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Rev. latinoam. quím vol.38 n.2 Naucalpan de Juárez Aug. 2010
Secondary metabolites and pharmacology of Foeniculum vulgare Mill. Subsp. Piperitum
Mahmoud I. Nassarª*, Elsayed A. Aboutablb, Yousrya A. Makledc, EzzelDIN A. ElKhrisyª, Abeer F. Osmanª
ª Chemistry of Natural Compounds Department, National Research Centre, 12622 Dokki, Cairo, Egypt. *Corresponding author: Tel. 20222337651; Fax: 20233370931. Email: email@example.com
b Pharmacognosy Department, Faculty of Pharmacy, Cairo University, KasrelAini St., 11562 Cairo, Egypt.
c Pharmaceutical and Medicinal Chemistry Department, National Research Centre, 12622 Dokki, Cairo, Egypt.
Received March 2010.
Accepted August 2010.
From hexane extract of Foeniculum vulgare Mill. Subsp. piperitum the fatty acids, hydrocarbons and sterols were identified. The furocoumarins imperatorin, psoralen, bergapten, xanthotoxin and isopimpinellin were isolated from the methylene chloride extract. The flavonoids isorhamnetin 3Oαrhamnoside, quercetin and kaempferol were isolated from the ethyl acetate extract, whereas quercetin 3Orutinoside, kaempferol 3Orutinoside and quercetin 3O βglucoside were isolated from the methanol extract. The crude hexane, methylene chloride, ethyl acetate and methanol extracts of this plant showed antinociceptive and antiinflammatory activity.
Keywords: Foeniculum vulgare Mill. Subsp. piperitum, coumarins, flavonoids, antinociceptive, antiinflammatory.
Se realizó la investigación de la constitución química y la actividad biológica de Foeniculum vulgare Mill. Subsp. Piperitum. En el extracto de nhexano de Foeniculum vulgare Mill. Subsp. Piperitum. se identificaron ácidos grasos, hidrocarburos y esteroles. Las furocumarinas imperatorina, psoraleno, bergapteno, xantotoxina y isopimpinelina se aislaron del extracto de cloruro de metileno. Los flavonoides 3Oαramnosido de isoramnetina, quercetina y kaempferol se aislados del extracto de acetato de etilo, en tanto que la 3Orutinósido de quercetina, el 3Orutinósido de kaempferol y 3Oβglucósido de quercetina se aislaron del extracto metanólico. Los extractos crudos de nhexano, cloruro de metileno,acetato de etilo y metanol presentaron actividad antinociceptiva y antiinflamatoria.
Foeniculum vulgare Mill. subsp. piperitum (Ucria) Coutinho (Fam. Apiaceae) grows wildly in the Mediterranean coastal strip, Egypt (Tackholm, l974). It has been widely used as a folk remedy by the native people for treatment of various inflammatory ailments. Chemically, Foeniculum species are characterized by the presence of essential oils (Ozbek et al.,2003), sterols (Ivanov et al., 1979), coumarins (ElKhrisy et al., 1980; Kwon, et al., 2002) and flavonoids (Kunzemann et al., 1977; Parejo et al., 2004). Certain bioactivities have been attributed to some Foeniculum species; viz, antioxidant and antimicrobial activities for F. vulgare Mill. aerial parts (Ruberto et al., 2000), antiinflammatory and analgesic activities for the fruits of the same plant ( Eun and Jae, 2004). Volatiles reported from the fruits of F. vulgare subsp. piperitum comprise anethol, methyl chavicol, fenchone and limonene (Muckensturm et al., 1997), as well as piperitenone and piperitenone oxide (Badoc et al., 1994). Chlorogenic acid (Ishikawa et al., 1999), caffeic acid and cynarin (Scarpati, 1957) have also been isolated from the plant. The purpose of this study was the isolation and identification of chemical constituents from Foeniculum vulgare Mill. subsp. piperitum growing in Egypt, as well as the evaluation of antinociceptive and antiinflammatory activities of crude extracts of the plant.
The aerial parts and fruits of Foeniculum vulgare Mill. subsp. piperitum were collected from North Western Mediterranean coastal strip near ElSalloum, Egypt in October 2005. The plant material was identified by Prof. Dr. S.A. Kawashty, Phytochemistry and Plant Systematics Department, NRC, Cairo, Egypt. Voucher specimen (F121) is kept in Herbarium, Pharmacognosy Department Faculty of Pharmacy, Cairo University.
EXTRACTION AND ISOLATION
Successive extracts of the airdried powdered aerial parts (1.8 kg) and fruits (150 g) of Foeniculum vulgare Mill. subsp. piperitum were prepared using nhexane, methylene chloride, ethyl acetate and methanol, in succession. nHexane extracts (7 g) from each of the aerial parts and fruits were saponified (ElSaid and Amer,1965) to yield the unsaponifiable matter fractions (USM) (3.5 g and 4 g, respectively), and fatty acids fractions (FA) (2.0 g and 2.5 g, respectively). Methylation of FA was carried out by refluxing in 50 ml absolute methanol and 1.5 ml sulphuric acid for 2 h to give fatty acids methyl esters.
GLC of unsaponfiable matter (USM)
Agilent 6890N gas chromatograph (Hewlett Packard) equipped with FID (Flame Ionization Detector) was used. Column: capillary, 30 m length, 0.53 mm internal diameter, film thickness 0.5 pm, packed with 5% phenyl 95% dimethyl polysiloxane; temperature program: 80ºC, for 1min, increased at a rate of 8ºC/min.; final temperature, 250ºC (kept for 20 min). Injector temperature: 280ºC; detector temperature: 300ºC; carrier gas: nitrogen, flowrate 30 ml/min; H2 flowrate: 30 ml/min.; air flowrate, 300ml/min.
GLC of fatty acids methyl esters (FAME)
Agilent 6890N gas chromatograph (Hewlett Packard) equipped with FID was used. Column: capillary, 30 m length, HPINWAX Polyethylene glycol, 320 pm internal diameter, 0.25 μm film thickness; temperature program: 70ºC for 2min., increased at a rate of 4ºC/min. till 220ºC; carrier gas: nitrogen, flowrate, 30 ml/min.; air flowrate, 230 ml/min.
Isolation of coumarins from methylene chloride extract:
The solventfree methylene chloride extract (greenish brown, 37g) was subjected to silica gel CC (75 x 3cm, 150 g) and eluted with benzene and stepgradient benzene/ethyl acetate; 50 ml fractions being collected, monitored by TLC using solvent system benzene/ethyl acetate (8:2) and similar fractions were pooled. Fractions eluted with benzene afforded compound 1 (20 mg), while those eluted with benzene/ethyl acetate (9.5:0.5) afforded compound 2 (18 mg), Fractions eluted with benzene/ethyl acetate (9:1) gave a mixture (1.5 g) of compounds 3,4 and 5 which was subjected to silica gel CC (30 x 2 cm, 25g), eluted successively with benzene, benzene/ethyl acetate (9.5: 0.5) and benzene/ethyl acetate (9:1); yielding 60 subfractions (each, 20 ml). Subfractions eluted with benzene/ethyl acetate (9.5:0.5) afforded compounds 3 (20 mg), and 4 (22 mg), while those eluted with benzene/ ethyl acetate (9:1) afforded compound 5 (15 mg).
Isolation of flavonoids from ethyl acetate extract
The ethyl acetate extract (24 g) was subjected to polyamide 6S CC (100 x 4 cm, 200 g). Elution with water, followed by methanol (40 %, 60 %, 80 % and 100 %, in succession) yielded 130 fractions (200 ml, each) which were monitored by PC using BAW and 15 % AcOH as developing systems; similar fractions being pooled. Fractions eluted with 80%methanol yielded the isolate 6 (23 mg) which was further purified by Sephadex LH20 CC (30 × 2cm, 40g) using methanol as eluent. Fractions eluted with methanol afforded a mixture of two compounds which were separated and purified by Sephadex LH20 CC (30 × 2cm, 40g) using methanol as eluent, to give the compounds 7 (30 mg) and 8 (28 mg).
Isolation of flavonoids from the methanolic extract
The methanolic extract (77 g) was treated as the ethyl acetate extract. The fractions eluted with 40 % MeOH yielded the isolate 9 (22 mg), those eluted with 60% MeOH afforded the isolate 10 (30 mg), while fractions eluted with 80% MeOH yielded the isolate 11 (20 mg). Each isolate was further purified by Sephadex LH20 CC, using methanol as eluent.
Evaluation of Bioactivities
The powdered airdried aerial parts of F. volgare Sups. piperitum (1.2 kg) was successively extracted with nhexane, methylene chloride, ethyl acetate and methanol to yield 40, 25, 16 and 70 g of extractives, respectively. 1 mg of the individual extracts was equivalent to 30, 48, 75 and 17.1 mg dry plant, respectively. The extracts were suspended in 7% Tween 80 and biologically tested in different doses. All doses were expressed in terms of extract weight/animal body weight (Berhrens and Kerber, 1953).
Animals and Reference drugs
Adult male albino rats weighing (120150 g) and adult albino mice of both sex (2025 g) used in this study, were obtained from the animalbreeding unit of National Research Centre, Cairo. The animals were fed a standard laboratory diet composed of vitamin mix (1 %), mineral mix. (4 %), corn oil (10 %), sucrose (20 %), cellulose (0.2 %), casein (10.5 %) and starch (54.3 %) and allowed free access to water. Animal procedures were in accordance with the recommendations for the proper care and use of laboratory animals. This study was performed according to the international rules and to the guidelines of ethical comity of National Research Centre for experimental animal use. Acetylsalicylic acid (El Nasr Co., Egypt), Ibuprofen (Egyptian International Pharma. Industries Co., Egypt), Carrageenan (BDH, England) and Fluconazole (Pfizer INC., USA) were used in bioactivity testing.
Acute toxicity (LD50)
Acute toxicity (LD50) of each of the extracts from the plant was determined in mice (Finney, 1964). They were subcutaneously (S.C.) administered in doses ranging from 4 to 13 g/kg body weight. Animals were observed and mortality rates were recorded within the first 24 h after administration. Different extracts assayed up to 5.5 g/kg did not show toxicity. LD50 being: 6.75, 11.0, 6.92 and 15 g/kg for nhexane, methylene chloride, ethyl acetate and methanol extracts, respectively.
This activity was investigated using the writhing method in mice (Margarita et al., 1995). The tested extracts were administered in doses ranging 2002000 mg/kg as well as acetylsalicylic acid (200 mg/kg) as a peripheral antinociceptive reference drug (Margarita et al., 1995) and the control vehicle (7% v/v between 80 in normal saline) were S.C. injected to groups of six animals 15 minutes before the intraperitoneal injection of a freshly prepared acetic acid solution (2% w/v in saline, pH 2.7, 10 ml/kg) and transferred immediately to individual observation cages and the assay was observed over a period of 25 min. The number of abdominal writhes was counted and the percentage of protection was expressed as the following:
Where: C = mean writhing of the control group; T = mean writhing of the treated group
Antiinflammatory activity was studied using Carrageenaninduced rat's paw edema (Winter et al., 1963). Groups of 18 hfasted male rats (110130 g, 6 animals each) were orallydosed with either one of the tested extracts from Foeniculum vulgare 1h before induction of Carrageenan foot paw edema by subplanter injection of 0.05 ml of 1% suspension of Carrageenan in saline into the planter tissue of one hind paw. An equal volume of saline was injected into the other hind paw and served as control. Four hours later, the animals were sacrificed and the hind paws were rapidly amputated at the tibiotarsal joint and weighed (Margarita et al., 1995; ElAzzouny et al., 1995). The average weight of edema was estimated for the treated as well as the control group and the percentage inhibition of weight of edema was also evaluated. Ibuprofen (35 mg/kg) (Makhlouf and Maklad, 2004) was employed as a standard.
IDENTIFICATION: SPECTRAL DATA
GLC analysis of FAME (Table 1) revealed 15 compounds in the aerial parts and 14 compounds in the fruits. In the aerial parts and fruits, saturated fatty acids represent 68.43 and 75.03%, respectively; the major being: palmitic, undecanoic and myristic acids, while the unsaturated acids represent 22.01% and 20.32%, respectively; the major being pentadecadienoic and pentadecenoic acids. GLC of USM revealed 27 compounds in the aerial parts, as well as the fruits (Table 2). Hydrocarbons (56.51, 54.17%),, cholesterol (1.71, 0.84%), βsitosterol (5.52, 5.95%), campesterol (3.33, 4.04%) and stigmasterol (14.86, 19.04%) were identified in the aerial parts as well as the fruits, respectively.
Imperatorin (1): UV λmax (MeOH): 262 and 300 nm. EIMS: m/z 270 [M+, base peak, C16H14O4]; m/z 202 [M+ C5H9 ]; m/z 174 [M+ C5H9 CO]; m/z 146 [M+ C5H9COCO]; 1HNMR (CDCl3, 270 MHz): δ 7.76 (H4, d, J = 9.5 Hz), δ 7.73, (H5,s), δ 7.34 (H7, d, J = 2.5 Hz), δ 6.79 (H6,d, J = 2.5 Hz), δ6.35 (H3, d, J = 9.5 Hz), δ5.58 (H, t, J = 6.5 Hz), δ4.99 (two protons for OCH2, d, J = 7.1 Hz), δ1.69 and δ1.71 (singlets for the two methyls).
Psoralen (2): UV λmax (MeOH): 246 and 293 nm. EIMS: m/z 186 [M+, base peak, CHOI which loses three carbon monoxide molecules, successively, to give the fragments m/z: 158, 130 and 102. 1HNMR (CDCl3, 270 MHz): δ7.48 (H4, d, J = 9.5 Hz), δ7.36 (H5, s), δ7.35 (H9, s), δ7.13 (H7,d, J = 2.5 Hz), δ6.50 (H6 ,d, J = 2.5), δ 6.06 (H3,d, J = 9.5 Hz).
Bergapten (3): UV λmax (MeOH): 249, 259, 267 and 310 nm. EIMS: m/z 216 [M+, C12H8O3], m/z 201 [M+CH3 ], followed by four successive expulsions of carbon monoxide to give the fragments m/z: 173, 145, 117 and 89. 1HNMR (CDCl3, 270 MHz): δ8.13 (H4, d, J = 9.7 Hz), δ7.57 (H7, d, J = 2.5Hz), δ7.08 (H9, s), δ7.00 (H6, d, J = 2.5 Hz), δ 6.25 (H3, d, J = 9.7 Hz), δ 4.25 (singlet, 5OCH3).
Xanthotoxin (4): UV λmax (MeOH):240 and 298 nm. EIMS: m/z 216 [M+, C12H8O3], m/z 201 [M+CH3], followed by four successive expulsions of carbon monoxide to give the fragments: m/z 173, 145, 117 and 89. 1HNMR (CDCl3, 270 MHz): δ 7.76 (H4, d, J = 9.5 Hz), δ 7.67 (H7, d, J = 2.1 Hz), δ 7.32 (H5, s), δ6.80 (H6, d, J = 2.1 Hz), δ6.35 (H3, d, J = 9.5 Hz), δ 4.26 (singlet, 9OCH3).
Isopimpinellin (5): UV λmax (MeOH): 248, 267and 309 nm. EIMS: m/z 246 [M+, C13H10O5], m/z 231 [M+CH3], followed by two successive expulsions of carbon monoxide to give the fragments m/z 203 and 175, and loss of methyl group to give the fragment m/z 160. 1HNMR (CDCl3, 300 MHz): δ8.14 (H4, d, J = 9.5 Hz), δ7.64 (H7, d, J = 2.5 Hz), δ7.06 (H6, d, J = 2.5 Hz), δ6.36 (H3, d, J = 9.5 Hz) and two singlets at δ 4.13 and 4.09 for 5 and 9OCH3.
Isorhamnetin 3OαLrhamnoside (6):
EIMS: m/z 316 [Aglycone+H]+, +ve ESIMS: m/z 462 [M+H+, C22H22O11]. 1HNMR (DMSOd6, 270 MHz): δ12.63 (5OH,s), δ7.27 (H2', d, metacoupling with H6', J = 2.0Hz), δ7.25 (H6' d, J = 8.8 Hz), δ 6.85 (H5\ d, J = 8.3 Hz), δ 6.32 (H8, d, J = 2.0 Hz), δ 6.13 (H6, d, J = 2.0 Hz ), δ5.24 (broad singlet for anomeric proton of rhamnose), δ0.95 (CH3 methyl rhamnosyl protons, d, J = 5.3 Hz), δ3.95 (OCH3 at position 3', sharp singlet) (Chang et al., 1998).
Quercetin (7): UV spectral data suggested a flavonol type with free hydroxyl groups at positions 3, 5, 7, 3' and 4'. EIMS: m/z 303 [M+1]+, C15H10O7.
Kaempferol (8): UV spectral data suggested a flavonol type with free hydroxyl groups at positions 3, 5, 7 and 4'. EIMS: m/z 285 [M1] , C15H10O6.
Quercetin 3O rutinoside (9): 1HNMR: δ7.55 (H2' and H6' overlapping protons, d, J = 7.6 Hz), δ 6.85 (H5', d, J = 8.0 Hz), δ 6.38 (H8 , d, J = 1.8 Hz), δ 6.19 (H6, d, J = 1.8 Hz), ,δ5.32 (anomeric proton of glucose, J = 8.1 Hz), δ 4.37 (anomeric proton of rhamnose, broad singlet), δ0.99 (CH3rhamnosyl, d, J = 5.4 Hz).
Kaempferol 3O rutinoside (10): UV spectra with shift reagents indicated a flavonol type with free hydroxyl groups at positions 5, 7 and 4', while that at position 3 being substituted (Mabry et al., 1970). Complete acid hydrolysis afforded glucose, rhamnose and kaempferol, identified by CoPC in comparison with authentic samples. 1HNMR (DMSOd6, 270 MHz): δ7.61 (H2'and H6', d, J = 8.5 Hz) δ 6.77 (H3' and H5', d, J = 8.5 Hz) , δ6.20 (H8 , d, J = 1.8 Hz) , δ6.0 (H6, d, J = 1.8 Hz), δ 5.21 (anomeric proton of glucose, d, J = 7.6 Hz), δ 4.40 (anomeric proton of rhamnose, broad singlet) δ 1.04 (methyl rhamnosyl protons, d, J = 6.1 Hz). ESIMS: m/z 593 [M1]+, C21H20O12.
Quercetin 3Oβ glucoside (11): UV spectral data suggested a flavonol type with free hydroxyl groups at positions 5, 7, 3' and 4', while being substituted at position 3. Complete acid hydrolysis afforded glucose. 1HNMR: δ 7.66 (H6', d, J = 8.6 Hz), δ 7.55 (H2', d, J =2.0 Hz) , δ 6.84 (H5', d, J = 6.6 Hz), δ 6.38 (H8, d, J = 1.8 Hz), δ 6.17 (H6, d, J = 1.8 Hz), δ5.34 (anomeric proton of glucose, d, J = 7.5 Hz). ESIMS: m/z 464 [M+1] +, C21H20O12.
The methanolic extract of the aerial parts of Foeniculum vulgare subsp. piperitum exhibited the highest antinociceptive activity at a dose level of 2000 mg/kg (Table 3), while the activity exhibited by the ethyl acetate extract was at (800 mg/kg). On the other hand, nhexane extract (700 mg/kg) and methylene chloride extract (500 mg/kg) exhibited similar antinociceptive activities, being less than that of acetylsalicylic acid (200 mg/kg). Data presented in Table 4 revealed that the extracts under investigation exhibited significant antiinflammatory activity. The methanolic extract possessed the highest activity, where it significantly decreased the weight of edema induced by carrageenan in the rat paw at dose levels of 1500 and 2000 mg/kg, it exerted a protective effect of 28 and 47%, respectively, compared to the control value, while ibuprofen (35 mg/kg), used as a reference drug, exhibited a protective effect of 52.23 %. The present study showed that the four extracts of the aerial parts of the plant exhibited antinociceptive activity. Moreover, the potent antinociceptive activity expressed by the methanolic (2000 mg/kg) and ethyl acetate (800 mg/kg) extracts can be attributed to the content of phenolic constituents (Paulino et al., 2003). Also, the four extracts under investigation exhibited a significant antiinflammatory activity. The highest antiinflammatory activity of methanolic extract (2000 mg/kg) of the aerial parts of the plant can possibly be attributed to the presence of phenolic content. In conclusion, traditional application of F. vulgare Mill subsp. piperitum could be beneficial in the management of different inflammatory disease cases. Our current findings suggested that F. vulgare Mill subsp. piperitum contains a high amount of flavonoids, which exhibited a strong potency towards suppressing inflammation, as evidenced by our in vivo models.
Foeniculum vulgare Mill. Subsp. Piperitum to contain fatty acids, hydrocarbons and sterols in the nhexane extract. Furocoumarins; imperatorin, psoralen, bergapten, xanthotoxin and isopimpinellin were isolated from the methylene chloride extract. Flavonoids; isorhamnetin 3Oαrhamnoside, quercetin and kaempferol were isolated from the ethyl acetate extract, whereas quercetin 3Orutinoside, kaempferol 3Orutinoside and quercetin 3O βglucoside were isolated from the methanol extract. The crude extracts exhibited significant antinociceptive and antiinflammatory activity.
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