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https://doi.org/10.29013/AJT-20-5.6-22-25
Tabatadze Nino, Tsomaia Irma, Chikovani Anton, Gigoshvili Tamar, Sokhumi State University, Tbilisi, Georgia E-mail: nino_tabatadze@yahoo.com
BIOLOGICALLY ACTIVE SUBSTANCES OF CEPHALARIA GIGANTEA GROWING IN GEORGIA
Abstract. Among the biologically active substances, the roots of Cephalaria gigantea mainly contain alkaloids, phenolic compounds and triterpene glycosides. Studies have shown that the total amount of saponins is less than 14 glycosides. Eleven individual compounds have been isolated, named giganteosides A, B, C, D, E, F, G, H, I, J, J', K, L,
Antifungal and antiprotozoal activities of several giganteosides and enriched fractions have been evaluated. The alkaloid fraction has shown an antimalarial effect. Monodesmosides - giganteosides D and E were evaluated for their cytotoxicity against human non pigmented melanoma MEL-5 and human leukemia HL-60. Of exceptional importance is the anticonvulsive effect of the aqueous extracts of C. gigantea roots in pentylenetetrazol and audiogenic seizure models, with minimal toxic effects.
High-performance liquid chromatography method has been developed for the qualitative detection of the phenolic compounds. This method will be used for the standartization of the expected anticonvulsive drug form.
Keywords: Triterpene saponins, giganteoside, HPLC, antifungal, anticonvulsive activity.
Cephalaria gigantea (Ledeb.) Bobr. (fam. Dip-sacaceae) is an endemic plant of Caucasus. Among the biologically active substances, the roots of the plant mainly contain alkaloids, phenolic compounds and triterpene glycosides [1-2]. In folk medicine, the aqueous extract of the roots of Cephalaria has been used successfully to treat epilepsy [3].
In order to identify biologically active substances and to determine their pharmacological activities, we have developed an effective method of separation and purification the sum of saponins from the roots of the plant, fractionation and division into individual compounds.
Studies have shown that the total amount of sa-ponins is less than 14 glycosides. Eleven individual
compounds have been isolated, named giganteosides A, B, C, D, E, F, G, H, I, J, J', K, L, M, N. The chemical structures of individual molecules were established on the basis of physical-chemical methods, by 1D and 2D NMR experiments (1H, 13C, gs-COSY, gs-HMBC, gs-HMQC and gs-HSQC-TOCSY) and mass spectrometry (MALDI-TOF, ESI-HR-MS) [4-9].
Giganteosides are the derivative of two agly-cones - oleanolic acid and hederagenin.
Giganteoside D - a-L-Rhap(l^2)-ß-D-Xylp(l^3)- oleanolic acid;
Giganteoside E - a-L-Rhap(l^2)-a-L-Rhap(l*2)-ß-D-Xylp(l*2)-a-L-Arap(l*3)- hed-eragenin;
Giganteoside G - a-L-Rhap(l^2)-ß-D-Xylp (l^3)-oleanolic acid -28-O-ß-D-Glcp;
Giganteoside H - a-L-Rhap(l^2)-a-L-Rhap (U2)-ß-D-Xylp(U2)-a-L-Arap(U3)- hederagenin-28-O-ß-D-Glcp;
Giganteoside I-a-L-Rhap(l^4)-ß-D-Glcp (l^4)-[a-L-Rhap(l^2)]-a-L-Arap
(l*3)- oleanolic acid- 28-O-ß-D-Glcp(U6)-ß-D-Glcp;
Giganteoside J-ß-D-Glcp(U3) -a-L-Rhap (l^2)-a-L-Arap(l^3)- hederagenin - 28-O-ß-D-Glcp(l^6)-ß-D-Glcp;
Giganteoside J'- a-L-Rhap(l^3)-ß-D-Glcp (l ^3 ) -a-L-Rhap (l ->2 ) -a-L-Arap (l ->3 )- hederagenin - 28-O-ß-D-Glcp(l^6)-ß-D-Glcp;
Giganteoside K - a-L-Rhap(l^4)-ß-D-Glcp (l^4)-[a-L-Rhap(l^2)]-a-L-Arap
(l^3)- hederagenin - 28-O-ß-D-Glcp(l^6)-ß-D-Glcp;
Giganteoside L - a-L-Rhap(l^2)-ß-D-GlcAp (U3)-hederagenin-28-O-ß-D-Glcp(U6)-ß-D-Glcp;
Giganteoside M - ß-D-Galp(U2)-ß-D-GlcAp (l*3)- oleanolic acid 28-O-ß-D-Glcp(u6)-ß-D-Glcp;
Giganteoside N - ß-D-Galp(U2)-ß-D-GlcAp (l*3)- hederagenin - 28-O-ß-D-Glcp(u6)-ß-D-Glcp.
Saponins containing glucuronic acid in the sugar chain were reported for the first time in Cephalaria species.
It has been established that the roots of Cephalaria from different regions of Georgia, do not differ from each other in the content of biological active components.
Biological studies have shown the antifungal, antiprotozoal, cytotoxic and anticonvulsant effects of Cephalaria root's saponins extract, alkaloid fraction and individual glycosides [l0-l3].
The fungicidal and antiprotozoal activities of individual substances and enriched fractions from the roots of Cephalaria were studied in the Laboratory of Botany and Parasitology of the faculty of Phar-
macy at the University of Mediterranean (Marseille, France).
The antifungal activity of saponins has been determined against yeasts and dermatophytes with an agar dilution method as previously described [14]. The tested concentration range was 1-2 mg/ml for crude extract, and 0.06^g/ml to 200 ^g/ml for the individual compounds. The reference antifungal agents were amphotericin B for yeasts and ketokona-sole for dermatophytes. The minimal inhibitory concentration of saponins has been determined.
The highest antifungal activity was obtained with monodesmoside - giganteoside E (MIC12.5^g/ml), which confirms the higher activity of hederagenin derivatives compared with glycosides of oleanolic acid. The most sensitive yeast species were Candida glabrata and C, kefyr.
The antiprotozoal activities against Leishmania infantum and Trichomonas vaginalis have been evaluated as described in [15]. The inhibitory concentration (IC50) and letal dose (LD100) were determined with amphotericin B and metronidazole as references, respectively.
The most active product against Trichomonas vaginalis is a mixture (50/50) of monodesmosides -giganteosides D+ E (IC50 1-2.5^g/ml). Giganteoside E with aglycone as hederagenin has shown more strong activity (lC2.5-5^g/ml) than giganteoside D (lC5^g/ml).
No significant activity was detected against Leishmania infantum. The synergism of activity has been found in the case of mixture of two monodesmosides (50:50).
Monodesmosides - giganteosides D and E were evaluated for their cytotoxicity against human non pigmented melanoma MEL-5 and human leukemia HL-60 cell line by using the metabolic MTT or WST1 cell viability assay, campthotecin was used as positive control [16]. The tested compounds exhibited interesting antiproliferative effect on human cell lines with IC50 values in the range 3.15-7.5^M.
The leukemia HL-60 line was more sensitive to these monodesmosides and the most active product against HL-60 was giganteoside D (3.15 ^M) compared with giganteoside E (6.8 ^M). The cytotoxic activity may be related to the rhamnose residue of the sugar chain attached at C3 of the aglycone as it has been reported previously [17].
Preliminary phytochemical analysis showed the presence of 0,2% alkaloids in the plant's roots. These include 3 known alkaloids: gentianine, gentianidine, gentianaine. Alkaloid containing fractions of the roots of C. gigantea were active against Plazmo-dium falciparum, a parasite that causes malaria (lC50-27.4yl) [2].
The anticonvulsive effects of the aqueous extract of C. gigantea was studied using pentylentet-razol (PTZ) and audiogenic seizure models (The Krushinsky-Molodkina and Wistar rat strains). In PTZ-model onset of tonic-clonic seizures, latencies to the beginning of the seizure activity and mortality and similarlyin audiogenic seizures behavioral convulsive reactions, facial automatisms and latency to wild running were evaluated.
It was shown that the aqueous extract demonstrates anticonvulsant properties as in both model, as in both routs of administration -peroral and intraperitoneal. Extract appeared most effective in case of preliminary repetitive administration: in PTZ model seizures were eliminated in 9 of 10 animals and in audiogenic model convulsive response was no more observable during 7 days of treatment termination.
It was found also that sedative properties are characteristic of this antiseizure extract according to the sleeping prolongation in barbiturate treated mice. Safety of the extract was evaluated in toxicological acute and chronic experiments, and consequently it is considered a substance of moderate general toxicity.
Strong anticonvulsive effect and a very low toxicity of Cephalaria root's extract gives an opportunity for the creation of a new medicine of plant origin for prevention and treatment of psychosomatic forms of epilepsy.
Studies have shown higher concentration of phenolic compounds (both flavonoids and phenolic acids) in the roots extract of Cephalaria. Up to 9 compounds were identified.
High-performance liquid chromatography method has been developed for the qualitative detection of the phenolic compounds. The mixture of water (0,1%HCOOH) - acetonitrile (0,1%HCOOH) was used as a mobile phase with a gradient condition (acetonitrile 5% ^ 40%). All solutions were of HPLC purity, the concentrations of the sample and standards were 10 mg/mL and 1 mg/mL in MeOH, respectively. Solid phase - C18 reversed phase column Kinetex XB-Rp18, 250X4.6mm (Phenomenex), run time 25 min. UV detection was performed at 254, 280, 325 and 365 nm.
The following compounds were used as stand-arts: rutin, caffeic acid, chlorogenic acid, cumaric acid, ferulic acid, hyperoside, luteolin and querci-trin. On the basis on the retention time, using cor-respoding standards and UV spectra, there have been identified 3 main compounds - chlorogenic acid, caffeic acid and flavonoid glycoside quercitrin. The obtained result allow us to performed standar-tization of biological and chemical markers of the anticonvulsive drug form [18].
Thus, the Cephalaria gigantea is a very perspective medicinal source due to its large botanical resources in Georgia, a rich chemical composition and a wide range of biological activities.
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