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Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
New quinoline alkaloid from Ruta graveolens aerial parts and evaluation of the antifertility activity a
a
b
Josline Y. Salib , Sayed A. El-Toumy , Emad M. Hassan , Nabila H. a
c
Shafik , Sally M. Abdel-Latif & Ignacio Brouard
d
a
Chemistry of Tannins Department, National Research Centre, Dokki, Cairo, Egypt b
Medicinal and Aromatic Plants Research Department, National Research Centre, Dokki, Cairo, Egypt c
Pharmacognosy Department, Faculty of Pharmacy, Cairo University, Cairo, Egypt d
Instituto de Productos Naturales y Agrobiología, Av.Astrofisico F. Sanchez 3, 38206 La Laguna, Tenerife, Spain Published online: 04 Apr 2014.
To cite this article: Josline Y. Salib, Sayed A. El-Toumy, Emad M. Hassan, Nabila H. Shafik, Sally M. Abdel-Latif & Ignacio Brouard (2014): New quinoline alkaloid from Ruta graveolens aerial parts and evaluation of the antifertility activity, Natural Product Research: Formerly Natural Product Letters, DOI: 10.1080/14786419.2014.903395 To link to this article: http://dx.doi.org/10.1080/14786419.2014.903395
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Natural Product Research, 2014 http://dx.doi.org/10.1080/14786419.2014.903395
New quinoline alkaloid from Ruta graveolens aerial parts and evaluation of the antifertility activity Josline Y. Saliba*, Sayed A. El-Toumya, Emad M. Hassanb, Nabila H. Shafika, Sally M. Abdel-Latifc and Ignacio Brouardd
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a
Chemistry of Tannins Department, National Research Centre, Dokki, Cairo, Egypt; bMedicinal and Aromatic Plants Research Department, National Research Centre, Dokki, Cairo, Egypt; cPharmacognosy Department, Faculty of Pharmacy, Cairo University, Cairo, Egypt; dInstituto de Productos Naturales y Agrobiologı´a, Av.Astrofisico F. Sanchez 3, 38206 La Laguna, Tenerife, Spain (Received 11 November 2013; final version received 8 March 2014) Bioassay-guided isolation of methanol extract of Ruta graveolens L. leaves yielded a new quinoline alkaloid, (4S) 1,4-dihydro-4-methoxy-1,4-dimethyl-3-(3-methylbut-2enyl)quinoline 2,7-diol, and nine phenolic compounds including rutin as a major compound. Structures of the isolated compounds were determined by using chromatography, UV, HR-ESI-MS and 1D/2D 1H/13C NMR spectroscopy. The uterotonic activity of methanol extract fractions (ethyl acetate, n-butanol and aqueous fraction) as well as the isolated major compounds was tested in the isolated mouse uterus in vitro. The n-butanol-soluble fraction was found to demonstrate the most potent uterotonic activity in a dose-dependent manner, also the major isolated compound rutin revealed the occurrence of an uterotonic response, which was maximum at a concentration level of 0.25 mg/mL, accounting for 68.7% of that exhibited by the chosen concentration of oxytocin. Keywords: Ruta graveolens L.; polyphenolic metabolites; uterotonic activity
1. Introduction Despite many achievements in human health care in the twentieth century, many of the world’s population in developing countries lack regular access to affordable essential drugs. In contrast, traditional medicine is widely available and affordable, even in remote areas. Growth of human population has been underway for thousands of years and was never a problem until recently. Currently, overpopulation has led to series social and environmental problems such as poverty, overcrowded slums, crime, pollution of air and water and depletion of the protective ozone layer (Greep 1998). Family planning has been promoted through several methods of contraception, but due to serious adverse effects produced by synthetic steroidal contraceptives, attention has now been given to indigenous plants for possible contraceptive effects (Ghosh & Bhattacharya 2004) whereby several active chemical constituents accountable for uterotonic effects are discovered in various plant species from time to time. Ruta graveolens L. (Rutaceae) or common rue, native of the Mediterranean region but cultivated throughout Europe and many Asian countries, is an evergreen shrub with bluish-green leaves that emit a powerful odour and have a bitter taste. Ruta is cited in the ancient herbals and has deep roots in folklore. Ruta has a long history of use as a domestic remedy, being especially valued for its strengthening action on the eyes. Ruta contains flavonoids (notably rutin) that
*Corresponding author. Email: [email protected] q 2014 Taylor & Francis
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reduce capillary fragility, which might explain the plant’s reputation as an eye strengthener (Bown 1995). The whole herb is abortifacient, anthelmintic, antidote, antispasmodic, carminative, emetic, brings relief from giddiness and nervous headaches, strongly stimulant and mildly stomachic (Aliotta et al. 1994; Sallal & Alkafahi 1996; Aliotta et al. 2000; De Feo et al. 2002; Oliva et al. 2003). In Germany, extracts of R. graveolens are marketed for relief of cramps and rheumatism (Kutchan 1998). Photo-chemotherapy with a constituent from R. graveolens has been used to treat cutaneous T-cell lymphoma (Gasparro 1994). In Taiwan, the fresh aerial part of this plant is widely used to treat palpitation of the heart and circulatory disorders (Kong et al. 1989). Quinoline alkaloids (Paulini, Popp et al. 1991; Paulini, Waibel et al. 1991), coumarins (Rosza et al. 1989; Kostova et al. 1999; Kuzovkina et al. 2004), lignans (Reisch et al. 1970) and flavonoids (Bautz et al. 1989; Ulubelen et al. 1994; Hashemi et al. 2011) have been isolated from R. graveolens and exhibit a variety of pharmacological activities. This study deals with bioassay-guided fractionation using ethyl acetate, n-butanol and water of the methanol extract of Egyptian R. graveolens aerial parts. The n-butanol fraction demonstrated the most potent uterotonic activity in a dose-dependent manner (responses accounting for 13.76%, 48.62% and 67.58% of that exhibited by the chosen concentration of oxytocin for concentration levels of 0.25, 0.375 and 0.5 mg/mL, respectively). Fractionation of the n-butanol fraction resulted in the isolation and identification of seven polyphenolic compounds namely, quercetin-3-O-b-D glucopyranoside, quercetin-3-O-a-L -rhamnopyranoside, kaempferol-3-O-b-D -glucopyranoside, rutin, isorhamnetin, quercetin and kaempferol, with the new natural quinoline alkaloid, (4S) 1,4-dihydro-4-methoxy-1,4-dimethyl-3-(3-methylbut-2-enyl)quinoline-2,7-diol, as well as two phenolic acids caffeic and ferulic acids. 2. Results and discussion Fractionation of the n-butanol extract resulted in the isolation and identification of 10 compounds 1 – 10. The structure of the isolated compounds was determined using chromatography as well as conventional chemical and spectroscopic methods of analysis (UV, HR-ESI-MS and 1D/2D 1H/13C NMR spectroscopy). Compound 1, obtained as a yellow amorphous powder exhibited chromatographic properties (yellow spot on paper chromatogram under UV light). The presence of quinoline nucleus was indicated by its UV spectrum. It also yielded orange colour with Dragendorff reagent for an alkaloid. Its molecular formula was established as C17H23NO3 from its HR-ESI-MS at m/z 290.1363 [M þ H]þ. Its 1H NMR spectrum revealed five singlets for methyl groups each at d 1.19, 1.21, 1.48, 3.92 and 4.42, among which the presence of a methoxyl O – CH3 (d H 4.42) and – N –CH3 (d H 3.92) were confirmed by its DEPT and 1H – 13C COSY spectra. 1H NMR of 1 also displayed a doublet at d 7.75 (d,1H), a double of doublet at d 7.38 (dd, 1H) and another doublet at 7.28 (d,1H) indicating the presence of 1, 2, 4 system which has been confirmed by its coupling constants, 1H – 1H COSY and 1H – 13C COSY spectra. This suggested that one of the (OH) groups must be present on aromatic nucleus. Its HMBC correlation studies confirmed that the methyls at d 1.19 and 1.21 were gem dimethyls and attached to carbon at d 128.39 as revealed by 1H – 13C correlation spectrum. This particular carbon at d 128.39 was also attached to double bond (CvC) and displayed correlation with the carbon that appeared at d 93.78 whose 1 H NMR signals displayed a triplet at d 5.51 (1H) suggesting that both these units were linked together and to a methylene carbon which yielded a doublet signal at d 3.89 (2H). This confirmed the presence of 3-methyl but-2-enyl group. The HMBC correlation of CH3 group at dH 3.92 with carbons at dC 159.37 and dC 163.98 clearly indicated the N –CH3 group and further confirming the presence of quinoline nucleus. The presence of a methoxy group and a methyl group at C-4 was confirmed through the observed HMBC correlations which revealed the correlation of OCH3 group at (dH 4.42) with C-4 at (dC 70.37) and CH3 at (dH 1.48) with C-4 at (dC 70.37). The
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positive Cotton effect observable in the circular dichroism (CD) spectrum indicated that the absolute configuration at the 4-position of compound 1 was S. Thus, on the basis of the abovementioned discussion, 1 was assigned the constitution as (4S) 1,4-dihydro-4-methoxy-1,4dimethyl-3-(3-methylbut-2-enyl)quinoline-2,7-diol, which is a new natural product (Figure 1). The other nine known compounds (2 –10) were identified as quercetin-3-O-b-D -glucopyranoside (2), quercetin-3-O-a-L -rhamnopyranoside (3), kaempferol-3-O-b-D -glucopyranoside (4), rutin (5), isorhamnetin (6), quercetin (7), kaempferol (8) and the two phenolic acids, caffeic (9) and ferulic acids (10), by comparing their 1D and 2D NMR spectral data with the reported data in the literature (Agrawal 1989). Control of fertility using traditional antifertility plants has been practiced for many years in Africa. R. graveolens is one of the many plants used for fertility regulation all over the world. That is why this study revealed the in vitro uterotonic effect which was very evident of R. graveolens whose extracts exhibited such activity can be one of the factors rationalising the abortifacient effect of the crude drug reported in the literature. Results of preliminary trials for the tested extract in the concentration range 0.0125 – 1.25 mg/mL against the chosen concentration of oxytocin revealed an uterotonic responses exhibited by methanol extract. Different concentrations of the methanolic extract of R. graveolens were tested (Table 1). Thus, the methanolic extract exhibited a high and dose-related response, denoting highly effective ingredient(s) responsible for this type of action (Mitchell & Seeman 1998). The n-butanol-soluble fraction exhibited the strongest uterotonic activity doubling and significantly different from that of the water-soluble fraction and still displaying a dose-related response as the original methanolic extract, while the ethyl acetate-soluble fraction was found to be inactive and the results are shown in Table 2 and Figure 2. According to these results, it was suggested that the biological activity of R. graveolens methanolic extract was due to the compounds present in the n-butanol-soluble fraction and this activity may be enhanced by other compounds present in the water-soluble fraction.
H3CO 6
5
4
4a
5` CH3
CH3 3
2` HO
8a 8
1 N
3`
CH3
CH3
H3CO
1` CH3 4`
OH
CH3 HO
CH3
N
OH
CH3
Figure 1. Compound 1 and its HMBC correlation. Table 1. The uterotonic responses exhibited by different concentrations of the methanolic extract of R. graveolens. Tested sample Oxytocin Methanolic extracta
a b c
Concentration (mg/mL)
Mean response (cm) ^ s
Uterotonic activity (%)
3.75 £ 1026 0.5 0.375 0.25
7.17 ^ 0.907 5.47 ^ 0.850b 3.97 ^ 0.153b,c 2.83 ^ 0.058b,c
100 76.29 55.37 39.47
Dose-related response. Significantly different from control (oxytocin) at p # 0.05. Significantly different from the mean response caused by larger concentration(s) of the same extract at p # 0.05.
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Table 2. Comparison between the uterotonic effects exhibited by different concentrations of the n-butanolsoluble fraction. Tested sample
Concentration (mg/mL)
Oxytocin Butanol-soluble fractiona
Mean response (cm) ^ s
Uterotonic activity (%)
6.54 ^ 3.01 4.42 ^ 1.11 3.18 ^ 0.295b,c 0.9 ^ 0.534b,c
100 67.58 48.62 13.76
26
3.75 £ 10 0.5 0.375 0.25
a
Dose-related response. Significantly different from control (oxytocin) at p # 0.05. c Significantly different from the mean response caused by larger concentration(s) of the same fraction at p # 0.05. b
10 Mean response (cm)
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8 6 4 2 Soluble
0 Oxytocin
IIID2
IIID3
Tested sample
Figure 2. Uterotonic responses exhibited by the butanol and water-soluble fractions at 0.5 mg/mL concentration level against the chosen concentration of oxytocin.
After performing the biological study of the different extracts and fractions, we decide to study the n-butanol fraction phytochemically, since they proved to be the most biologically active fraction in an attempt to isolate the biologically active ingredients present. Rutin was found to be the major compound and revealed the occurrence of an uterotonic response (followed by self-inhibition) whose magnitude was closely related to concentration (Table 3), while the comparison between the uterotonic effects exhibited by rutin and related compounds at the same concentration of oxytocin is shown (Figure 3). Table 3. Comparison between the uterotonic effects exhibited by different concentrations of rutin. Drug Oxytocin Rutin
a b
Concentration (mg/mL) 26
3.75 £ 10 0.025 0.125 0.25 0.375 0.5
Mean response (cm)
Uterotonic activity (%)
9.17 ^ 1.6 1.3 ^ 0.529a,b 4.47 ^ 1.53a 6.3 ^ 1.71 4.53 ^ 0.907a 3.37 ^ 0.666a,b
100 14.18 48.75 68.7 49.4 36.75
Significantly different from control (oxytocin) at p # 0.05. Significantly different from the mean response caused by 0.25 mg/mL rutin at p # 0.05.
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Mean resones (cm)
10
8 6 4
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2
0 Oxytocin
Rutin
Quercetin
Quercitrin
Isoquercitrin
Tested compound
Figure 3. Uterotonic responses exhibited by rutin and related compounds at 0.25 mg/mL concentration level against the chosen concentration of oxytocin.
Reviewing the current literature, there is only one old report concerning the adrenaline like action of rutin on the smooth muscle of rabbit uterus (in situ) and intestine (Baraboi & Moldavskaya 1966). According to Susun Weed (1986), rutin can be used as a form of contraception or emergency contraception when taken as a tablet in doses of at least 500 mg daily for several days proceeding and following ovulation or after fertilising intercourse and continuing until the menstrual flow begins. It can be concluded from (Table 3) that maximum uterotonic activity of rutin is exhibited at 0.25 mg/mL concentration level when there is no significant difference between the uterotonic response caused by rutin and that caused by the chosen concentration of oxytocin. The diminution of activity with higher concentrations of the compound can be easily attributed to that the stimulation of uterine smooth muscle is always followed by inhibition and the ratio of stimulation to inhibition responses is related to concentration. This can also rationalise the conflict about its use during pregnancy, as the effect appears to be dose-dependent. 3. Experimental 3.1. Plant materials Leaves of R. graveolens were collected from the field (Experimental and Research Station of the Faculty of Pharmacy, Cairo University, Cairo, Egypt) in spring 2010 and dried under shade. The plants were authenticated by Dr Tarek Abdel-Salam, Professor of Botany, Department of Botany, Faculty of Science, Cairo University, Cairo, Egypt, and voucher specimens (No. 20 R) have been deposited in the herbarium, National Research Center, Cairo, Egypt. 3.2. General 1
H NMR and 13C NMR spectra were obtained on Bruker AMX-400, Avance 400, and Avance 300 spectrometers (Bruker, Rheinstetten, Germany) with standard pulse sequences operating at 400, 300 MHz in 1H NMR and 100, 75 MHz in 13C NMR. Chemical shifts are given in d values (ppm) using tetramethylsilane as the internal standard. UV spectra were recorded with Shimadzu UV-1601
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(Shimadzu, Tokyo, Japan). HR-ESI-MS was measured on a Micromass Autospec (70 eV) spectrometer (Micromass, Manchester, UK). CD spectra were obtained with a JASCO J-720 spectropolarimeter (JASCO, Tokyo, Japan). Column chromatography (CC) was carried out on Polyamid 6S and Sephadex LH20; CC: Polyamide 6S, (Riedel-De Haen, Hannover, Germany) and Sephadex LH-20 (Fluka Pharmazia, Uppsala, Sweden); PC was carried out on Whatman No. 1 and 3 mm paper using the following solvent systems: (1) BAW (n- BuOH/HOAc/H2O, 4:1:5); (2) H2O and (3) AcOH/H2O (15:85). De Jalon’s solution (for isolated uterus), Tween 80 (Fluka), oestradiol benzoate (Folone ampoules, Misr Company for Pharmaceutical Industries, SAE, Cairo, Egypt) and oxytocin (Syntocinon ampoules, Novartis Pharma, SAE, UK) were used.
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3.3. Extraction, fractionation and isolation Five hundred grams of the plant were reduced to a coarse powder extracted by methanol in a Soxhlet extractor at room temperature till exhaustion. The extract was dried under vacuum at 408C to yield 100 g of the methanol extracts of R. graveolens leaves. Eighty grams of the methanol extract were suspended in 500 mL distilled water and successively extracted in a separating funnel with ethyl acetate and n-butanol at room temperature till exhaustion leaving a residual water-soluble fraction. The three fractions were dried under vacuum at 408C and their net weights were calculated. Percentage yields (w/w) of the ethyl acetate, n-butanol and water-soluble fractions of R. graveolens methanolic extract were 9.86, 25.43 and 35.14, respectively. The n-butanol extract (15 g) was chromatographed on a Sephadex LH-20 column with water and water/ethanol mixtures as eluent to yield seven fractions. Fractions were further separated and purified using Whatman 3 mm paper and Sephadex LH-20 column to yield the pure compounds (4S) 1,4-dihydro-4-methoxy-1,4-dimethyl-3-(3-methylbut-2-enyl)quinoline-2,7diol (1), quercetin-3-O-b-D -glucopyranoside (2), quercetin-3-O-a-L -rhamnopyranoside (3), kaempferol-3-O-b-D -glucopyranoside (4), rutin (5), isorhamnetin (6), quercetin (7), kaempferol (8) and the two phenolic acids, caffeic (9) and ferulic acids (10). 3.3.1. (4S) 1,4-dihydro-4-methoxy-1,4-dimethyl-3-(3-methylbut-2-enyl)quinoline-2,7-diol (1) Rf-values £ 100: 78 (BAW), 80 (15% AcOH). UV lmax nm (MeOH): 332, 283, 256, 237 and 232 nm. HR-ESI-MS at m/z: 290.1363 [M þ H]þ. 1H NMR: d 7.75 (d, J ¼ 7 Hz, H-5),7.38 (dd, J ¼ 2 and 7 Hz, H-6), 7.28 (d, J ¼ 2 Hz, H-8), 5.51 (t, J ¼ 5.79 Hz, H-20 ), 4.42 (s, O – CH3), 3.92 (s, N –CH3), 3.89 (d, J ¼ 4.38, H-10 ), 1.48 (s, 4-CH3), 1.21 (s, H-40 ), 1.19 (s, H-50 ). 13C NMR: 163.98 (C-2), 103.61 (C-3), 70.37 (C-4),120.76 (C-4a), 124.43 (C-5), 106.72 (C-6), 160.87 (C7), 118.22 (C-8), 159.37 (C-8a), 28.81 (C-10 ), 93.78 (C-20 ), 128.39 (C-30 ), 25.91 (C-40 ), 21.77 (C50 ), 34.14 (N– CH3), 59.74 (O – CH3), 25.42 (4-CH3). 3.4. Animals Healthy adult virgin female albino rats of Sprague-Dawely strain weighing 140– 170 g were supplied by the Department of Hygiene and Animal Management, Faculty of Veterinary Medicine, Cairo University, Giza, Egypt. The animals were kept on standard laboratory diet and under hygienic conditions. 3.5. Uterotonic activity bioassay 3.5.1. Preparation of uterine tissues Virgin female albino mice were used for testing uteri-contracting (uterotonic) activity of the different fractions of the plant. The mice were injected intramuscularly with 0.1 mg/kg of
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oestradiol benzoate 24 h prior to the experiment, were sacrificed by cervical dislocation and the uterine horns were removed and placed in a dish containing physiological De Jalon’s solution with the following composition (g/L): NaCl, 9 g; KCl, 0.42 g; NaHCO3, 0.5 g; D -glucose, 0.5 g; CaCl2, 0.062 g (378C) connected to a transducer and the uterine response was left to stabilise for 20 min.
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3.5.2. Dose– response study In order to assess the uterotonic activity, 2 g of the dried extract were dissolved in De Jalon’s solution to produce 10% solution. The extract was preliminarily tested in the concentration range 0.0125 –1.25 mg/mL against 3.75 £ 1026 mg/mL [0.001875 IU/mL] of oxytocin as a standard (optimum concentration chosen after several trials) and the three most suitable concentrations were chosen for calculations. Before every test, the bath solution was drained completely and washed two to three times and filled with fresh solution. For calculations, the three chosen concentrations of the extract were tested in six different uterine horns against the chosen concentration of oxytocin and the mean response corresponding to each concentration was calculated and compared with the mean response corresponding to the used concentration of oxytocin. The method used was a modification of that adapted by Desta (1994). The data were subjected to statistical analysis using Student’s unpaired test (Armitage & Berry 1987). Two grams of each of the butanol- and water-soluble fractions were dissolved in 20 mL De Jalon’s solution while, in case of the ethyl acetate-soluble fraction, 2 g were dissolved in 20 mL 15% solution of Tween-80 (in De Jalon’s solution) to produce 10% solutions. The uterotonic activity of each fraction was assessed following the same method adopted earlier for testing the activity of the methanolic extract and using the same three concentrations chosen before for the methanolic extract against the chosen concentration of oxytocin as a standard for calculations. Rutin and other isolated compounds were tested for uterotonic activity following the same method adopted earlier for the methanolic extract and its butanol-soluble fraction at 0.025, 0.125, 0.25, 0.375 and 0.5 mg/mL concentration levels against the chosen concentration of oxytocin as a standard.
4. Conclusion In addition to the fact that the Egyptian variety of R. graveolens has been proved to possess an antifertility activity like their corresponding varieties of other geographical regions, it is the first time to relate the mechanism of action to specific compound isolated from crude extract. Thus, we recommend that the n-butanol fraction of R. graveolens together with its main active ingredient to be subjected to extensive clinical studies in order to be able to introduce them to the society as antifertility drugs that could meet the needs of many partners interested in family planning but searching for an effective, safe, cheap and available birth-control method.
Supplementary material Supplementary material relating to this article is available online, alongside Figures S1 – S5.
References Agrawal PK. 1989. Carbone-13 NMR of flavonoids. Amsterdam: Elsevier. Aliotta G, Cafiero G, De Feo V, Di Blasio B, Iacovino R, Oliva A. 2000. Allelochemicals from rue (Ruta graveolens L.) and olive (Olea europaea L.) oil mill waste waters as potential natural pesticides. Curr Top Phytochem. 3:167–177.
Downloaded by [University of Waikato] at 07:01 04 July 2014
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Aliotta G, Cafiero G, De Feo V, Sacchi R. 1994. Potential allelochemicals from Ruta graveolens L. and their action on radish seeds. J Chem Ecol. 20:2761–2775. Armitage P, Berry G. 1987. Statistical methods in medical research. Oxford: Blackwell. Baraboi VA, Moldavskaya SI. 1966. Pharmacology of some polyphenols possessing vitamin P activity. Vrachebnoe Delo. 1:71–73. Bautz C, Bohuslavizki KH, Hansel W, Koppenhofer E. 1989. Effects of Ruta on the excitation process in myelinated nerves. Plant Med. 55:649. Bown D. 1995. Encyclopaedia of herbs and their uses. New York: DK. De Feo V, De Simone F, Senatore F. 2002. Potential allelochemicals from the essential oil of Ruta graveolens. Phytochemistry. 61:573–578. Desta B. 1994. Ethiopian traditional herbal drugs. Part III: antifertility activity of 70 medicinal plants. J Ethnopharmacol. 44:199–209. Gasparro FP. 1994. Extracorporeal photochemotherapy: clinical aspects and the molecular basis for efficacy. Boca Raton, FL: CRC Press. Ghosh K, Bhattacharya TK. 2004. Preliminary study on the antiimplantation activity of compounds from Thespesia populnea. Indian J Pharmacol. 36:288–290. Greep RO. 1998. Whether the global population problem. Biochem Pharmacol. 55:385–386. Hashemi M, Sharifi-Mood B, Nezamdoost M, Moazeni-Roodi A, Naderi M, Kouhpayeh H, Taheri M, Ghavami S. 2011. Functional polymorphism of interferon-g (IFN-g) gene þ874T/A polymorphism is associated with pulmonary tuberculosis in Zahedan, southeast Iran. Prague Med Rep. 112:38–43. Kong YC, Lau CP, Wat KH, Ng KH, But PPH, Cheng KF, Waterman PG. 1989. Antifertility principle of Ruta graveolens. Plant Med. 55:176–178. Kostova I, Ivanova A, Mikhova B, Klaiber I. 1999. Alkaloids and coumarins from Ruta graveolens. Monatsh Chem. 130:703–707. Kutchan TM. 1998. Molecular genetics of plant alkaloid biosynthesis. In: Cordell GA, editor. The alkaloids: chemistry and biology. San Diego, CA: Academic Press; p. 258– 311. Kuzovkina I, Al_terman I, Schneider B. 2004. Specific accumulation and revised structures of acridone alkaloid glucosides in the tips of transformed roots of Ruta graveolens. Phytochemistry. 65:1095–1100. Mitchell J, Seeman P. 1998. Drug receptors. In: Kalant H, Roschlau WHE, editors. Principles of medical pharmacology. 6th ed. Oxford: Oxford University Press; p. 91– 98. Oliva A, Meepagala KM, Wedge DE, Harries D, Hale AL, Aliotta G, Duke SO. 2003. Natural fungicides from Ruta graveolens L. leaves, including a new quinolone alkaloid. J Agric Food Chem. 51:890–896. Paulini H, Popp R, Schimmer O, Ratka O, Roder E. 1991. Sogravacridon-chlorine: a potent and direct acting frameshift mutagen from the roots of Ruta graveolens. Plant Med. 57:59–61. Paulini H, Waibel R, kiefer J, Schimmer O. 1991. Gravacridonediol acetate, a new dihydrofuroacridone alkaloid from Ruta graveolens. Plant Med. 57:82– 83. Reisch J, Szendrei K, Novak I, Minker E. 1970. Natural product chemistry, 27 lignans of Ruta graveolens roots. Pharmazie. 25:435–436. Rozsa Z, Mester I, Reisch I, Szendrei K. 1989. Naphthoherniarin an unusual coumarin derivative from Ruta graveolens. Plant Med. 55:68–69. Sallal AJ, Alkofahi A. 1996. Inhibition of the hemolytic activities of snake scorpion venoms in vitro with plant extracts. Biomed Lett. 53:211–215. Ulubelen A, Ertugrul L, Birman H, Yigit R, Erseven G, Olgac V. 1994. Antifertility effects of some coumarins isolated from Ruta chalepensis and R. chalepensis var. latifolia in rodents. Phytother Res. 8:233–236. Weed S. 1986. Wise women herbal for the childbearing year. New York: Ash Tree Publishing Woodstock.
Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
New quinoline alkaloid from Ruta graveolens aerial parts and evaluation of the antifertility activity a
a
b
Josline Y. Salib , Sayed A. El-Toumy , Emad M. Hassan , Nabila H. a
c
Shafik , Sally M. Abdel-Latif & Ignacio Brouard
d
a
Chemistry of Tannins Department, National Research Centre, Dokki, Cairo, Egypt b
Medicinal and Aromatic Plants Research Department, National Research Centre, Dokki, Cairo, Egypt c
Pharmacognosy Department, Faculty of Pharmacy, Cairo University, Cairo, Egypt d
Instituto de Productos Naturales y Agrobiología, Av.Astrofisico F. Sanchez 3, 38206 La Laguna, Tenerife, Spain Published online: 04 Apr 2014.
To cite this article: Josline Y. Salib, Sayed A. El-Toumy, Emad M. Hassan, Nabila H. Shafik, Sally M. Abdel-Latif & Ignacio Brouard (2014): New quinoline alkaloid from Ruta graveolens aerial parts and evaluation of the antifertility activity, Natural Product Research: Formerly Natural Product Letters, DOI: 10.1080/14786419.2014.903395 To link to this article: http://dx.doi.org/10.1080/14786419.2014.903395
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Natural Product Research, 2014 http://dx.doi.org/10.1080/14786419.2014.903395
New quinoline alkaloid from Ruta graveolens aerial parts and evaluation of the antifertility activity Josline Y. Saliba*, Sayed A. El-Toumya, Emad M. Hassanb, Nabila H. Shafika, Sally M. Abdel-Latifc and Ignacio Brouardd
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a
Chemistry of Tannins Department, National Research Centre, Dokki, Cairo, Egypt; bMedicinal and Aromatic Plants Research Department, National Research Centre, Dokki, Cairo, Egypt; cPharmacognosy Department, Faculty of Pharmacy, Cairo University, Cairo, Egypt; dInstituto de Productos Naturales y Agrobiologı´a, Av.Astrofisico F. Sanchez 3, 38206 La Laguna, Tenerife, Spain (Received 11 November 2013; final version received 8 March 2014) Bioassay-guided isolation of methanol extract of Ruta graveolens L. leaves yielded a new quinoline alkaloid, (4S) 1,4-dihydro-4-methoxy-1,4-dimethyl-3-(3-methylbut-2enyl)quinoline 2,7-diol, and nine phenolic compounds including rutin as a major compound. Structures of the isolated compounds were determined by using chromatography, UV, HR-ESI-MS and 1D/2D 1H/13C NMR spectroscopy. The uterotonic activity of methanol extract fractions (ethyl acetate, n-butanol and aqueous fraction) as well as the isolated major compounds was tested in the isolated mouse uterus in vitro. The n-butanol-soluble fraction was found to demonstrate the most potent uterotonic activity in a dose-dependent manner, also the major isolated compound rutin revealed the occurrence of an uterotonic response, which was maximum at a concentration level of 0.25 mg/mL, accounting for 68.7% of that exhibited by the chosen concentration of oxytocin. Keywords: Ruta graveolens L.; polyphenolic metabolites; uterotonic activity
1. Introduction Despite many achievements in human health care in the twentieth century, many of the world’s population in developing countries lack regular access to affordable essential drugs. In contrast, traditional medicine is widely available and affordable, even in remote areas. Growth of human population has been underway for thousands of years and was never a problem until recently. Currently, overpopulation has led to series social and environmental problems such as poverty, overcrowded slums, crime, pollution of air and water and depletion of the protective ozone layer (Greep 1998). Family planning has been promoted through several methods of contraception, but due to serious adverse effects produced by synthetic steroidal contraceptives, attention has now been given to indigenous plants for possible contraceptive effects (Ghosh & Bhattacharya 2004) whereby several active chemical constituents accountable for uterotonic effects are discovered in various plant species from time to time. Ruta graveolens L. (Rutaceae) or common rue, native of the Mediterranean region but cultivated throughout Europe and many Asian countries, is an evergreen shrub with bluish-green leaves that emit a powerful odour and have a bitter taste. Ruta is cited in the ancient herbals and has deep roots in folklore. Ruta has a long history of use as a domestic remedy, being especially valued for its strengthening action on the eyes. Ruta contains flavonoids (notably rutin) that
*Corresponding author. Email: [email protected] q 2014 Taylor & Francis
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reduce capillary fragility, which might explain the plant’s reputation as an eye strengthener (Bown 1995). The whole herb is abortifacient, anthelmintic, antidote, antispasmodic, carminative, emetic, brings relief from giddiness and nervous headaches, strongly stimulant and mildly stomachic (Aliotta et al. 1994; Sallal & Alkafahi 1996; Aliotta et al. 2000; De Feo et al. 2002; Oliva et al. 2003). In Germany, extracts of R. graveolens are marketed for relief of cramps and rheumatism (Kutchan 1998). Photo-chemotherapy with a constituent from R. graveolens has been used to treat cutaneous T-cell lymphoma (Gasparro 1994). In Taiwan, the fresh aerial part of this plant is widely used to treat palpitation of the heart and circulatory disorders (Kong et al. 1989). Quinoline alkaloids (Paulini, Popp et al. 1991; Paulini, Waibel et al. 1991), coumarins (Rosza et al. 1989; Kostova et al. 1999; Kuzovkina et al. 2004), lignans (Reisch et al. 1970) and flavonoids (Bautz et al. 1989; Ulubelen et al. 1994; Hashemi et al. 2011) have been isolated from R. graveolens and exhibit a variety of pharmacological activities. This study deals with bioassay-guided fractionation using ethyl acetate, n-butanol and water of the methanol extract of Egyptian R. graveolens aerial parts. The n-butanol fraction demonstrated the most potent uterotonic activity in a dose-dependent manner (responses accounting for 13.76%, 48.62% and 67.58% of that exhibited by the chosen concentration of oxytocin for concentration levels of 0.25, 0.375 and 0.5 mg/mL, respectively). Fractionation of the n-butanol fraction resulted in the isolation and identification of seven polyphenolic compounds namely, quercetin-3-O-b-D glucopyranoside, quercetin-3-O-a-L -rhamnopyranoside, kaempferol-3-O-b-D -glucopyranoside, rutin, isorhamnetin, quercetin and kaempferol, with the new natural quinoline alkaloid, (4S) 1,4-dihydro-4-methoxy-1,4-dimethyl-3-(3-methylbut-2-enyl)quinoline-2,7-diol, as well as two phenolic acids caffeic and ferulic acids. 2. Results and discussion Fractionation of the n-butanol extract resulted in the isolation and identification of 10 compounds 1 – 10. The structure of the isolated compounds was determined using chromatography as well as conventional chemical and spectroscopic methods of analysis (UV, HR-ESI-MS and 1D/2D 1H/13C NMR spectroscopy). Compound 1, obtained as a yellow amorphous powder exhibited chromatographic properties (yellow spot on paper chromatogram under UV light). The presence of quinoline nucleus was indicated by its UV spectrum. It also yielded orange colour with Dragendorff reagent for an alkaloid. Its molecular formula was established as C17H23NO3 from its HR-ESI-MS at m/z 290.1363 [M þ H]þ. Its 1H NMR spectrum revealed five singlets for methyl groups each at d 1.19, 1.21, 1.48, 3.92 and 4.42, among which the presence of a methoxyl O – CH3 (d H 4.42) and – N –CH3 (d H 3.92) were confirmed by its DEPT and 1H – 13C COSY spectra. 1H NMR of 1 also displayed a doublet at d 7.75 (d,1H), a double of doublet at d 7.38 (dd, 1H) and another doublet at 7.28 (d,1H) indicating the presence of 1, 2, 4 system which has been confirmed by its coupling constants, 1H – 1H COSY and 1H – 13C COSY spectra. This suggested that one of the (OH) groups must be present on aromatic nucleus. Its HMBC correlation studies confirmed that the methyls at d 1.19 and 1.21 were gem dimethyls and attached to carbon at d 128.39 as revealed by 1H – 13C correlation spectrum. This particular carbon at d 128.39 was also attached to double bond (CvC) and displayed correlation with the carbon that appeared at d 93.78 whose 1 H NMR signals displayed a triplet at d 5.51 (1H) suggesting that both these units were linked together and to a methylene carbon which yielded a doublet signal at d 3.89 (2H). This confirmed the presence of 3-methyl but-2-enyl group. The HMBC correlation of CH3 group at dH 3.92 with carbons at dC 159.37 and dC 163.98 clearly indicated the N –CH3 group and further confirming the presence of quinoline nucleus. The presence of a methoxy group and a methyl group at C-4 was confirmed through the observed HMBC correlations which revealed the correlation of OCH3 group at (dH 4.42) with C-4 at (dC 70.37) and CH3 at (dH 1.48) with C-4 at (dC 70.37). The
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positive Cotton effect observable in the circular dichroism (CD) spectrum indicated that the absolute configuration at the 4-position of compound 1 was S. Thus, on the basis of the abovementioned discussion, 1 was assigned the constitution as (4S) 1,4-dihydro-4-methoxy-1,4dimethyl-3-(3-methylbut-2-enyl)quinoline-2,7-diol, which is a new natural product (Figure 1). The other nine known compounds (2 –10) were identified as quercetin-3-O-b-D -glucopyranoside (2), quercetin-3-O-a-L -rhamnopyranoside (3), kaempferol-3-O-b-D -glucopyranoside (4), rutin (5), isorhamnetin (6), quercetin (7), kaempferol (8) and the two phenolic acids, caffeic (9) and ferulic acids (10), by comparing their 1D and 2D NMR spectral data with the reported data in the literature (Agrawal 1989). Control of fertility using traditional antifertility plants has been practiced for many years in Africa. R. graveolens is one of the many plants used for fertility regulation all over the world. That is why this study revealed the in vitro uterotonic effect which was very evident of R. graveolens whose extracts exhibited such activity can be one of the factors rationalising the abortifacient effect of the crude drug reported in the literature. Results of preliminary trials for the tested extract in the concentration range 0.0125 – 1.25 mg/mL against the chosen concentration of oxytocin revealed an uterotonic responses exhibited by methanol extract. Different concentrations of the methanolic extract of R. graveolens were tested (Table 1). Thus, the methanolic extract exhibited a high and dose-related response, denoting highly effective ingredient(s) responsible for this type of action (Mitchell & Seeman 1998). The n-butanol-soluble fraction exhibited the strongest uterotonic activity doubling and significantly different from that of the water-soluble fraction and still displaying a dose-related response as the original methanolic extract, while the ethyl acetate-soluble fraction was found to be inactive and the results are shown in Table 2 and Figure 2. According to these results, it was suggested that the biological activity of R. graveolens methanolic extract was due to the compounds present in the n-butanol-soluble fraction and this activity may be enhanced by other compounds present in the water-soluble fraction.
H3CO 6
5
4
4a
5` CH3
CH3 3
2` HO
8a 8
1 N
3`
CH3
CH3
H3CO
1` CH3 4`
OH
CH3 HO
CH3
N
OH
CH3
Figure 1. Compound 1 and its HMBC correlation. Table 1. The uterotonic responses exhibited by different concentrations of the methanolic extract of R. graveolens. Tested sample Oxytocin Methanolic extracta
a b c
Concentration (mg/mL)
Mean response (cm) ^ s
Uterotonic activity (%)
3.75 £ 1026 0.5 0.375 0.25
7.17 ^ 0.907 5.47 ^ 0.850b 3.97 ^ 0.153b,c 2.83 ^ 0.058b,c
100 76.29 55.37 39.47
Dose-related response. Significantly different from control (oxytocin) at p # 0.05. Significantly different from the mean response caused by larger concentration(s) of the same extract at p # 0.05.
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Table 2. Comparison between the uterotonic effects exhibited by different concentrations of the n-butanolsoluble fraction. Tested sample
Concentration (mg/mL)
Oxytocin Butanol-soluble fractiona
Mean response (cm) ^ s
Uterotonic activity (%)
6.54 ^ 3.01 4.42 ^ 1.11 3.18 ^ 0.295b,c 0.9 ^ 0.534b,c
100 67.58 48.62 13.76
26
3.75 £ 10 0.5 0.375 0.25
a
Dose-related response. Significantly different from control (oxytocin) at p # 0.05. c Significantly different from the mean response caused by larger concentration(s) of the same fraction at p # 0.05. b
10 Mean response (cm)
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8 6 4 2 Soluble
0 Oxytocin
IIID2
IIID3
Tested sample
Figure 2. Uterotonic responses exhibited by the butanol and water-soluble fractions at 0.5 mg/mL concentration level against the chosen concentration of oxytocin.
After performing the biological study of the different extracts and fractions, we decide to study the n-butanol fraction phytochemically, since they proved to be the most biologically active fraction in an attempt to isolate the biologically active ingredients present. Rutin was found to be the major compound and revealed the occurrence of an uterotonic response (followed by self-inhibition) whose magnitude was closely related to concentration (Table 3), while the comparison between the uterotonic effects exhibited by rutin and related compounds at the same concentration of oxytocin is shown (Figure 3). Table 3. Comparison between the uterotonic effects exhibited by different concentrations of rutin. Drug Oxytocin Rutin
a b
Concentration (mg/mL) 26
3.75 £ 10 0.025 0.125 0.25 0.375 0.5
Mean response (cm)
Uterotonic activity (%)
9.17 ^ 1.6 1.3 ^ 0.529a,b 4.47 ^ 1.53a 6.3 ^ 1.71 4.53 ^ 0.907a 3.37 ^ 0.666a,b
100 14.18 48.75 68.7 49.4 36.75
Significantly different from control (oxytocin) at p # 0.05. Significantly different from the mean response caused by 0.25 mg/mL rutin at p # 0.05.
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Mean resones (cm)
10
8 6 4
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2
0 Oxytocin
Rutin
Quercetin
Quercitrin
Isoquercitrin
Tested compound
Figure 3. Uterotonic responses exhibited by rutin and related compounds at 0.25 mg/mL concentration level against the chosen concentration of oxytocin.
Reviewing the current literature, there is only one old report concerning the adrenaline like action of rutin on the smooth muscle of rabbit uterus (in situ) and intestine (Baraboi & Moldavskaya 1966). According to Susun Weed (1986), rutin can be used as a form of contraception or emergency contraception when taken as a tablet in doses of at least 500 mg daily for several days proceeding and following ovulation or after fertilising intercourse and continuing until the menstrual flow begins. It can be concluded from (Table 3) that maximum uterotonic activity of rutin is exhibited at 0.25 mg/mL concentration level when there is no significant difference between the uterotonic response caused by rutin and that caused by the chosen concentration of oxytocin. The diminution of activity with higher concentrations of the compound can be easily attributed to that the stimulation of uterine smooth muscle is always followed by inhibition and the ratio of stimulation to inhibition responses is related to concentration. This can also rationalise the conflict about its use during pregnancy, as the effect appears to be dose-dependent. 3. Experimental 3.1. Plant materials Leaves of R. graveolens were collected from the field (Experimental and Research Station of the Faculty of Pharmacy, Cairo University, Cairo, Egypt) in spring 2010 and dried under shade. The plants were authenticated by Dr Tarek Abdel-Salam, Professor of Botany, Department of Botany, Faculty of Science, Cairo University, Cairo, Egypt, and voucher specimens (No. 20 R) have been deposited in the herbarium, National Research Center, Cairo, Egypt. 3.2. General 1
H NMR and 13C NMR spectra were obtained on Bruker AMX-400, Avance 400, and Avance 300 spectrometers (Bruker, Rheinstetten, Germany) with standard pulse sequences operating at 400, 300 MHz in 1H NMR and 100, 75 MHz in 13C NMR. Chemical shifts are given in d values (ppm) using tetramethylsilane as the internal standard. UV spectra were recorded with Shimadzu UV-1601
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(Shimadzu, Tokyo, Japan). HR-ESI-MS was measured on a Micromass Autospec (70 eV) spectrometer (Micromass, Manchester, UK). CD spectra were obtained with a JASCO J-720 spectropolarimeter (JASCO, Tokyo, Japan). Column chromatography (CC) was carried out on Polyamid 6S and Sephadex LH20; CC: Polyamide 6S, (Riedel-De Haen, Hannover, Germany) and Sephadex LH-20 (Fluka Pharmazia, Uppsala, Sweden); PC was carried out on Whatman No. 1 and 3 mm paper using the following solvent systems: (1) BAW (n- BuOH/HOAc/H2O, 4:1:5); (2) H2O and (3) AcOH/H2O (15:85). De Jalon’s solution (for isolated uterus), Tween 80 (Fluka), oestradiol benzoate (Folone ampoules, Misr Company for Pharmaceutical Industries, SAE, Cairo, Egypt) and oxytocin (Syntocinon ampoules, Novartis Pharma, SAE, UK) were used.
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3.3. Extraction, fractionation and isolation Five hundred grams of the plant were reduced to a coarse powder extracted by methanol in a Soxhlet extractor at room temperature till exhaustion. The extract was dried under vacuum at 408C to yield 100 g of the methanol extracts of R. graveolens leaves. Eighty grams of the methanol extract were suspended in 500 mL distilled water and successively extracted in a separating funnel with ethyl acetate and n-butanol at room temperature till exhaustion leaving a residual water-soluble fraction. The three fractions were dried under vacuum at 408C and their net weights were calculated. Percentage yields (w/w) of the ethyl acetate, n-butanol and water-soluble fractions of R. graveolens methanolic extract were 9.86, 25.43 and 35.14, respectively. The n-butanol extract (15 g) was chromatographed on a Sephadex LH-20 column with water and water/ethanol mixtures as eluent to yield seven fractions. Fractions were further separated and purified using Whatman 3 mm paper and Sephadex LH-20 column to yield the pure compounds (4S) 1,4-dihydro-4-methoxy-1,4-dimethyl-3-(3-methylbut-2-enyl)quinoline-2,7diol (1), quercetin-3-O-b-D -glucopyranoside (2), quercetin-3-O-a-L -rhamnopyranoside (3), kaempferol-3-O-b-D -glucopyranoside (4), rutin (5), isorhamnetin (6), quercetin (7), kaempferol (8) and the two phenolic acids, caffeic (9) and ferulic acids (10). 3.3.1. (4S) 1,4-dihydro-4-methoxy-1,4-dimethyl-3-(3-methylbut-2-enyl)quinoline-2,7-diol (1) Rf-values £ 100: 78 (BAW), 80 (15% AcOH). UV lmax nm (MeOH): 332, 283, 256, 237 and 232 nm. HR-ESI-MS at m/z: 290.1363 [M þ H]þ. 1H NMR: d 7.75 (d, J ¼ 7 Hz, H-5),7.38 (dd, J ¼ 2 and 7 Hz, H-6), 7.28 (d, J ¼ 2 Hz, H-8), 5.51 (t, J ¼ 5.79 Hz, H-20 ), 4.42 (s, O – CH3), 3.92 (s, N –CH3), 3.89 (d, J ¼ 4.38, H-10 ), 1.48 (s, 4-CH3), 1.21 (s, H-40 ), 1.19 (s, H-50 ). 13C NMR: 163.98 (C-2), 103.61 (C-3), 70.37 (C-4),120.76 (C-4a), 124.43 (C-5), 106.72 (C-6), 160.87 (C7), 118.22 (C-8), 159.37 (C-8a), 28.81 (C-10 ), 93.78 (C-20 ), 128.39 (C-30 ), 25.91 (C-40 ), 21.77 (C50 ), 34.14 (N– CH3), 59.74 (O – CH3), 25.42 (4-CH3). 3.4. Animals Healthy adult virgin female albino rats of Sprague-Dawely strain weighing 140– 170 g were supplied by the Department of Hygiene and Animal Management, Faculty of Veterinary Medicine, Cairo University, Giza, Egypt. The animals were kept on standard laboratory diet and under hygienic conditions. 3.5. Uterotonic activity bioassay 3.5.1. Preparation of uterine tissues Virgin female albino mice were used for testing uteri-contracting (uterotonic) activity of the different fractions of the plant. The mice were injected intramuscularly with 0.1 mg/kg of
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oestradiol benzoate 24 h prior to the experiment, were sacrificed by cervical dislocation and the uterine horns were removed and placed in a dish containing physiological De Jalon’s solution with the following composition (g/L): NaCl, 9 g; KCl, 0.42 g; NaHCO3, 0.5 g; D -glucose, 0.5 g; CaCl2, 0.062 g (378C) connected to a transducer and the uterine response was left to stabilise for 20 min.
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3.5.2. Dose– response study In order to assess the uterotonic activity, 2 g of the dried extract were dissolved in De Jalon’s solution to produce 10% solution. The extract was preliminarily tested in the concentration range 0.0125 –1.25 mg/mL against 3.75 £ 1026 mg/mL [0.001875 IU/mL] of oxytocin as a standard (optimum concentration chosen after several trials) and the three most suitable concentrations were chosen for calculations. Before every test, the bath solution was drained completely and washed two to three times and filled with fresh solution. For calculations, the three chosen concentrations of the extract were tested in six different uterine horns against the chosen concentration of oxytocin and the mean response corresponding to each concentration was calculated and compared with the mean response corresponding to the used concentration of oxytocin. The method used was a modification of that adapted by Desta (1994). The data were subjected to statistical analysis using Student’s unpaired test (Armitage & Berry 1987). Two grams of each of the butanol- and water-soluble fractions were dissolved in 20 mL De Jalon’s solution while, in case of the ethyl acetate-soluble fraction, 2 g were dissolved in 20 mL 15% solution of Tween-80 (in De Jalon’s solution) to produce 10% solutions. The uterotonic activity of each fraction was assessed following the same method adopted earlier for testing the activity of the methanolic extract and using the same three concentrations chosen before for the methanolic extract against the chosen concentration of oxytocin as a standard for calculations. Rutin and other isolated compounds were tested for uterotonic activity following the same method adopted earlier for the methanolic extract and its butanol-soluble fraction at 0.025, 0.125, 0.25, 0.375 and 0.5 mg/mL concentration levels against the chosen concentration of oxytocin as a standard.
4. Conclusion In addition to the fact that the Egyptian variety of R. graveolens has been proved to possess an antifertility activity like their corresponding varieties of other geographical regions, it is the first time to relate the mechanism of action to specific compound isolated from crude extract. Thus, we recommend that the n-butanol fraction of R. graveolens together with its main active ingredient to be subjected to extensive clinical studies in order to be able to introduce them to the society as antifertility drugs that could meet the needs of many partners interested in family planning but searching for an effective, safe, cheap and available birth-control method.
Supplementary material Supplementary material relating to this article is available online, alongside Figures S1 – S5.
References Agrawal PK. 1989. Carbone-13 NMR of flavonoids. Amsterdam: Elsevier. Aliotta G, Cafiero G, De Feo V, Di Blasio B, Iacovino R, Oliva A. 2000. Allelochemicals from rue (Ruta graveolens L.) and olive (Olea europaea L.) oil mill waste waters as potential natural pesticides. Curr Top Phytochem. 3:167–177.
Downloaded by [University of Waikato] at 07:01 04 July 2014
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Aliotta G, Cafiero G, De Feo V, Sacchi R. 1994. Potential allelochemicals from Ruta graveolens L. and their action on radish seeds. J Chem Ecol. 20:2761–2775. Armitage P, Berry G. 1987. Statistical methods in medical research. Oxford: Blackwell. Baraboi VA, Moldavskaya SI. 1966. Pharmacology of some polyphenols possessing vitamin P activity. Vrachebnoe Delo. 1:71–73. Bautz C, Bohuslavizki KH, Hansel W, Koppenhofer E. 1989. Effects of Ruta on the excitation process in myelinated nerves. Plant Med. 55:649. Bown D. 1995. Encyclopaedia of herbs and their uses. New York: DK. De Feo V, De Simone F, Senatore F. 2002. Potential allelochemicals from the essential oil of Ruta graveolens. Phytochemistry. 61:573–578. Desta B. 1994. Ethiopian traditional herbal drugs. Part III: antifertility activity of 70 medicinal plants. J Ethnopharmacol. 44:199–209. Gasparro FP. 1994. Extracorporeal photochemotherapy: clinical aspects and the molecular basis for efficacy. Boca Raton, FL: CRC Press. Ghosh K, Bhattacharya TK. 2004. Preliminary study on the antiimplantation activity of compounds from Thespesia populnea. Indian J Pharmacol. 36:288–290. Greep RO. 1998. Whether the global population problem. Biochem Pharmacol. 55:385–386. Hashemi M, Sharifi-Mood B, Nezamdoost M, Moazeni-Roodi A, Naderi M, Kouhpayeh H, Taheri M, Ghavami S. 2011. Functional polymorphism of interferon-g (IFN-g) gene þ874T/A polymorphism is associated with pulmonary tuberculosis in Zahedan, southeast Iran. Prague Med Rep. 112:38–43. Kong YC, Lau CP, Wat KH, Ng KH, But PPH, Cheng KF, Waterman PG. 1989. Antifertility principle of Ruta graveolens. Plant Med. 55:176–178. Kostova I, Ivanova A, Mikhova B, Klaiber I. 1999. Alkaloids and coumarins from Ruta graveolens. Monatsh Chem. 130:703–707. Kutchan TM. 1998. Molecular genetics of plant alkaloid biosynthesis. In: Cordell GA, editor. The alkaloids: chemistry and biology. San Diego, CA: Academic Press; p. 258– 311. Kuzovkina I, Al_terman I, Schneider B. 2004. Specific accumulation and revised structures of acridone alkaloid glucosides in the tips of transformed roots of Ruta graveolens. Phytochemistry. 65:1095–1100. Mitchell J, Seeman P. 1998. Drug receptors. In: Kalant H, Roschlau WHE, editors. Principles of medical pharmacology. 6th ed. Oxford: Oxford University Press; p. 91– 98. Oliva A, Meepagala KM, Wedge DE, Harries D, Hale AL, Aliotta G, Duke SO. 2003. Natural fungicides from Ruta graveolens L. leaves, including a new quinolone alkaloid. J Agric Food Chem. 51:890–896. Paulini H, Popp R, Schimmer O, Ratka O, Roder E. 1991. Sogravacridon-chlorine: a potent and direct acting frameshift mutagen from the roots of Ruta graveolens. Plant Med. 57:59–61. Paulini H, Waibel R, kiefer J, Schimmer O. 1991. Gravacridonediol acetate, a new dihydrofuroacridone alkaloid from Ruta graveolens. Plant Med. 57:82– 83. Reisch J, Szendrei K, Novak I, Minker E. 1970. Natural product chemistry, 27 lignans of Ruta graveolens roots. Pharmazie. 25:435–436. Rozsa Z, Mester I, Reisch I, Szendrei K. 1989. Naphthoherniarin an unusual coumarin derivative from Ruta graveolens. Plant Med. 55:68–69. Sallal AJ, Alkofahi A. 1996. Inhibition of the hemolytic activities of snake scorpion venoms in vitro with plant extracts. Biomed Lett. 53:211–215. Ulubelen A, Ertugrul L, Birman H, Yigit R, Erseven G, Olgac V. 1994. Antifertility effects of some coumarins isolated from Ruta chalepensis and R. chalepensis var. latifolia in rodents. Phytother Res. 8:233–236. Weed S. 1986. Wise women herbal for the childbearing year. New York: Ash Tree Publishing Woodstock.
