Short communication: antinociceptive effect of Russelia equisetiformis leave extracts: identification of its active constituents.
Russelia equisetiformis, (Schlecht and Cham) belongs to the family Scrophulariacae. Phytochemical study of the plant revealed the presence of triterpenes of lupane type (Burns et al., 2001). The extract of the whole plant has been found to possess anti-microbial activity (Awe et al., 2004a). Various triterpenoids obtained from the plant have also been reported to exhibit analgesic and anti-inflammatory effects (Awe et al., 2004b). No systematic study has been carried out on the plant in order to establish the chemical principle responsible for the reported activities. We now report the isolation of one of the chemical principles and a new phenylethanoid glycoside from the ethylacetate fraction of the plant. This study establishes the antinociceptive activity of the fractions from R. equisetiformis, and Russetinol glycoside as the major compound responsible for the activity.
Extraction and isolation
IR spectral was obtained with BUCK 500 M spectrometer.[.sup.1]H and [.sup.13]C NMR were measured with Bruker Avance DPX 300 spectrometer ([.sup.1]H at 300 MHz, [.sup.13]C at 75MHz), the chemical shift being reported in ppm with TMS as an internal standard and spectral run in [DMSO-[d.sub.6]. Electron impact mass spectra were recorded with a Finnigan LQC Deca mass spectrometer in the electron ionization (EI) positive and negative mode.
The whole part of R. equisetiformis, was collected from Bodija, Ibadan, Western Nigeria in October 2003. The plant was kindly identified by Mr. E.A. Ogundayile, a technologist in the herbarium, Department of Botany, University of Ibadan, where a voucher specimen is deposited with voucher no. 106698.
Extraction and isolation
A 400 g powdered air-dried whole plant material of R. equisetiformis was extracted with 50% aqueous ethanol in the cold for 72 h; it was filtered and concentrated to dryness in the rotary evaporator. The crude ethanolic extract was re-dissolved in water and partitioned successively with four organic solvents to obtain n-hexane, dichloromethane, ethylacetate and n-butanol fraction.
These fractions were screened for antinociceptive activity using the method described in the section on antinociceptive activity. The ethylacetate fraction was found to be most active. The dried ethylacetate extract (2.63 g) was subjected to bioactivity-guided fractionation using the accelerated gradient chromatography technique with silica gel as adsorbent. Elution was started with 100% n-hexane, and increasing polarity sequentially in a step of 2% between n-hexane-chlor-oform-ethylacetate and methanol. A total of 141 test tube fractions (15 ml each) were collected, similar fractions were combined according to the tlc characteristics to afford nine different fractions which were coded [AW.sub.1]-[AW.sub.9] These were assayed for biological activity. Fraction [AW.sub.7] eluted with ethylacetate and methanol (70:30) was found most active. Repeated fractionation of [AW.sub.7] (l.6 g) using LH-20 Sephadex column chromatographic method and eluting with 50% toluene-ethanol with increasing polarity in step of 10% (v/v) resulted in the isolation of two compounds. Compound 1 coded as [AW.sub.7]C (58 mg), is a yellow amorphous solid, with deep colour intense when sprayed with vanillin--sulphuric acid reagent and has an [R.sub.f] value of 0.56 in EtOAc-MeOH (4:1).
Compound 2 coded as [W.sub.8]A (14 mg), was obtained as yellow amorphous solid and minor constituent with [R.sub.f] value of 0.48 in EtOAc-MeOH (4:1). The structures of the two compounds were established using the available spectroscopic techniques including the high-resolution electron spray ionization mass spectrum, COSY[.sup.1]H, [.sup.13]C, HMBC, HMQC, DEPT, IR and MS.
This was carried out as described by Koster et al. (1959). Male Swice Mice (19-23 g) were used. Mice were given 2% DMSO (10 ml/kg) intraperitoneally (i.p.) (negative control). Other groups of animals were treated with solvent fractions at 5 mg/kg, ethylacetate tlc fractions at 2 mg/kg and Indomethacin at 5 mg/kg i.p. (positive control); 30 min later, animals in all groups were injected with 0.6% acetic acid (10 ml/kg) (i.p.). After 5 min the number of writhing (abdominal constriction arching of the back, development of tension in the abdominal muscles, elongation of the body and extension of the forelimbs) were counted.
This was carried out as described by Janssen et al. (1963). (Mice were given 2% DMSO (10 ml/kg) intraperitoneally (i.p.) (negative control). Other groups of animals were treated with solvent fractions at 5 mg/kg and ethylacetate tlc fractions at 2 mg/kg i.p; 30 min later, tail reaction response was done with one-third of the tail immersed in a water bath heated at 55[degrees]C. The response latency between the onset of immersion and the with-drawal of the tail was recorded. A cut of time of 30 s was used in order to prevent damage to the tail. Morphine (1 mg/kg) was used as standard drug.
Results and discussion
Preliminary phytochemical analysis showed that R. equisetiformis contains saponins, cardiac glycoside, triterpenes and flavonoids. The ethylacetate fraction of the aqueous alcoholic extract caused 73.6% inhibition of the acetic acid induced abdominal contraction in mice while n-butanol, n-hexane and dichloromethane fractions caused 67.5%, 64.3% and 20.4% reduction in writhing respectively. In the tail flick test, n-hexane showed the highest activity with ethylacetate and n-butanol with weak activity. Six out of the nine TLC fractions of the ethylacetate caused significant inhibition (p<0.050 of acetic acid induced abdominal contraction in mice. The effects of the isolated compound on acetic acid induced writhing and tail-flick response in mice are shown in Tables 1-4. In the chemical induced pain, the ethylacetate fraction showed the highest activity. It is therefore possible that the active component(S) reside in the polar fractions. It has been reported that in chemical induced pain pro-inflammatory mediators such as bradykinin stimulate pain nociceptors (Keele, 1969; Collier et al., 1968; Keele and Armstrong, 1964).
Table 1. Effect of solvent fractions of ethanolic extract of Russelia equisetiformis on acetic-acid induced writhing test Treatment Dose (mg/kg) No. of % [writhing.sub.b] inhibition Control (DMSO, -- 31.4 [+ or- ] 2.33 - 2%) lOmg/kg n-hexane 5 11.2 [+ or -] 0.83 64.3 Dichloromethane 5 25.0 [+ or -] 1.63 20.4 Ethylacetate 5 8.3 [+ or -] 0.54 73.6 n-butanol 5 10.2 [+ or -] 0.67 67.5 Indomethacin 5 8.33 [+ or -] 0.54 73.4 (a) Values are statistically significant at p<0.05, comparing each sub-fraction for indomethacin treatment values with 2% DMSO (control) values, using Student's t-test. (b) Values are recorded as mean [+ or -] SEM of five independent observations. Table 2. Effect of solvent fractions of ethanolic extract of Russelia equisetiformis on tail-flick test Treatment Dose Reaction time (s)a % (mg/kg) inhibition Control (DMSO, 2%) -- 2.0 [+ or -] 0.73(b) -- lOmg/kg n-hexane 5 6.3 [+ or -] 2.31 68.3(b) Dichloromethane 5 5.8 [+ or -] 2.13(b) 65.5(b) Ethylacetate 5 2.5 [+ or -] 0.92 25.0 n-butanol 5 2.5 [+ or -]0.92 25.0 Indomethacin 5 9.2 [+ or -] 3.38(b) 78.3(b) (a) Values are recorded as mean [+ or -] SEM of five independent observations.(b) Values are statistically significant at p<0.05, comparing each sub-fraction or morphine treatment values with 2% DMSO (control) values, using Student's t-test. Table 3. Effect of compound [AW.sub.7] C Russetinol from Russelia equisetiformis on acetic acid induced writhing Treatment Dose No. of % (mg/kg) writhing (a) inhibition Control -- 26.2 [+ or -] 1.75 -- (DMSO, 2%) [AW.sub.7]C 1 18.6 [+ or -] 1.21 (b) 30.60 [AW.sub.7]C 2 0.4 [+ or -] 0.03 (b) 98.51 [AW.sub.7]C 4 0.0 [+ or-] 0.0 (b) 100.0 Indomethacin 5 7.6 [+ or -] 0.57 (b) 71.64 (a) Values are recorded as mean [+ or -] SEM of five independent observations. (b) Values are statistically significant at p<0.05, comparing each does level of [AW.sub.7] C or indomethacin with 2% DMSO (control) values, using Student's t-test. Table 4. Effect of compound [AW.sub.7] C Russetinol from Russelia equisetiformis on tail-flick response in mice Treatment Dose Reaction % (mg/kg) time (s)(a) inhibition Control -- 2.0 [+ or -] 0.18 -- (DMSO, 2%) [AW.sub.7]C 1 1.8 [+ or -] 0.18 10.0 [AW.sub.7]C 2 2.8 [+ or -] 0.25 40.0 [AW.sub.7]C 4 3.2 [+ or -] 0.29 (b) 60.0 Morphine 1 8.7 [+ or -] 0.78 (b) 79.3 (a) Values are recorded as mean [+ or -] SEM of six independent observations. (b) Vaiues are statistically significant at p<0.05, comparing compound [AW.sub.7] C treatment values with morphine with DMSO (control) values, using Student's t-test.
In thermal-induced pain, n-hexane fraction showed the highest activity indicating the central involvement of the components of this fraction. The crude extract showed an interaction with opioids receptors (Keele, 1969). This observation was substantiated by the fact that like morphine, the antinociception produced by the crude extract was almost prevented by the non-selective antagonist naloxone (Awe et al., 2004b). It is also possible that the compounds responsible for this action reside in the n-hexane fraction, however through this study, fractions AWRE.sub.1-7, showed significant (p<0.05) antinociceptive activity in chemical-induced pain while [AWRE.sub.4] and [AWRE.sub.7] showed antinociceptive activity in thermal-induced pain.
Two phenolic compounds were isolated from the ethylacetate fraction of the whole plant part extracted with 50% aqueous ethanol. This molecular formula of compound 1 obtained as a yellow amorphous powder was determined as [C.sub.29][H.sub.36][O.sub.15] by El-MS. This El-MS (negative-ion mode) of 1 displayed a pseudo-molecular ion peak [[M-H].sup.+] at m/z 623. IR [[upsilon].sub.max] KBr (cm).sup.-1): 3424, 2937, 1825, 1594, 1363. and [sup.1.H-NMR spectrum exhibited the presence of eight olefinic protons, one methyl doublet signals, two anomeric proton signals ([delta]HI' 4.39 and [delta]HI" 5.20) confirmed by two anomeric carbons ([delta]CI' 103.20 and [delta]CI" 102.03) indicating that the compound contains two sugar moieties (glucose and rhamnose). Furthermore, the [sup.1.H-NMR spectrum indicated the presence of two ABX coupling systems (ranging from [delta] 6.58 to 7.08), one belonging to the trans-caffeoy 1 moiety ([[delta].sub.C2"' 7.08, [delta].sub.C5"' 6.80, [delta].sub.C6"' 6.97)) and the other belonging to the 3, 4-dihydroxyphenyl moiety ([[delta].sub.C2].6.72, [[delta].sub.C5] 6.69, [[delta].sub.C6].6.58)). The [.13.sup]C-NMR and DEPT spectra further indicated that the compound 1 contains two sugar moieties, one methyl belonging to rhamnose sugar moiety, one non-oxygenated methylene, two oxygenated methylene of which one belongs to the glucose moiety and the other oxygenated -[CH.sub.2-] carbon (C-[alpha]) of the side chain of 3, 4-dihydroxyphenyl moiety is clearly apparent between the sugar carbons, the anomeric carbons at [delta]C 102.03, [delta]C 103.20, eight methines ([delata]c-2 116.15, [delta]c-5 115.35, [delta]c-6 120.30, [delta]c-22"' 114.29, [delta]c-5"' 115.55, [delta]c-6"' 122.23, [delta]c-[beta]"' 147.03, [delta]c-y"' 113.73) and seven quaternary carbons ([delta]c-1 130.52, [delta]c-3 143.66, [delta]c-4 145.12, Sc-a'" 167.33, Sc-V" 126.69, [delta]c-3"' 145.82, [delta]c-4 148.77).
The structure of the trans-caffeoyl moiety was established by [.sup.1]H NMR data showing two vicinal olefinic protons [delta]H 7.61 d,J = 15.87Hz and [delta]H 6.29, d, j = 15.89 Hz.
Crucial [.sup.1]H-[.sup.13]C long-range correlations as determined from HMBC spectrum included cross-link of the proton at [delta] 4.94 (H-4) of the glucose to C-[alpha]' ([delta] 167.33), C-3' ([delta] 80.67), C-5' ([delta] 75.21), C-6' ([delta] 61.38) and the proton at [delta] 4.39 (H-1) of the glucose to C-[alpha] ([delta] 71.25) and the proton at [delta] 5.20 (H-1) of the rhamnose to C-3' ([delta]) 80...67) of the glucose.
The coupling constant ([J.sub.H1-H2] = 7.86 Hz) of the glucose anomeric proton resonating at 4.39 ppm is in accordance with a [beta]-configuration. Furthermore, it is a well-known observation that glucobioses, only the anomeric proton can be observes above 4 ppm. Thus, the higher shift of the 4.94 ppm triplet attributed to H-4 of the glucose, also reflects the caffeic acid substitution on C-4 of the glucose. No other effects were observed on the remaining protons by this substitution. Upon consideration of the above results, compound 1 was determined as Russetional glycoside isolated from R. equisetiformis.
The molecular formula of compound 2 obtained as a yellow amorphous powder was determined as [C.sub.23][H.sub.26][O.sub.10] by EI-MS (negative-ion mode) of 1 displayed a pseudo-molecular ion peak [[M-H].sup.plus] at m/z 461.
The [.sup.1]H-NMR spectrum exhibited the presence of eight olefinic protons, one methyl doublet signals, one anomeric proton signal ([[delta].sub.HI]" 5.21) confirmed by one anomeric carbon ([[delta].sub.CI"] 102.07) indicating that the compound contains one sugar moiety (Rhamnose). Furthermore, the [.sup.1]H-NMR spectrum indicated the presence of two ABX coupling systems (ranging from [delta] 6.79 to 7.08), belonging to the trans-caffeoyl moeity ([delta.sub.C2"'] 7.08, [delta.sub.C5"'] 6.79, [delta.sub.6.98]). The [.sup.13]C NMR and DEPT spectra further indicated that the compound 2 contains one sugar moiety, one methyl belonging to rhamnose sugar moiety, the anomeric carbons at [delta]c 102.07, eight methines ([deltac-2 114.30, [delta]c-5 115.50, [delta]c-6 120.26, [delta]2"' 114.26, [delta]c-5"' 115.53, [delta]c-6"' 122.20. [[delta]c-[beta]"' 145.83, [delta]c-y"' 113.73) and seven quaternary carbons ([delta]c-1"'126.69, [delta]c-3"' 145.14, [delta]c-4"' 148.79).
The structure of the trans-caffeoyl moiety was established by [.sup.1]H-NMR data showing two vicinal olefinic protons [[delta].sub.H] 7.59 d, J= 14.91 [H.sub.z] and [[delta].sub.H] 6.29, J = 15.87 [H.sub.z.] Based on these data the structure of compound 2 was established as 2-(3, 4-dihydroxylphenyl)-ethyl-5-O-trans-caffeoly)-[beta]-D-rhamnopyranoside and was given the name Russelianoside A.
In addition to the preliminary phytochemical screening of Russelia equisetiformis for triterpenes, and cardiacglycoside, the present study provides the first report on the isolation of the phenolic compounds from the whole plant extracts. The compound exhibits a high antinociceptive effect as compared to standard drugs. The spectrum of activity of the isolated compound suggests the possible utilization of Russelia equisetiformis as a valuable crude drug.
The authors are grateful to Prof. A.O. Ogundaini, Professor of Pharmaceutical Chemistry, Faculty of Pharmacy, Obafemi Awolowo University, and Ile-ife, Nigeria for making his laboratory available for the isolation work and NABSA for providing NMR facilities.
Awe, E.O., Adeniyi, O.A., Olajide, O.A., Wahkeel, O.K., Abiodun, O.O., Makinde, J.M., 2004a. Studies on antibacterial properties at Russelia equisetiformis (Schlecht and Chan) Scrophulariacae. Sci. Focus 6, 131-133.
Awe, E.O., Makinde, J.M., Olajide, O.A., Wahkeel, O.K., 2004b. Evaluation of anti-inflammatory and analgesic activity of Russelia equisetiformis. Inflammopharmacology 12, 399--405.
Burns, D., Reynolds, W.F., Reese, P.B., Enriquez, R.G., 2001. Phytochemical screening of Russelia equisetiformis. Maqgn Reson. Chem. 387, 488-493.
Collier, H.O.J., Dineen, L.C., Johnson, C.A., Schneider, C., 1968. Abdominal constriction response and its suppression by analgesic drugs in mouse. Br. J. Pharmacol. Chemother. 32, 295-310.
Janssen, P.C., Niemegeers, Dony, J., 1963. The inhibitory effects of phentannyl (R-4263) and other morphine-like analgesics on the warn water induced tail withdrawal reflex in rats. Arzneim. Foursch. 13, 502-507.
Keele, C.A., 1969. Clinical and pathological aspects of kinins in man. Proc. R. Soc. Biol. 173, 361-369.
Keele, C.A., Armstrong, D.M., 1964. Substances Causing Pain and Itch. Edward Arnold, London.
Koster, R.M., Anderson, M., De-beer, E.J., 1959. Acetic-acid for analgesic screening. Fed. Proc. 18, 412.
E.O. Awe (a) *, A. Adeloye (b), T. Idowu (c) O.A. Olajide (d), J. Makinde (d)
(a) Department of Veterinary Pharmacology and Therapeutics, College of Veterinary Medicine, University of Agriculture, Abeokuta, Nigeria
(b) Department of Chemistry, Faculty of Science, Obafemi Awolowo University, Ile-ife, Nigeria
(c) Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Obafemi Awolowo University, Ile-ife, Nigeria
(d) Department of Pharmacology and Therapeutics, College of Medicine, University of Ibadan, Nigeria
Received 5 December 2006; accepted 11 January 2007
* Corresponding author.
E-mail address: firstname.lastname@example.org (E.O. Awe).
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|Author:||Awe, E.O.; Adeloye, A.; Idowu, T.; Olajide, O.A.; Makinde, J.|
|Publication:||Phytomedicine: International Journal of Phytotherapy & Phytopharmacology|
|Date:||Apr 1, 2008|
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