Pak. j. sci. ind. res. Ser. A: phys. sci. 2018 61A(3) 126-131 Studies of Chemical Constituents from Eremostachys loasifolia Muhammad Imranab*, Uzma Rasheed Mughalc, Muhammad Iqbald, Riaz Hussaine, Wasif Iqbalf, Rafy Yaqeeng and Sadia Ferheeng a Department of Chemistry, Ghazi University Dera Ghazi Khan, Pakistan b School of Chemistry, University of Bristol, United Kingdom c Departmentof Chemistry, GC University Faisalabad, Layyah Campus, Pakistan d Department of Biochemistry& Biotechnology, The Islamia University of Bahawalpur, Bahawalpur, Pakistan e Departmentof Chemistry, University of Education Lahore, D. G. Khan Campus, Pakistan f Science of Dental Materials, Mohammad Bin Qasim Medical and Dental College, Port Qasim, Karachi, Pakistan g PCSIR Laboratories Complex, Karachi-75280, Pakistan (received November 9, 2017; revised August 8, 2018; accepted August 15, 2018) Abstract. The chemical constituent analysis on the whole plant Eremostachys loasifolia (Family: Lamiaceae) resulted 13 compounds of flavonoids class for the first time from this species namely Loasifolin (1), Eremoside A (2), Eremoside B (3), Kaempferol (3,4',5,7-tetrahydroxyflavone) (4), 5-Hydroxy-6,7,8,4'tetramethoxyflavone (5), 5-Hydroxy-3',4',6,7,8-pentamethoxyflavone (6), Apigenin (4',5,7-trihydroxyflavone) (7), Luteolin (3',4',5,7-tetrahydroxyflavone) (8), Apigenin 7-O-b-D-glucopyranose (9) 4-Methyl Kaempferol (3,5,6-trihydroxy-4methoxy flavone) (10) 5-Hydroxy-7,4-dimethoxy flavones (11), 6,7-Dimethoxy-4,5hydroxyflavone (12), 3,5,7-Trihydroxy-3´,4´-dimethoxyflavone (13). Further, all isolated compounds showed antioxidant activity. The structures of isolated compounds were determined using different spectroscopic techniques including NMR, UV/Vis., IR and MS. Keywords: Eremostachys loasifolia, antioxidant activity, flavonoids pounds are Loasifolin (1), Eremoside A (2), Eremoside B (3), Kaempferol (3,4',5,7-tetrahydroxyflavone) (4), 5-Hydroxy-6,7,8,4'-tetramethoxyflavone (5), 5-Hydroxy3',4',6,7,8-pentamethoxyflavone (6), Apigenin (4',5,7trihydroxyflavone) (7), Luteolin (3',4',5,7-tetrahydroxyflavone) (8), Apigenin 7-O-b-D-glucopyranose (9) 4'Methyl Kaempferol (3,5,6-trihydroxy-4methoxy flavone) (10) 5-Hydroxy-7,4-dimethoxy flavones (11), 6,7-Dimethoxy-4,5-hydroxyflavone (12) and 3,5,7-Trihydroxy3´,4´-dimethoxyflavone (13). The structures of all isolated compounds are showed in Fig. 1. Introduction The plant genus Eremostachys is member of the family Labiatae (Lamiaceae) which comprises of eighty species and distributed in mostly Afghanistan and Russia region (Li and Hedge, 1995). Plants of this genus are used for medicinal purpose in China and Asia for the purpose of local analgesic, for the treatment of allergies, headache, liver diseases and skin diseases (Ali and Nasir, 1990). The genus compounds also show strong antidepressant activity, cytotoxic activity and free radical scavenging activity (Delazar et al., 2004a; 2004b). Eremostachys loasifolia is one of the important species of this genus that can be found in Balochistan, and in the northern province of Pakistan. The previous study of the genus showed the presence of essential diterpenes, iridoids, fatty acids, oils, flavonoids, and aromatic glucosides (Modaressi et al., 2009; Muhammad et al., 2006; Said et al., 2002). In this plant previously, no systematic pharmacological studies have so far been carried out and therefore chemical analysis of this species was studied because of its medicinal importance. From the results of present experimental work thirteen flavonoids isolated for the first time from E. loasifolia. The com- Materials and Methods The Al precoated sheets (Merck diameter 20 × 20 cm,) with silica gel were used for TLC and stationary phase in glass column for chromatographic (230-400 and 80200 mesh size) silica gel was used. Hitachi UV-3200 spectrometer for the TLC spot location determination was used under wavelength at 254 and 366. Polarimeter DIP-360 JASCO was used for the determination of optical rotations of the compounds. Infra Red 460 Shimadzu spectrometer were recorded for IR. JMSHX-110 mass spectrometers were used for EI-MS and HR-EI-MS measurement, Bruker spectrometers (400 MHz) for the the proton-NMR,13C-NMR. The activity *Author for correspondence: E-mail: imranchemist@gmail.com 126 127 Chemical Constituents from Eremostachys loasifolia i.e., antioxidant of all isolated compounds1-13 was carried out by the same protocol as described in discussion (Wavy et al., 2003; Yoshi Rawa et al., 2002). chloroform:methanol was re-chromatographed over FCC and eluted with chloroform:methanol to obtain (2) and (3). Plant material. Eremostachys loasifolia complete plant was collected from Lakpass, Quetta Valley, Balochistan, Pakistan and identified by Balochistan University, Plant Taxanomist, where No. el. Rbt. 01. 2005 (specimen voucher) was alloted in the departmental herbarium. Loasifolin (1), Eremoside A (2), Eremoside B (3), for these new compounds spectroscopic data are already published (Mughal et al., 2010) but activity are reported first time. The data including both physical and spectral of compounds 4-13 were comparable with the literature values (Imran et al., 2012; Ahmed et al., 2006; Sadikum et al., 1980; Redalli et al., 1980; Dawson et al., 1966). Extraction, fractionation and purification. Freshly collected Eremostachys loasifolia plant (20 kg) was dried and extracted at r.t with ethanol (4 ´ 30 L, 08 days each). 750 g residue was attained from these combined ethanolic portion after reduced pressure evaporation (400 g). The ethanolic plant residue 750 g was dissolved in water (500 mL) and further extracted into petroleum ether (80 g), CH2Cl2 (65 g), ethyl acetate (105 g), n-butanol (164 g) and H2O soluble portion. Ethyl acetate (105 g) soluble portion was exposed to chromatography separation FCC as stationary phase silica gel (different mesh size) and mobile phase petroleum ether, chloroform and methanol depending upon polarity of constituents. From first large column five major fractions obtained namely E1 to E-5. The E1 fraction was chromatographed over FCC silica gel and obtained three fractions (A-1 to A-3). The (4.0:6.0) petroleum ether:CHCl3 fraction A-1 on further chromatography gives (4). The E-2 portion (7.0:3.0) petroleum ether-CHCl3 was finally purified by CC via solvent (2.3:7.7) system petroleum ether -CHCl3 giving compounds 5 and 6. The petroleum ether-CHCl3 (1.0:9.0). The portion E3 was chromatography using eluting system (1.5:8.5) with petroleum ether -CHCl3 to pay for 7 and two compounds mixture that was separated by preparative TLC (1.8: 8.2) solvent petroleum ether -CHCl3 to give compound 8 and 9. The (9.8:0.2) CHCl3: MeOH fraction A-2, gives compound 10. The third fraction hexane and chloroform (8.5:1.5) upon further FCC analysis gives compound 11. The fraction E-4 obtained from (9.5:0.6) Chloroform-MeOH was chromatographed using eluent system (9.3:0.7) Chloroform-MeOH to afford 12 (9 mg) and (9.2:0.8) 13 (11 mg). The fraction E-5, obtained from pure chloroform was subjected to FCC give rise to three sub fractions. The first fraction further chromatographed by (7.5:2.5) of chloroform: methanol mobile phase solvent system gives pure compound 1. The second portion (9.8:0.2) Results and Discussion The chemical constituents were extracted with ethanol and then crude extract (ethanol) was suspended in water and partitioned into petroleum ether, CH2Cl2, EtOAc, n-BuOH and water fractions on the basis of respective solubility. Further analysis especially chromatographic were performed on EtOAc portion because of its strong crude activity. Series of column and flash chromatographic analyses were performed on the EtOAc soluble part and as a result 13 compounds separated and identified. These are namely, Loasifolin (1), Eremoside A (2), Eremoside B (3), Kaempferol (3,4',5,7-tetrahydroxy-flavone) (4), 5-Hydroxy-6,7,8,4'-tetramethoxyflavone (5), 5-Hydroxy-3',4',6,7,8-pentamethoxy-flavone (6), Apigenin (4',5,7-trihydroxy-flavone) (7), Luteolin (3',4',5, 7-tetrahydroxy-flavone) (8), Apigenin 7-O-bD-glucopyranose (9) 4-Methyl Kaempferol (3,5,6trihydroxy-4-methoxy-flavone) (10) 5-Hydroxy-7,4'dimethoxy-flavones (11), 6,7-Dimethoxy-4',5-hydroxyflavone (12), 3,5,7-Trihydroxy-3´,4´-dimethoxy-flavone (13). The purified flavonoids showed antioxidant properties (Table 1). The structures of all isolated compounds were shown in Fig. 1. The structures of compounds 1-3 were already published in detail with our previous articles. 5 compound Kaempferol (4) was obtained as light yellow powder from EtOAc-soluble part. It UV (lmax) spectrum and IR (cm-1) spectrum absorption bands are the typical flavonoidal nature. The HREI-MS showed the molecular ion peak at m/z 286 consistent with the formula C15H10O6. Furthermore, the 1H NMR spectrum indicated the presence of para-substituted benzene ring with AA'BB' pattern. The 13C NMR (BB and DEPT) spectra showed the characteristic signals of a flavonoidal skeleton. On the basis of these evidences and comparison with published data its named as Kaempferol. 128 Muhammad Imran et al. Compound (5) was obtained as yellow crystalline powder from the chloroform portion. It gave violet colour with FeCl3 for a phenol. The UV (lmax) spectrum and IR (cm-1) spectrum showed absorption bands of aromatic moieties. The EI-MS showed the fragment peaks at m/z 358. The HREI-MS showed the molecular ion peak at m/z 358 indicating the molecular formula C19HI807. From 1 H-NMR spectrum data presence of para substituted ring revealed. Furthermore the presence of four methoxy moieties were also observed at d 3.84, 3.87, 3.90 and 3.93. The 13C-NMR (BB and DEPT) spectra showed well resolved total nineteen carbon signals comprising four methyl, five methine and ten quaternary carbon atoms. Compound (6) was obtained as yellow solid from the EtOAc soluble part. It gave violet colour with FeCl3 for a phenol. The UV (lmax) spectrum and IR (cm-1) spectrum absorption bands indicating aromatic skeleton. The high resolution EI-MS showed the molecular ion peak at m/z 388 which deduced the molecular formula C20H2008. Its 1H-NMR showed at the aromatic(ABX system) region i.e., of five methoxyl protons and four protons in the. The 13C-NMR showed well resolved total twenty signals comprising of 11 quaternary carbon, 4 methine and 5 methyl atoms. Compound (7) was obtained as pale yellow form from the CH3Cl portions showing violet colour with FeCl3 Table 1. Antioxidant activity of compounds 1-13 Compound DPPH assay (IC50)a Cytochrome-c reduction assay (IC50)a (µM) 1 2 3 4 5 6 7 8 9 10 11 12 13 Gallic acidb 25.3 33.1 32.5 24.2 26.1 26.3 23.9 24 25.7 25.1 25.9 26.1 24.8 20.9 24.1 26.5 25.1 23.2 24.2 24.1 23.2 23.5 23.9 23.5 24.5 25.2 23.1 17.3 a = results in IC50 values (µM); b = positive control. All analysis were average of four experiments. for a phenol. The UV (lmax) spectrum and IR (cm-1) spectrum showed presence of flavone moieties. The molecular ion peak observed at m/z 270 showing the molecular formula C15H10O5. The para-disubstituted benzene ring as an AA'BB' pattern observed in its 1HNMR spectrum. Its 13C-NMR spectra showed the typical carbon pattern of flavonoid. The compound (8) was obtained as light yellow powder from the CH3Cl portions giving violet colour with FeCl3 for a phenol. The UV (lmax) spectrum and IR (cm-1) spectrum absorption bands absorption bands of conjugated aromatic system. The MS spectra in hard ionization mode showed the molecular ion peak at m/z 286 which deduced the molecular formula C15H10O6. The tri-substituted ring pattern was observed in its 1HNMR spectrum. The 13C-NMR showed fifteen carbon 15 signals consisting of 9 quaternary and 6 methine carbon atoms. Compound (9) was obtained as a yellow amorphous powder. The UV (lmax) spectrum and IR (cm-1) absorption bands were similar to flavone. The MS gives M.F C21H21O10 showing peak at m/z 433. The chelated hydroxyl group observed in its IH-NMR spectrum because of H-bonding. The sugar moiety in b configuration were also observed. The 13C-NMR spectra showing twenty one carbon signal out of these 1 methylene 12 methine and 8 quaternary carbons. Compound (10) was obtained as a light yellow solid. Its mass spectra of showed molecular ion peak at m/z 300 with the molecular formula C16H12O6. The UV (lmax) spectrum and IR (cm-1) spectrum absorption bands absorption bands of flavone system. The total six signals of H with ring B is symmetrical, free methoxyl group at position C-4 in the downfield region in its 1H-NMR spectra. Compound (11) was obtained as a light yellow solid. Its mass spectra of showed molecular ion peak at m/z 298 with M.F C17H14O5. The UV (lmax) spectrum and IR (cm-1) spectrum absorption bands absorption bands of flavone skeleton. The seven signals of H in the downfield region i.e., aromatic in its 1H-NMR spectra. The EI-MS data of compound (12) showed molecular ion peak at m/z 314. Its molecular formula observed in HREI-MS 314 with the M.F C17H14O6. The UV (lmax) spectrum and IR (cm-1) spectrum absorption bands absorption bands of benzene rings. The six signals of H in the downfield region i.e., aromatic in its 1H-NMR spectra. Chemical Constituents from Eremostachys loasifolia Compound (13) showed the UV (lmax) spectrum and IR (cm-1) spectrum absorption bands absorption bands of aromatic species. Its EI-MS confirmed M.F as C17H14O7 showing mass peak at m/z 330. The two OCH3 signals and five H present in the aromatic region in its 1H-NMR. The 17 carbon signals were observed in 13C-NMR spectra including ten quaternary five methine, and two methyl carbons. All the physical and spectral data of isolated compounds correspondence with the published data (Imran et al., 2012; Ahmed et al., 2006; Sadi Rum et al., 1980; Redalli et al., 1980; Dawson et al., 1966). Antioxidant activity of compound 1-13. The determination of antioxidant activity of compounds 1-13, DPPH followed by cytochrome-c-reduction tests were followed. The TPA-induced (HL-60 cell culture system) free radical formation with the positive control and showed free radical scavenging activity in the DPPH assay (Wavg et al., 2003; Yoshi Rawa et al., 2002). All isolated compounds showed significant antioxidant activity compared against standards (Table 1). Spectral data. Kaempferol (3,4',5,7-tetrahydroxyflavone) (4). EI-MS m/z:286; HREI-MS: m/z 286.0488 [M]+(calcud. for C15H10O6, 286.0474). Light yellow powder (19 mg) M. P: 277 °C ; UV (MeOH) 213, 267, 337; IR (KBr) umax cm-1: 3417, 2928, 1661, 1611 and 1178; 1H NMR (500 MHz CD3OD) (dppm): (J in Hz) 6.95 (d, H each, J = 8.6, H-3'5'), 6.37 (d, H, J = 2.1, H8), 8.06 (d, 1H each, J = 8.6, H-2'6'), 6.18 (d, H, J = 2.0, H-6). 5-Hydroxy-6,7,8,4'-tetramethoxyflavone (5). EI-MS m/z (rel. int.): 358; HREI-MS m/z: 358.1043 (calcud. for C19H18O7, 358.1049). Yellowish solid (23); M.P: 181 °C; IR (KBr) nmaxcm-1: 3345, 1600, 888; 1H-NMR (300 MHz CDCl3,) d: (J in Hz) 12.61 (OH), 6.61 (s, H, H-3), 7.88 (d, twoH, J = 10.0 Hz, H-2'6'), 7.03 (d, twoH, J = 10.2, 3.0, H-3'5'), 3.94, 3.91, 3.86, 3.85 (s, each tweleveH, -OCH3-6, 7, 8, 4'). 5-Hydroxy-3',4',6,7,8-pentamethoxyflavone (6). EIMS m/z: 388; HREI-MS m/z: 388.1153 (calcud. for C20H20O8, 388.1158). Light yellowish crystal; (25 mg); M.P: 146 °C; IR (KBr) nmaxcm-1: 3511, 1681, 1575; 1HNMR (500 MHz C5D5N,) d: (J in Hz) 13.26 (5-OH)6.85 (s, H, H-3), 7.87 (d, H, J = 2.1, H-2'), 7.11 (d, H, J = 8.6, H-5'), 7.92 (dd,H, J = 8.5, 2.0 Hz, H-6'), 3.58, 3.99, 3.83x3, 4.00, (15H, s, -OCH3-6, 7, 8, 3', 4'). Apigenin (4',5,7-trihydroxyflavone)(7). EI-MS m/z: 270; HREI-MS: m/z 270.0525 [M]+ (calcud. for C15H10O5, 129 270.0527). Yellow powder (35 mg) Melting point: 314 °C; UV(MeOH) 338, 266, 214; IR (KBr) umax cm-1: 3416, 1670, 1610 1179; 1H-NMR (500 MHz C5D5N,) (dppm), (J in Hz) 7.21 (d, H each, J = 9.1, H-3'5'), 6.92 (s, H, H-3), 7.91 (d, H each, J = 9.2, H-2'6') 6.81 (d, 1H, J = 2.1, H-8), 6.75 (1H, d, J = 2.0 , H-6). Luteolin (3',4',5,7-tetrahydroxyflavone) (8). EI-MS m/z: 286; HREI-MS: m/z 286.0475 [M]+ (calcud. for C15H10O6, 286.0469). Light yellow powder (21 mg); Melting point: 324 °C ;UV (MeOH)351, 259, 211; IR (KBr) umax cm-1: 3426, 1674, 1607; 1H-NMR ,(300 MHz (CD3OD) (dppm): (J in Hz) 7.37 (dd, H, J = 9.1, 2.2, H-2'), 6.87 (d, H, J = 9.1, H-3'), 7.39 (d,H, J = 2.1 H6'), 6.52 (s,1H), 6.41 (d, H, J = 2.2, H-8), 6.75 (d, H, J = 2.2, H-6). Apigenin 7-O-b-D-glucopyranose (9). EI-MSm/z: 270 [M-glucose]+; HRFAB-MS: m/z 433.1139 [M+H]+ (calcud. for C21H21O10, 433.1128). Pale Yellow (30 mg); Melting point: 228 °C; UV(MeOH) 337, 265, 215; IR (KBr) umax cm-1: 3425, 1668, 1637, 1618; 1H NMR (500 MHz C5D5N,) (dppm): (J in Hz) 7.21 (d, H each, J = 9.1, H-3'5'), 7.86 (d, H each, J = 9.1, H-2'6'), 7.08 (d, H, J = 2.1, H-8), 6.88 (s, H, H-3), 6.84 (d, H, J = 2.1, H-6), 5.85 (d, H, J = 7.5, H -1"), 4.57 (dd,H, J = 1.6, 1.6, H -6"), 4.37 (dd, H, J = 1.6, 1.6, H -6"), 4.40 (H, m, H -3"), 4.35 (m, H, H -2"); 4.23 (m, H, H -5"),4.35 (m, H). 4'-Methyl Kaempferol (3,5,6-trihydroxy-4methoxy flavone) (10). El-MS m/z: 300.2; HREI-MS: m/z 300.0541 (calcud. for C16H12O6; 300.0634). White crystals (18 mg); M.P 227 °C; UV (MeOH): 287, 327; 1 H-NMR (500 MHz CDCl3,) d: (J in Hz) 3.92 (s, threeH, OCH3) 7.25 (d, twoH, 9.1, H-3', 5'), 7.95 (d, twoH, 9.1, H-2', 6'),6.94 (br.s, H, H-8), 6.87 (br. s, H, H-6). 5-Hydroxy-7, 4'-dimethoxy flavones (11). HREIMS; /z 298.0625 (calcud. for C17H14O5, 298.0839), Yellow solid (26 mg); M.P. 174 °C; UV (MeOH): 270, 328; 1H-NMR (400 MHz CDCl3,) d: (J in Hz) 3.87 (s, 3H, OCH3 at C-7) and 3.86 (s, 3H, OCH3); 7.25 (d, H, J = 9.2, H2',6'), 7.01 (d, twoH, J = 9.1, H-3',5'), 6.57 (s, H, H-3), 6.45 (d,H, J = 2.1, H-8), 6.35 (d, H, J =2.1, H-6). 6, 7-Dimethoxy-4',5-hydroxyflavone (12). EI-MS m/z: 314; HREI-MS: m/z 314.0769 [M] + (calcud. for C17H14O6; 314.0788). Yellowish crystals (15 mg); M.P. 201 °C; UV (MeOH): 215, 274, 343; IR (KBr) nmax cm-1: 3456, 1671, 1182; 1H-NMR (400 MHz CDCl3,) d: (J in Hz) 3.99 (threeH, s, OMe), 3.87 (3H, s, OMe) 130 Muhammad Imran et al. OH 3' (1) HO 8 H3CO 8 HO 2 OH 4' 4' 1' O 9 7 2' 3' (7) 7 1 10 O 1' 2 5' 5' 6' 6' 6 3 10 5 OH 6' HO 4' O 1' 6' 5' 10 1 O 2 1' 4 6 OH 3' OH 9 O OH 9 4 5 3 5 7 4' 2' 6 O 1 8 HO 6'' H 10 8 7 H HO O 5' (8) O 2' OH O 2'' HO (9) OH 4'' OH 5'' O 1'' 4' HO HO OH 3'' 5' 6' HO 6' 5' O 1 10 O 8 O 3' 2' HO 7 MeO 6 H 2' 3' MeO HO O 1' 4' 6'' H 10 HO O 5'' O 1'' HO 8 10 O HO OH O 4' OH 2' 7 2'' OH 4'' OH 3'' OH OCH3 OH 3' (4) HO (10) O 1 8 6' 5' O 3 5 9 7 (11) 1 O 2 1' 6' 4 9 H3CO 1 O 2 7 OH 5 5 6 OH 4' 2' OCH3 5' 3 6 3' 3 4 9 OH 4 6 1' 4' OH 2' 5 MeO 2 1' H (3) 3 O (2) MeO 3' 4 9 5 4 OH MeO 6 OCH3 1' 3 O OH 3' (5) OCH3 8 H3CO 7 4' 2' (12) 1' O 9 6' 6 H3CO H3CO 4' 2’ 5' 2 O OCH3 1 O 2 7 OH 1' 3 10 4 5 H3CO O OH 3 6 OH (6) 2' OCH3 H3CO 8 7 9 OCH3 3' 4' OCH3 1' O 5' 2 O (13) HO O O 6' H3CO 6 3 10 OH OH 4 5 O Scheme 1. Structures of Isolated Compounds (1-13). OH O Chemical Constituents from Eremostachys loasifolia 7.02 (twoH, d, J = 8.8, H-3',5') 7.83 (twoH, d, H-2',6') and 6.58 (twoH, s, H-8, H-3). 3,5,7-Trihydroxy-3',4'-dimethoxyflavone (13). EI-MS m/z: 330; HR-EI-MS m/z: 330.0741 (calcud for C17H14O7, 330.0739). Yellow powder 933 mg; UV (MeOH): 269 and 370; IR (KBr) nmax cm-1: 3275, 1651, 1609 and 867; 1 H-NMR (300 MHz CDCl3,): d: (J in Hz) 3.85 (threeH, s, OMe), 3.74 (threeH, s, OMe); 7.65(d, H, J = 1.9 H2'), 7.60 (dd, H, H-6'), 6.35 (d,H, J = 2.2, H-8), 6.19 (d, H, J = 2.2 H-6) and 6.84 (d, H, J = 8.7, H-5'). Conclusion Bioassy guided separation of secondary metabolites freshly collected from Eremostachys loasifolia plant was carried out first time with the aid of advanced chromatographic and spectroscopic techniques. This study gives thirteen mostly flavonoids metabolites showing significant antioxidant activity. All physical and spectral data of isolated compounds were comparable with already reported structures. 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