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1 Article DOI: /bbeq B&E Intraspecific variability of biologically active compounds of different populations of Tribulus terrestris in Thracian floristic region Antoaneta Ivanova 1, Irina Lazarova 1, Pepa Mechkarova 1, Ivanka Semerdjieva 2, Ljuba Evstatieva 3 1 Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences, Sofia, Bulgaria 2 Agricultural University, Faculty of Agronomy, Plovdiv, Bulgaria 3 Institute of Biodiversity and Ecosystem Research, Bulgarian Academy of Sciences, Sofia, Bulgaria Correspondence to: Antoaneta Ivanova ivanova111@yahoo.com ABSTRACT Tribulus terrestris, commonly known as Puncture vine, is a member of the Zygophyllaceae family. The steroidal saponins are considered to be the factor responsible for the biological activity of the products derived from this plant. The content of the main steroidal saponins protodioscin, prototribestin, dioscin and the flavonoid glucoside rutin in T. terrestris populations from 19 localities in the Thracian floristic region were analyzed using an HPLC method. Seven localities from Haskovo Plain and Pazardzhik-Plovdiv Plain showed the highest content of Pd, which varied from to mg /g and Pt -from 7.29 to mg/g with a total of kg dry mass exploitation reserve. Cluster analysis was performed to reveal the possible similarities among the investigated habitats. The economically important populations can be successfully used in our country and international markets for the preparation Tribestan and some food supplements containing extracts from T. terrestris. Biotechnol. & Biotechnol. Eq. 2011, 25(2), Keywords: Tribulus terrestris, Bulgaria, Thracian floristic region, steroidal saponins, HPLC Introduction Tribulus terrestris, commonly known as Puncture vine, is a member of the Zygophyllaceae family, and an annual herb found in the warm regions of Europe, Asia, America, Africa and Australia (2). Tribulus terrestris contains a mixture of different compounds such as steroidal saponins, flavonoids, cinammic acid amides, lignanamides and alkaloids (11). Many pharmaceutical preparations and food supplements with these active components are commercially available (1, 3, 14, 15). For example, the rich fraction of this herb is used for the treatment of sexual impotence, blood pressure in USA and Europe, coronary heart diseases in China, eye problems, edema, etc. (6, 9). The furostanol saponins protodioscin (Pd) and prototribestin (Pt) have been found to be responsible for the general stimulating activity of the saponin fraction of Tribestan, especially on the reproductive system (6, 7, 14). They are major in all of the samples. Tribulus terrestris is widely distributed in Bulgaria. There are 20 floristic regions in the country and the species is established in 10 of them (8). The saponin fraction of the plant is used for preparation of the drug Tribestan and food suplements against oligospermia and sexual dysfunctions in men and women (10, 13). The steroidal saponins- protodioscin, prototribestin and dioscin, and the flavonoid glucoside rutin are some of the main compounds responsible for the medicinal properties of the plant which are present in the Bulgarian samples. For the production of Tribestan SOFARMA Pharmaceutical Company uses about 150 tons of Tribulus terrestris annually, mainly from Turkey and the Thracian floristic region in Bulgaria. Detailed information of the ecological and resource characteristic of the species from this region have been given by Semerdjieva and Evstatieva, 2010 (12), but there are limited studies on the saponin content from it (4, 5). The aim of this work was to determine the variability of the main steroidal saponins and the flavonoid glucoside rutin in Tribulus terrestris populations from 19 localities in the Thracian floristic region and to define the most perspective ones for collection and production of high quality drugs and food supplements. The relationship between the ecological conditions and the presence of biologically active compounds was estimated. Materials and Methods Plant material The diversity of the topography, climate and soils in the Tharacian floristic region includes microregions of the Pazardzhik-Plovdiv Plain, Karlovo Valley, Stara Zagora Plain and Haskovo Plain. The nineteen samples of Tribulus terrestris from these microregions were subjected to analysis of phytochemical variability of the main steroidal saponins (protodioscin, prototribestin and dioscin) and the flavoniod glucoside rutin using an HPLC method (Table 1, Fig. 1). The samples were collected at the flowering-seeding stage (July- 2357

2 August). The aerial parts of the leaves and fruits at a 1:1 ratio were used as samples for phytochemical analysis. Cluster analysis was performed in order to uncover similarities in the investigated main components in the samples (Fig. 3). Voucher specimens of the samples were deposited in the Herbarium of the Institute of Botany, Bulgarian Academy of Sciences, Sofia. Analytical method An HPLC system La Chrom Elite consisting of L-2130 pump equipped with a gradient controller and UV detector L-2400 was used. The separation was performed on 250 x 4.6 mm i. d., 5 µm, Inertsil ODS-2 column (Tokyo, Japan) with MetaGuard Pursuit direct connect guard column from Varian was used for all separations. The mobile phase which consisted of phosphoric acid buffer with ph-3 (A) and acetonitrile (B) was used for gradient elution. The flow rate was adjusted to 1.0 ml/min, the detection wavelength was 203 nm. All separations were performed at ambient temperature. TABLE 1 The localities in the microregions of the Thracian floristic region Microregions Town Village Pazardzhik- Plovdiv Plain Karlovo Valley Stara Zagora Plain Haskovo Plain 1.Pazardzhik 3.Stamboliyski 5.Asenovgrad 9.Stara Zagora 10.Chirpan 11.Simeonovgrad 14.Harmanli 16.Lyubimets Fig. 1. Location of the studied samples from T. terrestris 2.Varvata 4.Karadzhalovo 6.Ognyanovo 7.Vedrare 8.Marino Pole 12.Zdravets 13.Dobrich 15.Balgarin 17.G.Dobrevo 18.Mezek 19.Raykova Mogila Sample preparation The finely powdered plant material (1g, leaves and fruits, 1:1) was extracted three times with 5.0 ml of 50% aqueous acetonitrile by sonication for 15 min. The extracts were combined, after filtration in a ml volumetric flask and the volume was adjusted to 20 ml with the solvent used for extraction. Prior to injection, all samples were filtered through a 0.45-mm Chromafil 0-45/25 Machery-Nagel. Every sample solution was injected in triplicate with an injection volume of 20ml. Cluster analyses An unsupervised technique was applied, cluster analysis, to discover similarities within the obtained concentration data for the main compounds-protodioscin, prototribestin, dioscin and rutin. Squared Euclidean distance was always used as the interval measure for clustering using distinct linkage methods: between-groups linkage, within-groups linkage and Ward s method. All statistical manipulation and graphical display was done with STATISTICA 7 for Windows (StatSoft, Inc., Tulsa, OK, USA). Results and Discussion The results from the quantitative determination of protodioscin, prototribestin, dioscin and rutin in Tribulus terrestris from different populations of the Thracian floristic region were presented in Table 2 and Fig. 2. Two of the investigated samples from the Haskovo Plain (Balgarin, Harmanli), two from the Plovdiv-Pazardzhik Plain (Karadzhalovo, Pazardzhik) and one from Stara Zagora contained all the bioactive compounds under investigation. The presence of dioscin (Ds) in them was presented from 1.35 to 1.94 mg/g. In all the other localities the quantity was less than the limit of detection (Table 2, Fig. 2). The results from the quantitative determination of protodioscin, prototribestin and rutin showed that the compounds dominated mainly in the localities from the microregion of the Haskovo Plain (Simeonovgrad, Balgarin, Lyubimets, G. Dobrevo and Raykova mogila) as well as two localities from the Pazardzhik-Plovdiv Plain (Asenovgrad and Pazardzhik). Apparently, all samples containing high concentrations of the steroidal saponins also had a high concentration of rutin- from 1.96 to 3.69 mg/g. The results from the phytochemical analysis showed a remarkable difference in the furostanol saponins protodioscin (Pd) and prototribestin (Pt). The above mentioned five localities from the Haskovo Plain showed the highest content, which varied from to mg /g Pd and from to mg/g f Pt, and the two localities from the Pazardzhik-Plovdiv Plain- from to Pd and 7.29 to 8.93 mg/g Pt. These localities were characterized with a high concentration of aerial and soil moisture and high average monthly temperatures during the vegetation of the plant. One sample from the Pazardzhik-Plovdiv Plain (Ognyanovo), one from the Karlovo Valley (Marino Pole) and 2358

3 one from the Haskovo Plain (Dobrich) contained the lowest concentration of the two saponins- from 3.41 to 3.75 mg/g Pd and 1.49 to 2.33 mg/g Pt. The content in the samples taken from all the other nine localities distributed in the fourth Thracian floristic region varied from 5.25 to mg/g Pd and 3.22 to 7.98 mg/g Pt. These high results were in accordance with our previous studies for this region (4, 5). Generally, our investigations showed that the highest content of the main saponins in the Thracian floristic region was observed in the habitats in the lower reaches of the Maritza River. The content of the compounds was less in the localities remote from the river (localities Ognyanovo, Marino Pole and Dobrich). The correlation between high concentrations of the biologically active compounds and the exploration resources fresh/dry mass was estimated in the investigated localities from the Thracian floristic region. Our investigations from the Thracian floristic region by Semerdjieva and Evstatieva (12) showed that it could give about 90 tons of dry drugs annually. The most important localities with high-yield plant resources were distributed again near the Maritza River and its flows such as Asenovgrad (11430 kg), Simeonovgrad (11300 kg), Varvara (9430 kg), G. Dobrevo (2500 kg), Balgarin (1630 kg), Harmanli (1540 kg), Raykova Mogila (1280 kg) etc. Probably the high concentration of the investigated saponins and rutin and the resource capacity could be due to the good ecological conditions Mediterranean influence, determined high average monthly temperature and rainfalls during the vegetation of the species and soil. Vital significance had the agroclimatic factors, fertilization, growth density, harvesting time, etc. With the accession of Bulgaria to the European Union the standardization of natural resources used in pharmaceutical practices and as dietary supplements, as well as the strict adherence to specified parameters have become mandatory. The international herbal market has placed similar requirements for their quality. More of the populations in the region have been included in the protected area of NATURA 2000 as the Directive 92/43 of Comparative analysis of the main components of Tribulus terrestris in the Thracian floristic region TABLE 2 Locality Exploitation Resources Fresh/dry mass (Semerdjieva, Rutin Protodioscin Prototribestin Dioscin Evstatieva, 2010) kg mg/g DW Pazardzhik-Plovdiv Plain 1 Pazardzhik 3250/ ± ± ± ± Varvara 33000/ ± ± ±0.07 < LOD a 3 Stamboliyski 8500/ ± ± ±0.17 < LOD 4 Karadzhalovo 1500/ ± ± ± ± Asenovgrad 40000/ ± ± ±0.01 < LOD 6 Ognyanovo 960/ ± ± ±0.06 < LOD Karlovo valley 7 Vedrare 19500/5570 < LOD 11.68± ±0.03 < LOD 8 Marino Pole 4400/ ± ± ±0.08 < LOD Stara Zagora Plain 9 Stara Zagora 3360/ ± ± ± ± Chirpan 1125/ ± ± ±0.02 < LOD Haskovo Plain 11 Simeonovgrad 34000/ ± ± ±0.39 < LOD 12 Zdravets 10800/ ± ± ±0.12 < LOD 13 Dobrich 7500/ ± ± ±0.04 < LOD 14 Harmanli 5400/ ± ± ± ± Balgarin 5700/ ± ± ± ± Lyubimets 840/ ± ± ±0.47 < LOD 17 G.Dobrevo 7500/ ± ± ±0.09 < LOD 18 Mezek 1140/ ± ± ±0.11 < LOD 19 Raykova Mogila 4500/ ± ± ±0.35 < LOD a limit of detection 2359

4 EEC on the Habitats and could be used again rationally for the pharmaceutical industry and for export. Fig. 2. Chemical profile of biologically active compounds in Tribulus terrestris from 19 localities in the Thracian floristic region The aggregate cluster analysis of the main compounds from 19 populations in the Thracian floristic region showed the following characteristic feature (Fig. 3). Simeonivgrad G. Dobrevo Balgarin Lyubimets Raykova mogila Pazardzhik Asenovgrad Chirpan Vedrare Zdravets Varvara Harmanli Stara Zagora Stamboliyski Mezek Karadzhalovo Ognyanovo Dobrich Marino pole Tree Diagram for 19 Cases Ward`s method Euclidean distances Linkage Distance Fig. 3. Dendrogram obtained with Ward s method of linkage. It shows the distribution of biologically active compounds in Tribulus terrestris from 19 localities of the Thracian floristic region Two cluster groups could be identified in Fig. 3. The populations of the first group were characterized by the highest content of the main investigated compounds. Three subgroups were formed with the same degree of similarity: first- populations of G. Dobrevo and Balgarin, second- Lyubimets, Raykova Mogila, Pazardzhik and Asenovgrad and third- Chirpan-Vedrare. The last subgroup was characterized with lower concentrations of Pt, Pd, Ds and Ru. The population of Simeonovgrad differed from the first subgroup with the presence of the highest content of the investigated biologically active compounds. The populations of the second group were separated into two subgroups. The populations of Ognyanovo, Dobrich and Marino Pole showed the highest degree of similarity and were characterized with the lowest content of Pd, Pt, Ds and Ru. The second subgroup included the populations Zdravets, Varvara, Harmanli, Stara Zagora, Stamboliyski, Mezek and Karadzhalovo. They showed a differently high degree of similarity. Conclusions The populations in the Thracian floristic region were characterized with a high content of the saponins protodioscin, prototribestin and the flavonoid rutin and high-yield plant resources. Five populations from Haskovo Plain showed the highest content of Pd, which varied from to mg/g Pd and from to mg/g Pt. Comparatively near to them were two populations from the Pazardzhik-Plovdiv Plain- Pd from to and Pt from 7.29 to 8.93 mg/g. The best populations for collection of the botanical drug were shown to be Simeonovgrad, Georgi Dobrevo, Balgarin and Raykova Mogila, distributed in the lower reaches of the Maritza River. They were characterized with the highest content of the studied saponins and the mass of exploitation reserves. The economically important populations could be used successfully in our country and international markets for preparation of Tribestan, for some food supplements containing extracts from T. terrestris and for selection under cultivation. Acknowledgments This work was supported by the National Science Fund of Bulgaria (BNSF), grant DO REFERENCES 1. Adimoelja A. and Adaikan P.G. (1997) Int. J. Impotence Res., 9, Al-Bayati F.A. and Al-Mola H.F. (2008) J. of Zhejiang University Science, 9(2), Cai L., Wu Y., Zhang J., Pei F., Xu Y., Xie S., Xu D. (2001) Planta Med., 67, Dinchev D., Evstatieva L., Platikanov S., Galambosi B. (2010) Compt. Rend. Acad. Bulg. Sci., 63, Dinchev D., Janda B., Evstatieva L., Oleszek W., Aslani M.R., Kostova I. (2008) Phytochemistry, 69, Kostova I. and Dinchev D. (2005) Phytochemistry Rev., 4, Kostova I., Dinchev D., Rentsch G.H., Dimitrov V., Ivanova A. (2002) Z. Naturforsch. 57C(1-2), Lazarova I., Ivanova A., Mechkarova P.D., Peev D., Valyovska N. (2011) Biotechnol. & Biotechol. Eq., in press. 9. Mulinacci N., Vignoloni P., La Marca G., Pieraccani G., Innocenti L., Vincieri F.F. (2003) Chromatographia, 57, Roman M. / tribosten-lab.pdf Analytical test report 2360

5 11. Saleh N.A.M., Ahmed A.A., Abdalla M.F. (1982) Phytochemistry, 21, Semerdjieva I. and Evstatieva L. (2010) Biotechnol. & Biotechol. Eq., 24(special edition 2/on-line), Tomova M. (1987) Farmacia (Sofia), 37(6), (in Bulgarian) 14. Tomova M., Gyulemetova R., Zarkova S., Peeva S., Pangarova T.,Simova M. (1981) First International Conference on Chemical, Biotechnological and Biologically Active Natural Products, Proceedings Varna, September 3, pp Xu Y.-X., Chen H.-S., Liang H.-Q., Gu Z.-B., Liu W.-Y., Leang W.-N., Li T.-J. (2000) Planta Med., 66,

CHAPTER 8 HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) ANALYSIS OF PHYTOCHEMICAL CONSTITUENTS OF M. ROXBURGHIANUS AND P. FRATERNUS PLANT EXTRACTS

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