Secondary metabolites from two species of tolpis and their biological activities

Molecules 2012, 17, 12895-12909; doi:10.3390/molecules171112895 molecules ISSN 1420-3049 www.mdpi.com/journal/molecules Article Secondary Metabolites from Two Species of Tolpis and Their Biological Activities Jorge Triana 1,*, Mariana López 1, Francisco Javier Pérez 1, Milagros Rico 1, Aroa López 1, Francisco Estévez 2,3, María Teresa Marrero 2, Ignacio Brouard 4 and Francisco León 4,* 1 Departamento de Química, Unidad Asociada al CSIC, Universidad de Las Palmas de Gran Canaria, Campus de Tafira, 35017, Las Palmas de Gran Canaria, Spain; E-Mails: mlope[email protected] (M.L.); [email protected] (F.J.P.); m[email protected]c.es (M.R.); [email protected] (A.L.) 2 Departamento de Bioquímica, Unidad Asociada al CSIC, Universidad de Las Palmas de Gran Canaria, Plaza Dr. Pasteur s/n, 35016, Las Palmas de Gran Canaria, Spain; E-Mails: [email protected] (F.E.); mariatem[email protected]m (M.T.M.) 3 Fundación Canaria Instituto Canario de Investigación del Cáncer, Torre Agustín Arévalo-7ª Planta (PCTT-ULL) Avda de la Trinidad s/n 38204 San Cristóbal de La Laguna-S/C de Tenerife, Spain 4 Instituto de Productos Naturales y Agrobiología, Consejo Superior de Investigaciones Científicas (CSIC), Avda. Astrofísico F. Sánchez, 3, 38206 La Laguna, Spain; E-Mail: [email protected].es * Authors to whom correspondence should be addressed; E-Mails: [email protected] (J.T.); [email protected] (F.L.); Tel.: +34-922-256847 (ext. 224) (F.L.); Fax: +34-922-260135 (F.L.). Received: 27 September 2012; in revised form: 24 October 2012 / Accepted: 26 October 2012 / Published: 1 November 2012 Abstract: Phytochemical research of two Tolpis species, T. proustii and T. lagopoda, led to the isolation of three new compounds: 30-chloro-3β-acetoxy-22α-hydroxyl-20(21)- taraxastene (1), 3β,22α-diacetoxy-30-ethoxy-20(21)-taraxastene (2) and 3β,28-dihydroxy11α-hydroperoxy-12-ursene (3). The structures of the new compounds were elucidated by means of extensive IR, NMR, and MS data and by comparison of data reported in the literature. The in vitro antioxidant activities of the extracts were assessed by the DPPH and ABTS scavenging methods. The cytotoxicity of several known compounds and its derivatives was also assessed against human myeloid leukemia K-562 and K-562/ADR cell lines. Keywords: Asteraceae; Tolpis proustii; Tolpis lagopoda; ursane-triterpenes; taraxastane-triterpenes OPEN ACCESS Molecules 2012, 17 12896 1. Introduction The Tolpis genus (Asteraceae: Cichorioideae, Cichoriinae) consists of some 20 species distributed throughout Europe, North Africa, Canary Islands, Cape Verde and Asia, this genus being represented in the Canary Islands by around ten species [1]. Although a wide-ranging study of different species of Tolpis from the Canary Islands and from other Macaronesian archipelagos has been carried out from the botanical point of view [2,3], only one phytochemical report has previously appeared on the isolation and structural elucidation of aromatic compounds, triterpenes, and sterols from T. webbi and T. spp [4]. As a part of our continuing search for novel, plant-derived biological agents and our systematic investigation of the composition of Canarian endemic plants, the present work describes the isolation and structural elucidation of the constituents of the ethanolic extracts of the aerial parts of T. proustii Pitard in Pitard and Proust and T. lagopoda C.Sm. ex Buch. The constituents of these extracts were purified by CC, MPLC and preparative TLC. The structures of the known compounds were confirmed by comparison of their spectroscopic data with those reported in the literature. From T. proustii seventeen compounds were isolated including two taraxan-triterpenoid 30-chloro3β-acetoxy-22α-hydroxyl-20(21)-taraxastene 1, and 3β,22α-diacetoxy-30-ethoxy-20(21)-taraxastene 2 a new ursan-triterpenoid 3β,28-dihydroxy-11α-hydroperoxy-12-ursene 3. From T. lagopoda, eight compounds were isolated including a 3β,22α-diacetoxy-30-ethoxy-20(21)-taraxastene 2 (Figure 1). Figure 1. Compounds 1–3 isolated of the T. proustii and T. lagopoda. The high level of solar radiation and the high temperature prevailing in the region of the Canary Islands (opposite the northwest coast of Africa) force plants to develop defence mechanisms against ultraviolet radiation and excessive production of free radicals through the accumulation of antioxidant metabolites [5]. This prompted us to evaluate the antioxidant activity of the crude extract of T. proustii and T. lagopoda, and that of some of the isolated compounds. Furthermore, one of the most important mechanisms by which tumor cells resist to cytotoxic effects of a variety of chemotherapeutic drugs is overexpression of the human multidrug resistance (MDR1) gene and its product, P-glycoprotein [6]. Here we have evaluated the effects of natural compounds and derivatives on the growth of the human leukemia K-562 and the P-glycoprotein-overexpressing K-562/ADR cell lines. Molecules 2012, 17 12897 2. Results and Discussion 2.1. Structure Elucidation of Compounds 1–3 The positive EIMS spectrum of compound 1 showed a molecular ion at m/z 518/520 (3:1) in agreement with the presence of a chlorine atom in the structure, and with the formula C32H51O3Cl for this compound. In the same spectrum, the ions at m/z 500/502 [M-H2O]+, 458/460 [M-CH3COOH]+ suggested that this compound contained a hydroxyl and an acetyl group, respectively. Confirmed by the IR spectrum with absorptions of a hydroxyl 3446 cm−1 and a carbonyl 1730 cm−1 group. Its HREIMS experiment indicated the molecular formula C32H51O337Cl (calcd. for [M]+ 520.3497; found 520.3497) and C32H51O335Cl (calcd. for [M]+ 518.3527; found 518.3517). All spectral data suggested that 1 was a pentacyclic triterpene with a trisubstituted double bond in the E ring [7] with a 20(21)-taraxastane structure. The 1H and 13C-NMR (Table 1) spectra of 1 showed the presence of the oxygenated methine proton at δH 3.35 (1H, d, J = 6.6 Hz) and an unusual chloro atom at C-30 at δC 47.6 (Table 1). The structure elucidation and NMR assignments were therefore based primarily on the results of COSY, HSQC, HMBC, and NOESY experiments (Figure 2). Table 1. 1Hand 13C-NMR data for compounds 1, 1a and 2 a. 1 1a 2 Position H C H C H C 1 1.65 * 38.5 1.65 * 38.7 1.60 * 38.5 2 1.50 * 23.7 1.54 * 21.7 1.55 * 23.7 3 4.41 dd (6.1,10.8) 81.0 4.43 dd (6.4,10.0) 81.1 4.43 dd (5.1 10.4) 81.0 4 - 38.3 - 38.0 - 37.8 5 0.75 * 55.4 0.74 * 55.6 0.75 * 55.4 6 1.45 * 1.35 * 18.2 1.44 * 1.34 * 18.0 1.30 * 18.2 7 1.35 * 34.2 1.34 * 34.4 1.35 * 34.2 8 - 41.2 - 41.3 - 41.1 9 1.25 * 50.3 1.26 * 50.6 1.25 * 50.4 10 - 37.1 - 37.3 - 37.1 11 1.20 * 21.6 1.21 * 21.4 1.20 * 21.6 12 1.60 * 1.20 * 27.6 1.60 * 27.7 1.58 * 27.5 13 0.95 * 38.6 0.95 * 1.66 * 38.6 14 - 42.3 - 42.4 - 42.3 15 1.72 * 1.05ddd (2.5,4.0,13.05) 26.7 1.71 * 1.02 * 29.9 1.50 * 26.6 16 0.95 * 1.85 dt (9.0,13.0) 29.7 0.95 * 29.9 1.60 * 29.9 17 - 37.8 - 37.4 - 37.2 18 1.45 * 40.5 1.44 * 41.5 1.45 * 41.5 Molecules 2012, 17 12898 Table 1. Cont. 1 1a 2 Position H C H C H C 19 2.00 t (7.0) 31.6 2.03 tbr (7.0) 31.7 1.79 q (6.6) 32.3 20 - 144.9 - 146.5 - 147.7 21 5.89 d (6.5) 126.3 5.87 d (6.4) 123.1 5.75 d (6.3) 119.2 22 3.35 dbr (6.6) 73.3 4.51 d (6.4) 75.3 4.55 d (6.3) 75.3 23 0.78 s 28.0 0.79 s 28.1 0.81 s 28.0 24 0.77 s 16.6 0.78 s 16.7 0.80 s 16.5 25 0.82 s 16.4 0.82 s 16.7 0.81 s 16.4 26 0.99 s 16.1 0.98 s 16.2 0.96 s 16.1 27 0.93 s 14.7 0.91 s 14.7 0.92 s 14.6 28 0.63 s 17.8 0.70 s 17.9 0.70 s 18.2 29 0.99 d (6.5) 22.2 0.97 d (7.6) 22.1 0.96 d (6.6) 22.6 30 4.15 d (11.2) 3.89 d (11.2) 47.6 4.14 d (11.2) 3.89 d (11.6) 47.5 3.96 d (12.6) 3.72 d (12.6) 72.3 OH 3.14 m OAc 1.97 s 21.4 171.4 1.97 s 1.98 s 21.3 21.3 171.1 171.1 1.97 s 1.98 s 21.3 21.3 171.1 171.1 OEt 1.13 t (6.9) 3.35 m 15.2 65.9 a δ in ppm and J (in Hz) are in parentheses. Recorded in CDCl3 at 400 MHz and 125 MHz for 1H and 13C-NMR, respectively. * overlapped. Figure 2. Selected correlations of 1. Double-ended arrows indicate NOESY, and single arrows indicate HMBC (C to H) correlations. The most important HMBC and NOESY correlations are shown in Figure 1. Treatment of 1 with Ac2O-pyridine gave a diacetyl derivative 1a for which its HREIMS experiment indicated the molecular formula C34H53O437Cl (calcd. for [M]+ 562.3603; found 562.3624) and C34H54O435Cl (calcd. for [M]+ 560.3654; found 560.3644). The data implied the presence of a double bond between C-20 and C-21, the chloro atom at C-30, and the acetyl group at C-3 and hydroxyl group at C-22. H-22 was assigned in β-orientation on the basis of the coupling constant with the vinylic proton at C-21 and cross-peak in the NOESY experiment with the CH3-28. From the above findings, the structure of Molecules 2012, 17 12899 30-chloro-3β-acetoxy-22α-hydroxyl-20(21)-taraxastene was assigned to 1, and it was named chlorotolpidiol. To the best of our knowledge, this is the first example of a pentacyclic triterpene of the taraxastane-ursane series with a chloro functionality. Compound 2 was obtained as a colourless amorphous solid and its molecular formula was determined by a HRESIMS experiment as C36H58O5 (calcd. for [M+Na]+ 593.4182; found 593.4176). The IR spectrum revealed the absorption bands for a carbonyl group (1732 cm−1) and double bond (2872 cm−1). The 1H-NMR spectrum (Table 1) exhibited six singlet methyl groups at δH 0.70, 0.80, 0.81 (6H), 0.92 and 0.96, a secondary methyl group at δH 0.96 (3H, d, J = 6.6 Hz) attributed to C-29, a vinyl proton at δH 5.75 (1H, d, J = 6.3 Hz), two acetyl groups at δH 1.97 s and 1.98 s, two oxymethine signals at δH 4.43 (1H, dd, J = 5.1, 10.4 Hz) and 4.55 (1H, d, J = 6.3 Hz), an oxymethylene signal at δH 3.96 (1H, d, J = 12.6 Hz) and 3.72 (1H, d, J = 12.6 Hz) and an ethoxy group at δH 3.35 (2H, m) and 1.13 (3H, t, J = 6.9 Hz). The extra acetyl signal at C-22 was observed, since the oxygenated methine proton of 1 at δH 3.35 (1H, d, J = 6.6 Hz) was displaced to δH 4.55 (1H, d, J = 6.3 Hz) in 2. Moreover, the halogenated group was replaced by a ethoxyl group at C-30 since the carbon in 2 was displaced to low field at δC 72.3 (Table 1). The structure of 2 was determined by a combination of COSY, DEPT, HSQC, HMBC, and NOESY experiments. Based on the above data, the new compound tolpidiol A 2 was identified as 3β,22α-diacetoxy-30-ethoxy-20(21)-taraxastene. Compound 3 was purified as its diacetate 3a by treatment with acetic anhydride (Ac2O) in pyridine, 3a was isolated as a colourless amorphous solid and its HRESIMS experiment indicated the molecular formula C34H54O6 (calcd. for [M+Na]+ 581.3818; found 581.3801). The IR spectrum revealed the absorption bands for carbonyl 1728 cm−1, and hydroxyl 3391 cm−1 groups, the presence of these groups was confirmed by the 1H and 13C-NMR spectra (Table 2). The 1H-NMR spectrum of 3a showed signals for five tertiary methyl groups at δH 0.81(6H, br, s), 1.00, 1.02 and 1.11, and two secondary methyl groups at δH 0.86 (3H, d, J = 6.4 Hz) and δH 0.88 (3H, d, J = 7.3 Hz) suggesting a pentacyclic triterpene with an ursane skeleton. An olefinic proton at δH 5.30 (1H, d, J = 3.1 Hz) was assigned to H-12, two oxygenated methines at δH 4.45 (1H, dd, J = 3.0, 9.6 Hz) and 4.46 (1H, dd, J = 5.0, 9.5 Hz) corresponding to H-3 and H-11 respectively, the latter showing vicinal correlation in the COSY experiment with the olefinic proton H-12, while the proton H-9 δH 1.81 (1H, d, J = 9.5 Hz) indicated the presence of a hydroperoxide at C-11. The presence of an oxygenated methylene was confirmed by the signals at δH 3.56 (1H, d, J = 11.0 Hz) and 3.93 (1H, d, J = 11.0 Hz). The 13C-NMR (Table 2) and DEPT data indicated the presence of two ester carbonyl groups, nine methyl carbons, nine aliphatic methylenes, two olefinic carbons, and seven methine carbons. Thus, the position of acetyl groups in compound 3a was assigned by a HMBC correlation between the signal at δC 171.0 and that at δH 4.45; and the signals at δC 171.3 and δH 3.93. The coupling constant between H-9 and H-11 (J = 9.5 Hz) established the α-orientation of the hydroperoxide at C-11. The EI-MS data of 3a showed direct loss of H2O m/z 540 and H2O2 m/z 524, confirming the presence of the hydroperoxide. The structure elucidation and NMR assignments were based primarily on the results of HSQC, HMBC, and COSY experiments which allowed the complete assignment of all Hand C-atoms, and the NOESY (Figure 3) data provided the configuration of compound 3a. Therefore, the structure of 3 was established as 3β,28-dihydroxy-11α-hydroperoxy-12-ursene. To the best of our knowledge, compound 3 is a novel triterpenoid, which we named tolpidiol B. Molecules 2012, 17 12900 Table 2. 1H and 13C-NMR data for compound 3a a. Position H C 1 0.85 m 2.08 dt (3.5, 7.0) 39.4 2 1.58 * 23.7 3 4.45 dd (3.0,9.6) 80.6 4 - 38.0 5 0.88 m 55.3 6 1.45 * 1.31 * 18.1 7 1.45 m 1.25 m 33.3 8 - 43.2 9 1.81 d (9.5) 48.8 10 - 37.8 11 4.46 dd (5.0, 9.5) 81.6 12 5.30 d (3.1) 125.8 13 - 144.5 14 - 42.0 15 1.60 * 0.90 m 26.2 16 1.16 * 1.92 td (3.5,9.0) 23.3 17 - 36.9 18 1.45 m 53.7 19 1.35 m 39.0 20 1.28 m 39.3 21 1.40 m 30.4 22 1.31 m 1.52 dt (3.0, 6.5) 35.5 23 0.81 s 28.2 24 0.81 s 16.7 25 1.02 s 16.8 26 1.00 s 18.0 27 1.11 s 22.2 28 3.56 d (11.0) 3.93 d (11.0) 71.0 29 0.86 d (6.4) 17.4 30 0.88 d (7.3) 21.3 OAc 1.97 s 1.97 s 21.0 21.3 171.0 171.3 a δ in ppm and J (in Hz) are in parentheses. Recorded in CDCl3 at 400 MHz and 125 MHz for 1Hand 13C-NMR, respectively. * overlapped. Molecules 2012, 17 12901 Figure 3. Selected correlations of 3a. Bond bolded indicate COSY, Double-ended arrows indicate NOESY, and single arrows indicate HMBC (C to H) correlations. Compound 1 could derive from the known triterpene acetyl-ptiloepoxide 4 [8,9] which was identified by us from T. proustii as an inseparable mixture. Triterpenes containing an epoxide at the Δ21-22 position are known and have been isolated before from a Tolpis species [4]. Based on this, we envisioned the formation of compound 1 by chlorination of the double bond, followed by isomerization and β opening of the epoxide, and protonation, maintaining the α-orientation at the C-22 observed in the precursor compound acetyl-ptiloepoxide (Figure 4). Figure 4. Tentative forming process of compound 1. Although the number is relatively small, several halogenated triterpenes and other higher terpenes have been described mainly from marine sources [10]. However, the presence of chlorinated triterpenoids in terrestrial plants is very rare and just few cases have been reported [11–14]. Initially, compound 1 seemed to be an artifact of the isolation process, but Chen et al. [14] proved that to obtain chlorinated compounds, a chlorine source such as CHCl3 with HCl is necessary. During the isolation process, no chlorinated solvents were used (see Experimental). In the chromatographic separation, dichloromethane was used in the preparative TLC, which was not enough to interact with the possible precursor (ptiloepoxide) of 1. Compound 2 was isolated as a presumed artifact; this compound was probably obtained from 1, by a nucleophilic substitution reaction due to the use of hot EtOH during the extraction process. Additionally, from T. proustii 15 known compounds were isolated, including aromatic compounds: scopoletin [15] aesculetin [15] and apigenin [16]; the diterpene phytene-1,2-diol [17] and the triterpenoids stigmasterol [18], ergosterol peroxide [19], ursolic acid [20], lupan-20(29)-ene-3,30diol [21], 21-hydroxytaraxasterol [22], 11-oxo-β-amyrin [23], 3-acetoxy-urs-12-ene-1,11-diol [24], 21,22-epoxy-20-hydroxy-20(30)-dihydrotaraxasterol [4], 3-acetoxy-21,22-epoxytaraxastan20α-ol [25], 22-oxo-20-taraxasten-3-ol [9], -amyrin [26]. From T. lagopoda seven known Molecules 2012, 17 12902 compounds were isolated, including aromatic compounds: 2,4′-dihydroxy-4-methoxybenzophenone [4] and triterpenoids: stigmasterol [18], ergosterol peroxide [19], a mixture of 7-oxo-β-sitosterol and 7-oxo-stigmasterol [26,27], ursolic acid [20], and α-amyrin [28]. Their structures were confirmed by comparison of their spectral data with those reported in the literature. 2.2. Antioxidant Activities Natural antioxidants that are present in plants are responsible for inhibiting or preventing the deleterious consequences of oxidative stress. In Table 3 the relative antioxidant efficiency of both Tolpis extracts against the DPPH radical is shown. Antioxidants suppress the absorbance at 515 nm on a time scale dependent on the antioxidant activity of extracts. The RSA of the crude extract of T. proustii (59.6%) was higher than that of T. lagopoda (41.4%). FRAP assay was used to study the ability of the antioxidants in the extracts to reduce ferric iron to the ferrous form. The same behaviour as for the DPPH assay was observed, T. lagopoda being less active than T. proustii (4.1 and 18.1 µmol of Fe(III) reduced to Fe(II) per gram of dry plant respectively) (Table 3). On the other hand, the free radical scavenging and ferric reducing power assays revealed that aesculetin (isolated from T. proustii) showed the highest antioxidant activities as compared with those of α-tocopherol and BHA (Table 4). Aesculetin gave a RSA value of 100% with a t1/2 (time required for 50% scavenging of DPPH radical in the specified concentration of antioxidant) of 22.5 seconds, while BHA and α-tocopherol showed RSA of 21.9 and 17.7% respectively after 20 min. Aesculetin (at concentration 0.1 mg mL−1) showed also higher antioxidant activities than both extracts (at concentration 10 mg mL−1), because the extracts are complex mixtures that include active components at lower levels. Moreover, the crude extracts tend to have more interfering substances that may interact with the antioxidants, decreasing their effectiveness. The antioxidant activities found in this study indicated that aesculetin, as well as both extracts, are ideal for use in the health food industry. Because of the high content of aesculetin found in the T. proustii extract (566.8 mg), this extract may be considered to be a natural source of aesculetin with diverse potential therapeutic uses. Table 3. Antioxidant activity of crude extracts derived from T. proustii and T. lagopoda. Assays T. proustii T. lagopoda RSA a 59.6 ± 0.4 41.4 ± 0.1 FRAP b 18.1 ± 0.4 4.1 ± 0.2 FRAP c 93 ± 2 41 ± 1 a % inhibition ± standard deviation of three measurements. b µmol of Fe(III) reduced to Fe(II) per gram of dry plant ± standard deviation of three measurements. c µmol of Fe(III) reduced to Fe(II) per gram of ethanolic residue ± standard deviation of three measurements. Table 4. Antioxidant activity of aesculetin, α-tocopherol and butylated hydroxyanisol (BHA). Assays Aesculetin 0.1 mg mL−1 α-Tocopherol 0.1 mg mL−1 BHA 0.1 mg mL−1 DPPH a 100 ± 0 17.7 ± 0.1 21.9 ± 0.6 FRAP b 9.4 ± 0.7 0.97 ± 0.03 3.13 ± 0.05 a % inhibition ± standard deviation of three measurements. b µmol of Fe(III) reduced to Fe(II) per mg of compound ± standard deviation of three measurements. Molecules 2012, 17 12903 2.3. Cytotoxic Activity K-562 and K-562/ADR cells which are sensitive or resistant to doxorubicin, respectively, were incubated with the compounds shown in Table 5 to evaluate their potential cytotoxicity. After 72 h, cell survival was determined by the MTT assay and the IC50 values are summarized in Table 5. Among the different compounds ursolic acid and 22-oxo-20-taraxasten-3β-ol exhibit the strongest effects in mitochondrial reduction of tetrazolium salts to formazan, while the ursolic derivatives and the 1,2-diacetoxyphytene exhibit the weakest effects. Furthermore, K-562 and K-562/ADR cells exhibit comparable sensitivity to compounds ursolic acid and 22-oxo-20-taraxasten-3β-ol (Table 5). These results suggest that the overexpression of the drug efflux protein, P-glycoprotein does not confer resistance against these compounds. Table 5. Effects of some compounds and derivatives isolated from T. proustii and T. lagopoda on the growth of the human leukemia cell lines. Compound IC50 (M) K562 K562/ADR Ursolic acid 40.6 ± 3.6 49.2 ± 3.1 Ursolic acid methyl ester 59.3 ± 15.5 64.0 ± 14.5 Acetyl ursolic acid 99.5 ± 20.5 >100 Acetyl ursolic acid methyl ester >100 >100 Aesculetin 63.2 ± 3.2 77.0 ± 5.1 Aesculetin acetyl 68.6 ± 17.1 70.3 ± 19.2 Aesculetin diacetyl 62.3 ± 6.6 59.5 ± 4.5 11-Oxo-β-amyrin >100 >100 22-Oxo-20-taraxasten-3-ol 30.0 ± 10.0 43.0 ± 7.0 1,2-Diacetoxyphytene >100 >100 Cells were cultured for 72 h and the IC50 values were calculated as described in the Experimental section. The data shown represent the means ± SEM of 3–5 independent experiments with three determinations in each. 3. Experimental 3.1. General Experimental Procedures Optical rotations: Perkin-Elmer model 343 polarimeter. IR Spectra: Bruker model IFS-55 spectrophotometer. 1H and 13C-NMR spectra: Bruker model AMX-500 and AMX-400 spectrometers with standard pulse sequences, operating at 500 and 400 MHz for 1H-, and 125 MHz for 13C-NMR, CDCl3 was used as solvent and TMS as internal standard. EI–MS: Micromass model Autospec (70 eV) spectrometer. The constituents of the ethanolic extracts were separated by gravity column chromatography, medium pressure liquid chromatography (MPLC) and preparative TLC. Column chromatography (CC): silica gel SiO2; (70–230 mesh, Merck), column fractions were monitored by TLC (silica gel 60 F254), Medium pressure column chromatography (MPLC): silica gel Merck (40–63 m). Prep. TLC: silica gel 60 PF254 + 366 plates (20 × 20 cm, 1-mm thickness). 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