Linum mucronatum: Organ to Organ Linastrum (Mohagheghzadeh et al., 2003). All Lignan Variations studied from genus that produce Abdolali Mohagheghzadeha,*, Azra Gholamia, podophyllotoxin (PTOX) show the production Mohammad Soltania, Shiva Hemmatia,b, of 6-methoxypodophyllotoxin (MPTOX) besides and A. Wilhelm Alfermannb PTOX in different ratio (Mohagheghzadeh et al., 2003; Konuklugil et al., 1999, 2001). Lignans are a Department of Pharmacognosy, Faculty of Pharmacy, present at different levels of abundance in Shiraz University of Medical Sciences and Health Services, P.O. Box 71345-1583, Shiraz, I.R. Iran. parts including roots, stems, leaves, petals and Fax: +98-711-2426070. E-mail: [email protected] flowers (Mohagheghzadeh et al., 2003). b Institut für Entwicklungs- und Molekularbiologie The presence of PTOX and MPTOX was re- der Pflanzen, Heinrich-Heine-Universität Düsseldorf, ported in L. mucronatum ssp. armenum cell cul- Universitätsstr. 1, D-40225 Düsseldorf, Germany tures previously (Konuklugil et al., 2001). Accord- * Author for correspondence and reprint requests ing to the previous work (Mohagheghzadeh et al., Z. Naturforsch. 60c, 508Ð510 (2005); 2003), the section Syllinum was divided into three received January 24, 2005 sub-groups. Our evaluation consists of the analysis The percentage of podophyllotoxin (PTOX) and its and determination of lignans in different parts of congener lignans were measured by HPLC in Linum L. mucronatum Bertol. ssp. mucronatum, one of mucronatum ssp. mucronatum () fresh plant or- gans. The highest amounts of PTOX (0.595 ð 0.060% g/g species belonging to the sub-group b of section dry wt) and 6-methoxypodophyllotoxin (MPTOX) Syllinum. (1.491 ð 0.125% g/g dry wt) were found in the plant sexual organs. Whereas, the highest levels of -peltatin, 5Ј-demethoxy-MPTOX and yatein were found in not de- Results and Discussion veloped buds, petals and sepals, respectively. The percentage contents of PTOX and related Key words: Aryltetralin Lignans, Linaceae, Linum mucronatum ssp. mucronatum lignans in different parts of L. mucronatum ssp. mucronatum are summarized in Table I. The results show a variation in the distribution and relative abundance of lignans from plant organs. The high- Introduction est amounts of PTOX (0.595% g/g dry wt) and Linaceae, one of the families, MPTOX (1.491% g/g dry wt) were found in the sex- consists of genera including Anisadenia, Cliococca, ual organs (p < 0.001). Furthermore, considerable Hesperolinon, Linum, Radiola, Reinwardtia, Scle- amounts of MPTOX were found in petals and not rolinon and Tirpitzia (Heywood, 1985). The genus developed buds (0.951 and 0.946% g/g dry wt, Linum is the largest genus in this family consisting respectively). MPTOX has been reported from dif- of a large group of herbs and sub-shrubs divided ferent plant parts of Linum flavum and agar-grown into the sections Syllinum, Cathartolinum, Dasyli- results showed that roots and stems are im- num, Linum and Linastrum according to classical portant accumulation sites for MPTOX (Wichers based on morphological characters et al., 1990). Like the other yellow flower Linum (Ockendon and Walters, 1968). The occurrence of spp. belonging to the sub-group b of the section Syl- lignans in the Linum genus is documented since linum (Mohagheghzadeh et al., 2003), the L. mucro- 1975 (Weiss et al., 1975; Berlin et al., 1986; natum plant is rich in MPTOX. The highest levels of Broomhead and Dewick, 1990; Wichers et al., -peltatin (-PLT, 0.928% g/g dry wt), 5Ј-demeth- 1990), aryltetralin lignans occur in the section Syl- oxy-MPTOX (0.994% g/g dry wt) and yatein (YAT, linum (Weiss et al., 1975; Broomhead and Dewick, 1.480% g/g dry wt) were found in not developed 1990; Mohagheghzadeh et al., 2003; Konuklugil buds, petals and sepals, respectively. Lignan study et al., 1999, 2001), whereas arylnaphthalene lig- in different organs will guide us to more elucidation nans (Mohagheghzadeh et al., 2002) and simple of the biosynthetic pathway in the plant. On the acyclic, furanofuran- and dibenzylbutyrolactone- whole, L. mucronatum ssp. mucronatum is rich in type lignans (Meagher et al., 1999) have been aryltetralin lignans and the plant cultivation would found in the section Linum, and dibenzylbutyro- be an interesting method for the production of aryl- lactone lignans has been recorded in species of tetralin lignans in the large scale.

0939Ð5075/2005/0500Ð0508 $ 06.00 ” 2005 Verlag der Zeitschrift für Naturforschung, Tübingen · http://www.znaturforsch.com · D Notes 509

Experimental Plant material

(*) Linum mucronatum Bertol. ssp. mucronatum Ψ Ψ Ψ Ψ Ψ Ψ Ψ Ψ Ψ (Linaceae) plant materials were collected in April 2003 from Tang-e-Abol Hayat, Shiraz-Kazeroun 0.087 0.185 0.028 0.005 0.043 road, Fars province, Iran, at an altitude of 1000 m. ð ð ð ð ð Voucher specimens (No. 237) were deposited at Ð t 1.480 Ð 0.212 0.049 0.121 Ð 0.077 the herbarium of Department of Pharmacognosy, Faculty of Pharmacy, Shiraz University of Medical

(**), Sciences. The samples were identified by Iraj Meh- Ќ regan (Institut für Spezielle Botanik und Bota- (*),

(***) nischer Garten, Johannes Gutenberg-Universität Ψ Ψ Ψ Ќ Ψ Ќ Ψ Ψ Mainz, Germany). Different parts of the perennial herbaceous 0.142 0.016 0.151 0.056 0.154 0.125 0.052 0.015 0.065 plant were separated carefully based on the mor- ð ð ð ð ð ð ð ð ð phology for the lignan assay as follows: roots pe- < 0.05.

p rennial thick, slightly branched and twisted; stems 0.701 0.215 0.342 0.140 0.109 up to 40 cm high, with peripheral sterile branches (**) 0.951 (**) 1.491 at base bearing imbricate, spatulate leaves; fertile Ќ Ќ stems bearing alternate 1Ð3 veined leaves and

< 0.01, (***) ending to cymes of yellow flowers; stem leaves p . (***), (***), 5Ð30 mm long and 2Ð6 mm width, simple, linear- a,b Ќ Ψ Ψ Ψ Ψ Ψ Ψ oblong, mucronate to acute, basal ones smaller than upper ones; not opened buds lanceolate, 0.006 0.027 0.140 0.052 0.672 0.038 0.946 0.057 0.046 0.001 petals are hidden by lanceolate sepals; flowers ac- < 0.001, (**) ð ð ð ð ð ð ð ð tinomorph; receptacle bearing 5 sepals, 5 petals, 5 p mucronatum -Demethoxy- Significance MPTOX Significance YAT Significance

Ј stamens alternating with 5 staminoids, and a 0.208 Ð 0.994 0.599 0.854 = 3), ANOVA test. 5-loculate superior ovary becomes a ð spherical ssp. n capsule; sepals lanceolate, acute, 7Ð9 mm long and 1Ð2 mm width, membranous at margins; petals S.D. ( (***) 0.656

ð yellow, 15Ð28 mm long, connected with claws at Ψ differences MPTOX differencesΨ Ψ differences differences Ψ Ψ Ψ base. 0.054 0.061 0.133 1.066 0.268 0.053 0.072 0.064 Quantitative analysis ð ð ð ð ð Fresh plant materials were frozen and then lyo- Linum mucronatum e -PLT Significance 5 d Ð 0.306 0.350 t 0.061 0.854 t Ð 0.928 philized by freeze-dryer. Extraction and determi-

c nation of lignans were performed as described by (**) Ј

Ќ Empt et al. (2000). PTOX, -PLT, 5 -demethoxy-

(***) MPTOX, MPTOX and YAT were determined at (*), , , , , , Rt = 9.15 min, 10.12 min, 12.53 min, 13.03 min and Ќ differences Ψ Ψ Ψ Ψ Ψ Ψ Ψ Ψ 26.26 min, respectively. Calibration curves for PTOX and -PLT standards were drowning (inter- 0.025 0.036 0.034 0.013 0.060 0.166 0.033 0.008 0.008 day and intra-day injections) in the range of 0.9Ð ð ð ð ð ð ð ð ð ð 400 µg/ml and 0.7Ð200 µg/ml, respectively. 5Ј-De- methoxy-MPTOX, MPTOX and YAT were quanti- fied according to commercial PTOX (Roth 3946.1). Accuracy of each quantified lignan was admitted by retention time, co-chromatography and meas-

: significant different from other indicated values in the same column; (*) uring on-line UV spectra using a Thermo Quest , HPLC system (Egelsbach, Germany) equipped Ќ , -Demethoxy-MPTOX, MPTOX and YAT were quantified according to commercial PTOX. Ј with a spectra system KO 6000 LP photodiode Values calculated on dry plant5 materials (% g/gΨ dry wt) are mean Trace (t < 0.001). No peak at the retention time of this compound was detected. Petal 0.280 Table I. Lignan contents in different organsPlant part of Root PTOX Significance 0.125 a b c d e Peripheral branches 0.081 Leaves 0.061 ReceptacleSepals 0.124 0.096 Bud (developed) 0.139 Sexual organs 0.595 Bud (not developed) 0.201 array detector. 510 Notes

Berlin J., Wray V., Mollenschott C., and Sasse F. (1986), nans, isolariciresinol and pinoresinol in flaxseed meal. Formation of -peltatin-A methylether and coniferin J. Agric. Food Chem. 47, 3173Ð3180. by root cultures of Linum flavum. J. Nat. Prod. 49, Mohagheghzadeh A., Schmidt T. J., and Alfermann 435Ð439. A. W. (2002), Arylnaphthalene lignans from in vitro Broomhead A. J. and Dewick P. M. (1990), Aryltetralin cultures of Linum austriacum. J. Nat. Prod. 65,69Ð71. lignans from Linum flavum and Linum capticum. Mohagheghzadeh A., Hemmati S., Mehregan I., and Phytochemistry 29, 3839Ð3844. Alfermann A. W. (2003), Linum persicum: lignans and Empt U., Alfermann A. W., Pras N., and Petersen M. placement in Linaceae. Phytochemistry Rev. 2, 363Ð (2000), The use of plant cell cultures for the pro- 369. duction of podophyllotoxin and related lignans. J. Ockendon D. J. and Walters S. M. (1968), Linum. In: Appl. Bot. 74, 145Ð150. Flora Europaea (Tutin T. G., Heywood V. H., Burges Heywood V. H. (1985), Flowering Plants of the World. N. A., Moore D. M., Valentine D. H., Walters S. M., Croom Helm, London, pp. 207Ð208. and Webb D. A., eds.). Cambridge University Press, Konuklugil B., Schmidt T. J., and Alfermann A. W. Cambridge, pp. 206Ð211. (1999), Accumulation of aryltetralin lactone lignans in Wichers H. J., Harkes M. P., and Aroo R. R. J. (1990), cell suspension cultures of Linum nodiflorum L. Occurrence of 5-methoxypodophyllotoxin in plants, Planta Med. 65, 587Ð588. cell cultures and regenerated plants of Linum flavum. Konuklugil B., Schmidt T. J., and Alfermann A. W. Plant Cell Tissue Organ Cult. 23,93Ð100. (2001), Accumulation of lignans in suspension cul- Weiss S. G., Tin-Wa M., Perdue R. E., and Farnsworth tures of Linum mucronatum ssp. armenum (Bordz.) N. R. (1975), Potential anticancer agents II. Antitu- Davis. Z. Naturforsch. 56c, 1164Ð1165. mor and cytotoxic lignans from Linum album (Lina- Meagher L. P., Beecher G. R., Flanagan V. P., and Li ceae). J. Pharm. Sci. 64,95Ð98. B. W. (1999), Isolation and characterization of the lig-

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