepa L.). Natural Product Research, 32(10), 1193–1197. Loesberg, C., Spence, S., Nijkamp, F. P., & Houslay, M. D. (1994). Dietary linoleic acidinduced changes in respiratory beta-adrenergic receptor function and the form of arrhenius plots of isoprenaline- and prostaglandin E2-stimulated adenylate cyclase activity in a model for atopy. Cellular Signalling, 6(2), 187–199. Maghsoodia, R., Afsharb, V., Rabania, M., Mahmoodic, M., & Ebrahimzadeh, M. A. (2018). Antinociceptive effects of methanolic extract of Allium paradoxum in mice in hot-plate and writhing tests. International Pharmacy Acta, 1(1) (in press). Masoumeh, S. H., Mosayyeb, N. A., Nasri, H., Hadi, M., & Mahmoud, R. K. (2014). InVitro evaluation of hydroalcoholic extract of Allium ampeloprasum on rat ileum contractions. Journal of Isfahan Medical School, 31(268), 2238–2249. Mnayer, D., Fabiano-Tixier, A. S., Petitcolas, E., Hamieh, T., Nehme, N., Ferrant, C., ... Chemat, F. (2014). Chemical composition, antibacterial and antioxidant activities of six essentials oils from the Alliaceae family. Molecules, 19(12), 20034–20053. Mohammadi, S., Zarei, M., Mahmoodi, M., Zarei, M. M., & Nematian, M. A. (2015). In vivo antinociceptive effects of Persian Shallot (Allium hirtifolium) in male rat. Avicenna Journal of Neuropsychophysiology, 2(1), e27149. Morales, P., Ferreira, I. C. F. R., Carvalho, A. M., Sánchez-Mata, M. C., Cámara, M., & Tardío, J. (2012). Fatty acids profiles of some Spanish wild vegetables. Food Science and Technology International, 18(3), 281–290. Mosavat, S. H., Ghahramani, L., Sobhani, Z., Haghighi, E. R., & Heydari, M. (2015). Topical Allium ampeloprasum subsp Iranicum (leek) extract cream in patients with symptomatic hemorrhoids. Journal of Evidence-Based Complementary & Alternative Medicine, 20(2), 132–136. Mousavi, R., Fashi, Z. H., Jahromy, M. H., & Rasooli, R. (2017). Potential of the Allium jesdianum extract in suppression of anxiety and depression in mice. British Biomedical Bulletin, 5(2), 302. Najda, A., Błaszczyk, L., Winiarczyk, K., Dyduch, J., & Tchórzewska, D. (2016). Comparative studies of nutritional and health-enhancing properties in the “garliclike” plant Allium ampeloprasum var. ampeloprasum (GHG-L) and A. sativum. Scientia Horticulturae, 201, 247–255. Nencini, C., Franchi, G. G., & Micheli, L. (2010). Cardiovascular receptor binding affinity of aqueous extracts from Allium species. International Journal of Food Sciences and Nutrition, 61(4), 433–439. Pertovaara, A. (2006). Noradrenergic pain modulation. Progress in Neurobiology, 80, 53–83. Pitchaiah, G., Kumar, A., & Kumar, T. R. (2015). Anxiolytic and anticonvulsant activity of methanolic extract of allium cepa Linn (Onion) bulbs in Swiss albino mice. Journal of Pharmacognosy and Phytochemistry, 4(3), 131–135. Rahimi-Madiseh, M., Heidarian, E., Kheiri, S., & Rafieian-Kopaei, M. (2017). Effect of hydroalcoholic Allium ampeloprasum extract on oxidative stress, diabetes mellitus and dyslipidemia in alloxan-induced diabetic rats. Biomedicine and Pharmacotherapy, 86, 363–367. Ranjan, S., Jadon, V. S., Sharma, N., Singh, K., Parcha, V., Gupta, S., & Bhatt, J. P. (2010). Anti-inflammatory and analgesic potential of leaf extract of Allium stracheyi. Journal of Applied Sciences Research, 6(2), 139–143. Roghani, M., & Aghaie, M. (2007). The effect Allium ampeloprasum feeding on serum level of glucose, triglyceride, and total cholesterol of diabetic rats. Koomesh, 8(2), 73–78. Sakakibara, H., Yoshino, S., Kawai, Y., & Terao, J. (2008). Antidepressant-like effect of onion (Allium cepa L.) powder in a rat behavioral model of depression. Bioscience, Biotechnology, and Biochemistry, 72(1), 94–100. Saldanha, A. A., Siqueira, J. M., Castro, A. H. F., Matos, N. A., Klein, A., Silva, D. B., ... Soares, A. C. (2017). Peripheral and central antinociceptive effects of the butanolic fraction of Byrsonima verbascifolia leaves on nociception-induced models in mice. Inflammopharmacology, 25(1), 81–90. Santos, C. C. de M. P., Salvadori, M. S., Mota, V. G., Costa, L. M., de Almeida, A. A. C., de Oliveira, G. A. L., ... de Almeida, R. N. (2013). Antinociceptive and antioxidant activities of phytol in vivo and in vitro models. Neuroscience Journal Article ID 949452. Singh, K., Gangwar, R. K., Singh, G., Jadon, V. S., & Ranjan, S. (2014). Studies on the analgesic potential of leaf extracts of Allium humile on swiss albino mice. ER, 83(9–2), 1677–1681.
There is no conflict of interest to declare. References Acharya, C., Choubey, A., & Acharya, M. (2013). Biologically active saponin from seeds of Allium ampeloprasum. Journal of Chemistry and Materials Research, 3(6) ISSN 22250956. Adão, C. R., Da Silva, B. P., & Parente, J. P. (2011). A new steroidal saponin with antiinflammatory and antiulcerogenic properties from the bulbs of Allium ampeloprasum var. porrum. Fitoterapia, 82(8), 1175–1180. Afify, E. A., & Andijani, N. M. (2017). Potentiation of morphine-induced antinociception by propranolol: The involvement of dopamine and GABA systems. Frontiers in Pharmacology, 8, 794. An, X., Zhang, X., Yao, H., Li, H., & Ren, J. (2015). Effects of diallyl disulfide in elephant garlic extract on breast cancer cell apoptosis in mitochondrial pathway. Journal of Food and Nutrition Research, 3(3), 196–201. Bareemizadeh, F., Ghasempour, H., Maassoumi, S. M., Karimi, N., Taran, M., & Ghasempour, M. (2014). In vitro antimicrobial activity of Allium ampeloprasum L. var. atroviolaceum Regel. International Journal of Biosciences, 4(5), 80–84. Bentley, G. A., & Starr, J. (1986). The antinociceptive action of some b-adrenoceptor agonists in mice. British Journal of Pharmacology, 88, 515–521. Brochet, D., Micó, J. A., Martin, P., & Simon, P. (1986). Antinociceptive activity of betaadrenoceptor agonists in the hot plate test in mice. Psychopharmacology (Berl), 88, 527–528. Carroll, I., Mackey, S., & Gaeta, R. (2007). The role of adrenergic receptors and pain: The good, the bad, and the unknown. Seminars in Anesthesia, Perioperative Medicine and Pain, 26, 17–21. Chebil, L., Humeau, C., Anthoni, J., Dehez, F., Engasser, J.-M., & Ghoul, M. (2007). Solubility of flavonoids in organic solvents. Journal of Chemical and Engineering Data, 52, 1552–1556. Dange, S., Mathew, J., Datta, A., Tilak, A., & Jadhav, M. (2016). Evaluation of the analgesic efficacy of garlic shoots extract in experimental pain models in mice. International Journal of Basic and Clinical Pharmacology, 5(6), 2393–2396. Dey, P., & Khaled, K. L. (2015). An Extensive review on Allium ampeloprasum: A magical herb. International Journal of Science and Research, 4(7), 371–377. Dhingra, D., & Kumar, V. (2008). Evidence for the involvement of monoaminergic and GABAergic systems in anti-depressant-like activity of garlic extract in mice. Indian Journal of Pharmacology, 40(4), 175–179. García-Herrera, P., Morales, P., Fernández-Ruiz, V., Sánchez-Mata, M. C., Cámara, M., Carvalho, A. M., ... Tardio, J. (2014). Nutrients, phytochemicals and antioxidant activity in wild populations of Allium ampeloprasum L., a valuable underutilized vegetable. Food Research International, 62, 272–279. Gilhotra, N., & Dhingra, D. (2016). GABAergic and nitriergic influence in antianxiety-like activity of garlic in mice. Journal of Applied Pharmaceutical Science, 6(4), 77–85. Jaffal, S., & Abbas, M. (2018). Analgesic action of Achillea biebersteinii methanolic flower extract is mediated by interaction with cholinergic receptor in mouse pain models. Inflammopharmacology (in press). Janapati, Y. K. (2012). Sedative and hypnotic activity of bulbs of Allium cepa LINN. International Journal of Pharmaceutical Sciences and Research, 4(12), 4650–4655. Janssen, C. F., Maiello, P., Wright, M. J., Jr., Kracinovsky, K. B., & Newsome, J. T. (2017). Comparison of atipamezole with Yohimbine for antagonism of xylazine in mice anesthetized with ketamine and xylazine. Journal of the American Association for Lab Animal Sciences, 56(2), 142–147. Jayanthi, M. K., & Jyoti, M. B. (2012). Experimental animal studies on analgesic and antinociceptive activity of Allium sativum (Garlic) powder. The Indian Journal of Research and Reports in Medical Sciences, 2(1), 1–6. Khaksarian, M., Meshkat Alsadat, M. H., Farazifard, R., & Safarpour, F. (2008). A study of chemistry and antinociceptive properties of medicinal plant Allium jesdianum leaves and the probable role of opioidergic system. Yafteh, 9(4), 21–26. Kim, S., Kim, D. B., Jin, W., Park, J., Yoon, W., Lee, Y., ... Yoo, M. (2018). Comparative studies of bioactive organosulphur compounds and antioxidant activities in garlic
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Analgesic effect of Allium ampeloprasum: Evidence for the involvement of beta-adrenergic system
T
Manal A. Abbas Faculty of Pharmacy and Medical Sciences, Al-Ahliyya Amman University, 19328 Amman, Jordan
A R T I C LE I N FO
A B S T R A C T
Keywords: Allium ampeloprasum Analgesic Antinociceptive Beta-adrenergic receptor Antidepressant Anxiolytic
Allium ampeloprasum (leek) is a bulbous perennial edible vegetable closely related to garlic (A. sativum L.). In this study, the antinociceptive, antidepressant and anxiolytic effects of A. ampeloprasum were studied. A. ampeloprasum inhibited abdominal cramps in writhing test and increased latency time in hot-plate and tail-flick tests. In formalin test, A. ampeloprasum inhibited paw licking in both early and late phases. Propranolol, but not naloxone or yohimbine, reversed this effect. A. ampeloprasum decreased the number of lines crossed in open field test but has no effect on the time spent in open arm in elevated plus maze or the immobility time in forced swimming test. GC–MS analysis resulted in the identification of 18 compounds with phytol acetate, linoleic acid and tricosane as major constituents. In conclusion, the analgesic action of A. ampeloprasum was mediated by interaction with β-adrenergic receptor. No antidepressant or anxiolytic effects were exerted by A. ampeloprasum.
1. Introduction Allium genus has over 500 species (Mohammadi, Zarei, Mahmoodi, Zarei, & Nematian, 2015) which differ in maturing, color and taste. However, they are similar in biochemical content (Dey & Khaled, 2015). The presence of organosulfur compounds is responsible for the characteristic taste, aroma, and lachrymatory effects of the Allium species (Najda, Błaszczyk, Winiarczyk, Dyduch, & Tchórzewska, 2016). Allium ampeloprasum (leek) is a bulbous perennial plant belonging to the family Amaryllidaceae (Acharya, Choubey, & Acharya, 2013). The native range of this edible plant is the Mediterranean region (south Europe, northern Africa to west Asia) (Dey & Khaled, 2015). However, it is cultivated in many countries (Rahimi-Madiseh, Heidarian, Kheiri, & Rafieian-Kopaei, 2017). Leek is closely related to garlic (A. sativum L.) (Najda et al., 2016). It is rich in organosulphur compounds especially γglutamyl peptides. Also, it contains alliin and allicin but at lower levels compared to garlic (Kim et al., 2018). In addition to the presence of saponins (Adão, Da Silva, & Parente, 2011), A. ampeloprasum leaves and bulbs contain higher levels of polyphenolics, phenolic acid, flavonoids, and tannins than garlic (Najda et al., 2016). In addition to culinary purposes, A. ampeloprasum is used for several medical purposes (Mohammadi et al., 2015). It has been reported that A. ampeloprasum has antibacterial (Bareemizadeh et al., 2014), anticancer (An, Zhang, Yao, Li, & Ren, 2015), anti-hemorrhoidal (Mosavat, Ghahramani, Sobhani, Haghighi, & Heydari, 2015) and antiulcer effects (Adão et al., 2011). Also, it exerted hypoglycemic, hypolipidemic, and
anti-oxidative effects in alloxan-induced diabetic model in rats (RahimiMadiseh et al., 2017). In addition, steroidal saponins in the bulbs exerted anti-inflammatory effects similar to dexamethasone (Adão et al., 2011). Antinociceptive action was reported in many Allium species like garlic (A. sativum L.) (Dange, Mathew, Datta, Tilak, & Jadhav, 2016), A. hirtifolium (Mohammadi et al., 2015), A. paradoxum (Maghsoodia, Afsharb, Rabania, Mahmoodic, & Ebrahimzadeh, 2018), A. humile (Singh, Gangwar, Singh, Jadon, & Ranjan, 2014), A. stracheyi (Ranjan et al., 2010) and A. jesdianum (Khaksarian, Meshkat Alsadat, Farazifard, & Safarpour, 2008). According to Acharya et al., A. ampeloprasum has the same medicinal virtues as garlic, but in a much milder and less effective form (Acharya et al., 2013). Since garlic has well documented analgesic effects in different animal models of pain, this study was conducted to investigate the antinociceptive action of A. ampeloprasum in mice. In addition, antidepressant and anxiolytic effects were investigated in different behavioral models. 2. Materials and methods 2.1. Plant collection, extraction and chemical identification of constituents A. ampeloprasum was collected from Subaihi, Jordan in May 2015. The plant was authenticated by Prof. Barakat Abu-Irmaileh/ The University of Jordan. A voucher specimen was deposited in Graduate Studies Laboratory at Al-Ahliyya Amman University (Voucher number
E-mail address: [email protected]. https://doi.org/10.1016/j.jff.2019.03.046 Received 6 December 2018; Received in revised form 2 March 2019; Accepted 25 March 2019 1756-4646/ © 2019 Published by Elsevier Ltd.
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2.6. Hot-plate test
Alliaceae#5-2015). Purple inflorescences of A. ampeloprasum growing in the wild were immediately dipped and extracted in acetone for 3 days at room temperature. The choice of acetone as a solvent was due to its water miscibility since flowers were extracted while they were fresh and because it has low boiling point so that the specimen was exposed to temperatures not exceeding 40 ˚C, and because acetone is useful in extracting biologically active compounds like flavonoids (Chebil et al., 2007). After evaporation of the solvent, the extract was stored at −20 °C. A. ampeloprasum extract was dissolved in sterile normal saline immediately before use and administered intraperitoneally (i.p). Gas chromatography–mass spectrometric (GC–MS) analysis was performed utilizing a Varian CP-3800 GC/MS/MS-220 (Saturn, the Netherlands) system, equipped with a DB-5 GC capillary column (95% dimethyl polysiloxane, 5% diphenyl, 30 m × 0.25 mm i.d., 0.25 μm film 1 thicknesses). The analysis method was as in Jaffal and Abbas (2018).
The hot-plate test was used to determine the latencies in pain reaction as in Dange et al. (2016). The temperature of the beaker on a hotplate was adjusted at 55 ± 1 °C. The test was done only once for each mouse. The time between the animal’s placement and first jumping was recorded as an indication for the latency of pain reaction. Latency time was measured after 45 min and 90 min. A cutoff time of 60 sec was determined to avoid tissue damage. 2.7. Paw licking test Paw licking test was carried out by injecting twenty microliters of formalin (2.5%) intraplantarly to the right hind paw of the animal as in Jaffal and Abbas (2018). In all tests, animals were pretreated i.p with vehicle (sterile distilled water, negative control), 150 mg/kg, 300 mg/ kg A. ampeloprasum flower extract or 30 mg/kg diclofenac sodium (positive control) 30 min prior to formalin injection. Antagonists used were naloxone (5 mg/kg, injected 30 min before extract), propranolol (10 mg/kg, given 20 min before extract) and yohimbine (1 mg/kg, given in 15 min before extract). Choice of antagonist dose and administration time was according to previous studies (Afify & Andijani, 2017; Janssen, Maiello, Wright, Kracinovsky, & Newsome, 2017; Saldanha et al., 2017). Nociception was determined by counting the time of licking the injected paw, moving while lifting the leg or exhibiting flinching behavior. Counting was performed during the early phase (the first 5 min) and during the late phase (25–30 min after formalin injection). The percentage inhibition was calculated using this formula:
2.2. Drugs and chemicals Clomipramin hydrochloride was purchased from Novartis, Switzerland. Diazepam was purchased from Medochemie LTD, Cyprus. Naloxone hydrochloride and Yohimbine were purchased from Tocris Bioscience (UK), Diclofenac sodium was from Novartis, Switzerland. Propranolol hydrochloride was from APM, Jordan. All drugs were dissolved in sterile normal saline and administered i.p. 2.3. Experimental animals All experiments were conducted in agreement with the Jordanian Animal Welfare By-Law No. (11) of the year 2010 and approved by the ethical committee at Al-Ahliyya Amman University (ethical approval number AAU-2/4/2018). Male BALB/c mice (weight: 20–25 g) were used in all experiments. Mice were brought from the animal house at Al-Ahliyya Amman University, Jordan. Animals were kept at 23 ± 2 °C with 12 h lightness and 12 h darkness. Water and pellets of food were available ad libitum. Before tests, the animals were acclimatized to the experimental room for at least 2 h. In each of the following experiments, 9 mice per group were used.
average time of paw licking in control − average time of paw licking in extract % inhibition = − treated animals × 100% average number of paw licking in control 2.8. Open field test (OFT) The open field was performed as in Mousavi, Fashi, Jahromy, and Rasooli (2017). Briefly, a 30 × 40 × 20 cm high arena divided into 20 equal squares was used. The ambulatory locomotion of each mouse was recorded for 3 min by video camera. The number of squares crossed by each animal and the number of rearing (i.e. the number of times the animal stood on the hind paws) was counted. A square crossing is considered when the animal totally crosses from one square to the next. The arena was cleaned with ethanol and dried after each experiment. The experimental animals were i.p treated with vehicle, A. ampeloprasum extract (150, and 300 mg/kg) or diazepam (2 mg/kg) 30 min before evaluation.
2.4. Writhing test The writhing test was conducted by injecting acetic acid solution (1%, 10 ml/kg) i.p, 30 min after receiving the vehicle, A. ampeloprasum flower extract (150 mg/kg or 300 mg/kg) or 30 mg/kg diclofenac sodium as in Jaffal and Abbas (2018). Ten minutes after acetic acid injection, the number of writhes was counted for 20 min. A contraction of the abdominal muscles with elongation of the body and extension of the forelimbs is considered a writhe. The percentage inhibition of abdominal cramps was calculated using this formula:
2.9. Elevated plus maze (EPM)
% inhibition
The EPM was performed as in Pitchaiah, Kumar, and Kumar (2015). Briefly, a maze consisted of two open (10 × 5 cm) and two closed (10 × 5) arms, extending from a center platform (5 × 5 cm) and elevated to a height of 50 cm above the floor was used. Mice were individually placed on the center of the maze facing a closed arm, and the time spent in closed and open arms were recorded during a 5 min by video camera. The maze was wiped clean with ethanol and dried after each trial. The experimental animals were i.p treated with vehicle, A. ampeloprasum extract (150, and 300 mg/kg) or diazepam (4 mg/kg) 30 min before evaluation in the maze.
average number of writhes in control =
− average number of writhes in extract − treated animals average number of writhes in control × 100%
2.5. Tail-flick test The tail-flick test was performed as in Dange et al. (2016) by immersing the tail in water at 55 ± 1 °C. Latency time was measured after 45 min, 90 min and 180 min. Different animals were used for measuring tail flick for each time. A cutoff time of 10 sec was determined.
2.10. Forced swim test (FST) FST was performed as in Mousavi et al. (2017) with minor 2
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modifications. The mouse was placed into a glass container (25 cm in height and 10 cm in diameter) containing 19 cm-deep water at 24–25 °C and left there for 5 min. Video recording started two minutes from the placement of mouse in water and lasted for 3 min to record immobility time. The mice were considered immobile when they remained floating in the water, without struggling, making only very slight movements necessary to keep the head above water. Mice were divided into 4 groups: one group (the control group) was treated with normal saline, 2 groups received i.p injection of A. ampeloprasum flower extract (300 or 150 mg/kg) and the forth group received clomipramin hydrochloride (40 mg/kg); used as a reference drug. Treatments were given 30 min before video recording. 2.11. Statistical analysis For all measured parameters, one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test was conducted using GraphPad Prism version 6. 3. Results
Fig. 1. Writhing test. *Significant difference compared with the vehicle-treated control, P < 0.05.
3.1. GC–MS analysis GC–MS analysis of the acetone extract of A. ampeloprasum inflorescence resulted in the identification of 18 compounds. The major constituents were found to be phytol acetate (33.94%), linoleic acid (11.96%) and tricosane (11.40%) (Table 1). 3.2. Writhing test Both doses of A. ampeloprasum (150 and 300 mg/kg) decreased the number of writhings in a significant manner compared to non-treated group (Fig. 1). Abdominal cramps produced by acetic acid were inhibited by 78.6% and 93.5% using 150 mg/kg and 300 mg/kg of A. ampeloprasum flower extract, respectively compared to 43.9% inhibition produced by diclofenac sodium (30 mg/kg). 3.3. Hot-plate test and tail-flick tests A significant increase in latency time in hot-plate was observed after 45 min using 150 and 300 mg/kg A. ampeloprasum flower extract (Fig. 2). In tail-flick test, 300 mg/kg A. ampeloprasum increased latency time after 45 min as well as after 90 min and 180 min (Fig. 3). Propranolol reversed the effect of A. ampeloprasum extract in hot plate test (Fig. 2). In tail flick test, propranolol antagonized the action of A. Table 1 Results of GC–MS analysis of A. ampeloprasum. No
Retention index
Compound
%
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
867 889 904 1057 1164 1211 1344 1346 1364 1567 1833 1878 1960 2041 2100 2133 2218 2300
Allyl propyl sulfide Allyl (E)-1-propenyl sulfide 3,4-Dimethylthiophene Thiophene (2-butyl) Methyl (E)-1-propenyltrisulfide Dimethyltetrasulfide 5-methyl-1,2,3,4-tetrathianene Methyl methylthiomethyl trisulfide Allyl methyl tetrasulfide Ionone < Dimethyl > Cyclopentadecanolide Cubitene Hexadecanoic acid Cyclic octaatomic sulfur Heneicosane < n- > Linoleic acid Phytol acetate < (E)- > Tricosane < n- >
2.88 2.65 4.29 1.41 1.80 1.30 1.82 1.24 2.17 4.05 6.49 3.39 5.40 2.11 1.63 11.96 33.94 11.40
Fig. 2. Hot plate test. * Significant difference compared with the vehicle-treated control, P < 0.05. **Significant difference between A. ampeloprasum (150 mg/ kg) and propranolol with A. ampeloprasum (150 mg/kg), P < 0.05.
ampeloprasum extract partially. However, the effect was not statistically significant (Fig. 3). Diclofenac sodium increased latency in both hotplate and tail-flick tests significantly.
3.4. Paw licking test (Formalin test) Both doses of A. ampeloprasum (300 and 150 mg/kg) inhibited significantly paw licking in the early phase of formalin test (Fig. 4). Percentage inhibition in the early phase was 90.3%, 70.7.8% and 52.7% for high dose (300 mg/kg), low dose (150 mg/kg) of A. ampeloprasum and diclofenac sodium (30 mg/kg), respectively. In late phase, both doses of A. ampeloprasum produced significant inhibition (Fig. 5). The percentage inhibition was 82.9%, 85.5% and 64.0% for high dose of A. ampeloprasum (300 mg/kg), low dose (150 mg/kg) and diclofenac 3
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Fig. 3. Tail flick test. *Significant difference compared with the vehicle-treated control, P < 0.05.
Fig. 5. Formalin test (late phase). *Significant difference compared with the vehicle-treated control, P < 0.05. **Significant difference between A. ampeloprasum (150 mg/kg) and propranolol with A. ampeloprasum (150 mg/kg), P < 0.05.
clomipramin hydrochloride (standard drug) decreased it significantly (Table 2). In OFT, both doses of A. ampeloprasum decreased the number of lines crossed and rearing significantly (Table 2). Similarly, both doses of A. ampeloprasum decreased the total time spent in open arm while diazepam increased it significantly (Table 2).
4. Discussion GC–MS analysis of the acetone extract of A. ampeloprasum inflorescence resulted in the identification of 18 compounds. The major constituents were found to be phytol acetate (33.94%), linoleic acid (11.96%) and tricosane (11.40%). It should be noted that the studied plant in this work was collected from the wild and not cultivated. Similar to our results, García-Herrera et al. (2014) found a predominance of polyunsaturated fatty acids, mainly linoleic acid, in wild leek (A. ampeloprasum). Also according to Morales et al. (2012), linoleic acid constituted 53.45% of the A. ampeloprasum bulbs and bottom of the leaves. Phytol and ethyl linoleate were previously isolated from leek bulbs (Mnayer et al., 2014) and tricosanoic acid was isolated from the bulb and bottom of the leaves (Morales et al., 2012). On the other hand, the essential oil of the aerial parts of A. ampeloprasum L. var, atroviolaceum contained D-limonene (26.9%), beta-pinene (25.3%), 9-octadecanoic acid (17.3%) and hexadecanoic acid (14.9%) as major constituents (Bareemizadeh et al., 2014). The difference in the findings of this study and our study can be explained by the difference in plant part analyzed, differences in geographical region and time of plant collection as well as differences between wild and cultivated plants. In the present study, A. ampeloprasum extract was effective in alleviating pain in chemical and thermal models. Up to our best knowledge, this study represents the first investigation of analgesic effect of A. ampeloprasum flower inflorescence in normal laboratory animals. Both the lower dose of A. ampeloprasum used in this study (150 mg/ kg) and the higher dose (300 mg/kg) were more effective than 30 mg/ kg diclofenac sodium in reducing abdominal cramps. Similar results were obtained by other Allium species like A. stracheyi (Ranjan et al., 2010), A. hirtifolium (Mohammadi et al., 2015), A. paradoxum
Fig. 4. Formalin test (early phase). *Significant difference compared with the vehicle-treated control, P < 0.05. **Significant difference between A. ampeloprasum (150 mg/kg) and propranolol with A. ampeloprasum (150 mg/kg), P < 0.05.
sodium (30 mg/kg), respectively. Involvement of opioid receptor was not evident since the opioid antagonist naloxone didn’t abolish the effect of A. ampeloprasum extract. Similarly, pretreatment with yohimbine, an alpha-adrenergic receptor antagonist didn’t reverse A. ampeloprasum action. On the other hand, propranolol reversed A. ampeloprasum action in both early and late phases (Figs. 4 and 5). 3.5. OFT, EPM and FST In FST, A. ampeloprasum has no effect on immobility time while 4
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Table 2 Results of FST, OFT and EPM (values are represented as mean ± SE). Test
Control (vehicle only) A. ampeloprasum (150 mg/kg) A. ampeloprasum (300 mg/kg) Standard drug◊
OFT
OFT
EPM
FST
Number of lines crossed
Number of rearing
Time spent in open arm (sec)
Immobility time (sec)
66.62 ± 6.71 9.00* ± 2.88 3.37* ± 0.73 99.5* ± 5.98
22.88 ± 2.00 3.83* ± 0.90 2.50* ± 0.64 22.0 ± 1.11
5.57 ± 2.51 0.0 ± 0.00 0.0 ± 0.00 33.2* ± 2.69
161.75 ± 4.02 146.85 ± 9.31 151.14 ± 5.27 109.57* ± 9.91
◊
Clomipramin hydrochloride (40 mg/kg) in FST, diazepam (2 mg/kg) in OFT and diazepam (4 mg/kg) in EPM. * Significant difference compared with the vehicle-treated control, P < 0.05.
could be one of the active constituents mediating the effect of this plant. Also, it is possible that more than one active constituent act synergistically to produce the analgesic effect. In a recent clinical study, A. ampeloprasum cream was applied topically to treat hemorrhoids. A significant decrease was achieved in the grade of bleeding severity and defecation discomfort. However, no significant change was observed in pain scores (Mosavat et al., 2015). This cream contained fresh aqueous liquid obtained from cutting A. ampeloprasum subsp iranicum. Our study demonstrated the effectiveness of flower acetone extract in reducing pain in different pain models. Therefore, the use of A. ampeloprasum flower extract to treat hemorrhoids is suggested to alleviate pain as well as discomfort in patients. In OFT, A. ampeloprasum decreased the number of lines crossed and rearing significantly. This may indicate the presence of sedative action of A. ampeloprasum. Sedative properties have been documented in several Allium species such as onion (A. cepa) (Janapati, 2012) and A. jesdianum extract that decreased the number of lines crossed in OFT (Mousavi et al., 2017). In our investigation, no anxiolytic action of A. ampeloprasum was observed as both doses of A. ampeloprasum had no effect on the total time spent in open arm in EPM while diazepam increased it significantly. This indicates that A. ampeloprasum differs from onion (A. cepa) (Pitchaiah et al., 2015) and garlic (Gilhotra & Dhingra, 2016) that exerted anxiolytic activity in EPM model. Unlike garlic (A. sativum), A. jesdianum, and onion (A. cepa), A. ampeloprasum exerted no antidepressant-like activities in FST (Dhingra & Kumar, 2008; Mousavi et al., 2017; Sakakibara, Yoshino, Kawai, & Terao, 2008). The Forced swimming test is the most widely used model for assessing antidepressant activity. Drugs with antidepressant activity reduce that parameter (immobility time), while depressant drugs exert the opposite effect (Mousavi et al., 2017).
(Maghsoodia et al., 2018) and A. humile (Singh et al., 2014) in acetic acid-induce writhing model. Also, A. sativum (garlic) shoot extract reduced writhing induced by 4% NaCl (Dange et al., 2016). In thermal pain models, A. ampeloprasum increased the latency time in hot-plate and tail-flick tests. Similarly, garlic shoot extract exerted antinociceptive action in tail flick and hot plate models of experimental pain (Dange et al., 2016). Also, A. jesdianum leaf-extract increased tail flick and hot plate latencies (Khaksarian et al., 2008). In Hot plate test, A. paradoxum extract increased pain threshold compared to control specifically in 30th minute of the test (Maghsoodia et al., 2018). Also, A. hirtifolium had antinociceptive effect in tail-flick test (Mohammadi et al., 2015). In formalin test, both doses of A. ampeloprasum flower extract inhibited significantly paw licking in the early and late phases. Interestingly, A. sativum bulb powder completely abolished the early phase using 300 mg/kg dose and was also effective in the late phase (Jayanthi & Jyoti, 2012). In our study, the same dose of A. ampeloprasum extract produced 90.3% and 85.5% inhibition in the early and late phases of formalin test, respectively. Similarly, A. hirtifolium decreased pain score in both phases of formalin test (Mohammadi et al., 2015). On the other hand, treatment of diabetic rats for one month with A. ampeloprasum extract attenuated hyperalgesia in chronic phase of the formalin test only (Roghani & Aghaie, 2007). The presence of phytol in A. ampeloprasum extract may explain, at least partially, its antinociceptive activity since the analgesic effects of phytol were reported earlier (Santos, 2013). Concerning the mechanism of action of A. ampeloprasum extract, the results of our study exclude the possibility of involvement of opioid receptor but suggests an interaction with beta; but not alpha, adrenergic receptors. It is well known that adrenergic systems modulate nociceptive transmission which is mediated through their respective receptors (Pertovaara, 2006). Alpha-adrenergic receptors were implicated in the mediation of noradrenergic analgesia (Carroll, Mackey, & Gaeta, 2007). However, this was not the case in A. ampeloprasum analgesic action since the alpha-adrenergic antagonist yohimbine failed to antagonize it. Up to our best knowledge, no previous report exists concerning the involvement of alpha-adrenergic receptors in A. ampeloprasum pharmacological activities. On the other hand, beta-adrenergic receptors mediated the analgesic effect of A. ampeloprasum in this study and in the action of this plant in decreasing ileum spasms in vitro (Masoumeh, Mosayyeb, Nasri, Hadi, & Mahmoud, 2014). Several lines of evidence exist suggesting that beta-adrenergic agonists exert central effects in mice when given i.p producing analgesic effects in hot plate test (Brochet, Micó, Martin, & Simon, 1986) and abdominal constriction test (Bentley & Starr, 1986). The interaction of A. ampeloprasum with beta adrenergic receptor sub-types remains to be investigated in the future since in vivo and in vitro studies are lacking. Other members of the garlic family like A. neapolitanum and A. subhirsutum interacted with beta-2 receptors in vitro (Nencini, Franchi, & Micheli, 2010). According to Loesberg, Spence, Nijkamp, and Houslay (1994), linoleic acid, one of the major constituents of A. ampeloprasum, induced changes in respiratory beta-adrenergic receptor function. Therefore, it
5. Conclusions Wild A. ampeloprasum inflorescence extract has significant analgesic effects in chemical and thermal acute pain models. Its mechanism of action involves interaction with beta-adrenergic receptor. On the other hand, it is devoid of anxiolytic and antidepressant actions.
Ethical statement All experiments were conducted in agreement with the NIH guiding principles in the care and use of animals and the Jordanian Animal Welfare By-Law No. (11) of the year 2010.
Acknowledgments The authors thank Prof. Barakat E. Abu-Irmaileh at the University of Jordan for plant authentication. This work was published with the support of Al-Ahliyya Amman University. 5
Journal of Functional Foods 57 (2019) 1–6
M.A. Abbas
Conflict of interest
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