Mamadalieva et al. Phytopharmacology 2013, 4(1), 1-18 Antiproliferative, antimicrobial and antioxidant activities of the chemical constituents of Ajuga turkestanica Nilufar Z. Mamadalieva1*, Mahmoud Z. El-Readi2, Elisa Ovidi3, Mohamed L. Ashour4, Razan Hamoud5, Shamansur S. Sagdullaev1, Sakhnoza S. Azimova1, Antonio Tiezzi3, Michael Wink5 1 Institute of the Chemistry of Plant Substances AS RUz, Laboratory of Chemistry of Glycosides, Tashkent, Uzbekistan. 2 Department of Biochemistry, Faculty of Pharmacy, Al-Azhar University, Assiut, Egypt. 3 Department for the Innovation in Biological, Agro-food and Forest systems, Laboratory of Plant Cytology and Biotechnology, Tuscia University, Viterbo, Italy. 4 Department of Pharmacognosy, Faculty of Pharmacy, Ain Shams University, Cairo, Egypt. 5 Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Heidelberg, Germany. * Corresponding author: nmamadalieva@yahoo.com; Phone: +998 71 262 59 13; Fax: +998 71 262 73 48 Received: 12 July 2012, Revised: 11 August 2012, Accepted: 13 August 2012 Abstract Ajuga turkestanica Rgl. Brig (Lamiaceae) is a medicinal plant from Uzbekistan. Methanol, chloroform, butanol, and water extracts as well as isolated phytoecdysteroids and iridoids were evaluated for their antioxidant, cytotoxic and antibacterial activities. Water and butanol extracts exhibited good antioxidant activity with IC50 values of 7.24 ± 0.82 and 14.57 ± 1.64 µg/mL. The chloroform extract showed potent cytotoxic effects against the cancer cell lines HeLa, HepG-2, and MCF-7 with IC50 values of 7.13 ± 0.85, 9.03 ± 0.92, and 10.77 ± 1.44 µg/mL, respectively. Compared to the extracts, isolated phytoecdysteroids and iridoids showed weak cytotoxic activity. The chloroform extract has antimicrobial properties even against multiresistant strains like Staphylococcus aureus MRSA 1000/93 and Streptococcus pyogenes ATCC 12344. The methanol and chloroform extracts of A. turkestanica were further investigated for their GLC-volatile components using GLC/FID and GLC/MS. Pregna-4,9 (11)-dien-20-ol-3-on-19-oic acid lactone (19.58%), 20methyl-pregna-5,17-dien-3β-ol (12.93%), 3,7-dioxocholan-24-oic acid (10.53%) and betulin (10.18%) were detected as the major compounds. Keywords: Ajuga turkestanica; phytoecdysteroids; iridoids; HPLC; GLC; activity Introduction More than 45 species of the genus Ajuga L. (Lamiaceae) are found in temperate regions of the Old World and have been used in folk medicine because of their anthelmintic, antifungal, hypoglycemic, antitumor, and antimicrobial properties (Mabberley, 2008; Israili et al., 2009). Plants of the genus Ajuga produce a variety of biological active secondary meta1 © 2013 Inforesights Publishing UK Mamadalieva et al. Phytopharmacology 2013, 4(1), 1-18 bolites including phytoecdysteroids, iridoids, neoclerodane diterpenoids, sterols, withanolid-es, anthocyanins, flavonoids, ionones, and quinones (Turkoglu et al., 2010). In the flora of Uzbekistan, Ajuga is represented by two species: Ajuga genevensis L., and Ajuga turkestanica Rgl. Brig. (Sokolov et al., 1991). A. turkestanica is an endemic perennial plant and grows in areas at 600-1000 m above sea level in Southern Pamir-Аlay mountains on Southwest slobes of the Hissar Mountain (Ganiev et al., 1990; Sokolov et al., 1991). A. turkestanica has been widely used in folk medicine for enhancement of muscular strength, against heart disease, muscle and stomach aches (Grace et al., 2008). A. turkestanica produces a rich amount of bioactive phytoecdysteroids: 20-hydroxyecdysone (0.25% of dry weight), turkesterone (0.22% ) (Usmanov et al., 1973, 1975; Abdukadyrov et al., 2005), cyasterone (Usmanov et al., 1971), 22-acetylcyasterone (Usmanov et al., 1978), ajugalactone (Saatov et al., 1977), ajugasterone B (Usmanov et al., 1977), -ecdysone, ecdysone 2,3-monoacetonide (Saatov et al., 1999). Further secondary metabolites are iridoid glucosides, such as harpagide, 8-О-acetylharpagide (Kotenko et al., 1994) and carbohydrates (Abdukadyrov et al., 2004). From aerial parts six neo-clerodane diterpenoids were isolated: 14,15-dihydroajugachin B, 14-hydro-15-methoxyajugachin B, chamaepitin, ajugachin B, ajugapitin, and lupulin A (Grace et al., 2008). The phytoecdysteroids show low in vivo toxicity to vertebrates (LD50 values of 20hydroxyecdysone is 6.4 g/kg and >9 g/kg, i.p. and p.o. to mice). Since ecdysteroids function as moulting hormones in insects, they can be considered as natural insecticides. Some phytoecdysteroids strengthened lactation especially in conditions of hypolactation (Khalitova et al., 1998), and possess the hypoglycemic activity (Kutepova et al., 2001). The iridoids harpagide (5) and 8-О-acetylharpagide (6) promote bile secretion (Syrov et al., 1986). These iridoids can be found in many other plants and have been used in phytomedicine against inflammation, pain, and microbial infections (Van Wyk and Wink, 2004). In the present study, we report on the chemical composition of polar and non-polar extracts and the antimicrobial, antioxidant and cytotoxic activities of root extracts (methanol, chloroform, butanol and water) from A. turkestanica in comparison to four isolated phytoecdysteroids 20-hydroxyecdysone (syn. ecdysterone) (1), turkesterone (2), cyasterone (3), 22acetylcyasterone (4), and two iridoid glucosides harpagide (5), 8-О-acetylharpagide (6) (Fig. 1). HO OH HO RO O HO HO OH R HO O O HO OH HO H 1 R=H 2 R=OH OH R1O H HO O H O HO O 3 R=H 4 R=Ac O OH OH OH 5 R=H 6 R=Ac Figure 1. Chemical structures of the phytoecdysteroids and iridoids from A. turkestanica. 2 © 2013 Inforesights Publishing UK Mamadalieva et al. Phytopharmacology 2013, 4(1), 1-18 Materials and methods Plant material Roots of A. turkestanica were collected in the Surkhan-Darya region of Uzbekistan in the summer of 2009 and identified at the Department of Herbal Plants (Institute of the Chemistry of Plant Substances, Uzbekistan) by Dr. O.A. Nigmatullaev (voucher specimen number 20077092). Preparation of A. turkestanica extracts Roots were air-dried at room temperature before grinding them to a powder with a Warring blender. After grinding, 100 g of plant material was extracted with solvents of methanol, chloroform, butanol and water; yields of these extracts were 5.1, 1.6, 4.2 and 8.3%, respectively (from the weight of the air dried roots). Extraction with each solvent was carried out for one day at room temperature. The solvent was evaporated in a rotary vacuum evaporator at 40 °C. The extracts were then kept under refrigerated conditions until further use. Chemicals and reagents Cell culture media, supplements, and dimethylsulfoxide (DMSO) were purchased from Roth (Karlsruhe, Germany) and Greiner Labortechnik (Frickenhausen, Germany). Doxorubicin ( ≥ 98 %) and quercetin ( ≥ 98 %) were obtained from Gibco (Invitrogen, Karlsruhe, Germany). Authentical phytoecdysteroids and iridoids were obtained from the Institute of the Chemistry of Plant Substance, Tashkent, Uzbekistan. The purity of the tested compounds were > 95 %, as determined by HPLC. HPLC analysis The contents and quantity of the phytoecdysteroids and iridoids from the roots of A. turkestanica were investigated by HPLC. Chromatographic profiles of A. turkestanica extracts were generated using a high performance liquid chromatograph LC-10ATvp connected to a UV-VIS detector SPD-10Avp (Shimadzu Co, Kyoto, Japan). A. turkestanica extracts were diluted to 1 mg/ml, filtered through 0.22 µm and 20 µl were injected. For separation of these extracts, a Nucleosil 100-5 C18 column with a size 250 mm × 4 mm (Macherey-Nagel Gm bH & Co, KG) was used. Elution was carried out by a mobile phase consisted of A (water) and solvent B (acetonitrile) and the gradient profile was as follows: from 0% B to 5% B in 8 min, from 5% B to 85% B at 8-30 min, from 95% B to 100% B% at 30-35 min and at 100% B% until 40 min. Flow rate was 1 ml/min and detection was at 247 nm and 200 nm (Abdukadyrov et al., 2005). The quantifications of 20‐hydroxyecdysone (1), turkesterone (2), cyasterone (3), harpagide (5) and 8‐О‐acetylharpagide (6) in the extracts of A. turkestanica were carried out using a calibration curve of corresponding standards at different concentra‐tions. GLC/FID analysis High-resolution GLC analyses were carried out on a Focus GC (Thermo Fisher Scientific, Milan, Italy) equipped with TR1-MS fused bonded column (30 m × 0.25 mm × 0.25 µm) (Thermo Fisher Scientific®, Florida, USA) and FID detector; carrier gas was nitro3 © 2013 Inforesights Publishing UK Mamadalieva et al. Phytopharmacology 2013, 4(1), 1-18 gen (1.5 ml/ min). The operating conditions were: initial temperature 40 ºC, for 1 min isothermal followed by linear temperature increase till 230 ºC at a rate of 4 ºC/min 230 ºC, then 5 min isothermal. Detector and injector temperatures were 300 ºC and 220 ºC, respectively. The split ratio was 1:20. Chrom-card® chromatography data system ver. 2.3.3 (Thermo Electron Corp®, Florida, USA) was used for recording and integrating of the chromatograms. GLC/MS analysis The analyses were carried out on Focus GC (Thermo Fisher Scientific, Milan, Italy) equipped with the same column and conditions mentioned for GLC/FID. The capillary column was directly coupled to a quadrupole mass spectrometer Polaris Q (Thermo Electron Corp®, Milan, Italy).The injector temperature was 220 ºC. Helium carrier gas flow rate was 1.5 ml/min. All the mass spectra were recorded under the following conditions: filament emission current, 100 mA; electron energy, 70 eV; ion source, 250 ºC; diluted samples were injected with split mode (split ratio, 1:15). Compounds were identified by comparison of their spectral data and retention indices with Wiley Registry of Mass Spectral Data 8th edition, NIST Mass Spectral Library (December 2005), our own laboratory database and the literature (Adams, 2004; Budzikiewicz et al., 1964; Nibret and Wink, 2010). Antioxidant activity DPPH* radical-scavenging activity The antioxidant and radical scavenging activities of the isolated compounds and extracts were evaluated according to Brandwilliams et al. (1995) using diphenyl picryl hydrazyl (DPPH*). Equal volumes of sample solutions containing 0.02–10 mg/mL of the samples and 0.2 mM methanolic solution of DPPH* were pipetted into 96-well plates. The absorbance was measured against a blank at 517 nm using a Tecan Safire II Reader after incubation in the dark for 30 min at room temperature compared with DPPH* control after background subtraction. Quercetin was used as a positive control. The percent inhibition was calculated from three different experiments using the following equation: RSA (%) = [(Abs517control - Abs517sample)/ Abs517control] × 100 where RSA = radical scavenging activity; Abs517 = absorption at 517 nm; control = nonreduced DPPH*. Cytotoxicity studies Cell cultures HeLa (cervical cancer), HepG-2 (hepatic cancer), and MCF-7 (breast cancer) human cell lines were maintained in DMEM complete media (L-glutamine supplemented with 10% heat-inactivated fetal bovine serum, 100 U/mL penicillin, and 100 µg/mL streptomycin) in addition to 10 mM non-essential amino acids. Cells were grown at 37 °C in a humidified atmosphere of 5% CO2. All experiments were performed with cells in the logarithmic growth phase. 4 © 2013 Inforesights Publishing UK Mamadalieva et al. Phytopharmacology 2013, 4(1), 1-18 Cytotoxicity assay Sensitivity of the cancer cells to drugs was determined in triplicate using the 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell viability assay (Mosmann, 1983). The extracts and individual substances were dissolved in dimethylsulfoxide (DMSO) and further serially diluted with the medium in two-fold fashion into ten different concentrations so as to attain final concentrations ranging from 0.977 to 500 µg/mL for extracts and from 0.977 to 500 µM for isolated substances, in 96-well plates; each well contained 100 µL medium. The concentration of the solvent, DMSO, did not exceed 0.05% in the medium that contained the highest concentration of extract or compound tested. Wells containing the solvent and wells without the solvent were included in the experiment. Exponentially growing cells were seeded in a 96-well plate (2×104 cells/well), the cells were cultivated for 24 h and then incubated with various concentrations of the serially diluted tested samples at 37 °C for 24 h and then with 0.5 mg/mL MTT for 4 h. The formed formazan crystals were dissolved in 100 µL DMSO. The absorbance was detected at 570 nm with a Tecan Safire II Reader. The cell viability rate (%) of three independent experiments was calculated by the following formula: Cell viability rate (%) = ((OD of treated cells − OD of media (blank) / (OD of control cells − OD of media (blank)) × 100 % where OD = optical density Antimicrobial activity Test microorganisms The antimicrobial activity was evaluated against standard strains which included Gram-positive bacteria such as methicillin-resistant Staphylococcus aureus MRSA ATCC 10442, vancomycin- resistant Enterococcus VanB VRE ATCC 31299 and Streptococcus pyogenes ATCC 12344, two clinical isolates Staphylococcus aureus MRSA 1000/93 and Enterococcus VanB VRE 902291, Gram-negative bacteria such as Escherichia coli ATCC 25922, Klebsiella pneumonia ATCC 700603, and Pseudomonas aeruginosa ATCC 27853, and yeasts such as Candida albicans ATCC 90028, and Candida glabrata ATCC MYA 2950. All microorganism cultures were supplied by Medical Microbiology Laboratory, Hygiene Institute, Heidelberg University, Germany. Culture media Columbia with 5% sheep blood (BD) and Mueller-Hinton Broth (MHB) (Fluka) were used in bacterial tests. All bacterial cultures were incubated at 37 °C for 24 h. CHROM agar Candida (BD) and Sabouraud Dextrose broth (SDB) (Oxid) were used in fungal tests. All fungal cultures were incubated at 25 °C for 48 h. Inoculum preparation One or two bacterial or fungal colonies from an 18-24 h agar plate were suspended in saline to a turbidity matching 0.5 McFarland ≈1×108 CFU/mL; 1:100 dilution was performed 5 © 2013 Inforesights Publishing UK Mamadalieva et al. Phytopharmacology 2013, 4(1), 1-18 from this suspension using 900 µL broth to get 1×106 CFU/mL. Diffusion method 1×106 CFU/mL of bacterial and fungal suspensions was spread on Colombia with 5% sheep blood and CHROM agar Candida, respectively. Wells with diameter of 6 mm were cut off and delivered with 40 µL of each extract (40 mg/ml) and of each pure substance (0.5 mM). DMSO, ampicillin (1 mg/mL), vancomycin (1 mg/mL), and nystatin (1 mg/mL) were used as controls. All the plates were observed for zone of inhibition at 37 °C for 24 h (bacteria) and at 25 °C for 48 h (yeasts). Determination of minimum inhibitory concentration (MIC) and minimum microbicidal concentration (MMC) Microdilution method was used to determine MIC as described by NCCLS (2006). Plant extracts were first of all dissolved in DMSO 5% to concentration of 8 mg/8 mg/ml and the pure substances to the concentration of 1 mM and then were diluted two-fold with MHB (bacteria) and SDB (fungi) in 96-well plates to obtain a range of concentrations between (80.015 mg/mL) for plant extracts, and between (1000 and 1.5 µM) for pure substances. The bacterial and fungal suspensions of 1×106 CFU/mL were subsequenly added and the plates were incubated at 37 °C for 24 h (bacteria) and at 25 °C for 48 h (yeasts). MIC was defined as the first concentration did not give visible turbidity comparing to a negative control. Each test was performed in duplicate for each extract and substance. 3 µL of each clear well was inoculated in appropriate agar media and incubated in the appropriate conditions. MMC was determined as the concentration that did not yield growth on agar after incubation. Statistical analysis All experiments were carried out three times unless mentioned in the text. Continuous variables were presented as mean ± SD. IC50 values were calculated using a four parameter logistic curve (SigmaPlot 11.0) and all the data were statistically evaluated using Student’s ttest or the Kruskal–Wallis test (GraphPad Prism 5.01; GraphPad Software, Inc., San Diego, USA) followed by Dunn’s post-hoc multiple comparison test when the significance value is <0.05 using the same significance level. Results HPLC analysis of the extracts of A. turkestanica Chromatographic profiles for butanol, methanol, chloroform and water extracts were generated by HPLC (Fig. 2). HPLC analysis revealed the presence of phytoecdysteroids and iridoids as the most abundant metabolites. The following phytoecdysteroids and iridoids could be identified unequivocally: 20-hydroxyecdysone (1) (tR=17.7 min), turkesterone (2) (tR=16.7 min), cyasterone (3) (tR=19.0 min), harpagide (5) (tR=12.7 min), and 8-О-acetylharpagide (6) (tR=15.6 min) (Fig. 2). The composition of the extracts is reported in Table 1. 6 © 2013 Inforesights Publishing UK Mamadalieva et al. Phytopharmacology 2013, 4(1), 1-18 7 © 2013 Inforesights Publishing UK Mamadalieva et al. Phytopharmacology 2013, 4(1), 1-18 Figure 2 Representative HPLC chromatogram of A. turkestanica (A) methanol, (B) butanol, (C) water and (D) chloroform extracts. Peaks correspond to: 1: 20-hydroxyecdysone, 2: turkesterone, 3: cyasterone, 5: harpagide, 6: 8-О-acetylharpagide. Table 1. Composition of water, methanol, butanol and chloroform extracts from A. turkestanica Compound Retention time (tR, min) 20-Hydroxyecdysone (1) Turkesterone (2) Cyasterone (3) Harpagide (5) 8-О-Acetylharpagide (6) 17.7 16.7 19.0 12.7 15.6 Content of extracts (mg/ml) Water 0.01 0.012 n/d 0.061 0.148 Butanol 0.065 0.193 0.027 0.016 0.098 Methanol 0.046 0.114 n/d 0.049 0.125 Chloroform 0.034 n/d n/d n/d 0.002 n/d – not determined Table 2. Identified compounds in chloroform extracted volatiles of the roots of A. turkestanica. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Retention time (min) 36.36 38.06 39.11 40.43 40.51 40.63 40.89 42.32 42.49 43.42 44.64 44.68 46.81 45.00 48.89 49.83 50.91 51.55 19 52.19 20 52.39 N Compound name 1,2,7,8,8a,9,10,10a-Octahydro-2,2,7,7-tetramethylphenanthrene 4β-18-Norkaur-16-ene Abieta-9(11),8(14),12-trien-12-ol (Ferruginol) Unknown Abieta-6,8,11,13-tetraen-12-yl acetate Totarol Stigmast-5-en-3-ol (β-Sitosterol) 16α,17-Epoxypregn-4-ene-3,20-dione* 3,17-Dihydroxypregn-5-en-20-one Unknown 20-Methyl-pregna-5,17-dien-3β-ol Unknown 16-Dehydropregnenolone Pregnane-3,11,20-trione* 5-Pregnen-3β-ol-7,20-dione* 3,7-Dioxocholan-24-oic acid* Pregna-4,9 (11)-dien-20-ol-3-on-19-oic acid Ajuforrestine A* 11b-Hydroxy-3,11a-dimethyl-1,9-dioxo3a,4,5,5a,5b,9,10,11,11a,11b,12,13-dodecahydro-3Hnaphtho[2',1':4,5]indeno[1,7a-c]furan-12-yl acetate* 4,18-Epoxy-6,19-dihydroxy-13-cleroden-15,16-olide-19- 8 Retention indexa (RI) 2045 2144 2205 2280 2286 2293 2308 2391 2401 2455 2526 2537 2542 2550 2582 2587 2673 2731 0.58 0.66 0.24 0.47 0.41 0.36 0.33 0.57 0.76 0.23 12.93 0.78 2.19 0.98 0.19 10.53 19.58 3.33 2933 1.55 3103 0.98 Abundance b % © 2013 Inforesights Publishing UK Mamadalieva et al. Phytopharmacology 2013, 4(1), 1-18 21 22 23 24 25 26 a 53.68 53.74 53.97 54.18 54.65 55.42 acetate* Stigmast-4-en-3-one Unknown 4,4-Dimethylcholesta-7,9(11)-dien-3-ol Olean-12-en-3-one Betulin Barrigenol 3206 3225 3283 3370 >3400 >3400 3.34 0.83 8.43 1.76 10.18 4.37 - the Kovats index was calculated on TR1-MS column; b - average of three analyses; * - tentatively identified GLC analysis The chloroform extract was analysed in more detail because of its pronounced biological activities. Results of the GLC analysis of the chloroform extract are presented in Table 2 and Fig. 3. A total of 22 components were identified in this extract. The most abundant components were mainly sterols and oxo steroids, and triterpenes. Furthermore, abieta-, nor- and cleroden diterpenes; meroterpene and polycyclic aromatic hydrocarbons: pregna-4,9 (11)dien-20-ol-3-on-19-oic acid (19.58%), 20-methyl-pregna-5,17-dien-3β-ol (12.93%), 3,7dioxocholan-24-oic acid (10.53%), betulin (10.18%), 4,4-dimethylcholesta-7,9(11)-dien-3-ol Figure 3. Chromatogram of a chloroform extract of A. turkestanica by GLC. 9 © 2013 Inforesights Publishing UK Mamadalieva et al. Phytopharmacology 2013, 4(1), 1-18 Figure 4. Structures of selected identified compounds from the root extract of A. turkestanica (8.43%), barrigenol (4.37%), stigmast-4-en-3-one (3.34%), ajuforrestine A (3.33%) and 16dehydropregnenolone (2.19%) were identified unambiguously as major compounds (Fig. 4). Antioxidant test The DPPH* radical-scavenging activities of the reference substance (quercetin), extracts and isolated secondary metabolites are documented in Table 3 and Fig. 5. All ecdysteroids and iridoids exhibited weak DPPH* radical scavenging activity with IC50 values above 100 µM. Among the tested extracts, water and butanol extracts had a higher antiradical capacity with a IC50 value 6.13 ± 0.71 and 12.23 ± 1.42 µg/mL, respectively. Cytotoxicity analysis A cytotoxicity screening of the methanol, chloroform, butanol extracts and isolated phytoecdysteroids 1-4, and iridoid glucosides 5, 6 was carried out in HeLa, HepG-2, and MCF-7 cells. The IC50 values of corresponding extracts and isolated secondary metabolites are reported in Table 4 and Fig. 6. The phytoecdysteroids and iridoids showed a moderate in- 10 © 2013 Inforesights Publishing UK Mamadalieva et al. Phytopharmacology 2013, 4(1), 1-18 Table 3. Antioxidant activity of phytoecdysteroids, iridoids, and extracts isolated from A. turkestanica using the DPPH* radical scavenging assay. The data are represented as IC50 values (mean ± SD). Compounds and extracts IC50 (μg/mL) Extracts Water Butanol Methanol Chloroform 6.13 ± 0.71 12.23 ± 1.42 57.84 ± 4.19 100.5 ± 8.42 Phytoecdysteroids 20-Hydroxyecdysone (1) Turkesterone (2) Cyasterone (3) 22-Acetylcyasterone (4) 142.90 ± 10.43 140.92 ± 12.01 106.31 ± 9.12 114.23 ± 9.84 Iridoids Harpagide (5) 8-О-Acetylharpagide (6) 173.48 ± 15.72 294.94 ± 30.25 Control Quercetin 3.37 ± 0.77 Table 4. Antiproliferative activities of phytoecdysteroids, iridoids and extracts isolated from A. turkestanica on HeLa, HepG-2 and MCF-7 cell lines. The data are represented as IC50 values (mean ± SD). Sample HeLa IC50 of compounds and extracts (μg/mL) HepG-2 MCF-7 Extracts Water Butanol Methanol Chloroform 234.25 ± 18.34 133.11 ± 10.24 72.34 ± 2.78 7.13 ± 0.85 144.42 ± 10.07 119.96 ± 8.57 75.04 ± 5.80 9.03 ± 0.92 193.04 ± 10.59 130.56 ± 10.53 81.94 ± 2.59 10.77 ± 1.44 Phytoecdysteroids 20-Hydroxyecdysone (1) Turkesterone (2) Cyasterone (3) 22-Acetylcyasterone (4) 85.57 ± 3.25 75.17 ± 4.14 77.24 ± 10.15 67.49 ± 8.47 57.10 ± 10.77 63.01 ± 7.53 52.03 ± 7.85 71.38 ± 2.74 73.81 ± 10.71 105.21 ± 10.96 82.07 ± 11.69 115.45 ± 0.38 Iridoids Harpagide (5) 8-О-Acetylharpagide (6) 58.31 ± 10.58 61.59 ± 8.17 51.79 ± 12.85 68.14 ± 11.35 94.96 ± 19.07 86.09 ± 12.04 Control Doxorubicin (µg/ml) 1.07 ± 0.11 0.39 ± 0.04 0.28 ± 0.02 hibition of cell proliferation with IC50 values above 50 µg/mL. Methanol and chloroform extracts exhibited the highest level of cytotoxicity. Especially the chloroform extract strongly inhibited cell growth in all tested cell lines (IC50 = 7.13 ± 0.85 µg/mL in HeLa, 9.03 ± 0.92 µg/mL in HepG-2, and 10.77 ± 1.44 µg/mL in MCF-7 cells). Antimicrobial test The extracts were tested for antimicrobial activity against several human pathogenic bacteria and yeasts at various concentrations, ranging from 8 to 0.015 mg/mL. The correspo- 11 © 2013 Inforesights Publishing UK Mamadalieva et al. Phytopharmacology 2013, 4(1), 1-18 Figure. 5 Dose-response curve for DPPH* scavenging activity of A H2O, BuOH, MeOH, and CHCl3 extracts of A. turkestanica, B 20-hydroxyecdysone, turkesterone, C cyasterone, 22-acetyl-cyasterone, and D harpagide, 8O-acetylharpagide. The data shown are means ± SD obtained from three independent experiments. nding MIC and MMC values are reported in Table 5. Pure phytoecdysteroids and iridoids were tested at concentrations from 1.5 to 1000 µM. Growth of Enterococcus VanB VRE ATCC 902291, E. VanB VRE ATCC 31299, Staphylococcus aureus MRSA ATCC 1000/93, and S. aureus MRSA ATCC 10442 were not inhibited by any of the isolated secondary metabolites. Only compounds 1, and 4-6 showed a MIC of 0.5 mM against E. VanB VRE ATCC 31299, and compound 1 inhibited S. aureus MRSA ATCC 10442 (MIC 0.5 mM). Compounds 1-6 have MIC values corresponding to 0.5 mM in both S. pyogenes ATCC 12344 and C. albicans ATCC 90028. Other phytoecdysteroids and iridoids were inactive against Candida glabrata ATCC MYA 2950 except cyasterone (3) (MIC > 0.5 mM and MMC > 0.5 mM). Klebsiella pneumonia ATCC 700603 was inhibited by all the compounds (MIC = 0.25 mM), except compound 1 (MIC = 0.5 mM). P. aeruginosa ATCC 27853 was inhibited by the phytoecdysteroid 4, which showed the strongest activity (MIC = 0.125 mM), whereas other compounds showed MIC values from 0.25 to 0.5 mM (Table 5). The chloroform extract showed strong antimicrobial activity against S. aureus MRSA ATCC 1000/93 and S. pyogenes (MIC = 0.06 mg/mL and MMC = 0.03 mg/mL), respectively. C. glabrata was not inhibited by any of the tested plant extracts, whereas only chloroform extract showed a weak inhibition (MIC>4 mg/mL) against this yeast. Butanol and methanol extracts (MIC = 4 - 8 mg/mL) were less active. 12 © 2013 Inforesights Publishing UK Mamadalieva et al. Phytopharmacology 2013, 4(1), 1-18 Table 5a. Minimum inhibitory concentrations (MIC) and minimum microbicidal concentrations (MMC) of the extracts and compounds from the plant A. turkestanica against different pathogens Staphylococcus aureus MRSA ATCC 10442 Enterococcus Vanb VRE ATCC 31299 Staphylococcus aureus MRSA ATCC 1000/93 Enterococcus Vanb VRE ATCC 902291 Streptococcus pyogenes ATCC 12344 8 >8 0 4 >4 3.15±1.15 >4 >4 7.1±0.1 >4 >4 NA NA NA NA NA NA 3.8±0.2 2 8 3.4±0.2 >4 >4 6.05±0.05 8 8 4.05±0.05 4 8 3.9±0.1 4 >4 6.85±0.15 0.06 0.5 5.1±0.1 >8 >8 3.1±0.1 >4 >4 3.8±0.1 >4 >4 4.8±0.2 4 >4 8.8±0.1 4 >8 4.8±0.1 2 4 5.9±0.1 2 4 8.2±0.2 0.03 0.25 I.z. 0 0 NA MIC* MMC* I.z. MIC* MMC* I.z. MIC* MMC* 0.5 >0.5 NA NA NA NA NA NA 0.5 >0.5 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 0.5 >0.5 3.05±0.05 0.5 >0.5 4.1±0.1 0.5 >0.5 I.z. NA 3 NA NA 3.9±0.1 MIC* MMC* NA NA 0.5 >0.5 NA NA NA NA 0.5 >0.5 I.z. NA 0 NA NA 3.95±0.05 MIC* MMC* I.z. MIC* MMC* I.z. MIC MMC I.z. MIC MMC I.z. MIC MMC NA NA NA NA NA 14.5±0.5 25 >25 10±0.2 0.8 12.5 NT NT NT 0.5 >0.5 0 0.25 0.5 15 1 7 NT 25 >50 NT NT NT NA NA NA NA NA 13.5±0.5 50 >50 NT 7 12.5 NT NT NT Sample A. t. H2O extract A. t. BuOH extract A. t. MeOH extract A. t. CHCl3 extract 20-Hydroxyecdysone (1) Turkesterone (2) Cyasterone (3) 22-O-Acetylcyasterone (4) 8-О-Acetylharpagide (5) Harpagide (6) Ampicillin a Vancomycin Nystatine a a I.z. MIC MMC I.z. MIC MMC I.z. MIC MMC I.z. MIC MMC 0 NA NA NA NA NA NA NA NA NA NA NA NA NT NT NT 4.9±0.1 0.5 >0.5 3.9±0.1 0.5 >0.5 25±1 0.05 0.1 15±1 0.1 0.4 NT NT NT A.t. – A. turkestanica; I.z. – inhibition zone, mm; MIC - mg/mL; MIC* - mM; MMC - mg/mL; MMC* - mM; NT – not tested; NA – not active; a = MIC and MMC values in µg/ml Discussion A. turkestanica accumulates high levels of phytoecdysteroids (Abdukadyrov et al., 2005; Usmanov et al., 1975; Saatov et al., 1977) and has therefore been exploited as an indu strial source for the production of phytoecdysteroids. The main major phytoecdysteroids are 20-hydroxyecdysone (1) and turkesterone (2). The chemical analysis of the present study confirms already reported (Abdukadyrov et al., 2005; Usmanov et al., 1971, 1975, 1978; Saatov et al., 1977). Due to the substantial bioactivity of the CHCl3 extract (Table 3-5), such extract is characterized by both HPLC and GLC. In addition to compounds 1 and 6, chloroform extract contains a number of oxo sterols (pregna-4,9 (11)-dien-20-ol-3-on-19-oic acid lactone, 3,7-dioxocholan-24-oic acid and 16δ-pregnenolone), sterols (20-methyl-pregna-5,17-dien-3βol and 4,4-dimethylcholesta-7,9(11)-dien-3-ol) and triterpenes (betulin and barrigenol) (Table 2). 13 © 2013 Inforesights Publishing UK Mamadalieva et al. Phytopharmacology 2013, 4(1), 1-18 Table 5b. Minimum inhibitory concentrations (MIC) and minimum microbicidal concentrations (MMC) of the extracts and compounds from the plant A. turkestanica against different pathogens. Escherichia coli ATCC 25922 Klebsiella pneumonia ATCC 700603 Pseudomonas aeruginosa ATCC 27853 Candida albicans ATCC 90028 Candida glabrata ATCC MYA 2950 I.z. MIC MMC I.z. MIC MMC I.z. MIC MMC I.z. MIC MMC 0 8 >8 0 8 >8 3.15±0.15 8 >8 3.3±0.1 8 >8 0 8 >8 0 8 >8 4.85±0.15 4 >4 4.8±0.2 8 >8 4.5±0.1 8 >8 3 4 8 4.4±0.2 4 8 4.9±0.1 4 >4 0 8 >8 0 4 >4 3.8±0.2 >4 >4 5.75±0.25 2 >4 NA NA NA NA NA NA NA NA NA 3.9±0.1 >4 >4 I.z. 3.1±0.1 3 3.4 3.9±0.1 NA MIC* MMC* I.z. MIC* MMC* I.z. MIC* MMC* 1 >1 3 1 >1 3±0.1 1 >1 0.5 >0.5 3.8±0.2 0.25 0.5 3.9±0.1 0.25 0.5 0.25 0.5 3 0.5 1 3.05±0.15 0.5 1 >0.5 >0.5 4.8±0.2 0.5 >0.5 3 0.5 >0.5 NA NA NA NA NA 3.9±0.1 >0.5 >0.5 I.z. 3.05±0.15 3.85±0.15 3.4±0.4 3 NA MIC* MMC* 1 >1 0.25 0.5 0.125 0.25 >0.5 >0.5 NA NA I.z. 3.05±0.05 3 3.1±0.1 3.85±0.15 NA MIC* MMC* I.z. MIC* MMC* I.z. MIC MMC I.z. MIC MMC I.z. MIC MMC 0.5 >0.5 3 1 >1 14 12.5 25 NA NA NA NT NT NT 0.25 0.5 3.2±0.1 0.25 0.5 25 25 NT 25 50 NT NT NT 0.25 0.5 3.1±0.1 0.25 0.5 NA NA NA NA NA NA NT NT NT 0 0 NA NA NA NT NT NT NT NT NT 10±1.2 0.2 0.4 NA NA NA NA NA NT NT NT NT NT NT 12±1 0.2 0.2 Sample A. t. H2O extract A. t. BuOH extract A. t. MeOH extract A. t. CHCl3 extract 20-Hydroxyecdysone (1) Turkesterone (2) Cyasterone (3) 22-O-Acetylcyasterone (4) 8-О-Acetylharpagide (5) Harpagide (6) Ampicillin a Vancomycin a Nystatine a A.t. – A. turkestanica; I.z. – inhibition zone, mm; MIC - mg/mL; MIC* - mM; MMC - mg/mL; MMC* - mM; NT – not tested; NA – not active; . a = MIC and MMC in µg/ml These phytochemicals were completely different from those of previously published studies performed on lipophilic extractions of other Ajuga species (Azizan et al., 2002; Baser et al., 1999; 2001; Javidnia et al., 2010; Velasco-Negueruela et al., 2004; Sa-jjadi et al., 2004). Isolated phytoecdysteroids and iridoids of A. turkestanica were ineffective for DPPH radical scavenging activity (Table 3). This is not surprising because the structure of ecdysteroid molecules is unlikely to exert an antioxidant effect, as compared to the common antioxidative flavonoids (Harborne and Williams, 2000). In our experiments polar extracts such as butanol and methanol extracts were more active; this activity could be due to phenolics or other antioxidants that were not identified in our analysis. This finding is in agreement with other studies (Miliauskas et al., 2005; Turkoglu et al., 2010). 14 © 2013 Inforesights Publishing UK Mamadalieva et al. Phytopharmacology 2013, 4(1), 1-18 As shown in Table 4 none of the isolated secondary metabolites exhibited a high cytotoxicity (IC50 above 100 µM or 50 µg/ml). The higher activity of the chloroform extract is probably due to additional compounds that were identified by GLC and GLC-MS (Fig. 3). Since individual substances of the CHCl3 extract were not available, we could not identify the underlying cytotoxic principle. Lagova and Valueva (1981) found that 20-hydroxyecdysone was ineffective to inhibit the growth of several tumour types, whereas it stimulated that of mammary gland carcinomas. In this specific case, because ecdysteroids structurally resemble sex hormones, they may bind to steroid hormone receptors in mammals and stimulate hormone-dependent tumors. Takasaki et al. (1999) reported that phytoecdysteroids and iridoids from Ajuga decumbens have anticancer properties. In their study cyasterone, polypodine B, decumbesterone A, especially 8-О-acetylharpagide (6) showed strong tumour preventive activities in vivo in a mouse-skin model, using 7,12-dimethylbenz[a]anthracene as tumour initiator and TPA as promoter. As shown in Table 5, phytoecdysteroids and iridoids of A. turkestanica had weak antimicrobial activity against Gram-positive bacteria, C. glabrata ATCC MYA 2950, except S. pyogenes ATCC 12344. Only cyasterone (3) showed activity (MIC > 0.5 mM and MMC > 0.5 mM) against C. glabrata ATCC MYA 2950. Compounds 1-6 showed stronger activity against all Gram-negative bacteria. Acetyl group containing phytoecdysteroids such as 4 and 5 inhibited the growth of bacteria used. The maximum inhibition was observed against K. pneumonia ATCC 700603 and P. aeruginosa ATCC 27853 having of MIC values of 0.1250.25 mM. According to Shirshova et al. (2006) and Volodin et al. (1999) true phytoecdysteroids such as ecdysone, inokosterone and 20-hydroxyecdysone (1) have weak antimicrobial activity. But the introduction of acetyl groups into the molecule 1 can increase antimicrobial activity against Bacillus cereus, Proteus rettgeri and Saccharomyces cerevisiae in the sequence from 2-aсetate-20-hydroxyecdysone<2,3,22-tri-acetate-20-hydroxyecdysone < 2,3,22,25tetraacetate-20-hydroxyecdysone (Shirshova et al., 2006; Politova et al., 2001). Whereas iridoid glucosides are rather inactive, some iridoid aglycones from the Cymbaria mongolica showed antibacterial activity against Bacillus subtilis, Escherichia coli and Staphylococcus aureus. Among them, 1β-methoxylmussaenin A possessed significant activity similar to that of chloramphenicol (Dai et al., 2002). Iridoid glucosides are stored inactive prodrugs in plant vacuoles; only after treatment with beta-glucosidase an aglycone is formed in which the lactol rings opens. Then two reactive aldehyde groups are generated; these can interfere with amino groups of proteins and nucleic acids (Wink, 2008; Wink and van Wyk, 2008). Our antimicrobial tests revealed that the isolated ecdysteroids are hardly antimicrobial. However, the chloroform extract has antimicrobial activity even against multiresistant strains with known resistance against antibiotics, like Staphylococcus aureus MRSA ATCC 1000/93 and Streptococcus pyogenes ATCC 12344 (Table 5). We assume that cytotoxic and antibacterial activities of the chloroform extract of A. turkestanica may be due to the presence of nonpolar compounds such as oxo sterols, sterols, diterpenes, and triterpenes. According to the literature, some of pregnene and pregnadiene derivatives were potential inhibitors of 5α-reductase type II, inhibited cell proliferation of LNCap and PC-3 prostate cancer cells and were the most active in the 5AR2 inhibitory test (Kim and Ma, 2009). Also some triterpenes such as betulin could act as potent antitumour 15 © 2013 Inforesights Publishing UK Mamadalieva et al. Phytopharmacology 2013, 4(1), 1-18 promoters, being active against colorectal (DLD-1), breast (MCF-7), prostate (PC-3) and lung (A549) cancer cell lines (Gauthier et al., 2009). In conclusion, whereas isolated ecdysteroids and iridoid glucosides of A. turkestanica do not function as antioxidants or substantial cytotoxic or antimicrobial agents, the chloroform extract with more lipophilic compound was more active. In a next step further studies should be performed on the isolation and identification of the active compounds of the chloroform extract of A. turkestanica. Conflict of interest The authors declare no conflict of interest. Acknowledgements N.Z. Mamadalieva gratefully acknowledges the support of the UNESCO-L’OREAL and DAAD research grant. References Abdukadyrov IT, Khodzhaeva MA, Turakhodjaev MT, Mamatkhanov AU. (2004). Carbohydrates from Ajuga turkestanica. Chemistry of Natural Compounds 40, 85-86. Abdukadyrov IT, Yakubova MR, Nuriddinov KhR, Mamatkhanov AU, Turakhodjaev MT. (2005). Ecdysterone and turkesterone in Ajuga turkestanica determined by HPLC. Chemistry of Natural Compounds 41, 475-476. Adams RP. (2004). Identification of essential oil components by gas chromatography/quadrupole mass spectroscopy. 3rd edn. Illinois: Allured Pub Corp. Azizan J, Fallah-Bagher-Shaidaei H, Kefayati H. (2002). Chemical constituents of the essential oil of Ajuga chamaepitys growing in Iran. Journal of Essential Oil Research 14, 344-345. Baser KHC, Erdemgil Z, Ozek T, Demirci B. (1999). Compositions of essential oils from two varieties of Ajuga chamaepitys subsp. chia from Turkey. Journal of Essential Oil Research 11, 203-205. Baser KHC, Kurkcuoglu M, Erdemgil FZ. (2001). The essential oil of Ajuga bombycina from Turkey. Chemistry of Natural Compounds 37, 242-244. Brandwilliams W, Cuvelier M, Berset C. (1995). Use of a free-radical method to evaluate antioxidant activity. Lebensmittel Wissenschaft und Technologie 28, 25-30. Budzikiewicz H, Djerassi C, Williams DH. (1964). Structure Elucidation of Natural Products by Mass Spectrometry, Volume II: Alkaloids, Steroids, Terpenoids, Sugars, and Miscellaneous Classes. Holden-Day, Inc., San Francisco, London, Amsterdam. Dai J-Q, Liu Z-L, Yang L. (2002). Non-glycosidic iridoids from Cymbaria mongolica. Phytochemistry 59, 537-542. Ganiev ShG, Khamidkhodjaev SA, Djuharova MX, Saatov Z. (1990). Ajuga turkestanica from Baysuntay (Baysun mountin). Uzbek Biology Journal 30-32 [in Russian]. Gauthier C, Legault J, Lavoie S, Rondeau S, Tremblay S, Pichette A. (2009). Synthesis and cytotoxicity of bidesmosidic betulin and betulinic acid saponins. Journal of Natural Products 72, 72-81. Grace MH, Cheng DM, Raskin I, Lila MA. (2008). Neo-clerodane diterpenes from Ajuga turkestanica. Phytochemistry Letters 1, 81-84. 16 © 2013 Inforesights Publishing UK Mamadalieva et al. Phytopharmacology 2013, 4(1), 1-18 Harborne JB, Williams CA. (2000). Advances in flavonoid research since 1992. Phytochemistry 55, 481-504. Javidnia K, Miri R, Soltani M, Khosravi AR. (2010). Chemical constituents of the essential oil of Ajuga austro-iranica Rech. f. (Lamiaceae) from Iran. Journal of Essential Oil Research 22, 392-394. Israili ZH, Lyoussi B, (2009). Ethnopharmacology of the plants of genus Ajuga. Pakistan Journal of Pharmaceutical Sciences 22, 425-462. Khalitova YD, Syrov VN, Akhmedkhodjaeva KhS, Mamatkhanov AU. (1998). Possible use of the extract of Ajuga turkestanica as a remedy contributing to lactation. Doklady Akademii Nauk Respubliki Uzbekistan 8, 35-38 [in Russian, with an English abstract]. Kim S, Ma E. (2009). Synthesis of pregnane derivatives, their cytotoxicity on LNCap and PC-3 cells, and screening on 5α-reductase inhibitory activity. Molecules 14, 4655-4668. Kotenko LD, Yakubova MR, Mamatkhanov U, Turakhozhaev MT. (1994). Control of the production of the total iridoids from Ajuga turkestanica. Chemistry of Natural Compounds 30, 769-770. Kutepova TA, Syrov VN, Khushbaktova ZA, Saatov Z. (2001). Hypoglycemic activity of the total ecdysteroid extract from Ajuga turkestanica. Pharmaceutical Chemistry Journal 35, 608-609. Lagova ND, Valueva IM. (1981). Effect of ecdysterone isolated from Rhaponticum carthamoides on the growth of experimental tumors. Eksperimental'naya Onkologiya 3(4), 69-71. Mabberley D. (2008). The Plant-Book, 3rd edn. Cambridge University Press. Cambridge, New York. Miliauskas G, van Beek TA, de Waard P, Venskutonis RP, Sudhölter EJR. (2005). Identification of radical scavenging compounds in Rhaponticum carthamoides by means of LC-DAD-SPENMR. Journal of Natural Products 68, 168-172. Mosmann T. (1983). Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. Journal of Immunological Methods 65, 55-63. National Committee for Clinical Laboratory Standards. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, Approved Standard. In, M2-A9. Wayne, Pennsylvania, USA: NCCLS, 2006. Nibret E, Wink M. (2010). Volatile components of four Ethiopian Artemisia species extracts and their in vitro antitrypanosomal and cytotoxic activities. Phytomedicine 17, 369-374. Politova NK, Kovler LA, Volodin VV, Luksha VG, Pshunetleva EA. (2001). Chemical modification and membrane-acting property of 20-hydroxyecdysone and its derivatives. Khimiya Rastitelnogo Syr’ya 2, 69-81 [in Russian]. Saatov Z, Usmanov BZ, Abubakirov NK. (1977). Phytoecdysones from Ajuga turkestanica. IV. Chemistry of Natural Compounds 13, 359. Saatov Z, Agzamkhodzhaeva DA, Syrov VN. (1999). Distribution of phytoecdysteroids in plants of Uzbekistan and the possibility of using drugs based on them in neurological practice. Chemistry of Natural Compounds 35, 186-191. Sajjadi SE, Ghannadi A. (2004). Volatile oil composition of the aerial parts of Ajuga orientalis L. from Iran. Zeitschrift für Naturforschung C 59, 166-168. Shirshova T, Politova N, Burtseva S, Beshlei I, Volodin VV. (2006). Antimicrobial activity of natural ecdysteroids from Serratula coronata L. and their acyl derivatives. Pharmaceutical Chemistry Journal 40(5), 268-271. Sokolov PD. (1991). Plant resources of USSR. Nauka Publ. Sankt Petersburg. 6, 11-13. Syrov VN, Nabiev AN, Sultanov MB. (1986). Action of phytoecdysteroids on the bile-secretory function of the normal liver and in experimental hepatitis. Farmakologiia Toksikologiia [in Russian, with an English abstract]. 49, 100-103. Takasaki M, Tokuda H, Nishino H, Konoshima T. (1999). Cancer chemopreventive agents (antitumor-promoters) from Ajuga decumbens. Journal of Natural Products 62, 972-975. Turkoglu S, Turkoglu I, Kahyaoglu M, Celık S. (2010). Determination of antimicrobial and antioxidant activities of Turkish endemic Ajuga chamaepitys (L.) Schreber subsp. euphratica P.H. Davis (Lamiaceae). Journal of Medicinal Plants Research 4(13), 1260-1268. 17 © 2013 Inforesights Publishing UK Mamadalieva et al. Phytopharmacology 2013, 4(1), 1-18 Usmanov BZ, Gorovits MB, Abubakirov NK. (1971). Phytoecdysones from Ajuga turkestanica. Chemistry of Natural Compounds 7, 520. Usmanov BZ, Gorovits MB, Abubakirov NK. (1973). Phytoecdysones from Ajuga turkestanica. II. Chemistry of Natural Compounds 9, 125-126. Usmanov BZ, Gorovits MB, Abubakirov NK. (1975). Phytoecdysones from Ajuga turkestanica. III. The structure of turkesterone. Chemistry of Natural Compounds 11, 484-487. Usmanov BZ, Saatov Z, Abubakirov NK. (1977). Phytoecdysones from Ajuga turkestanica. V. Chemistry of Natural Compounds 13, 595. Usmanov BZ, Rashkes YV, Abubakirov NK. (1978). Phytoecdysones of Ajuga turkestanica. VI. 22Acetylcyasterone from Ajuga turkestanica. Chemistry of Natural Compounds 14, 175-178. Velasco-Negueruela A, Perez-Alonso MJ, Pala-Paul J, Inigo A, Sanz J. (2004). Volatile constituents of the essential oil of Ajuga chamaepitys (L.) Schreber. ssp. Chamaepitys from Spain. Journal of Essential Oil Research 16, 372-373. Volodin VV, Shirshova TI, Burtseva SA, Melnik MV. (1999). Biological activity of 20-hydroxyecdysone and its derivatives. Plant Resources 2, 76-81 [in Russian, with an English abstract]. Van Wyk BE, Wink M. (2004). Medicinal Plants of the World. Medicinal Plants of the World. 480 pp. Briza Publications, Pretoria. Wink M. (2008). Evolutionary advantage and molecular modes of action of multi-component mixtures used in phytomedicine. Current Drug Metabolism 9, 996-1009. Wink M, Van Wyk BE. (2008). Mind-Altering and Poisonous Plants of the World. 464 pp. Briza Publications, Pretoria. 18 © 2013 Inforesights Publishing UK