PolyphenolsStachys in and Betonica Species (Lamiaceae) Vassya Bankova3 *, Jordanka Koeva-Todorovskab, Tatyana Stambolijskab, Maria-Desislava Ignatova-Grocevab, Daniela Todorova3 and Simeon Popov3 a Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences, Sofia 1113, Bulgaria. Fax: 003592-700-225. E-mail: [email protected] b Faculty of Biology. Sofia University, Sofia 1000, Bulgaria * Author for correspondence and reprint requests Z. Naturforsch. 54c, 876-880 (1999); received March 15/June 6, 1999 Lamiaceae,Stachys, Betonica, Phenylethanoids, Flavonoids, Chemotaxonomy Above-ground parts and roots from four Stachys species (S. germanica L., S. sylvatica L. and the Balkan endemicsS. thracica Dav. and S. plumosa Griseb.) as well as o f three Betonica species (B. officinalis L. and the Balkan endemicsB. bulgarica Deg. et Neic. and B. scardica Griseb.) were screened for phenols (phenylethanoid glycosides, flavonoid glycosides and the phenolic diterpene betolide). Three phenylethanoid glycosides, a flavonoid glycoside and the phenolic diterpene betolide were isolated and identified, most of them for the first time in the investigated species. The results obtained support the view thatStachys and Betonica are well separated genera. Introduction S. thracica (Strandja Mt., village Bogoslov, 23.05.1994), B. bulgarica (Middle Balkan Mt., Kor- Betonica and Stachys species are widely used in udere, 20.07.1993 and 25.07.1994; (85963), B. scar­ folk medicine and recently in the official medicine dica (Rudina Mt., Borovski dol, 11.08.1993; (Ovcharov et al., 1992). Most of the biologically (99060), B. officinalis (near the city of Sofia, Kny­ active substances in these plants are polyphenols, azhevo, 2.08.1992; (99064). The numbers in brack­ compounds often used in taxonomic studies. B e­ ets correspond to the voucher specimens of the tonica and Stachys are represented in Bulgaria plants, determined by Dr. J. Koeva-Todorovska with 4 and 18 species, respectively (Koeva-Todor- and deposited in the herbarium of the Faculty of ovska, 1979). The separation of the Linnei’s genus Biology, Sofia University (SO). Betonica from Stachys is based on well defined dif­ ferences in the general morphological characteris­ tics and especially in the morphology of Isolation caryotypes and pollen of both genera (Koeva-To- Dried ground roots from S. germanica (620 g) dorovska, 1979; 1988). were extracted twice with 3 1 ethanol. The ethanol It was of interest to investigate the polyphenol extract was concentrated in vacuo, diluted with composition of some Stachys and Betonica species water and extracted successively with petrol ether distributed in Bulgaria, especially the endemic (3x) and ethyl acetate (3x). The ethyl acetate ex­ ones, and to use the phytochemical evidence to tract was subjected to column chromatography on clarify this classification problem. silica gel with mobile phase dichloroethane - methanol with increasing percentage of methanol. Materials and Methods After repeated column chromatography on silica gel with mobile phases dichloroethane-methanol Plant material and dichloroethane-methanol-water, 1 (20 mg), 2 Above-ground parts and roots from S. germ an­ (14 mg) and 4 (17 mg) were isolated. From the ae­ ica (near the city of Sofia, Knyazhevo, 5.08.1992; rial parts (800 g) by the same way 1, 2 and 4 (990663), S. sylvatica (near the city of Sofia, Kny­ were isolated. azhevo, 4.08.1992; (99065), S. plumosa (Znepole Dried ground roots from B. bulgarica (45 g) region, village Gorno Uyno, 12.08.1993; (99061); were extracted twice with 0.25 1 ethanol. The etha­ Osogovo Mt., village Bogoslov, 1.08.1994; (99066), nol extract was concentrated in vacuo, diluted with 0939-5075/99/1100-0876 $ 06.00 © 1999 Verlag der Zeitschrift für Naturforschung, Tübingen • www.znaturforsch.com • D V. Bankova et al. ■ Polyphenols in Stachys L. and Betonica L. Species 877 water and extracted successively with petrol ether ether-ethyl acetate 4:1 Rf = 0.60, and chloroform- (3x) and ethyl acetate (3x). From the petrol ether acetone 8:1 R f = 0.75. extract, using silica gel column chromatography with mobile phase hexane-ethyl acetate with Analysis of phenolics increasing polarity, 36 mg of 5 were isolated. The Sample preparation same way as described above from the ethyl ace­ tate extract, 1 (110 mg), 2 (38 mg) and 3 (77 mg) Of each extract (petrol ether and ethyl acetate) were isolated. a part corresponding to 1 g plant material was The extraction procedure was applied for S. syl- weightened and dissolved in 1 ml of chloroform vatica (82 g aerial parts, 25 g roots), S. plumosa for the petrol ether extracts and in 2 ml of metha­ (20 g aerial parts, 15 g roots), S. thracica (60 g ae­ nol/chloroform 1:1 v/v for the ethyl acetate ex­ rial parts, 10 g roots), B. bulgarica (165 g aerial tracts. parts, 45 g roots), B. scardica (75 g aerial parts, 25 g roots) and the following substances were iso­ Preparation of the standard solutions lated: from S. sylvatica: aerial parts - 1, 2 and 4, The standard solutions of the isolated pure com­ roots - 1 and 2; from S. plumosa: aerial parts - pounds (purity >95%, 1 H-NMR) 1, 2, 3, 4 and 5 1, 2 and 4, roots - 1, 2 and 4; S. thracica: aerial were prepared by dissolving 2 mg of pure com­ parts - 1, 2 and 4, roots - 1, 2 and ; B. bulgarica: pound in 2 ml of solvent. The solvent was metha­ aerial parts - 1, 2 and 3, roots - 1, 2, 3 and 5; B. nol/chloroform 1:1 v/v for 1, 2, 3 and 4, and chloro­ scardica: aerial parts - 1, 2 and 3, roots - 1, 2, 3 form for 5. and 5. TLC semiquantitative analysis of petrol ether Identification extracts The identification of the isolated compounds An aliquot (30 |il) of the sample solutions to­ was carried out by measuring their UV, ’H and gether with 12 [_il of the standard solution of 5 were 13C NMR spectra and comparing the results with applied to silica gel plates, developed with petrol published data, as follows. For compound 1: UV, ether-ethyl acetate 4:1 and chloroform-acetone *H and 13C NMR spectra identical with data pub­ 8:1. The evaluation of the spots was made after lished by Ikeda et al., 1994 for acteoside; for com­ viewing the plates in UV light (254 nm) and after pound 2: UV, 'H and 13C NMR spectra identical charring. with data published by Ikeda et al., (1994) for mar- tinoside; for compound 3: UV, 'H and 13C NMR TLC semiquantitative analysis of ethyl acetate spectra identical with data published by Miyase et extracts al. (1990) for forsythoside B; for compound 4: UV An aliquot (20 ^il) of the sample solutions to­ (incl. those with shift reagents), and l3C NMR gether with 12 ^1 of the standard solutions of 1, 2, spectra identical with data published by El-Ansari 3 and 4 were applied to silica gel plates and devel­ et al. (1991) for 4’-0-methylisoscutellarein 7-0- oped with three mobile phases: chloroform-metha- (2”-0-6”’-0-acetyl-ß-D-allopyranosyl-ß-D-gluco- nol-water 80:20:1, 30:20:4 v/v and chloroform-ace- pyranoside); for compound 5: UV, 1H,13C NMR tic acid-methanol 18:1:3 v/v. The evaluation of the and mass spectra identical with data published by spots was made after viewing the plates in UV Tkachev et al. (1987) for betolide. light (254 and 366 nm) and after charring with sul­ The Rf values on silica gel plates for the isolated furic acid. compounds are as folllows: In mobile phase chloroform-methanol-water Results and Discussion 80:20:1 v/v, 1 - 0.15; 2 - 0.25; 3 - 0.07; 4 - 0.32. Chloroform-methanol-water 30:20:4 v/v, 1 - 0.73; Seven species were investigated: Betonica offici­ 2 - 0.80; 3 - 0.65; 4 - 0.90. chloroform-acetic nalis L., the Bulgarian endemic B. bulgarica Deg. acid-methanol 18:1:3 v/v, 1 - 0.60; 2 - 0.68; 3 - et Neic., the Balkan endemic B. scardica Griesb., 0.51; 4 - 0.75. Compound 5 in mobile phase petrol Stachys germanica L., S. thracica Dav. (Balkan en- 878 V. Bankova et al. ■ Polyphenols in Stachys L. and Betonica L. Species 1.R = R1 = h ; 2. R = Me, R 1 = H. 3. R = H; R-j = Apiose Fig. 1. Phenolic components of Stachys and Be- tonic species.: 1 acteoside (Ikedaet al., 1994); 2 martinoside (Ikeda et al., 1994); 3 forsythoside S (Miyase et al., 1990); 4 7-0-(2”-0-6”,-0-acetyl-ß- D-allopyra-nosyl-ß-D-glucopyranoside) (El-Ansari Fig. 1 et al., 1991); 5 betolide (Tkachev et al., 1987). demic), S. sylvatica L. and S. plumosa Griesb. (Bal­ were subjected to column chromatography on sil­ kan endemic). Roots and above-ground parts were ica gel to afford pure phenylethanoid glycosides, investigated separately. Part of the species (see Ta­ identified by comparing their 'H and 13C NMR: ble I) were investigated in two consecutive years. acteoside 1 (Ikeda et al., 1994), martinoside 2 The MeOFI extracts of the investigated species (Ikeda et al., 1994) and forsythoside B 3 (Miyase showed considerable differences in the polyphenol et al., 1990) (from Stachys and Betonica species, composition. The concentrated methanolic ex­ see Table I.) and the flavonoid glycoside 4 (El-An- tracts were extracted subsequently with petroleum sari et al., 1991) (from Stachys species only). The ether and ethyl acetate. The ethyl acetate extracts petroleum ether extract was separated on silica gel V. Bankova et al. ■ Polyphenols in Stachys L. and Betonica L. Species 879 Table I. Chemical composition ofBetonica and Stachys Acteoside 1, martinoside 2 and forsythoside B 3 species.