Pharmaceutical Biology 2004, Vol. 42, Nos. 4–5, pp. 349–352
Flavonoids from Cressa cretica A.A. Shahat1,2, N.S. Abdel-Azim1, L. Pieters2 and A.J. Vlietinck2 1 2
Pharmacognosy and Chemistry of Medicinal Plants Department, National Research Centre, Dokki, Cairo, Egypt; Pharmacentical Sciences Department, University of Antwerp, Antwerp, Belgium
Abstract The aerial parts of Cressa cretica L. yielded five flavonoids that were identified as quercetin (1), quercetin-3-O-glucoside (2), kampferol-3-O-glucoside (3), kampferol-3-Orhamnoglucoside (4), and rutin (5). All of the isolated flavonoids were identified by spectroscopic methods (UV, FAB-MS, 1H NMR and 13C NMR) and in comparison with literature data. The isolated flavonoids, except quercetin, are reported here for the first time from Cressa cretica L. Keywords: Convolvulaceae, Cressa cretica, flavonoids.
Introduction Cressa cretica L. (Convolvulaceae) is known in Arabic as Molleih or Nadewa (Täkholm, 1974). C. cretica is a remarkable salt-tolerant plant, common in coastal areas. C. cretica is mentioned as an Ayurvedic drug, known in indigenous medicine in India as Rudanti (Satakopan & Karandikar, 1961). It is reported to be antibilious, antitubercular, and an expectorant (Rizk & El-Ghazaly, 1995; Satakopan & Karandikar, 1961). The plant is alternately used as an anthelmentic, stomachic, tonic, for aphrodisiac purposes, enrichment of the blood, and is useful in constipation, leprosy, asthma, and urinary discharges (Chopra et al., 1956). The plant is traditionally used in Bahrain as an expectorant and an antibilious agent (Rizk & El-Ghazaly, 1995). Phytochemical screening of the plant growing in Qatar revealed the presence of alkaloids, coumarins, and sterols (Rizk, 1995). In a previous investigation, syringaresinol glucoside and dicaffeoyl quinic acids were isolated. In addition, the antiviral activity of different extracts from the plant was reported (Shahat et al., 1999). In view of the medicinal
importance of C. cretica, we undertook a systematic chemical investigation of the plant growing in Egypt.
Materials and Methods General FAB-MS spectral analysis in negative or positive mode was performed on a VG70-SEQ Hyprid Mass Spectrometer. All NMR spectra were run on a Bruker DRX-400 instrument. The chemical shifts were reported in d values (ppm) with TMS as the internal standard. Carbon multiplicities were determined in DEPT-135 and DEPT-90 experiments. 1H and 13 C NMR spectra were recorded in CD3OD. UV spectra were recorded on a UVIKON 931 double beam UV-Vis spectrophotometer in the region of 200–500 nm. UV detection was at 254 and 336 nm. Thin-layer chromatography (TLC) was performed on Merck precoated silica gel 60 F254 plates. Column chromatography was carried out using Merck silica get 60 (230–400 mesh) as adsorbent. The solvent system for TLC was ethyl acetate : acetic acid : formic acid : water (30 : 0.8 : 1.2 : 8), and the plates were sprayed by diphenyl boric acid–ethanolamine complex (NA reagent) (Markham, 1982). Plant material The whole plant of C. cretica L. was collected at Helwan, South Cairo, Egypt, in October 1998 and was identified by Dr. M. Elgebaly, Department of Chemotaxonomy, National Research Centre (NRC), Cairo, Egypt. A voucher specimen has been deposited at the Herbarium of the NRC, Cairo, Egypt.
Accepted: February 4, 2004 Address correspondence to: A.A. Shahat. Pharmaceutical Sciences Department, University of Antwerp, B-2610, Antwerp, Belgium. E-mail:
[email protected] DOI: 10.1080/13880200490519622
© 2004 Taylor & Francis Ltd.
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Extraction and isolation The air-dried, powdered whole plant of C. cretica L. (1 kg) was defatted with n-hexane and then extracted subsequently with CHCl3, CH2Cl2/MeOH (1 : 1), and water. The CH2Cl2/ MeOH extract was evaporated under reduced pressure and dissolved in aqueous MeOH 20%. The aqueous methanolic solution was partitioned first against CHCl3, EtOAc, and then against n-BuOH. The EtOAc fraction was subjected to column chromatography (CC) on Sephadex LH-20 using propanol with increasing amounts (10%) of MeOH; four fractions (Cr1–Cr4) were collected after monitoring with TLC. The spots were detected with UV before and after spraying with NA reagent. The fraction Cr2 was submitted to CC (silica gel 60), the separation was initiated with CHCl3, and polarity was gradually increased with steps of 5% MeOH. The flavonoid-containing fraction was eluted with CHCl3/MeOH (2 : 8) and then subjected to PTLC to give compounds 1–3 (Fig. 1). The fraction eluted with CHCl3/MeOH (3 : 7) was also subjected to PTLC using the same solvent followed by CC on Sephadex LH-20 and eluted with MeOH to give compounds 4 and 5 (Fig. 1). Flavonoid compounds
428, 340, 271 nm, (NaOAc) 388, 310, (NaOAc/H3B3) 386, 326, 262 nm. Compound 2 (quercetin-3-O-b-glucoside) Compound 2 was isolated as an amorphous brown solid with Rf = 0.32 and color reaction (dark purple to yellow in UV + NH3 and orange color with NA reagent under UV). The negative FAB-MS showed a molecular ion (M-H]- at m/z 463 and 301 in accordance with a flavonol containing one hexose. UV l max (MeOH) 350, 282, 257 nm, (NaOH) 409, 350, 330, 272 nm, (AlCl3) 430, 333, 275 nm, (AlCl3/HCl) 403, 323, 270 nm, (NaOAc) 381, 293, 264 nm, (NaOAc/H3BO3) 379, 319, 262. 1H NMR (CD3OD, 400 MHz): d 7.70 (H, d, J = 2.1 Hz, H-2¢), d 7.56 (H, dd, J = 8.4, 2.1 Hz, H-6¢), d 6.90 (1H, d, J = 8.4 Hz, H-5¢), d 6.80 (1H, d, J = 2.3 Hz, H-8) d 6.36 (1H, d, J = 2.1 Hz, H-6), d 5.12 (1H, d, J = 7.5 Hz, H1≤) d 3.70–3.20 (5H, sugar). 13C NMR (CD3OD, 100 MHz): d 158.40 (C-2), d 135.60 (C-3), d 179.40 (C-4), d 163.00 (C5), d 99.80 (C-6), d 166.00 (C-7), d 94.70 (C-8), d 159.00 (C-9), d 105.70 (C-10), d 123.10 (C1¢), d 117.50 (C-2¢), d 145.90 (C-3¢), d 149.80 (C4¢), d 116.00 (C5¢), d 123.20 (C6¢), d 104.30 (C-1≤), d 78.10 (C-2≤), d 75.70 (C-3¢≤), d 71.20 (C-4¢≤), d 78.30 (C-5¢≤), d 62.50 (C-6¢≤).
Compound 1 (quercetin) Compound 1 was an amorphous yellow solid, changing to orange with NA reagent, Rf = 0.93, negative FAB-MS [MH]- 301. UV l max (MeOH) 371, 257 nm, (NaOH) 424, 325 dec., 287 nm. (AlCl3) 445, 362, 272, 252 nm, (AlCl3/HCl)
Figure 1.
Structures of compounds 1–5.
Compound 3 (kampferol-3-O-b-glucoside) Rf = 0.41, negative FAB-MS [M-H]- 447, [M-H-162]- 285. UV l max (MeOH) 350, 318, 281 nm, (NaOH) 401, 346, 325, 308 nm, (AlCl3) 350, 318, 281 nm, (AlCl3/HCl) 396,
Flavonoids from Cressa cretica 375, 346 nm. 1H NMR (CD3OD, 400 MHz): d 8.02 (2H, d, J = 8.6 Hz, H-2¢, H-6¢), d 6.87 (2H, d, J = 8.6 Hz, H-3¢, H-5¢), d 6.40 (1H, s, H-8), d 6.20 (1H, s, H-6) d 5.10 (1H, s, H-1≤), d 3.60–3.30 (5H, sugar). 13C NMR (CD3OD, 100 MHz): d 179.50 (C-4), d 166.20 (C-7), d 163.01 (C-5), d 161.60 (C4¢), d 159.20 (C-2), d 158.50 (C-9), d 135.50 (C-3), d 132.20 (C-2¢, C-6¢), d 122.70 (C-1¢), d 116.00 (C-5, C-5¢), d 105.00 (C-10), d 104.20 (C-1≤), d 100.00 (C-6), d 94.80 (C-8), d 78.30 (C-3≤), d 78.00 (C-5≤), d 75.30 (C-4≤), d 62.50 (C-6≤). Compound 4 [kampferol-3-O-a-rhamnosyl(1-6)-b-O-glucoside] Rf = 0.12, FAB-MS 593 [M-H]-, 447 [M-H-136]-, 285 [MH-136-162]-. 1H NMR (CD3OD, 400 MHz): d 8.09 (2H, d, J = 8.8 Hz, H-2¢, H-6¢), d 6.90 (2H, d, 8.8 Hz, H-3¢, H-5¢), d 6.4 (1H, s, H-8), d 6.23 (1H, s, H-6), d 5.1 (1H, d, J = 7.3 Hz H-1≤), d 4.5 (1H, a broad s, H-1¢≤), d 3.9–3.4 (10 H, rhamnose and glucose). 13C NMR (CD3OD, 100 MHz): d 179.40 (C-4), d 166.70 (C-7), d 162.99 (C-5), d 161.60 (C-4¢), d 159.55 (C-9), d 58.68 (C-2), d 135.50 (C-3), d 132.40 (C-2¢, C-6¢), d 122.08 (C-1¢), d 116.20 (C-3¢, C-5¢), d 104.70 (C10), d 102.48 (C-1≤, C-1¢≤), d 100.29 (C-6), d 95.12 (C-8), d 77.80 (C-3≤), d 77.20 (C-5≤), d 75.70 (C-2≤), d 73.9 (C4¢≤), d 72.30 (C-4≤), d 72.10 (C-2¢≤), d 71.53 (C-3¢≤), d 69.78 (C-5¢≤), d 68.70 (C-6≤), d 17.9 (C-6¢≤). Compound 5 (rutin) Compound 5 was crystalline yellow, changing to orange with NA reagent under UV. Rf = 0.12, negative FAB-MS displayed [M-H]- at m/z 609, 301. These results were according to the molecular weight calculated for aglycone + rhamnose + glucose. UV l max (MeOH) 360, 284 nm, (NaOH) 412, 330, 330, 272 nm, (AlCl3) 434, 320, 274 nm, (AlCl3/HCl) 405, 315, 270 nm, (NaOAc) 382, 317, 264 nm, (NaOAc/H3BO3) 382, 320, 264.
Results and Discussion Compound 1 color reactions (yellow, changing to orange with NA reagent) indicated the presence of 3¢, 4¢-dihydroxy group in the B-ring (Merkham, 1989). Chromatographic behavior and the UV spectral analysis with the usual shift reagents indicated the presence of free hydroxyl groups at C3, C-5, and C-4¢. The absorption peak at 371 nm is characteristic for flavonol (3-OH free). The additional peak at 327 nm with NaOH indicated the presence of 7-OH group (Markham 1982). The molecular ion of compound 1 in FABMS [M + H]+ at m/z 301 was consistent with a molecular formula C15H10O7 (molecular weight of quercetin is 302). Finally, the identity of quercetin was established by direct cochromatography with authentic sample. A dull brown spot on silica gel plate, changing to orange with NA reagent and UV analysis in the presence of the
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usual shift reagent suggested that compound 2 is flavonol 3substituted. The UV spectra 354 nm band I in methanol indicated a C3-O-substituted flavonol skeleton. The bathochromic shift (56 nm) on addition of NaOH without decrease of intensity confirmed the presence of free 4¢-OH, the presence of 7-OH group was indicated due to the bathochromic shift (11 nm) with NaOAc (band II) relative to pure methanol spectrum, and the bathochromic shift (25 nm) (band I) with NaOAc/ H3BO3 suggested the presence of O-dihydroxy groups in Bring. The 5-OH group was confirmed by the bathochromic shift in band I with AlCl3/HCl (41 nm) relative to the MeOH spectrum. These data were confirmed by 1H and 13 C NMR. The 1H and 13C NMR spectra indicated the presence of a quercetin moiety and sugar unit (Markham & Chari, 1982). The quercetin moiety exhibited seven signals of the nonoxygenated flavonoid aglycone at d 6.36 (d) and d 99.8; 6.8 (d) and d 94.7; d 7.7 (d) and 117.5; d 6.9 (d) and 116.0; d 7.56 (dd) and d 123.2; d 105.7; and d 123.1 for CH-6, CH-8, CH2¢, CH-5¢, CH-6¢, C-10; and C-1¢, respectively, and the oxygenated carbons at d 158.4, 135.6, 163.0, 166.0, 159.0, 145.9, and 149.8 for C-2, C-3, C-5, C-7, C-9, C-3¢, and C-4¢, respectively. The peak at downfield d 179.5 was corresponding to the carbonyl group. In the 1H NMR, the doublet at d 5.33 (diaxial coupling J = 7.5 Hz) was assigned to the anomeric proton of hexose and suggested the glycosidic b-linkage. The FAB-MS spectra at m/z 463 and m/z 301, which were ascribed to [M-H]- and [M-H-162]- ions, respectively, suggested the presence of one hexose moiety in the molecule. Acid hydrolysis of compound 2 gave glucose and quercetin, which were identified by co-chromatography with an authentic sample. Based on these data, compound 2 was identified as quercetin-3-b-O-glucoside. For compound 3, the UV spectrum at 350 nm in MeOH showed an absorption peak indicating a C3-O-substituted flavonol skeleton (Markham, 1982). The bathochromic shift on addition of AlCl3 and NaOH indicated the presence of free OH groups at C-5 and C-4¢, respectively. The four aromatic protons at d 8.02 for H-2¢ and H-6¢ and d 6.87 for H-3¢ and H-5¢ assigned for the B-ring protons of a 4¢-substituted, and the two doublets at d 6.8 and 6.36 assigned for C-8 and C6, respectively. The 13C NMR showed 15 signals attributed to the aglycone and hence 6 glucose that must be attributed to the sugar moiety. Moreover, the DEPT experiment confirmed the occurrence of 1 CH2, 11 CH, and 9 fully substituted carbons. The FAB-MS spectrum displayed [M-H]- at m/z 447, corresponding to the glycoside: other significant peak was visible at m/z 285 [M-H-hexose]-. In the 1H NMR, the anomeric proton (H-1≤) of the hexose appeared as doublet at d 5.18 (J = 7.2 Hz). This chemical shift confirmed that glucose is attached to the aglycone moiety and the diaxial coupling J = 7.2 Hz between H-1 glucose and H-2 glucose indicated the b-configuration (Merkham 1989). Acid hydrolysis of compound 3 afforded glucose (co-TLC) and aglycone,
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which was identified as kampferol by comparison TLC, UV, and FAB-MS with literature data. According to these data, compound 3 was identified as kampferol-3-O-bglucoside. The UV spectrum of compound 4 in MeOH showed the same absorption peaks of compound 3, indicating it is a flavonol glycoside. The negative FAB-MS spectrum showed a molecular ion [M-H]- at m/z 593, [M-H-146]- and [M-H-146-162]-. This indicated the successive elimination of rhamnose and glucose, respectively, and supported a 3glycosylated flavonol structure with rhamnose as the terminal unit. The sugars were supported by acid hydrolysis to yield kampferol, rhamnose, and glucose and were identified by co-TLC with reference samples. The interglycosidic linkage was determined by 1H and 13C NMR spectroscopy. 13 C NMR of C-2 was observed at downfield shift (d 158.68) compared with the C-2 of kampferol as well as the upfield shift for C-3 (d 135.5) linked to the sugar moiety (Markham & Chari 1982). The interglycosidic (1Æ6) linkage was confirmed from the downfield shift of C-6 of the glucose moiety by 6.2 ppm relative to compound 4 (C-6 of glucose is d 62.5). The protons at positions 6 and 8 were deduced from the 1H NMR spectrum, at d 6.23 and 6.4, respectively. This was confirmed from the 13C NMR resonance at d 100.29 and 95.12, respectively. The protons at 3¢, 5¢ and 2¢, 6¢ were confirmed by the two ortho coupled doublets at d 6.9 and 8.09 (J = 8.8 Hz) and corresponded to the carbons resonating at d 116.2 and 132.4, respectively. The anomeric protons at d 5.1 (J = 7.3 Hz) and 4.5 (a broad s) assigned to H-1 of b-glucose and a-rhamnose moieties, for which the corresponding carbons resonated at d 102.48. Acid hydrolysis of compound 4 gave glucose, rhamnose, and quercetin as aglycone as shown by co-chromatography and literature data. Therefore, compound 4 was identified as kampferol-3-O-b-rhamnoglucoside. Compound 5 was identified as rutin from the spectral data of UV and the negative FAB-MS. UV spectra suggested
O-dihydroxy flavonol substituted at position 3. The free hydroxyl groups at positions 5, 7, and 4¢ were deduced from the bathochromic shift in band 1 with AlCl3/HCl, NaOAc (band II) and NaOH (band I), respectively. Negative FAB-MS resulted in the presence of fragment ion m/z 609 [M-H-]-, 301 [M-H-308-]- indicating loss of glucose and rhamnose. Acid hydrolysis of compound 5 afforded quercetin, glucose, and rhamnose. Co-TLC with reference samples confirmed this identification.
References Chopra RN, Nayar SL, Chopra IC (1956): Glossary of Indian Medicinal Plants. New Delhi, Council of Industrial Research, p. 80. Markham KR, Chari VM (1982): In: Harborn JB, Mabry TJ, eds., The Flavonids: Advances in Research. London, Chapman & Hall. Markham KR (1982): Techniques of Flavonoid Identification. London, Academic Press. Rizk AM (1982): Constituents of plants growing in Qatar. Fitoterapia 52: 35–44. Rizk AM, El-Ghazaly, GA (1995): Medicinal and Poisonous Plants of Qatar. Scientific and Applied Research Centre, University of Qatar, p. 101. Roa KB, Bhat GG, Syamasundar J (1987): Cressa cretica, a saltextruding plant in salt-affected soils. J Curr Res (Univ. Agric. Sci. Bangalore) 7: 186. Satakopan S, Karandikar GK (1961): Rudanti: A pharmacognostic study – Cressa cretica Linn. J Sci Ind Res Section C, 20: 156–160. Shahat A, Abdel-Azim NS, Hammouda FM, Apres S, De Bruyne T, Pieters L, Vanden Berghe D, Vlietinck AJ (1999): 2000 Years of Natural Products Research – Past, Present and Future, Amsterdam, The Netherlands. Täckholm V (1974): Students Flora of Egypt, 2nd ed., p. 433.
