Bioactive compounds from Tipuana tipu growing in Egypt

Journal of American Science 2013;9(10) http://www.jofamericanscience.org Bioactive compounds from Tipuana tipu growing in Egypt Yhiya M. Amen*, Amani M. Marzouk, Mona G. Zaghloul, Mohamed S. Afifi Pharmacognosy Department, Faculty of Pharmacy, Mansoura University, 35516, Egypt *yhiyaamen@gmail.com Abstract: Six compounds were isolated from methanolic extracts of the leaf and the bark of Tipuana tipu growing in Egypt. The isolated compounds were identified as 1-Nonadecanol (1), β-sitosterol (2), Alpinumisoflavone (3), βsitosterol glucoside (4), Protocatechualdehyde (5) and Protocatechuic acid (6), based on different spectroscopic data (UV, IR, NMR and MS). This is the first report for the isolation of compounds 1 & 3-6 from Tipuana tipu. Compound 1 is isolated for the first time from family Fabaceae. Compounds 2 and 3 showed a promising anticancer activity, while compound 5 displayed a remarkable in vivo anti-inflammatory activity compared to indomethacin. [Amen YM, Marzouk AM, Zaghloul MG, Afifi MS. Bioactive compounds from Tipuana tipu growing in Egypt. J Am Sci 2013;9(10):334-339]. (ISSN: 1545-1003). http://www.jofamericanscience.org. 44 Keywords: Tipuana tipu, 1-Nonadecanol, Alpinumisoflavone, Protocatechualdehyde, Protocatechuic acid, anticancer, anti-inflammatory. 1. Introduction Family Leguminosae (Fabaceae) is one of the three largest families of flowering plants, exceeded only by the Compositae and Orchidaceae (Harborne et al., 1971). This family comprises 727 genera and ca. 19325 species. It was divided into three subfamilies; Mimosoideae, Caesalpinioideae and Papilionoideae (Lewis et al., 2005). The Genus Tipuana (Benth.) Benth. (subfamily Papilionoideae) was originally assigned to Machaerium, but now is excluded from that genus (Ducke, 1930). It comprises only one species: Tipuana tipu (Hickey and king, 1997). Tipuana tipu (Benth.) Lillo [syn T. tipu (Benth.) Kuntze] is a South American tree which is indigenous to Argentina, Uruguay, Paraguay, Bolivia and Brazil. The tree is mainly an ornamental tree. It can be used as a supplementary food to small ruminants (e.g. goats), mainly the dried leaves proved to have high nutritive value (Norton and Waterfall, 2000). Plants in genus Machaerium are used in South America by indigenous population to treat diarrhea (Heinrich et al., 1992), menstruation cramps (Ginzbarg, 1977), coughs (Joly et al., 1987) and aphthous ulcers of the mouth. Certain Machaerium species were reported to have antimicrobial (Waage et al., 1984), antigiardial (ElSohly et al., 1999) and antitumor activities (Seo et al., 2001). Little phytochemical studies were carried out on T. tipu including those of Braga De Oliveira et al. (1971) who reported the isolation of formononetin, βsitosterol and stearic acid, while Kansoh et al. (2009) studied the volatile oil content and lipoidal matter of the flowers and pods. The present research was conducted to isolate the chemical constituents of Tipuana tipu growing in Egypt and evaluate its potential use in pharmacy and medicine. http://www.jofamericanscience.org 2. Results and Discussion 2.1. Characterization of compounds Six compounds (Fig. 1) were isolated from methanolic extracts of the leaf and the bark of Tipuana tipu (Benth.) Lillo growing in Egypt. Compounds 2 and 4 were identified by m.p., cochromatography with authentic samples and comparing to literature data. Compound 1 was isolated as colorless needle crystals. Its EI-MS showed a quasi-molecular ion peak at m/z 285 [M+ H]+, compatible with the molecular formula C19H40O. APT spectrum showed nine signals, which were discriminated into a CH3 resonance at δ 14.1 and a signal due to a primary alcoholic group at δ 62.5. The other signals were assigned to aliphatic CH2 groups, which resonated at δ 22.7 – 32.5 (see experimental section 3.3.1). These data suggested compound 1 to be an aliphatic straight chain primary alcohol. 1H-NMR spectrum revealed a downfield signal at δ 3.47 (t, J = 6.9 Hz, 2H) assigned for CH2OH group that was confirmed by absorption band at 3423 cm-1 (alcoholic OH stretching) and 1062 cm-1 (C-O stretching of primary alcohol) in IR spectrum (Silverstein et al., 2005), in addition to a signal at δ 0.78 (t, J = 7.3 Hz, 3H) assigned for the terminal CH3 group. An upfield broad signal resonated at δ 1.16 (34H, m) was assigned to H3-H18 and confirmed by absorption band at 720 cm-1 (C-H bending of repeated methylene groups of straight chain paraffins) in IR spectrum (Silverstein et al., 2005). From the above data, compound 1 was concluded to be 1-Nonadecanol which was previously isolated from Convolvulus lanatus, family Convolvulaceae (El-Nasr et al., 1984), but isolated here for the first time from T. tipu as well as from family Fabaceae. 334 editor@americanscience.org Journal of American Science 2013;9(10) Compound 3 was isolated as yellow needles. EI-MS spectrum (positive mode) showed a molecular ion peak at m/z 336 [M]+ corresponding to a molecular formula C20H16O5. It displayed an intense UV absorption maximum at max 283 nm, typical of an isoflavone nucleus (Mabry et al., 1970). This was supported by 1H- and 13C-NMR spectral data (Table 1). In 1H-NMR spectrum, the most downfield of aromatic signals (δ 7.80, s, H-2) and signals at δ 152.6 for C-2, 123.1 for C-3 and 181.0 for C-4 in 13CNMR spectrum (Table 1) (Agrawal et al., 1989; Mabry et al., 1970). A pair of doublets in the 1H-NMR spectrum centered at δ 5.59 and 6.69 (J = 10.1 Hz) together with a sharp singlet integrated for six protons at δ 1.45, suggested the presence of 2'', 2''- dimethylpyrano- substituent and the set of signals at δ 28.4, 77.4, 128.3 and 115.6 in 13C-NMR spectrum provided further support for the presence of gemdimethylpyran moiety. The failure of NaOAC to produce a significant bathochromic shift (max 282 nm) in the UV absorption maximum compared to MeOH spectrum, together with the 1H- NMR singlet at δ 6.31 assigned to H-8 indicated a trisubstituted A ring. The signals at δ 106.0, 159.6 and 95.0 could be assigned to carbons C-6, C-7 and C-8 respectively. Thus, the gem-dimethylpyran moiety was suggested to be fused in a linear manner on ring A not in angular position, and this assignment was supported by inspection of the characteristic 13C-NMR signals for C-5, C6, C-7 and C-8 at the two possible positions (Agrawal et al., 1989). The 5-hydroxy substituent was deduced from the bathochromic shift (+12 nm) in the UV absorption maximum observed with AlCl3 after 30 min. 1H-NMR spectrum also displayed a pair of broad doublets (J = 8.2 Hz), each integrating for two protons, at δ 6.85 and 7.35, which were assigned to H-3΄, H-5΄ and H-2΄, H-6΄ of the para-disubstituted ring B. The relatively upfield resonance (δ 6.85) of H-3΄ & H-5΄ suggested the presence of an oxygenated substituent at C-4΄, most probably as a hydroxyl group. The structure of (3) was confirmed by comparison with the reported data (Stewart et al., 2000) as alpinumisoflavone. It was first isolated by Jackson et al. (1971) from Laburnum alpinum J. Presl. (Fabaceae).It was further reported in different species of the genus Erythrina (Fabaceae): from E. lysistemon, E. stricta, E. variegata, E. poeppigiana, E. senegalensis, E. indica and E. arborescens (Nkengfack et al., 2001; Rahman et al., 2010); from other genera belonging to family Fabaceae as Calopogonium mucunoides, Crotalaria ferruginea and Millettia thonningii (Perrett et al., 1995). Alpinumisoflavone was also reported from family Moraceae from Ficus chlamydocarpa, F. nymphaefolia and from Rinorea welwitschii, family Violaceae (Stewart et al., 2000). Table 1.13C and 1H-NMR spectral data of compound 3* 13 1 C H # APT 2 CH 152.7 7.80, s 3 C 123.1 -------4 C 181.0 -------http://www.jofamericanscience.org http://www.jofamericanscience.org 5 6 7 8 9 10 1' 2' 3' 4' 5' 6' 2'' 3'' 4'' Me2-2'' C C C CH C C C CH CH C CH CH C CH CH CH3 156.0 106.0 159.6 95.0 157.0 105.7 123.6 130.4 115.7 157.4 115.7 130.4 77.4 128.3 115.6 28.4 --------------6.31, s ---------------------7.35 (d, J= 8.2) 6.85 (d, J= 8.2) -------6.85 (d, J= 8.2) 7.35 (d, J= 8.2) -------5.59 (d, J= 10.1) 6.69 (d, J= 10.1) 1.45, s *Chemical shifts (δ) are expressed in ppm and coupling constants (J) in Hz. 13C and 1H-NMR were measured in CDCl3 at 100 and 400 MHz, respectively. Compound 5 was isolated as colorless needles. Absorption bands at 1595 and 1451 cm-1 (C=C aromatic) in IR spectrum and UV absorption maxima at max 313, 278 and 233 nm suggested the presence of aromatic ring (Silverstein et al., 2005). 1H-NMR spectrum (Table 2) clearly indicated the presence of three aromatic signals in the range of δ 6.91 – 7.32. This was confirmed by 13C-NMR spectrum which revealed the presence of signals due to a benzene ring resonating between δ 115.2 – 153.5, in addition to a signal at δ 192.9 assigned to a carbonyl carbon of aldehydic group.This was supported by absorption bands at 1670 cm-1 (aryl aldehyde), a pair of bands at 2734 and 2853 cm-1 (C-H stretching vibrations in aldehydes) in IR spectrum. A doublet observed at δ 6.91 (J= 8.0 Hz, 1H) was coupled to another doublet at δ 7.31 (J= 8.0 Hz, 2H) and could be attributed to two ortho-coupled protons at C-5 and C-6. The other singlet at δ 7.32 which was coincident with H-6 (d, J= 8 Hz) could be assigned to proton at C-2 and supported by two signals in 13C-NMR spectrum for two oxygenated carbons at δ 147.0 and 153.5. By comparing these NMR spectral data (Table 2) with those reported by Pauletti et al. (2012), the structure of compound 5 was confirmed to be protocatechualdehyde which was previously isolated from the leaf of Musanga cecropioides, family Urticaceae (Ayinde et al., 2007). Compound 6 was isolated as white crystals. EI-MS spectrum showed a molecular ion peak at m/z 154 corresponding to a molecular formula C7H6O4. The UV absorption maxima in MeOH at max 307, 298 and 260 nm and the absorption bands in IR at 1600 and 1468 cm-1 suggested also an aromatic nucleus. All the NMR spectral data of 6 (Table 2) almost resembled those of 5 described above. The differences appeared in ¹³C-NMR spectrum which displayed a quaternary carbon signal at δ 170.7 assigned to a carbonyl function of carboxylate supported by 335 editor@americanscience.org Journal of American Science 2013;9(10) IR absorption bands at 3213 cm-1 (br, OH) and 1677 cm-1 C=O carboxylate) (Silverstein et al., 2005). Based on 1HNMR spectrum of both compounds, the substitution pattern of the aromatic ring remains the same except for the presence of a carboxyl group replacing the aldehyde group. The identity of (6) was confirmed as Protocatechuic acid through comparison with published literature data (Nova et al., 2012). Table 2.13C and 1H-NMR spectral data of compounds 5 and 6** # 5 6 13 1 13 1 C H C H 1 130.6 -------123.7 -------2 116.0 7.32, s 117.7 7.46, s 3 147.0 -------145.9 -------4 153.5 -------151.2 -------5 115.2 6.91 (d, J= 8.0) 115.6 6.81 (d, J= 8.0) 6 126.2 7.31 (d, J= 8.0) 123.7 7.44 (d, J= 8.0) 7 192.9 -------170.7 -------- ** Chemical shifts (δ) are expressed in ppm and coupling constants (J) in Hz. 13C and 1H-NMR were measured in CD3OD at 100 and 400 MHz, respectively. 2.2. Biological activity 2.2.1 Anticancer activity β-sitosterol (2) showed a weak activity towards CNS SF-539, ovarian OVCAR-4 and renal UO-31 cancer cell lines with cell promotion percentages of 82.09, 83.24 and 85.91% respectively, while it showed a fairly good activity against Non-small cell lung cancer HOP-62 cell line with cell promotion percentage 73.78%. Alpinumisoflavone (3) was active against different leukemia cell lines. It showed a high activity against CCRF-CEM (acute lymphoblastic leukemia, nuclear lysate) with cell promotion percentage 48.83%, while it was less active against MOLT-4 (acute lymphoblastic leukemia, of peripheral blood origin) and HL-60(TB) (promyelocytic leukemia) cell lines with cell promotion percentages of 73.85% and 84.51%, respectively. It also, exhibited a good activity against renal SN12C cancer cell line with cell promotion percentage 67.33 % and a weak activity against breast MCF7 cancer cell line with cell promotion percentage of 82.82%. 2.2.2 Anti-inflammatory activity The results of the anti-inflammatory screening (Table 3) revealed variable activities of the different compounds. Protocatechualdehyde (5) displayed a remarkable antiinflammatory activity comparable to that of indomethacin while protocatechuic acid (6) was less active. 1Nonadecanol, β-sitosterol, alpinumisoflavone and βsitosterol glucoside displayed weak activity compared to the standard drug. http://www.jofamericanscience.org http://www.jofamericanscience.org Table 3. Results of screening of the anti-inflammatory activity of the isolated compounds† Treatment Control only) (Carrageenan % of increase in paw thickness 141.38 ± 4.33 % of inhibition of edema - Indomethacin (10 mg/kg) 79.44 ± 1.44 41.67 ± 0.21 1-Nonadecanol 106.5 ± 2.34 25.5 ± 0.22 β-sitosterol 97.35 ± 1.84 30.17 ± 0.17 Alpinumisoflavone 103.27 ± 3.83 β-sitosterol glucoside 124.69 ± 4.05 29.17 ± 0.17 7.83 ± 2.83 Protocatechualdehyde 69.54 ± 0.56 42.17 ± 0.17 Protocatechuic acid 90.42 ± 1.1 35.17 ± 0.17 † Values are expressed as mean ± SEM, n = 6 in each group, P<0.01 compared with the control. 3. Experimental 3.1. General 1 H and 13C NMR spectra were recorded on a Bruker DPX-400 spectrometer (400 and 100 MHz for 1H and 13C respectively). The melting points were measured on melting point apparatus Fisher-johns scientific Co., USA and were uncorrected. UV spectra were performed on a Shimadzu UV-1201 spectrophotometer (Japan). IR spectra were recorded in KBr disks using Infra-red spectrophotometer, Mattson 5000 FTIR (England). EI mass spectra (positive mode) were carried out on a JOEL JMS600 spectrometer (Japan). Male Wistar albino rats were provided by the Experimental Animal House, Pharmacology Department, Faculty of Pharmacy, Mansoura University. λ-carrageenan from Sigma-Aldrich (USA). All other chemicals used were of analytical grade.3.2. Plant material The leaves were collected from trees growing on Mansoura University campus, Egypt on May, 2011. The freshly collected leaves were air-dried in shade at room temperature. The plant identity was confirmed by staff members at Department of Horticulture, Faculty of Agriculture, Mansoura University, Egypt. A representative specimen deposited at the Department of Pharmacognosy, Faculty of Pharmacy, Mansoura University. The bark was collected from the same trees on August, 2012. 336 editor@americanscience.org Journal of American Science 2013;9(10) http://www.jofamericanscience.org Figure 1. Structures of isolated compounds (1) (2) (3) (4) (5) 3.3. Extraction and isolation The dried powdered leaves (6000 g) were percolated with MeOH (9 x 10 L). The combined extracts were concentrated to a syrupy consistency under reduced pressure at 40 oC. The residue (629.8 g) was suspended in distilled water and extracted successively with petroleum ether, methylene chloride, ethyl acetate and then finally with n-butanol. The different extracts were, separately, evaporated under reduced pressure to obtain petroleum ether fraction (fraction A, 70 g), methylene chloride fraction (fraction B, 7 g), ethyl acetate fraction (fraction C, 15 g) and nbutanol fraction (fraction D, 30 g). The dried powdered bark (8000 g) was percolated with MeOH (8 x 18 L). The combined extracts were concentrated to a syrupy consistency under reduced pressure at 40 oC. The residue (170 g) was suspended in distilled water and extracted successively with http://www.jofamericanscience.org (6) petroleum ether, methylene chloride, ethyl acetate and then finally with n-butanol. The different extracts were, separately, evaporated under reduced pressure to obtain petroleum ether fraction (fraction E, 27 g), methylene chloride fraction (fraction F, 22 g), ethyl acetate fraction (fraction G, 9 g) and n-butanol fraction (fraction H, 28 g). Fraction A was subjected to silica gel column chromatography and eluted with Petroleum ether – EtOAc gradient (0-80%). These subfractions were pooled based on similar Rf values and purified by rechromatography on silica gel columns and PTLC and by repeated crystallization to afford 1-Nonadecanol 1 (59 mg), β- sitosterol 2 (250 mg), Alpinumisoflavone 3 (26 mg) and β-sitosterol glucoside 4 (232 mg). Fraction G was subjected to silica gel column chromatography and eluted with Petroleum ether – 337 editor@americanscience.org Journal of American Science 2013;9(10) EtOAc gradient (50-100%) and EtOAc – MeOH (060%). Collected fractions were pooled based on similar Rf values and subjected to purification through column chromatography and repeated crystallization to afford Protocatechualdehyde 5 (24 mg) and Protocatechuic acid 6 (28 mg). 3.3.1 1-Nonadecanol (1) colorless needle crystals (Rf 0.51) on precoated silica gel plates GF254 using 10% EtOAC /pet. ether and colored brown upon spraying with vanillin/ H2SO4 spray reagent and heating at 110 ˚C for 1 min., m.p. 70-71˚C (lit. 60-61˚C), 1H-NMR (CDCl3, 400 MHz, δ ppm): 3.47 (t, J = 6.9 Hz, H-1), 1.44 (m, H-2), 1.16 (m, H-3 to H-18), 0.78 (t, J = 7.3 Hz, H-19).13C-NMR (CDCl3, 100 MHz, δ ppm): 62.5 (C-1), 32.5 (C-2), 31.9 (C-3), 29.4 – 29.7 (C-4 to C16), 25.8 (C-17), 22.7 (C-18), 14.1 (C-19). 3.3.2 Alpinumisoflavone (3) yellow needles, (Rf 0.22) on precoated silica gel plates GF254 using 20% EtOAC /pet. ether, m.p. 210-212˚C (lit. 210-212˚C), UV λmax MeOH 283 nm, λmax NaOAC 282 nm, λmax AlCl3 295 nm after 30 min.; 1H and 13C NMR (Table 1). 3.3.3 Protocatechualdehyde (5) colorless needles, (Rf 0.50) on precoated silica gel plates GF254 using 10% MeOH /CH2Cl2, m.p. 152-154 oC (lit. 152-154 ˚C), UV λmax MeOH 313, 278, 233 nm; IR (KBr, νmax): 3439, 2853, 2734, 1670, 1595, 1451 cm-1; 1H and 13C NMR (Table 2). 3.3.4 Protocatechuic acid (6) white crystals, (Rf 0.30) on precoated silica gel plates GF254 using 10% MeOH / CH2Cl2, m.p. 196-198 oC (lit. 198-200˚C). UV λmax MeOH 307, 298 and 260 nm; IR (KBr, νmax): 3213, 1677, 1600, 1468 cm-1; 1H and 13C NMR (Table 2). 3.4. Biological activity 3.4.1. Anticancer activity Compounds 2 and 3 were submitted to National Cancer Institute (NCI), USA, for evaluating their antitumor activities under the Developmental Therapeutic Program (DTP). The operation of this screen utilizes 60 different human tumor cell lines. The compounds were tested at a concentration of 10-5 M in the full NCI 60 cell-line panel. The cultures were incubated for 48 h. End point determinations were made with a protein binding dye, Sulforhodamine B. Results for each compound are reported as mean of the percentage growth of the treated cells when compared to the untreated control cells (Monks et al., 1991; Grever et al., 1992; Boyd and Paull, 1995).3.4.2. Antiinflammatory activity The antiinflammatory activity of the isolated compounds (1-6) was carried out using carrageenan-induced rat paw edema model (Winter et al., 1962) by employing carrageenan solution as the phlogistic agent. Male Wistar albino rats weighing 150–180 g were kept in the animal house under standard conditions of light and temperature with free access to food and water. After overnight fasting, the animals were randomly divided into three groups each http://www.jofamericanscience.org http://www.jofamericanscience.org consisting of six rats. The first group received only carrageenan. Another group received the standard drug indomethacin (10 mg/kg, i.p., 1% w/v in normal saline). The last group of rats was administered the test compounds in equal doses (25 mg/kg, i.p.; w/v). Thirty minutes after administration of the standard drug and test compounds, 0.1 ml of 1% w/v of λ-carrageenan (Type IV) in sterile normal saline was injected into the subplanter region of the right hind paw of each rat. After 4 hr, the paw thickness was measured with electronic digital calipers, and the difference in the paw thickness between the injected and the control was recorded for each rat. The edema was expressed as the percentage of increase in the paw thickness compared to the positive control and the percentage of inhibition of edema for each group. Data were expressed as mean ± standard error (S.E.M.). The statistical significance of difference between groups were assessed by means of analysis of variance (ANOVA) followed by Dunnet’s test. Corresponding author: Yhiya M. Amen, Department of Pharmacognosy, Faculty of Pharmacy, Mansoura University, Mansoura, 35516, Egypt. E-mail: yhiyaamen@gmail.com References 1. Agrawal, P., Thakur, R., Bansal, M. 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