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Anti-oedematous activities of the main triterpendiol esters of marigold (Calendula officinalis L.)
Journal of Ethnopharmacology 57 (1997) 139 – 144
Anti-oedematous activities of the main triterpendiol esters of marigold (Calendula officinalis L.) K. Zitterl-Eglseer a,*, S. Sosa b, J. Jurenitsch c, M. Schubert-Zsilavecz d, R. Della Loggia b, A. Tubaro b, M. Bertoldi 1,a, C. Franz a a
Institute for Botany and Food Science, Uni6ersity of Veterinary Medicine Vienna, Josef-Baumann-Gasse 1, A-1210 Wien, Austria b Institute of Pharmacology and Pharmacognosy, Uni6ersity of Trieste, Via L. Giorgieri 7, I-34 100 Trieste, Italy c Institute of Pharmacognosy, Uni6ersity of Vienna, Pharmaziezentrum, Althanstrasse 14, A-1090 Wien, Austria d Institute of Pharmaceutical Chemistry, Uni6ersity of Graz, Uni6ersita¨tsplatz 1, A-8010 Graz, Austria Received 18 April 1997; received in revised form 5 May 1997; accepted 13 May 1997
Abstract Separation and isolation of the genuine faradiol esters (1, 2) from flower heads of Marigold (Calendula officinalis L., Asteraceae) could be achieved by means of repeated column chromatography (CC) and HPLC for the first time. Structure elucidation of faradiol-3-myristic acid ester 1, faradiol-3-palmitic acid ester 2 and c-taraxasterol 3 has been also performed, without any previous degradation by means of MS, 1H-NMR, 13C-NMR and 2D-NMR experiments. The anti-oedematous activities of these three compounds were tested by means of inhibition of Croton oil-induced oedema of the mouse ear. Both faradiol esters showed nearly the same dose dependent anti-oedematous activity and no significant synergism appeared with their mixture. The free monol, c-taraxasterol, had a slightly lower effect. Furthermore, faradiol was more active than its esters and than c-taraxasterol and showed the same effect as an equimolar dose of indomethacin. © 1997 Elsevier Science Ireland Ltd. Keywords: Calendula officinalis L.; Marigold; Triterpendiol esters; Faradiol-3-myristic acid ester; Faradiol-3-palmitic acid ester; Taraxasterol; Anti-oedematous activity
* Corresponding author. 1 Permanent address: Department of Pharmaceutical Science, University of Modena, Via Campi 183, I-41 100 Modena, Italy.
1. Introduction Preparations of Marigold, Calendula officinalis L. (Asteraceae) for topical application are used
0378-8741/97/$17.00 © 1997 Elsevier Science Ireland Ltd. All rights reserved. PII S 0 3 8 - 8 7 4 1 ( 9 7 ) 0 0 0 6 1 - 5
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both in dermatology and cosmetics (Isaac, 1992, 1994). Besides traditional galenic formulations such as infusa, tinctures and fluid extracts in folk medicine flower heads of Calendula officinalis were extracted by hot lard, which is similar to the fat of the human skin, in order to obtain ointments for several dermatological diseases as wounds, ulcers, eczemas, burns, contusions, eruptions, varicose veins and haemorrhoids. The anti-inflammatory and wound-healing activity of the drug was documented in many experimental and clinical studies (Shipochliev et al., 1981; Peyrox et al., 1981; Perri de Carvalho et al., 1991; Lievre et al., 1992). Recent investigations demonstrated that a number of lipophilic compounds of hydroalcoholic (70%) and CO2-extracts account for the topical anti-inflammatory activity (Della Loggia et al., 1990, 1994). The main compounds of lipophilic extracts of flowers of Calendula officinalis are triterpendiols, which are 98% bound as 3-monoesters with higher fatty acids and occur almost exclusively in the chromoplasts (Wilkomirski and Kasprzyk, 1979). Structure elucidation of these esters has been performed formerly by chemical degradation, separation and characterization of the triterpene alcohols by their spectroscopic properties and of the corresponding fatty acids by comparison with authentic samples after methylation by means of gas chromatography (Kasprzyk and Pyrek, 1968; Wojciechowski et al., 1972; Pyrek and Baranowska, 1973; Della Loggia et al., 1994). Up to now the triterpenoid alcohols have been separated by means of column chromatography (CC) or preparative thin layer chromatography (TLC) and quantitatively determined by colorimetrical measurement of their CoCl2 complexes (Wojciechowski et al., 1972). The isolation of the genuine esters has never been performed. Therefore the biological activity of the esters was studied only on their mixture and the activity of the single compounds remained unknown, so far. In account of this knownledge, the aim of this work was to establish a separation of the genuine esters and to study their topical anti-oedematous properties.
2. Material and methods
2.1. Plant material TLC-screening: 1200 single plant samples of 20 commercially available origins of Calendula officinalis L. obtained from Austrian and German companies, namely, Austrosaat (5), Borntra¨ger (3), Chrestensen (1), Landes-Versuchsanlage fu¨r Spezialkulturen Wies/Stmk. (1), Landwirtschaftlich-chemische Bundesanstalt Wien/Korneuburg (3), Saatbau Linz (6) and Theiss (1) were investigated. Isolation of 1, 2 and 3: Calendula officinalis L. Cultivar: ‘Erfurter orangefarbige, gefu¨llt blu¨hende’ (Asteraceae) was grown from seeds in Mu¨hlbach and Korneuburg, Lower Austria and harvested in August 1993. Voucher specimens (CAL1061-1064/93) have been deposited in the Herbarium of the Institute for Botany, University of Veterinary Medicine, Vienna.
2.2. Reagents The following reagents were used: Dichloromethane p.a., n-hexane p.a., ethylacetate p.a., acetonitrile p.a., ethanol p.a., methanol CHROMASOLV (Riedel de Hae¨n), anisaldehyde, acetic acid (100%), sulfuric acid (100%). Faradiol was kindly provided by Prof. H. Becker, Saarbru¨cken.
2.3. Extraction TLC-screening: 100 mg of air-dried powdered flower heads of Calendula officinalis were extracted with 3 ml dichloromethane 15 min in an ultrasonic bath at room temperature and filtered. CC: 1000 g air-dried flower heads of Calendula officinalis were extracted exhaustively with dichloromethane. After removal of the solvent the residue was separated by CC.
2.4. Analytical and preparati6e methods TLC: Screening and CC-monitoring: Stationary phase: HPTLC, silica gel 60 F254 nm, MERCK, mobile phase: n-hexane/ethylacetate (8+ 2), detection: anisaldehyde reagent (Dequeker, 1964).
K. Zitterl-Eglseer et al. / Journal of Ethnopharmacology 57 (1997) 139–144
CC: Extract: 72.8 g, column dimensions: 100 × 7 cm, stationary phase: silica gel 60 TSC, 0.063 – 0.2 mm, mobile phase: n-hexane/ethylacetate, gradients: 100, 98+2, 96 +4, 94 + 6, 92 + 8, 90+ 10. SPE: Extract: 1.28 g, stationary phase: RP-18 material, mobile phase: methanol/water, gradients: 6+4, 8+ 2, 9+1, 10 +0. HPLC: PE Series 10, PE Series 10 LC Controller, PE UV/VIS LC290, PE Nelson model 1020, Okidata Microline 320 Printer and Kontron Instruments, LC Pump T-414, UV/VIS-Detector 432, Chromatography software: Borwin; Columns: LiChrosphere 100, RP-8, 5 mm, 15 × 250 and 4 ×250 mm, solvent: methanol/water, wavelength: 210 nm. MS: Shimadzu QP 1000, DI, 25°C, 20°C/min, EI: 70 eV: m/e (rel. int.%): 1, faradiol myristic acid ester 652 (M + ), 634 (6.0), 424 (16.7), 189 (100.0), 121 (41.3), 109 (44.6), 95 (57.1); 2, faradiol palmitic acid ester 680 (M+), 662 (10.4), 424 (42.9), 239 (15.6), 205 (41.1), 189 (98.0), 135 (79.7), 107 (80.4); 3, c-taraxasterol 426 (M+ ), 218 (96.4), 207 (69.4), 189 (99.6), 135 (52.2), 95 (92.2). NMR: 1H-NMR, 13C-NMR, 2D-NMR measurements were performed on a Bruker AC 200 and AMX 500 spectrometer. 1 faradiol myristic ester, 13C-NMR (CDCl3): d = 38.5 (C-1), 27.2 (C-2), 80.5 (C-3), 37.8 (C-4), 55.4 (C-5), 18.2 (C-6), 34.2 (C-7), 41.1 (C-8), 49.9 (C-9), 37.6 (C-10), 22.7 (C-11), 29.2 (C-12), 38.8 (C-13), 42.5 (C-14), 37.0 (C-15), 76.2 (C-16), 39.9 (C-17), 47.6 (C-18), 35.9 (C-19), 139.6 (C-20), 118.3 (C-21), 36.6 (C-22) 28.0 (C-23), 14.1 (C-24), 16.3 (C-25), 16.0 (C-26), 16.5 (C-27), 11.6 (C-28), 22.4 (C-29), 21.5 (C-30); acid moiety: 173.6, 34.8, 29.6, 29.4, 29.3, 29.2, 29.1, 25.1, 23.7, 16.3. 2 faradiol palmitic ester, 13C-NMR (CDCl3): d = 38.8 (C-1), 27.2 (C-2), 80.6 (C-3), 38.5 (C-4), 55.5 (C-5), 18.2 (C-6), 34.2 (C-7), 41.1 (C-8), 50.0 (C-9), 37.8 (C-10), 22.6 (C-11), 28.0 (C-12), 37.6 (C-13), 42.5 (C-14), 37.0 (C-15), 76.2 (C-16), 39.9 (C-17), 47.7 (C-18), 35.9 (C-19), 139.6 (C-20), 118.4 (C-21), 36.5 (C-22) 29.2 (C-23), 14.0 (C-24), 16.3 (C-25), 16.0 (C-26), 16.5 (C-27), 11.6 (C-28), 22.4 (C-29), 21.6 (C-30); acid moiety: 173.6, 34.8, 31.8, 29.6, 29.5, 29.4, 29.3, 29.2, 29.1, 25.1, 23.7, 16.3.
141
3 c-Taraxasterol, 1H-NMR (CDCl3/DMSO[d6]): d= 0.78 (H-1ax), 1.42 (H-1eq), 1.38 (H2ax), 1.44 (H-2eq), 2.99 (H-3), 0.52 (H-5), 1.19 (H-6ax), 1.32 (H-6eq), 1.17-1.26 (H-7ax and H-7eq), 1.11 (H-9), 1.08 (H-11ax), 1.37 (H-11eq), 1.05 (H12ax), 1.43 (H-12eq), 1.51 (H-13), 0.82 (H-15ax), 1.60 (H-15eq), 1.03 (H-16ax), 1.13 (H-16eq), 0.86 (H-18), 1.39 (H-19), 5.06 (H-21), 1.36 (H-22), 1.52 (H-22%), 0.79 (CH3-23), 0.58 (CH3-24), 0.82 (CH325), 0.86 (CH3-26), 0.77 (CH3-27), 0.55 (CH3-28), 0.81 (CH3-29), 1.43 (CH3-30). 13C-NMR: d= 38.5 (C-1), 27.0 (C-2), 78.1 (C-3), 38.5 (C-4), 55.0 (C-5), 17.9 (C-6), 33.9 (C-7), 41.1 (C-8), 50.4 (C-9), 36.7 (C-10), 21.1 (C-11), 27.2 (C-12), 38.8 (C-13), 41.8 (C-14), 26.4 (C-15), 36.5 (C-16), 34.0 (C-17), 48.4 (C-18), 35.9 (C-19), 139.4 (C-20), 118.5 (C-21), 41.9 (C-22), 27.7 (C-23), 15.1 (C-24), 15.9 (C-25), 15.7 (C-26), 14.4 (C-27), 17.3 (C-28), 22.1 (C-29), 21.2 (C-30).
2.5. Anti-oedematous acti6ity The Croton oil ear test was performed as already described (Tubaro et al., 1985). Groups of seven male Albino Swiss mice (28–30 g, Charles Rivers, Calco, Italy) were anaesthetized with Ketalar® and 15 ml of an acetonic solution containing the irritant (75 mg of Croton oil, Sigma) and the appropriate amount of the substances under testing were applied to the inner surface of the right ear of mice (surface, about 1 cm2), the left ear remaining untreated. Control animals recieved only the irritant. The animals were sacrificed by cervical dislocation 6 h later and a plug (6 mm in diameter) was removed from both the treated and the untreated ear. The difference in weight between the two plugs was taken as a measure of the oedematous response. At least two experimental groups of seven animals were used for each tested dose level.
3. Results and discussion A TLC-screening of 1200 individuals of Calendula officinalis from 20 origins showed the presence of faradiol monoesters in each plant, however in different quantities. Therefore a culti-
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var characterized by a TLC-fingerprint showing a high amount of all the faradiol esters could be chosen for the isolation. Since the cultivar was available in almost unrestricted amounts an optimization of the isolation procedure was possible, as well as the isolation of enough amounts of pure substances for chemical characterization and pharmacological experiments.
3.1. Isolation and characterization of 1, 2 and 3 The residue of the dichloromethane extract of flower heads of Calendula officinalis was purified by means of CC on silica gel monitored by TLC. Two fractions of CC containing the main compounds recorded by TLC (Rf =0.54 and Rf = 0.47) were further purified. The first fraction (1.28 g; Rf =0.54) was cleaned up with SPE-Clean-up-Columns on RP-18 material with methanol/water, the second fraction (Rf =0.47) by means of CC on silica gel with dichloromethane. Further separations were performed by repeated preparative HPLC. After extensive optimization experiments using different types of RP-materials and mixtures of acetonitrile/water, ethanol/water and methanol/water as mobile phases the following conditions were found as optimum for the semipreparative HPLC separation: Stationary phase: LiChrosphere 100, RP-8, 5 mm using methanol/water (9+ 1, isocratic) for the first CC-fraction and a gradient from methanol/water (8 + 2) to pure methanol for the second one. The first CC-fraction yielded 268.3 mg 1, 246.6 mg 2 and the second one 90.0 mg 3, each chromatographically pure. The characterization of faradiol-3-myristic acid ester 1, faradiol-3-palmitic acid ester 2 and c-taraxasterol 3 was confirmed by MS, FABMS, 1H-NMR, 13C-NMR and 2D-NMR studies (Figs. 1 and 2). The data obtained were found in good agreement with corresponding partial fractures of literature (Della Loggia et al., 1994; Reynolds et al., 1986; Doddrell et al., 1974; Wenkert et al., 1978; Saar, 1991). Copies of the original spectrae are available from the corresponding author.
Fig. 1. Structures of 1 faradiol myristic ester (R =myristyl) and 2 faradiol palmitic ester (R=palmityl).
3.2. Anti-oedematous acti6ity The anti-oedematous activity shown at the Croton oil ear test (Section 2) by the three isolated substances is presented in Table 1, in comparison with that of the esters mixture, that of faradiol and that of indomethacin as reference drug. The two esters 1 and 2 show almost the same anti-oedematous activity at the Croton oil ear test: 240 mg/cm2 of each of the compounds induce an inhibition of the oedema near to the 50% and doubling the administered dose an inhibition of 65% for 1 and 66% for 2 is obtained. No significant synergism appears with the mixture. The free monol 3 apparently shows the same activity of the two esters. However, on a molar base compound
Fig. 2. Structure of 3 c-taraxasterol.
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Table 1 Anti-oedematous activities of compounds isolated from Calendula officinalis L. Substance
Dose (mg/cm2)
Dose (mmole/ cm2)
Controls Faradiolmonoesters mixture
— 240 480 240 480 240 480 240 480 120 100
— 0.36c 0.72c 0.37 0.74 0.35 0.70 0.56 1.12 0.27 0.28
Faradiol-3-myristic acid ester (1) Faradiol-3-palmitic acid ester (2) c-Taraxasterol (3) Faradiol Indomethacin a
N, number of animals; esters.
b
N a Oedema (mg) mean9S.E.
14 14 14 14 14 14 14 14 14 14
7.4 9 0.3 3.3 90.4b 2.4 90.3b 4.0 9 0.4b 2.6 9 0.3b 4.1 9 0.4b 2.5 90.4b 3.8 9 0.2b 1.0 90.2b 2.0 9 0.2b 3.2 90.4b
Inhibition (%)
— 55 68 46 65 45 66 49 86 73 75
Statistically different from controls (t-test, PB0.05); c Calculated for an equimolar mixture of faradiol
3 results to be less active than 1 and 2, since 0.56 mmole/cm2 of 3 are needed to obtain almost the same effect obtained by 0.35 mmole/cm2 of 1 or 2. Furthermore, faradiol is more active than its esters and than taraxasterol and shows the same effect as an equimolar dose of indomethacin. Although the numeric values of oedema inhibitions obtained in chronologically separate experiments can hardly be compared, due to the variability of the animal response, the present data confirm the previous observation that the presence of the OH-group at C-16 enhances the activity, and that esterification at position 3 reduces it (Della Loggia et al., 1994). The activity reduction seen for the esters could also be due to kinetic reasons, since highly lipophilic compounds can be trapped in the epidermis, reaching a lower concentration at the underlying action site. The limited increase in activity observed after doubling the administered dose could support this hypothesis.
3.3. General conclusions The faradiol-3-myristic acid ester and faradiol3-palmitic acid ester represent main compounds in lipophilic extracts of flowers of Calendula officinalis. Their anti-oedematous properties contribute remarkably to the pronounced antiphlogistic activity of lipophilic extracts as well as free faradiol
and c-taraxasterol. However, the intensity of the activities was shown to be partially different. Therefore not only the amount of faradiol after degradation or that of the mixture of the faradiol esters should be established for purpose of quality control, but a method giving the amounts of faradiol-3-myristic acid ester and faradiol-3palmitic acid ester as well as the content of the free triterpene alcohols might be preferred.
Acknowledgements The authors are grateful to DI. Novak J. for providing us with plant material and Mag. Ruzicka V. for her contribution to separation of the compounds by means of CC. We thank Dr Theiss Naturwaren OHG, Homburg, Germany for supporting part of this work by a grant. HPLC and GC/MS apparatus were financed by the support of the Austrian ‘Bundesministerium fu¨r Wissenschaft und Forschung’. Bertoldi M. was granted by a scholarship of the University of Modena.
References Della Loggia, R., Becker, H., Isaac, O., Tubaro, A., Topical anti-inflammatory activity of Calendula officinalis extracts. Planta Medica 56, 658.
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Della Loggia, R., Tubaro, A., Sosa, S., Becker, H., Saar, St., Isaa, O., 1994. The role of triterpenoids in the topical anti-inflammatory activity of Calendula officinalis flowers. Planta Medica 60, 516–520. Dequeker, R., 1964. Over een niet gevvone Handelspolygala. Pharmaceutisch Tijdschrift voor Belgie 41, 39–47. Doddrell, D.M., Khong, P.W., Lewis, K.G., 1974. The stereochemical dependence of 13C chemical shifts in olean-12enes and urs-12-enes as an aid to structural assignment. Tetrahedron Letters 27, 2381–2384. Isaac, O., 1992 Die Ringelblume. Wissenschaftliche Verlagsgesellschaft Stuttgart, pp. 71–74. Isaac, O., 1994. Calendula officinalis L.—Die Ringelblume, Portrait einer Arzneipflanze. Zeitschrift fu¨r Phytotherapie 16, 357 – 370. Kasprzyk, Z., Pyrek, J., 1968. Triterpenic alcohols of Calendula officinalis L. flowers. Phytochemistry 7, 1631–1639. Lievre, M., Marichy, J., Baux, S., Foyatier, J.L., Perrot, J., Boissel, J.P., 1992. Controlled study of three ointments for the local management of 2nd and 3rd degree burns. Clinical Trials and Meta-Analysis 28, 9–12. Perri de Carvalho, P.S., Tagliavini, D.G., Tagliavini, R.L., 1991. Cutaneous cicatrization after topic application of calendulas cream and comfrey, propolis and honey associations in infected wound of skin. Revista Cieˆncia Biome´dica (Sao Paulo) 12, 39 – 50. Peyrox, J., Rossignol, P., Delaveau, P., 1981. Proprie´te´s antioede´mateuses et anti-hyperhe´miantes du Calendula offici-
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nalis L.. Plantes Me´dicinales et Phytothe´rapie 15, 210 – 216. Pyrek, J.St., Baranowska, E., 1973. Faradiol and arnidiol — revision of the structure. Tetrahedron Letters 11, 809 – 810. Reynolds, W.F., McLean, S., Poplawski, J., 1986. Total assignment of 13C and 1H spectra of three isomeric triterpenol derivatives by 2D NMR: An investigation of the potential utility of 1H chemical shifts in structural investigations of complex natural products. Tetrahedron 42, 3419 – 3428. Saar, St., 1991. Phytochemische Untersuchungen an Calendula officinalis. Diplomarbeit, Universita¨t Saarbru¨cken. Shipochliev, T., Dimitrov, A., Aleksandrova, E., 1981. Study on the anti-inflammatory effect of a group of plant extracts. Veterinary Sciences (Sofia) 18, 87 – 94. Tubaro, A., Dri, P., Delbello, G., Zilli, C., Della Loggia, R., 1985. The Croton oil ear test revisited. Agents Actions 17, 347 – 349. Wenkert, E., Baddeley, G.V., Burfitt, I.R., Moreno, L.N., 1978. Carbon-13 nuclear magnetic resonance spectroscopy of naturally-occurring substances. Organic Magnetic Resonance 11, 337 – 342. Wilkomirski, B., Kasprzyk, Z., 1979. Free and ester-bound triterpene alcohols and sterols in cellular subfractions of Calendula officinalis flowers. Phytochemistry 18, 253 – 255. Wojciechowski, Z., Bochenska-Hryniewicz, M., Kucharczak, B., Kasprzyk, Z., 1972. Sterol and triterpene alcohol esters from Calendula officinalis. Phytochemistry 11, 1165 – 1168.
Anti-oedematous activities of the main triterpendiol esters of marigold (Calendula officinalis L.) K. Zitterl-Eglseer a,*, S. Sosa b, J. Jurenitsch c, M. Schubert-Zsilavecz d, R. Della Loggia b, A. Tubaro b, M. Bertoldi 1,a, C. Franz a a
Institute for Botany and Food Science, Uni6ersity of Veterinary Medicine Vienna, Josef-Baumann-Gasse 1, A-1210 Wien, Austria b Institute of Pharmacology and Pharmacognosy, Uni6ersity of Trieste, Via L. Giorgieri 7, I-34 100 Trieste, Italy c Institute of Pharmacognosy, Uni6ersity of Vienna, Pharmaziezentrum, Althanstrasse 14, A-1090 Wien, Austria d Institute of Pharmaceutical Chemistry, Uni6ersity of Graz, Uni6ersita¨tsplatz 1, A-8010 Graz, Austria Received 18 April 1997; received in revised form 5 May 1997; accepted 13 May 1997
Abstract Separation and isolation of the genuine faradiol esters (1, 2) from flower heads of Marigold (Calendula officinalis L., Asteraceae) could be achieved by means of repeated column chromatography (CC) and HPLC for the first time. Structure elucidation of faradiol-3-myristic acid ester 1, faradiol-3-palmitic acid ester 2 and c-taraxasterol 3 has been also performed, without any previous degradation by means of MS, 1H-NMR, 13C-NMR and 2D-NMR experiments. The anti-oedematous activities of these three compounds were tested by means of inhibition of Croton oil-induced oedema of the mouse ear. Both faradiol esters showed nearly the same dose dependent anti-oedematous activity and no significant synergism appeared with their mixture. The free monol, c-taraxasterol, had a slightly lower effect. Furthermore, faradiol was more active than its esters and than c-taraxasterol and showed the same effect as an equimolar dose of indomethacin. © 1997 Elsevier Science Ireland Ltd. Keywords: Calendula officinalis L.; Marigold; Triterpendiol esters; Faradiol-3-myristic acid ester; Faradiol-3-palmitic acid ester; Taraxasterol; Anti-oedematous activity
* Corresponding author. 1 Permanent address: Department of Pharmaceutical Science, University of Modena, Via Campi 183, I-41 100 Modena, Italy.
1. Introduction Preparations of Marigold, Calendula officinalis L. (Asteraceae) for topical application are used
0378-8741/97/$17.00 © 1997 Elsevier Science Ireland Ltd. All rights reserved. PII S 0 3 8 - 8 7 4 1 ( 9 7 ) 0 0 0 6 1 - 5
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both in dermatology and cosmetics (Isaac, 1992, 1994). Besides traditional galenic formulations such as infusa, tinctures and fluid extracts in folk medicine flower heads of Calendula officinalis were extracted by hot lard, which is similar to the fat of the human skin, in order to obtain ointments for several dermatological diseases as wounds, ulcers, eczemas, burns, contusions, eruptions, varicose veins and haemorrhoids. The anti-inflammatory and wound-healing activity of the drug was documented in many experimental and clinical studies (Shipochliev et al., 1981; Peyrox et al., 1981; Perri de Carvalho et al., 1991; Lievre et al., 1992). Recent investigations demonstrated that a number of lipophilic compounds of hydroalcoholic (70%) and CO2-extracts account for the topical anti-inflammatory activity (Della Loggia et al., 1990, 1994). The main compounds of lipophilic extracts of flowers of Calendula officinalis are triterpendiols, which are 98% bound as 3-monoesters with higher fatty acids and occur almost exclusively in the chromoplasts (Wilkomirski and Kasprzyk, 1979). Structure elucidation of these esters has been performed formerly by chemical degradation, separation and characterization of the triterpene alcohols by their spectroscopic properties and of the corresponding fatty acids by comparison with authentic samples after methylation by means of gas chromatography (Kasprzyk and Pyrek, 1968; Wojciechowski et al., 1972; Pyrek and Baranowska, 1973; Della Loggia et al., 1994). Up to now the triterpenoid alcohols have been separated by means of column chromatography (CC) or preparative thin layer chromatography (TLC) and quantitatively determined by colorimetrical measurement of their CoCl2 complexes (Wojciechowski et al., 1972). The isolation of the genuine esters has never been performed. Therefore the biological activity of the esters was studied only on their mixture and the activity of the single compounds remained unknown, so far. In account of this knownledge, the aim of this work was to establish a separation of the genuine esters and to study their topical anti-oedematous properties.
2. Material and methods
2.1. Plant material TLC-screening: 1200 single plant samples of 20 commercially available origins of Calendula officinalis L. obtained from Austrian and German companies, namely, Austrosaat (5), Borntra¨ger (3), Chrestensen (1), Landes-Versuchsanlage fu¨r Spezialkulturen Wies/Stmk. (1), Landwirtschaftlich-chemische Bundesanstalt Wien/Korneuburg (3), Saatbau Linz (6) and Theiss (1) were investigated. Isolation of 1, 2 and 3: Calendula officinalis L. Cultivar: ‘Erfurter orangefarbige, gefu¨llt blu¨hende’ (Asteraceae) was grown from seeds in Mu¨hlbach and Korneuburg, Lower Austria and harvested in August 1993. Voucher specimens (CAL1061-1064/93) have been deposited in the Herbarium of the Institute for Botany, University of Veterinary Medicine, Vienna.
2.2. Reagents The following reagents were used: Dichloromethane p.a., n-hexane p.a., ethylacetate p.a., acetonitrile p.a., ethanol p.a., methanol CHROMASOLV (Riedel de Hae¨n), anisaldehyde, acetic acid (100%), sulfuric acid (100%). Faradiol was kindly provided by Prof. H. Becker, Saarbru¨cken.
2.3. Extraction TLC-screening: 100 mg of air-dried powdered flower heads of Calendula officinalis were extracted with 3 ml dichloromethane 15 min in an ultrasonic bath at room temperature and filtered. CC: 1000 g air-dried flower heads of Calendula officinalis were extracted exhaustively with dichloromethane. After removal of the solvent the residue was separated by CC.
2.4. Analytical and preparati6e methods TLC: Screening and CC-monitoring: Stationary phase: HPTLC, silica gel 60 F254 nm, MERCK, mobile phase: n-hexane/ethylacetate (8+ 2), detection: anisaldehyde reagent (Dequeker, 1964).
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CC: Extract: 72.8 g, column dimensions: 100 × 7 cm, stationary phase: silica gel 60 TSC, 0.063 – 0.2 mm, mobile phase: n-hexane/ethylacetate, gradients: 100, 98+2, 96 +4, 94 + 6, 92 + 8, 90+ 10. SPE: Extract: 1.28 g, stationary phase: RP-18 material, mobile phase: methanol/water, gradients: 6+4, 8+ 2, 9+1, 10 +0. HPLC: PE Series 10, PE Series 10 LC Controller, PE UV/VIS LC290, PE Nelson model 1020, Okidata Microline 320 Printer and Kontron Instruments, LC Pump T-414, UV/VIS-Detector 432, Chromatography software: Borwin; Columns: LiChrosphere 100, RP-8, 5 mm, 15 × 250 and 4 ×250 mm, solvent: methanol/water, wavelength: 210 nm. MS: Shimadzu QP 1000, DI, 25°C, 20°C/min, EI: 70 eV: m/e (rel. int.%): 1, faradiol myristic acid ester 652 (M + ), 634 (6.0), 424 (16.7), 189 (100.0), 121 (41.3), 109 (44.6), 95 (57.1); 2, faradiol palmitic acid ester 680 (M+), 662 (10.4), 424 (42.9), 239 (15.6), 205 (41.1), 189 (98.0), 135 (79.7), 107 (80.4); 3, c-taraxasterol 426 (M+ ), 218 (96.4), 207 (69.4), 189 (99.6), 135 (52.2), 95 (92.2). NMR: 1H-NMR, 13C-NMR, 2D-NMR measurements were performed on a Bruker AC 200 and AMX 500 spectrometer. 1 faradiol myristic ester, 13C-NMR (CDCl3): d = 38.5 (C-1), 27.2 (C-2), 80.5 (C-3), 37.8 (C-4), 55.4 (C-5), 18.2 (C-6), 34.2 (C-7), 41.1 (C-8), 49.9 (C-9), 37.6 (C-10), 22.7 (C-11), 29.2 (C-12), 38.8 (C-13), 42.5 (C-14), 37.0 (C-15), 76.2 (C-16), 39.9 (C-17), 47.6 (C-18), 35.9 (C-19), 139.6 (C-20), 118.3 (C-21), 36.6 (C-22) 28.0 (C-23), 14.1 (C-24), 16.3 (C-25), 16.0 (C-26), 16.5 (C-27), 11.6 (C-28), 22.4 (C-29), 21.5 (C-30); acid moiety: 173.6, 34.8, 29.6, 29.4, 29.3, 29.2, 29.1, 25.1, 23.7, 16.3. 2 faradiol palmitic ester, 13C-NMR (CDCl3): d = 38.8 (C-1), 27.2 (C-2), 80.6 (C-3), 38.5 (C-4), 55.5 (C-5), 18.2 (C-6), 34.2 (C-7), 41.1 (C-8), 50.0 (C-9), 37.8 (C-10), 22.6 (C-11), 28.0 (C-12), 37.6 (C-13), 42.5 (C-14), 37.0 (C-15), 76.2 (C-16), 39.9 (C-17), 47.7 (C-18), 35.9 (C-19), 139.6 (C-20), 118.4 (C-21), 36.5 (C-22) 29.2 (C-23), 14.0 (C-24), 16.3 (C-25), 16.0 (C-26), 16.5 (C-27), 11.6 (C-28), 22.4 (C-29), 21.6 (C-30); acid moiety: 173.6, 34.8, 31.8, 29.6, 29.5, 29.4, 29.3, 29.2, 29.1, 25.1, 23.7, 16.3.
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3 c-Taraxasterol, 1H-NMR (CDCl3/DMSO[d6]): d= 0.78 (H-1ax), 1.42 (H-1eq), 1.38 (H2ax), 1.44 (H-2eq), 2.99 (H-3), 0.52 (H-5), 1.19 (H-6ax), 1.32 (H-6eq), 1.17-1.26 (H-7ax and H-7eq), 1.11 (H-9), 1.08 (H-11ax), 1.37 (H-11eq), 1.05 (H12ax), 1.43 (H-12eq), 1.51 (H-13), 0.82 (H-15ax), 1.60 (H-15eq), 1.03 (H-16ax), 1.13 (H-16eq), 0.86 (H-18), 1.39 (H-19), 5.06 (H-21), 1.36 (H-22), 1.52 (H-22%), 0.79 (CH3-23), 0.58 (CH3-24), 0.82 (CH325), 0.86 (CH3-26), 0.77 (CH3-27), 0.55 (CH3-28), 0.81 (CH3-29), 1.43 (CH3-30). 13C-NMR: d= 38.5 (C-1), 27.0 (C-2), 78.1 (C-3), 38.5 (C-4), 55.0 (C-5), 17.9 (C-6), 33.9 (C-7), 41.1 (C-8), 50.4 (C-9), 36.7 (C-10), 21.1 (C-11), 27.2 (C-12), 38.8 (C-13), 41.8 (C-14), 26.4 (C-15), 36.5 (C-16), 34.0 (C-17), 48.4 (C-18), 35.9 (C-19), 139.4 (C-20), 118.5 (C-21), 41.9 (C-22), 27.7 (C-23), 15.1 (C-24), 15.9 (C-25), 15.7 (C-26), 14.4 (C-27), 17.3 (C-28), 22.1 (C-29), 21.2 (C-30).
2.5. Anti-oedematous acti6ity The Croton oil ear test was performed as already described (Tubaro et al., 1985). Groups of seven male Albino Swiss mice (28–30 g, Charles Rivers, Calco, Italy) were anaesthetized with Ketalar® and 15 ml of an acetonic solution containing the irritant (75 mg of Croton oil, Sigma) and the appropriate amount of the substances under testing were applied to the inner surface of the right ear of mice (surface, about 1 cm2), the left ear remaining untreated. Control animals recieved only the irritant. The animals were sacrificed by cervical dislocation 6 h later and a plug (6 mm in diameter) was removed from both the treated and the untreated ear. The difference in weight between the two plugs was taken as a measure of the oedematous response. At least two experimental groups of seven animals were used for each tested dose level.
3. Results and discussion A TLC-screening of 1200 individuals of Calendula officinalis from 20 origins showed the presence of faradiol monoesters in each plant, however in different quantities. Therefore a culti-
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var characterized by a TLC-fingerprint showing a high amount of all the faradiol esters could be chosen for the isolation. Since the cultivar was available in almost unrestricted amounts an optimization of the isolation procedure was possible, as well as the isolation of enough amounts of pure substances for chemical characterization and pharmacological experiments.
3.1. Isolation and characterization of 1, 2 and 3 The residue of the dichloromethane extract of flower heads of Calendula officinalis was purified by means of CC on silica gel monitored by TLC. Two fractions of CC containing the main compounds recorded by TLC (Rf =0.54 and Rf = 0.47) were further purified. The first fraction (1.28 g; Rf =0.54) was cleaned up with SPE-Clean-up-Columns on RP-18 material with methanol/water, the second fraction (Rf =0.47) by means of CC on silica gel with dichloromethane. Further separations were performed by repeated preparative HPLC. After extensive optimization experiments using different types of RP-materials and mixtures of acetonitrile/water, ethanol/water and methanol/water as mobile phases the following conditions were found as optimum for the semipreparative HPLC separation: Stationary phase: LiChrosphere 100, RP-8, 5 mm using methanol/water (9+ 1, isocratic) for the first CC-fraction and a gradient from methanol/water (8 + 2) to pure methanol for the second one. The first CC-fraction yielded 268.3 mg 1, 246.6 mg 2 and the second one 90.0 mg 3, each chromatographically pure. The characterization of faradiol-3-myristic acid ester 1, faradiol-3-palmitic acid ester 2 and c-taraxasterol 3 was confirmed by MS, FABMS, 1H-NMR, 13C-NMR and 2D-NMR studies (Figs. 1 and 2). The data obtained were found in good agreement with corresponding partial fractures of literature (Della Loggia et al., 1994; Reynolds et al., 1986; Doddrell et al., 1974; Wenkert et al., 1978; Saar, 1991). Copies of the original spectrae are available from the corresponding author.
Fig. 1. Structures of 1 faradiol myristic ester (R =myristyl) and 2 faradiol palmitic ester (R=palmityl).
3.2. Anti-oedematous acti6ity The anti-oedematous activity shown at the Croton oil ear test (Section 2) by the three isolated substances is presented in Table 1, in comparison with that of the esters mixture, that of faradiol and that of indomethacin as reference drug. The two esters 1 and 2 show almost the same anti-oedematous activity at the Croton oil ear test: 240 mg/cm2 of each of the compounds induce an inhibition of the oedema near to the 50% and doubling the administered dose an inhibition of 65% for 1 and 66% for 2 is obtained. No significant synergism appears with the mixture. The free monol 3 apparently shows the same activity of the two esters. However, on a molar base compound
Fig. 2. Structure of 3 c-taraxasterol.
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Table 1 Anti-oedematous activities of compounds isolated from Calendula officinalis L. Substance
Dose (mg/cm2)
Dose (mmole/ cm2)
Controls Faradiolmonoesters mixture
— 240 480 240 480 240 480 240 480 120 100
— 0.36c 0.72c 0.37 0.74 0.35 0.70 0.56 1.12 0.27 0.28
Faradiol-3-myristic acid ester (1) Faradiol-3-palmitic acid ester (2) c-Taraxasterol (3) Faradiol Indomethacin a
N, number of animals; esters.
b
N a Oedema (mg) mean9S.E.
14 14 14 14 14 14 14 14 14 14
7.4 9 0.3 3.3 90.4b 2.4 90.3b 4.0 9 0.4b 2.6 9 0.3b 4.1 9 0.4b 2.5 90.4b 3.8 9 0.2b 1.0 90.2b 2.0 9 0.2b 3.2 90.4b
Inhibition (%)
— 55 68 46 65 45 66 49 86 73 75
Statistically different from controls (t-test, PB0.05); c Calculated for an equimolar mixture of faradiol
3 results to be less active than 1 and 2, since 0.56 mmole/cm2 of 3 are needed to obtain almost the same effect obtained by 0.35 mmole/cm2 of 1 or 2. Furthermore, faradiol is more active than its esters and than taraxasterol and shows the same effect as an equimolar dose of indomethacin. Although the numeric values of oedema inhibitions obtained in chronologically separate experiments can hardly be compared, due to the variability of the animal response, the present data confirm the previous observation that the presence of the OH-group at C-16 enhances the activity, and that esterification at position 3 reduces it (Della Loggia et al., 1994). The activity reduction seen for the esters could also be due to kinetic reasons, since highly lipophilic compounds can be trapped in the epidermis, reaching a lower concentration at the underlying action site. The limited increase in activity observed after doubling the administered dose could support this hypothesis.
3.3. General conclusions The faradiol-3-myristic acid ester and faradiol3-palmitic acid ester represent main compounds in lipophilic extracts of flowers of Calendula officinalis. Their anti-oedematous properties contribute remarkably to the pronounced antiphlogistic activity of lipophilic extracts as well as free faradiol
and c-taraxasterol. However, the intensity of the activities was shown to be partially different. Therefore not only the amount of faradiol after degradation or that of the mixture of the faradiol esters should be established for purpose of quality control, but a method giving the amounts of faradiol-3-myristic acid ester and faradiol-3palmitic acid ester as well as the content of the free triterpene alcohols might be preferred.
Acknowledgements The authors are grateful to DI. Novak J. for providing us with plant material and Mag. Ruzicka V. for her contribution to separation of the compounds by means of CC. We thank Dr Theiss Naturwaren OHG, Homburg, Germany for supporting part of this work by a grant. HPLC and GC/MS apparatus were financed by the support of the Austrian ‘Bundesministerium fu¨r Wissenschaft und Forschung’. Bertoldi M. was granted by a scholarship of the University of Modena.
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