Intraspecific variability of great burnet (Sanguisorba officinalis L.) in respect of sterols content MAŁGORZATA MIRGOS 1*, EWELINA PRZYBYSZEWSKA 1, EWA CAPECKA 2, MIROSŁAW ANGIELCZYK 1, JAROSŁAW L. PRZYBYŁ 1, KATARZYNA BĄCZEK 1, ZENON WĘGLARZ 1 1 Department of Vegetable and Medicinal Plants Warsaw University of Life Sciences SGGW Nowoursynowska 166 02-787 Warsaw, Poland 2 Department of Vegetable and Medicinal Plants University of Agriculture in Krakow Al. 29 Listopada 54 31-425 Krakow, Poland * corresponding author: malgorzata_mirgos@sggw.pl S u m m a r y The content and composition of sterol compounds in wild growing great burnet (Sanguisorba officinalis L.) populations were determined. The herb and underground organs of this plant were collected from 10 natural sites in Poland. The HPLC analysis revealed the presence of 5 sterol compounds, namely β-sitosterol, β-sitosterol D-glucoside, campesterol, brassicasterol and stigmasterol in these raw materials. Among them, β-sitosterol D- glucoside was dominant one. The herb was richer in sterol compounds than underground organs. There were significant differences between the content of above mentioned compounds in these populations. Key words: great burnet, populations, sterol compounds, HPLC INTRODUCTION Great burnet (Sanguisorba officinalis L., Rosaceae) is a perennial growing up to 1.5 m high. In Poland it occurs in wet meadows and grassy banks, mainly in
Intraspecific variability of great burnet (Sanguisorba officinalis L.) in respect of sterols content 17 southern regions [1, 2]. The main raw materials from this plant are underground organs: comprising thick rhizome and fine roots. Its leaves are used less frequently [3, 4]. Both raw materials reveal astringent, antibacterial, analgetic and diuretic activities, and are used to treat skin disorders, digestive system, as well as ancillary treatment of haemorrhoids and colon cancer [5, 6]. Active compounds of this plant are mainly tannins, polyphenolic acids and saponins [4, 7-9]. Sterols as active compounds in the plant s underground organs are also mentioned [10]. The study aimed to define the content and composition of sterol compounds in the above- and underground organs of great burnet originating from natural sites. MATERIALS AND METHODS The plant material used for investigation was the above- and underground organs of great burnet, collected from ten natural sites of the species located in southern and eastern Poland (fig. 1, 2). The geographical coordinates of the sites were defined using GPS (tab. 1). The aboveground organs were collected in summer, at the beginning of the flowering stage, while the underground organs in autumn, towards the end of the vegetative period, in 2009. The raw materials were collected from 15 20 randomly chosen plants from each population. Raw materials were dried at 35 C and subjected for sterol analysis using HPLC. Figure 1. Natural site of great burnet Vol. 58 No. 3 2012
M. Mirgos, E. Przybyszewska, E. Capecka, M. Angielczyk, J. L. Przybył, K. Bączek, Z. Węglarz 18 Figure 2. Underground organs of great burnet Geographical coordinates of great burnet natural sites Ta b l e 1. No. Population Region Geographical coordinates 1. Siemiatycze Podlasie N 52 02.705 E 022 53.123 2. Łada Lubelszczyzna N 50 43.812 E 022 39.155 3. Tarnawa Mała Lubelszczyzna N 50 49.785 E 022 41.727 4. Goraj Lubelszczyzna N 50 42 855 E 022 40.545 5. Grabie Małopolska N 49 50.530 E 020 16.020 6. Kostrze Małopolska N 50 03.660 E 019 83.990 7. Zamoście Małopolska N 51 13.170 E 019 16.220 8. Pustelnik Sudety N 50 51.027 E 016 02.616 9. Kamienna Góra Sudety N 50 46.284 E 016 09.442 10. Marciszów Sudety N 50 53.330 E 015 30.490 One gram of raw material was extracted for 3.5 h with 100 ml of hexane in the Büchi B-811 Extraction System. After evaporation of solvent, the residue was dissolved in 10 ml of chloroform-methanol (4:1) mixture. The obtained extract was filtered through a Supelco IsoDisc PTFE 25 mm 0.45 μm and subjected to HPLC. The analysis was carried out using the Shimadzu chromatograph with SPD-M10A VP DAD detector and Phenomenex Luna C8 (2) 5 μm 250 mm 4.6 mm. The following conditions were applied: mobile phase A methanol (LabScan), mobile phase B ACN (LabScan), flow rate 1 ml min -1, temperature 30ºC. Peak identification was confirmed by comparison of retention time and spectral data with adequate parameters of standards purchased from ChromaDex. Quantification was based on the peak area
Intraspecific variability of great burnet (Sanguisorba officinalis L.) in respect of sterols content 19 at 210 nm for all substances (β-sitosterol, β-sitosterol D-glucoside, campesterol, brassicasterol, stigmasterol). The content of the determined compounds was calculated in mg 100g -1 dry matter. The analyseis were carried out in 3 replications. Statistical evaluation of obtained data was done by one-way ANOVA and Tukey s HSD test at α=0.05 using Stargraphics Plus for Windows v. 4.1 software. RESULTS AND DISSCUSSION The obtained results indicate the presence of five sterol compounds, namely β-sitosterol, β-sitosterol D-glucoside, campesterol, brassicasterol and stigmasterol in the herb and roots of investigated great burnet populations (fig 3, 4). The content of these compounds was much higher in herb as compared to underground organs. β-sitosterol D-glucoside was a dominant compound (on average present at 267.58 mg 100g -1 in the herb and at 191.62 mg 100g -1 in the underground organs). β-sitosterol and campesterol content in both raw materials was several times lower in comparison with β-sitosterol D-glucoside, while brassicasterol and stigmasterol were present only in trace amounts (tab. 2, 3). Up to now, only the presence of β-sitosterol in great burnet underground organs was mentioned [10]. Figure 3. HPLC chromatogram of herb extract Vol. 58 No. 3 2012
M. Mirgos, E. Przybyszewska, E. Capecka, M. Angielczyk, J. L. Przybył, K. Bączek, Z. Węglarz 20 Identified sterol compounds in the herb (mg 100 g -1 d. m.) Ta b l e 2. Population Sterol compound Siemiatycze Łada Tarnawa Mała Goraj Grabie Kostrze Zamoście Pustelnik Kamienna Góra Marciszów 1 2 3 4 5 6 7 8 9 10 Mean β-sitosterol 100.40 b 19.18 e 66.24 c 30.82 d 99.40 b 95.35 b 127.59 a 112.46 ab 39.02 d 17.56 e 70.80 β-sitosterol D-glucoside 209.66 d 465.92 a 376.85 b 251.10 c 225.61 cd 197.96 d 232.85 cd 289.72 c 211.08 d 215.00 d 267.58 Campesterol 282.54 a 31.02 f 56.00 d 123.92 b 54.05 d 46.21 e 43.10 e 67.60 c 111.84 b 36.92 f 85.32 Brassicasterol 11.10 a 2.28 e 2.72 e 2.20 e 7.28 b 4.95 c 3.35 d 10.48 a 3.24 d 2.90 e 5.05 Stigmasterol 8.70 c 12.80 bc 22.78 a 9.72 c 2.97 e 4.16 e 3.75 e 20.58 a 17.62 b 18.14 b 12.12 Values in rows marked with the same letters do not differ significantly at α=0.05, Tukey s HSD test. Identified sterol compounds in the underground organs (mg 100 g -1 d. m.) Ta b l e 3. Populations Sterol compound Siemiatycze Łada Tarnawa Mała Goraj Grabie Kostrze Zamoście Pustelnik Kamienna Góra Marciszów 1 2 3 4 5 6 7 8 9 10 Mean β-sitosterol 30.64 d 23.94 e 25.37 e 89.76 a 53.14 c 56.78 c 15.43 f 60.84 b 24.16 e 10.30 g 39.04 β-sitosterol D-glucoside 83.54 d 290.58 a 292.88 a 232.10 b 183.53 bc 105.31 c 18.05 f 227.64 b 309.98 a 172.58 bc 191.62 Campesterol 63.38 b 86.26 a 85.20 a 64.94 b 62.23 b 69.18 ab 70.37 ab 24.22 d 46.28 c 82.34 a 65.44 Brassicasterol 0.46 e 9.80 a 1.42 d 5.84 b 2.03 c 2.31 c 2.99 c 0.22 e 0.24 e 5.28 b 3.06 Stigmasterol 2.34 d 11.60 b 11.62 b 25.84 a 0.95 e 4.34 c 0.90 e 3.30 d 3.22 d 5.38 c 6.95 Values in rows marked with the same letters do not differ significantly at α=0.05, Tukey s HSD test.
Intraspecific variability of great burnet (Sanguisorba officinalis L.) in respect of sterols content 21 Figure 4. HPLC chromatogram of underground organs extract The obtained results showed large variation between 10 studied wild populations of great burnet in terms of identified sterol compounds. The content of the most commonly occurring sterol, β-sitosterol D-glucoside, ranged between 197.96 and 465.92 mg 100 g -1 in the herb, 18.05 and 309.98 mg 100 g -1 in underground organs; sitosterol content ranged between 17.56 and 127.59 mg 100 g -1 in herb, 10.30 and 89.76 mg 100 g -1 in underground organs; while campesterol content ranged between 36.92 and 282.54 in herb and 24.22 and 86.26 in underground organs (tab. 2, 3). Comparing results by region indicates that populations occurring in the Lublin region are characterised with a higher β-sitosterol D-glucoside content than those from other regions, while populations from the Małopolska region have a higher content of β-sitosterol. CONCLUSIONS Five sterol compounds were identified both in the herb and in the underground organs, with significantly higher content in the herb. The dominant sterol compound in both raw materials was β-sitosterol D-glucoside. Vol. 58 No. 3 2012
M. Mirgos, E. Przybyszewska, E. Capecka, M. Angielczyk, J. L. Przybył, K. Bączek, Z. Węglarz 22 The studied populations showed significant variation in terms of the content of all identified sterol compounds. ACKNOWLEDGEMENT The work was supported by Polish Ministry of Science and Higher Education, project No. R12 06803 REFERENCES 1. Broda B, Mowszowicz J. Przewodnik do oznaczania roślin leczniczych, trujących i użytkowych. Warszawa 1996. 2. Gibbons B, Brough P. Atlas roślin Europy Północnej i Środkowej. Warszawa 1995. 3. Mimaki Y, Fukushima M, Yokosuka A, Sashida Y, Furaya S, Sakagami H. Triterpene glycosides from the roots of Sanguisorba officinalis. Phytochem 2001; 57(5):773-9. 4. Van Vyk BE, Wink M. Rośliny lecznicze świata. 1st Polish ed. Wroclaw 2008. 5. Caia Y, Lub Q, Corke H. Antioxidant activity and phenolic compounds of 112 traditional Chinese medicinal plants associated with anticancer. Life Sci 2004; 74:2157-84. 6. Duke JA, Bogenschutz-Godwin MJ, ducellier J, Duke P-AK. Handbook of medicinal herbs. 2nd ed. Boca Raton, London, New York, Washington 2002. 7. Cheng DL, Cao XP. Pomolic acid derivatives from the root of Sanguisorba officinalis. Phytochem 1992; 31(4):1317-20. 8. Popov I, Andreeva N, Gavrilin MV. HPLC determination of tannins in raw materials and preparations of great burnet. Pharm Chem J 2003; 37(7):24-26. 9. Zhang F, Fu T-J, Peng S-L, Liu Z-R, Ding L-S. Two new triterpenoids from the roots of Sanguisorba officinalis L. J Integr Plant Biol 2005; 47(2):251-6. 10. Kühner K, Voight G, Hiller K, Rabe H, Franke P, Dube G, Habish D. Pharmazie 1985; 40:576-8. Zróżnicowanie wewnątrzgatunkowe Krwiściągu lekarskiego (SANGUISORBA OFFICINALIS l.) pod względem zawartości związków sterolowych MAŁGORZATA MIRGOS 1*, EWELINA PRZYBYSZEWSKA 1, EWA CAPECKA 2, MIROSŁAW ANGIELCZYK 1, JAROSŁAW L. PRZYBYŁ 1, KATARZYNA BĄCZEK 1, ZENON WĘGLARZ 1 1 Katedra Roślin Warzywnych i Leczniczych Szkoła Główna Gospodarstwa Wiejskiego w Warszawie ul. Nowoursynowska 166 02-787 Warszawa
Intraspecific variability of great burnet (Sanguisorba officinalis L.) in respect of sterols content 23 2 Katedra Roślin Warzywnych i Zielarskich Uniwersytet Rolniczy w Krakowie Al. 29 Listopada 54 31-425 Kraków *autor, do którego należy kierować korespondencję: malgorzata_mirgos@sggw.pl S t r e s z c z e n i e Badano skład i zawartość związków sterolowych w dziko rosnących populacjach krwiściągu lekarskiego. Ziele i organy podziemne zbierane były z roślin występujących na 10 stanowiskach naturalnych zlokalizowanych na terenie Polski. Analiza chromatograficzna wykazała obecność pięciu związków sterolowych w tych surowcach, a mianowicie β-sitosterolu, D-glukozydu β-sitosterolu, kampesterolu, brassikasterolu i stigmasterolu. Spośród nich D-glukozyd β-sitosterolu był związkiem dominującym, przy czym ziele okazało się bogatsze we wszystkie zidentyfikowane związki w porównaniu z organami podziemnymi. Stwierdzono istotne różnice pomiędzy populacjami odnośnie zawartości wyżej wymienionych związków sterolowych. Słowa kluczowe: krwiściąg lekarski, populacje, związki sterolowe, HPLC Vol. 58 No. 3 2012