|Iranian Journal of Pharmaceutical Research (2006)
Received: January 2006
Accepted: May 2006
Copyright ? 2005 by School of Pharmacy
Isolation and Identification of Calystegines in Root Cultures
of four Physalis Species
Mohammad Ebrahim Azemia, Mahmoud Mosaddeghb*, Abdol Majid Cheraghalic,
Forough Namjooyana and Birgit Dr?gerd
aPharmacognosy Department, Faculty of Pharmacy, Shaheed Beheshti Medical University of Science, Tehran, Iran. bTraditional Medicine and Materia Medica, Reacearch Center Tehran, Iran. cPharmacology Department, Baghyyatallah Medical University of Science, Tehran, Iran. dInstitute of Pharmaceutical Biology, Faculty of Pharmacy, Martin-Luther-University, Halle-Wittenberg, Germany.
Calystegines were isolated from the root cultures of Physalis divaricata, P. pubescens, P. philadelphia,and? P. philadelphia (solanaceae). Calystegines of P. divaricata were identified as calystegine A3, A5, B1 and B2, with concentrations of 6.99, 4.41, 8.52, and 14.7 μg/g in fresh mass, respectively. Root cultures of P.pubescens contain calystegine A3, B1 and B2, P. philadelphia only contains calystegine B2 and P. philadelphia root cultures solely consist of calystegine A3. Isolation and identification of calystegines have been achieved by ion exchange column chromatography and gas chromatography, using authentic samples.
Plants have been used as a drugs for centuries. The potential use of higher plants as a source of new drugs is still poorly explored. Of the estimated 250000-500000 plant species, only a small percentage has been investigated phytochemically and even a smaller percentage has been properly studied in terms of their pharmacological properties (1, 2).
P. divaricata is an annual of 15 to 45 cm height, sometimes with sparse hairs on the stem. The plant bears smooth, dark-green leaves and yellowish-white flowers. The fruit is a round, yellow to orange, about the size of a cherry, containing numerous flat kidney-shaped seeds.This plant is distributed in some parts of Iran (Lorestan and Fars provinces), Afghanistan, Pakistan and India.
The name Physalis derives from the Greek phusa (a bladder), for the five-cleft calyx greatly increasing in size after the corolla falls off, thus enclosing the fruit in a large, leafy bladder.
Calystegines are nortropane alkaloids with three to five hydroxyl groups in various positions. The name of these alkaloids derives from their first detection in transformed root cultures of Calystegia sepium (Convovulaceae) (3). They are also found in intact plant roots of C.sepium, C.arvensis and Atropa belladonna (Solanaceae) (4). Tropane alkaloids and their derivatives are formed in the roots and transported to the aerial parts of the plant (5, 6).
The biosynthesis of calystegine shares metabolic steps and enzymes of the classical tropane alkaloid formation, which is expressed in a number of Solanaceae and some Convolvulacea species (7). Calystegine biosynthesis via the tropane alkaloid pathway has been proven by enzymatic studies, gene isolation, NMR and GC-MS after labelled precursor feeding (4). Two enzymes and their corresponding gene sequences are known for calystegine biosynthesis (Figure 1). The genes were isolated from calystegine forming Solanaceae, namely from Solanum tuberosum, Hyoscyamus niger and Datura stramonium. should tropane alkaloids be also present in Physalis divaricata comparison of enzymes and genes involved in their biosynthesis to those of other Solanaceae may give clues on the diversity or relatedness of tropane alkaloid metabolism within the Solanaceae. For such studies, plant tissue cultures are of great advantage. From sterile cell or tissue cultures, enzyme and mRNA encoding them could be obtained more readily.
Calystegines are strong and selective glycosidase inhibitors. They share this activity with other hydroxylated nitrogen containing bicyclic compounds (3).? They also have a role in plant defense mechanism and plant-insect interactions. In usual alkaloid extraction procedures that contain a lipophilic extraction step from an aqueous alkaline medium, they remain in the aqueous phase. That may explain why they have not been found earlier in well-examined plants like Hyoscyamus or Atropa species (4). The structures of the first calystegines were published in 1990 (7).
In the course of ethnobotanic investigations of Iranian plants, we screened several Solanaceae species for cytotoxic activity. The results indicated significant cytotoxic activity of methanolic extract of Physalis divaricata D. Don. A particular interest in the analysis of alkaloids derived from the tropane biosynthetic pathway resulted from previous publications reporting calystegine in the fruits of Physalis alkekengi was arisen (8, 9). We therefore compared Physalis divaricata with three Physalis species with respect to its? capacity to form root cultures and to sythesize calystegines in the root cultures.
Physalis divaricata D. Don was collected in May 2003 from Fars (Iran), subsequently identified and the voucher specimen deposited at the Traditional Medicine and Materia Medica Reacearch Center, Tehran, Iran. P. divaricata?s dried seeds were used for preparing root cultures. Seeds of P.peruviana, P.philadelpica and P.pubescens were obtained from the Botanical Garden, Martin-Luther-University Halle-Wittenberg, Gemany.
Seeds were macerated first in H2O for 1 h and in EtOH 70% for 5 min subsequently. They were then transferred to a 6.5% sodium hypochlorite solution and kept for? 6.5% for 5 min to improve germination. After washing with H2O (4 times), they were cultured in water-agar 0.5%. After a few days, roots of the germinated seeds were cut and cultured in 100-ml flasks containing 25-ml of B5-medium supplemented with 1μM indol-3-butyric acid and 3% sucrose on a gyratory shaker at 90 rpm in the dark. Before extraction, the roots were transferred to 300-ml flasks containing 75-ml of hormone-free medium and subcultured every two weeks.
Extraction and derivatization
The extraction and GC measurements for calystegines were performed as described by Dr?ger (4). 2-5 g fresh mass of each root culture was extracted by 2?20 ml 50% (v/v) MeOH, using an Ultra-Turrax homogenizer and prior to being centrifuged at 4500 g for 10 min at 4oC. MeOH was evaporated by a rotary evaporator (50oC), and the remaining crude extract was adjusted to 1 ml /g fresh mass, using distilled water. For determination of calystegine concentration, 1 ml extract was applied on a cation exchange column (Ion exchange I LAB Merck,column10?65mm, ca 5ml ion exchange gel).The column was washed with H2O, until a neutral reaction of the elute was observed. Calystegines were eluted with 2 M NH3, and the elute concentrated in vacuo. Derivatization was necessary for GC analysis of calystegines. Further more, samples were dried by lyophilization. Trimethylsilyl ethers were formed with hexamethyldisilazane and trichlorosilane in pyridine at 80oC after 20 min.
Gas chromatograph HP 6890, column PHENOMENX SE52 (30?0.25 mm i.d., 0.25 μm film thickness) injection split ratio ca 1:20, detection simultaneous FID and PND, carrier gas Helium (1 ml /min), temperature program 160 oC, 2 min isotherm, 5oC/min up to 240 oC.
By comparing sample retention times with the related standard, calystegines were identified. Azobenzol was used as an internal standard in quantitative analysis, and the calystegines content were calculated by direct reference to Azobenzol peak area units. Detection limits were ca. 60 pmol calystegine with FID detection and ca 10 pmol with PND detection in a sample volume of 1 μl.
Result and discussion
Root cultures of Physalis divaricata contain more and greater amounts of calystegine A3, A5, B1 and B2 (Figure 1) with concentration of 6.99, 4.41, 8.52 and 14.70 μg/g fresh mass respectively, compared to other three investigated species (Table 1). GC chromatograms of Physalis pubescens with FID and PND detector are shown in Figure 2.
Based on tropane alkaloid biosynthesis pathway (Figure 3), it seems that tropinone reductase II (TRII), which catalyses the production of pseudotropine from tropinone in root cultures of P. divaricata, is much more active than the other three investigated Physalis species. This could explain the presence small amounts of tropane alkaloids in P.divaricata.
?The next step for further confirmation of tropane alkaloid biosynthesis pathway is to determine the amounts of tropinone, tropine and pseudotropine in these root cultures and compare these data with each other. This is being currentlycarried out and the results will be published soon after.
The authors wish to thank B. Zehzad for his kind help in the collection and identification of plant materials. Skillful technical assistance by B.Marx, U.K?del and D.Watzka is greatfully acknowledged.
Hamburger, M., Hostettmann, K.,1991. Phytochemistry bioactive in plants. The link between phytochemistry & medicine. Phytochemistry, 30, 3864-7.
Rates, S.M.K., 2001.Plants as a source of drugs, Toxicon, 39,603-613.
Dr?ger,B., Funck,C., Hohler,A., Mrachatz,G., Nahrstedt.A., Portsteffen,A., Schaal,A. and Schmidt,R., 1994.Calystegines as a new group of tropane alaloids in Solanaceae, Plant Cell ,Tissue and Organ Culture,38:235-240
Dr?ger, B. van Almsick, A. and Mrachatz, G.1995. Distribution of calystegines in several solanaceae. Planta Medica, 61, 577?579.
Rothe,G., Hachiya,A., Yamada,Y., Hashimoto,T., and Dr?ger, B.2003. Alkaloids in plants and root cultures of Atropa belladonna overexpressing putrescine N-methyl-transferase . Journal of Experimental Botany, Vol. 54, No. 390, pp 2065-2070
Rothe, G.; Garske, U.; Drager, B. 2001. Calystegines in root cultures of Atropa belladonna respond to sucrose, not to elicitation. Plant Science. 160(5): 1043-1053
Dr?ger, B.2004, Chemistry and biology of calystegines. Nat. Prod. Rep., 21,211-223
Asano,N., Kato,A., Oseki,K., Kizu,H., Matsui,K. (1995) Calystegins of Physalis alkekengi var. francheti (solanaceae), Structure determination and their glycosidase inhibitory activities. European Journal of Biochemistry 229, 369 - 376
Asano,N., Kato,A., Kizu,H., Matsui,K. (1996) 1-beta-amino-2-alpha,3-beta,5-beta-trihydroxycycloheptane from Physalis alkekengi var. francheti, Phytochemistry? 42, 719 ? 721