Evaluation of the anti-inflammatory and cytotoxic activities of naphthazarine derivatives from Onosma leptantha.Abstract The root extracts of Onosma leptanhtha were evaluated for their anti-iflammatory and cytotoxic activities. The cyclohexane cyclohexane (sī'kləhĕk`sān), C6H12, colorless liquid hydrocarbon. It is a cyclic alkane that melts at 6°C; and boils at 81°C;. It is nearly insoluble in water. extract, which appeared as the most active in both assays, has been further subjected to bioassay-directed fractionation fractionation /frac·tion·a·tion/ (frak?shun-a´shun) 1. in radiology, division of the total dose of radiation into small doses administered at intervals. 2. to afford the naphthazarine derivatives: [beta],[beta]-dimethylacrylshikonin (1), isovalerylshikonin (2) and acetylshikonin (3). The evaluation of the anti-inflammatory activity was performed on carrageenan-induced rat paw edema test. All the tested compounds proved to be active, while compound 3 showed the best anti-inflammatory effect. In addition, the cytotoxic activity of the extracts and isolated compounds, was also assayed against L1210 murine lymphoblastic leukemia cell line, and human fibrosarcoma fibrosarcoma /fi·bro·sar·co·ma/ (-sahr-ko´mah) a malignant, locally invasive, hematogenously spreading tumor derived from collagen-producing fibroblasts that are otherwise undifferentiated. HT-1080 cells. Compound 1 exhibited remarkable cytotoxic activity (390 nM for L1210 cells), which is superior to that of shikonin, which was used as control. [c] 2005 Elsevier GmbH. All rights reserved. Keywords: Onosma leptantha; Naphthazarines; Shikonin-derivatives; Anti-inflammatory activity; Cytotoxicity Introduction Root extracts of Boraginaceae species, like Alkana tinctoria in Europe, or Lithospermum erythrorhizon in the Orient, have been used widely for centuries, mostly for their anti-inflammatory activity. Shikonin and alkanin derivatives have been identified as the bioactive constituents of those plants (Papageorgiou et al., 1999). From genus Onosma, (Boraginaceae) only Onosma heterophylla roots appeared as the most thoroughly studied species (Papageorgiou et al., 1999). Onosma leptantha Heldr. (Boraginaceae) is a perennial plant, endemic in South Greece (Teppner, 1991). In the framework of our studies on Greek Boraginaceae plants, the aerial parts of O. leptantha have been previously studied, and have revealed the presence of five pyrrolizidine alkaloids alkaloids, n alkaline phytochemicals that contain nitrogen in a heterocyclic ring structure. They can have powerful pharmacological effects and are more often used in traditional medicine than in herbal treatments. (Kretsi et al., 2003). In continuation of our scientific study, we report herein the biological evaluation, as well as the isolation of some shikonin derivatives, from the roots of O. leptantha. Materials and methods Plant material Roots of O. leptantha Heldr., were collected on mount Taygetos (Greece) in July 1999. The plant has been collected by Dr. E. Kalpoutzakis and identified by Dr. T. Constantinidis, (Dept. of Botany, Agricultural University of Athens The Agricultural University of Athens (Greek Γεωπονικό Πανεπιστήμιο Αθηνών) is located in Athens, at the neighborhood of Votanikos. ). A voucher specimen of O. leptantha (Tg 008) has been deposited in the Herbarium herbarium, collection of dried and mounted plant specimens used in systematic botany. To preserve their form and color, plants collected in the field are spread flat in sheets of newsprint and dried, usually in a plant press, between blotters or absorbent paper. of Div. of Pharmacognosy pharmacognosy /phar·ma·cog·no·sy/ (fahr?mah-kog´nah-se) the branch of pharmacology dealing with natural drugs and their constituents. phar·ma·cog·no·sy n. and Natural Products Chemistry, University of Athens. Fractionation The air-dried roots of O. leptantha (0.7 kg) were pulverized and extracted with [C.sub.6][H.sub.12] (3 x 1.51) and C[H.sub.2][Cl.sub.2] (3 x 1.51) to give crude extracts (10.3 and 14.5 g, respectively). Two grams of the [C.sub.6][H.sub.12] extract was subjected to column chromatography over Silicagel 60F Merck (eluated with a gradient [C.sub.6][H.sub.12]-C[H.sub.2][Cl.sub.2] from 80:20 to 0:100) to obtain 270 fractions. All fractions were analyzed by TLC TLC total lung capacity; thin-layer chromatography. TLC abbr. 1. thin-layer chromatography 2. (silicagel 60[F.sub.254] Merck, C[H.sub.2][Cl.sub.2], detection vanillin/[H.sub.2]S[O.sub.4] reagent). Fractions 95-105 yielded [beta],[beta]-dimethylacrylshikonin (1), 60 mg, 151-170 isovaleryl shikonin (2) 23 mg, while fraction 211-220 gave acetylshikonin (3) 42 mg (Fig. 1). Structural analysis The structure of the isolated compounds was determined by spectroscopic spec·tro·scope n. An instrument for producing and observing spectra. spec  tro·scop methods. EIMS EIMS Environmental Information Management System (US EPA)EIMS Eudora Internet Mail Server EIMS Employment Information in the Mathematical Sciences EIMS European Innovation Monitoring System EIMS Electron Impact Mass Spectrometry were determined on a HP-6890 spectrometer. Optical rotations were measured with a Perkin-Elmer 341 polarimeter polarimeter: see polarization of light. . UV spectra were recorded on a Shimadzu-160A spectrophotometer spectrophotometer, instrument for measuring and comparing the intensities of common spectral lines in the spectra of two different sources of light. See photometry; spectroscope; spectrum. . NMR NMR: see magnetic resonance. spectra were recorded on Bruker DRX DRX X-Ray Diffraction DRX Digital Receiver DRX Discontinuous Reception DRX Discontinuously Reinforced Metal DRX Dynamic Range Extension DRX Dvd Rewritable 400 and Bruker AC 200 spectrometers. The signals in the [.sup.1]H- and [.sup.13]C-NMR spectra were assigned unambiguously using 2D NMR techniques. All chemical shift, and correlations observed were in accordance with those already described (Papageorgiou, 1979; Chien Chang et al., 2002). [FIGURE 1 OMITTED] Experimental animals Male Wistar rats (200-250 g each) were obtained from the Farm for Experimental Animals, Military Medical Academy in Belgrade. Groups of 5-10 animals were kept in plastic cages under standard laboratory conditions (room temperature, day and night light control, constant air humidity). The rats were allowed, before the experiment, to take commercial solidified food for rats (Veterinary Institute, Zemun) and tap water ad libitum. The animals deprived from the food 18-20 h before the experiment and with free access to water, were used in the experiment. Inflammatory agent A total of 0.1 ml of 0.5% carrageenan-saline solution was injected into the plantar surface of the right rat hind paw to induce inflammation. The same volume of saline was injected in the left paw that served as the control paw. Reference substance Indomethacin indomethacin /in·do·meth·a·cin/ (in?do-meth´ah-sin) a nonsteroidal antiinflammatory drug; used in the treatment of various rheumatic and nonrheumatic inflammatory conditions, dysmenorrhea, and vascular headache. (Galenika a.d., Belgrade, Serbia) was used as a reference substance. It was dissolved in DMSO DMSO dimethyl sulfoxide. DMSO n. Dimethyl sulfoxide; a colorless hygroscopic liquid obtained from lignin, used as a penetrant to convey medications into the tissues. DMSO, n. and administrated p.o. in doses of 1-8 mg/kg body wt. Assay of carrageenan-induced paw edema Groups of 6-10 animals were used for each dose. Extracts and isolated compounds (1, 2, 3) or indomethacin, were administrated 60 min before injection of carrageenan car·ra·geen·an or car·ra·geen·in n. Any of a group of closely related colloids derived from several red algae, widely used as a thickening, stabilizing, emulsifying, or suspending agent in pharmaceuticals. (right paw) and saline (left paw). The control group was treated in the same way by vehicle (DMSO--1 ml/kg). The extracts were dissolved in DMSO and administrated p.o. in doses of 50, 100 and 200 mg/kg b.w. through an orogastric tube. The isolated compounds were also dissolved in DMSO and administrated p.o. in doses of 7.5, 15 and 30 mg/kg body wt. Three hours after the injection of carrageenan and saline, the animals were sacrificed, and their paws were cut off for weighting. Inflammatory response intensity was calculated using the formula: Inflammatory response intensity (%) = ([DELTA]e/[DELTA]k) x 100 where [DELTA]k is the difference between paw weights in the control (DMSO-treated) group, and [DELTA]e the difference between paw weights in treatment groups. Maximal inflammatory response intensity was 100% (the control group) (Fig. 2). Statistics Data were analyzed by using the Mann-Whitney U-test. For the calculation of the mean effective dose (ED-50) of tested substances, the method of Litchfield and Wilcoxon (1949) was used. Differences between mean values were statistically significant for p<0.05. The effect was shown as mean[+ or -]SD. [FIGURE 2 OMITTED] Cytotoxicity assay The cytotoxicity assay was performed by a modification of the MTT-method (Harvala et al., 2002) against L1210 murine lymphoblastic leukemia cell line and human fibrosarcoma HT-1080 cells. Shikonin was used as a positive control. Results were expressed as I[C.sub.50]. Results and discussion A wide spectrum of pharmacological activities has been attributed to alkannin and shikonin (Chen et al., 2002). The cyclohexane and dichloromethane extracts of O. leptantha were investigated for their anti-inflammatory and cytotoxic activity. Bioassay-directed fractionation of the cyclohexane extract, which was the most active one, led to the isolation of: [beta],[beta]-dimethylacryl-shikonin (1), isovalerylshikonin (2) and acetylshikonin (3) (Fig. 1). Carrageenan-induced rat paw edema test, an experimental model of acute inflammation according to the method described by Oyanagui and Sato (1991), has been used for screening the anti-inflammatory activity of our extracts and isolated compounds. Subplantar injection of carrageenan solution induced the acute inflammatory edema with a maximum 3 h after application. All tested extracts, given in doses of 50, 100 and 200 mg/kg p.o., showed significant dose-dependent anti-inflammatory effect (Table la). The effect of the extracts, obtained with doses of 100 and 200 mg/kg, were comparable to that of indomethacin given in doses of 2-8 mg/kg. The most active was the cyclohexane extract of the roots with an ED-50 of 98.96 mg/kg (Table 2). The isolated naphthazarine derivatives (1, 2, 3), given in doses of 7.5, 15 and 30 mg/kg p.o., strongly suppressed carrageenan-induced paw edema (Table lb). Their potency was more than 10-fold greater than that of the cyclohexane extract of roots (relative potency = 11.48; Table 2). Data in Table 2 showed that acetylshikonin (3) exhibited the best anti-inflammatory effect, compared to the other tested substances. Its ED-50 value (6.91 mg/kg) was two times greater than ED-50 of indomethacin. In a dose of 30 mg/kg, (3) showed almost the same effect to that of indomethacin given in a dose of 8 mg/kg. In accordance to our results, it has been previously reported, by other authors, that shikonin derivatives and especially acetylshikonin, are strong anti-inflammatory agents (Seto et al., 1992). In a recently published report, studying bioactivities of shikonins, their anti-inflammatory effects have been ascribed to inhibition of the transcriptional activation of Tumour Necrosis Factor-[alpha] (TNF-[alpha]) promoter. In agreement to our results, their relative potency in inhibiting TNF-[alpha] promoter activation was 3>1>2 (Staniforth et al., 2004). Cytotoxic/cytostatic activities were assessed on a murine leukaemia and a human solid tumour cell line. Cyclohexane extract was more active against both cell lines. Among the isolated compounds (1, 2, 3), compound 1 exhibited the highest activity against both cell lines (I[C.sub.50]s: 390 nM for L1210 cells, and approx. 2 [micro]M for HT-1080), which is superior to that of shikonin (used as a control). Compound 2 possesses a moderate activity, being weaker than shikonin, while 3 is active only in high concentrations, similar to alkannin. In conclusion, our study has verified that shikonin derivatives 1, 2, 3 isolated from O. leptantha, possess multiple in vivo and in vitro activities such as suppressing inflammation and the viability of cancer cell lines. These novel bioactivities would provide greater insight into the medicinal value and therapeutic use of shikonins. Acknowledgements This study was supported, by the Special Account for Research Grants of the University of Athens and Ministry of Science, Technologies and Development of Serbia, Project No. 1568. References Chen, X., Yang, L., Oppenheim, J.J., Howard, M.Z., 2002. Cellular pharmacology studies of shikonin derivatives. Phytother. Res. 16, 199-209. Chien Chang, S., Wan Jr., S., Shyh-Yuan, L., Chia-Hung, L., Gum-Hee, L., Chang Ming, S., 2002. Antimicrobial activities of naphtazarins from Arnebia euchroma. J. Nat. Prod. 65, 1857-1862. Harvala, E., Aligiannis, N., Skaltsounis, A.L., Pratsinis, H., Lambrinidis, G., Harvala, C., Chinou, I.B., 2002. Chemical constituents of Inula verbascifolia ssp. methanea. J. Nat. Prod. 65, 1045-1048. Kretsi, O., Aligiannis, N., Skaltsounis, A.L., Chinou, I., 2003. Pyrrolizidine alkaloids from Onosma leptantha. Helv. Chim. Acta 86, 3136-3140. Litchfield, J.T., Wilcoxon, F.A., 1949. A simplified method of evaluating dose-effect experiments. J. Pharmacol. Exp. Ther. 96, 99-113. Oyanagui, Y., Sato, S., 1991. Inhibition by nilvadipine of ischemic Ischemic An inadequate supply of blood to a part of the body, caused by partial or total blockage of an artery. Mentioned in: Antiangiogenic Therapy, Subarachnoid Hemorrhage, Ventricular Fibrillation ischemic and carrageenan paw edema as well as of superoxide radical production from neutrophils and xanthine oxidase. Arzneim-Forsch/Drug Res. 41 (1, No. 5), 469-474. Papageorgiou, V.P., 1979. [.sup.1]H NMR spectra of natural occuring isohexenylnaphtazarin pigments. Planta Med. 37, 185-187. Papageorgiou, V.P., Assimopoulou, A.N., Couladouros, E.A., Hepworth, D., Nicolaou, K.C., 1999. The chemistry and biology of alkannin, shikonin, and related naphtahazarin natural products. Angew Chem. Int. Ed. 38, 270-300. Seto, Y., Motoyoshi, S., Nakamura, H., Imuta, J., Ishitoku, T., Isayama, S., 1992. Effect of shikonin and its derivatives, pentaacetylated shikonin (MDS-005) on granuloma granuloma /gran·u·lo·ma/ (gran?u-lo´mah) pl. granulomas, granulo´mata an imprecise term for (1) any small nodular delimited aggregation of mononuclear inflammatory cells, or (2) such a collection of modified macrophages formation and delayed-type allergy in experimental animals. Yakugaku Zasshi 112 (4), 259-271. Staniforth, V., Wang, S.Y., Shyur, L.F., Yang, N.S., 2004. Shikonins, phytocompounds from Lithospermum erythrorhizon, inhibit the transcriptional activation of human tumor necrosis factor tumor necrosis factor n. Abbr. TNF A protein that is produced in the presence of an endotoxin, especially by monocytes and macrophages, is able to attack and destroy tumor cells, and exacerbates chronic inflammatory diseases. alpha promoter in vivo. J. Biol. Chem. 279 (5), 877-5885. Teppner, H., 1991. In: Strid, A., Tan, K. (Eds.), Mountain flora of Greece. Edinburgh University Press Edinburgh University Press is a university publisher that is part of the University of Edinburgh in Edinburgh, Scotland. External links
T. Kundakovic (a,b), N. Fokialakis (b), S. Dobric (c), H. Pratsinis (d), D. Kletsas (d), N. Kovacevic (a), I. Chinou (b,*) (a) Department of Pharmacognosy, Faculty of Pharmacy, University of Belgrade The University of Belgrade (Serbian: Универзитет у Београду or Univerzitet u Beogradu) is the oldest and most important higher education institution in Belgrade , Belgrade, Serbia and Montenegro Serbia and Montenegro (sûr`bēə, mŏn'tənē`grō), Serbian Srbija i Crna Gora, former country of SE Europe, in the Balkan Peninsula, a short-lived union (2003–6) of the republics of Serbia and the much (b) Division of Pharmacognosy and Chemistry of Natural Products, School of Pharmacy, University of Athens, Athens, Greece (c) National Poison Control Centre, Military Medical Academy, Belgrade, Serbia and Montenegro (d) Laboratory of Cell Proliferation & Ageing, Institute of Biology The Institute of Biology (IoB) is a professional body for biologists, primarily those working in the United Kingdom. Membership currently stands around 14,000. It was founded in 1950, received a Royal Charter in 1979 and holds charitable status. , NCSR NCSR National Center for Scientific Research NCSR National Centre for Social Research (UK; formerly Social and Community Planning Research) NCSR National Compound Semiconductor Roadmap "Demokritos", Athens, Greece Received 13 October 2004; accepted 19 October 2004 *Corresponding author. Tel.: +30210 7274595; fax: +30210 7274115. E-mail addresses: chinou@pharm.uoa.gr, ichinou@pharm.uoa.gr (I. Chinou).
Table 1a. Influence of Onosma leptantha extracts on the inflammatory
response intensity (carrageenan-induced rat paw edema test)
Inflammatory response intensity (a)
Cyclohexane extract Dichioromethane
Treatment (mg/kg p.o.) (roots) extract (roots)
Control (DMSO - 1 ml/kg) 454.10[+ or -]101.86 (100%)
50 274.80[+ or -] 303.80[+ or -]
33.35 (a2) (60.5%) 31.79 (a2) (66.9%)
100 216.28[+ or -] 233.75[+ or -]
73.70 (a3) (47.6%) 78.35 (a2) (51.5%)
200 185.42[+ or -] 198.20[+ or -]
50.11 (a3,b2) 55.16 (a3,b2) (43.6%)
(40.8%)
Indomethacin (1 mg/kg) 326.45[+ or -]53.0 (a2) (71.9%)
Indomethacin (2 mg/kg) 187.80[+ or -]62.1 (a2) (41.4%)
Indomethacin (4 mg/kg) 222.25[+ or -]70.20 (a3) (48.9%)
Indomethacin (8 mg/kg) 115.00[+ or -]70.30 (a3) (25.3%)
(a) Results (X[+ or -]SD) are given as a difference in rat paw weights
(mg) and as % of control value that is taken as 100%; a1, a2,
a3--p<0.05; 0.01; 0.001 compared to the control; b1, b2--p<0.05; 0.01
compared to 50 mg/kg of corresponding group.
Table 1b. Influence of the pigment fractions I, II and III of the
cyclohexane extract of Onosma leptantha roots on inflammatory response
intensity (carrageenan-induced rat paw edema test)
Inflammatory response intensity (a)
Treatment (mg/kg p.o.) Compound 1 Compound 2
Control (DMSO - 1 ml/kg) 454.10[+ or -]101.86
(100.0%)
7.5 241.60[+ or -] 284.66[+ or -]
65.30 (a3) (53.2%) 106.08 (a2) (62.69%)
15 180.16[+ or -] 218.80[+ or -]
96.86 (a3) (39.67%) 65.90 (a3) (48.18%)
30 151.0[+ or -] 197.6[+ or -]
62.35 (a3) (33.25%) 24.17 (43.51%)
Indomethacin (1 mg/kg) 326.45[+ or -]
53.0 (a2) (71.9%)
Indomethacin (2 mg/kg) 187.80[+ or -]
62.1 (a2) (41.4%)
Indomethacin (4 mg/kg) 222.25[+ or -]
70.20 (a3) (48.9%)
Indomethacin (8 mg/kg) 115.00[+ or -]
70.30 (a3) (25.3%)
Inflammatory response intensity (a)
Treatment (mg/kg p.o.) Compound 3
Control (DMSO - 1 ml/kg)
7.5 225.20[+ or -]77.27 (a3) (49.59%)
15 153.20[+ or -]70.60 (a3) (33.74%)
30 116.28[+ or -]39.89 (a3) (25.61%)
Indomethacin (1 mg/kg)
Indomethacin (2 mg/kg)
Indomethacin (4 mg/kg)
Indomethacin (8 mg/kg)
(a) See under Table 1a; a1, a2, a3--p<0.05; 0.01; 0.001 compared to the
control; b1, b2--p<0.05; 0.01 compared to 50 mg/kg of corresponding
group.
Table 2. ED-50 values of tested extracts and isolated fractions compared
to indomethacin
ED-50 95% of confidence Relative
Extracts/compounds (mg/kg p.o.) limits potency (a)
Cyclohexane extract 98.96 23.32-419.96 1
Dichloromethane 124.63 28.24-550.03 0.79
extract
1 8.62 1.40-52.92 11.48
2 16.77 2.32-120.87 5.90
3 6.91 1.64-28.97 14.32
Indomethacin 3.08 0.96-9.86 32.13
(a) Relative potency = ED-50 of whole cyclohexane extract of roots/ED-50
of other tested extracts, fractions and indomethacin.
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