arian Journal of Medical Sciences | Vol. 26, Nos. 3 & 4, December 2031 STUDY OF CYTOTOXIC ACTIVITY OF DAPHNE MUCRONATA ROYLE GROWN IN IRAN Z, Amingholran’, R. Miri", K. Javidnia™, M. Davoodi™ “Department of Immunology, Faculty of Medicine,“ Department of Medicinal Chemistry iculty of Pharmacy, Shiraz University of Medical Seiences, Shiraz ABSTRACT Background: Plants ace a proven sourve of anitumor compounds and i is reasonable to assume that many suel substances remain co be discavered, In Irn foik medicine, ethanolic extriot of the aerial pets of Dapine mucronea Rayle isused sgains varcus kin disorders, Objective: In onder 0 evalva indigenous 0 tea. ¢ ami-nexplastic planis, we imtite’ a cytotexie study wing Dane mucronate that is Methods: The ejtoloxie activi of 180 fydmalcoholi: and ehlorotomic extacis of Daphne mucronate Royle were ‘examined on seven differen cell lines by MIT asay. Cell lies used tis Study mnhuced SK-Br-3 and MDA-MB-439 (east cancen, Hela (cervical eptheoid carcinoma), K362 (myelogenous leakemia), U937 (mosoblasic leuken) Ag.8 (mouse myeloma) and Yero (primary monkey kane} Resulls: The highest eytaonie activig: oF the hydrosteoelic exact oF DB. murine was found on breast caNRer ea Fires. 50 ygite of de extract inhibit proliferation of 24noue culures of MDA-MB-433 ang SK-r-) ell nes (795 ‘an 34% iuhibitinn, respectirely). The exact showed antileukemie setisity parveulaly agains, the U937 eel ne, A 50% inhibiion of cell proliferation due so {00 jgita) uF the exis ts 24-hour eulcure of U9¥? and 48-hour eucure of ‘Ag cell ines was obgerved. Despite the resul of MITT essay showing a reduction of Hela cell Line vial after 24 hou exposuze o 10-50 gin oF he extrac, @ slglfcaetStmulcory actly at concentrations moge dhan 400 g/t Wa ned No significant cytotouc eftect was detected in elution uw dhe Vero cel line. The chloevcomnie extract showed Weak eyiotoxie effex 08 NDA-MB438, 5K: 5-3 wad U9GT cells bu hal signiticae effec cn the ote cell Lines. Sronfcohelic exeeec of D. ewucronate showed amituiour setivity panicuanty against breast amd Feukomnia eel ins, len J Med Sei 2001; 26(984):146-151 Key Words * Cytotoxicity * MTT assay # cell line © Daphne mucronata Introduction conservative estimate, there are 250,000 species of plants worldwide. When this is coupled with the fact that there is no definitive method of selecting the proper starting material, screening for appropriate biological activity are necessary.” In Iranian folk medicine, ethanolic extract of aerial parts of Daphne mucronata Royle , (Thymelaeaceac) indigenous to Tran and Tearspendences R Mir, P.O.Bon: 71345119, Shira an eed a6 THT 200000, Rant 88-71-2290) Turkey is used against various skin disorders Pra arars i such as vitilligo."? The aerial parts of the Plants are a valuable source of useful anc- tumor compounds, and it is reasonable 10 assume that additional existing substances remain to be discovered." However, at a 146\47 Study of cytotoxic activity of Daphne mucronate Royle extract Daphne species (e.g. mezerewm) have been used in traditional medicine as a remedy for theumatism and as 2 purgative and abortifacient® On the other hand, literature reviews show that the different species of Dapine such as D. mecerewn, D. genkava, D. olidoies and D, odora have good cytocoxic effects, In earlier reports active compounds have been isolated and identified from this Daphne genera." Mererine obtained from D. mezertun has been shown (0 possess anti- Ieukemic activity. Odoriein is anew nematocidal compound drived from D. adora and daphnetin-8-glucoside from D. mucronata (accuminata) possesses cardiotoxie activity 2" In order to find an anti-cancer plant, we initiated a cytotoxic study on D. mucronata that grows in Iran, Materials and Methods Plant materials The plant was collected from Abadeh Fars Province (outh central part of Iran). ‘The plant was identified by the Departmen of Botany and voucher specimens were deposited in the Herbarium of the Faculty of Pharmacology, ‘Tehran University of Medical Sciences (Herbarium no. 6528). Aerial parts were dried in shade and chopped into stall pieces. The powder was divided ino (wo parts; the first part wes extracted with chloroform in 2 Soxtlet’s apparatus for 8 hour, The solvent was then removed under redaced pressure (yield—8% ww), The second part ‘was extracied by percolation in methanol/water (70:30) for 24 hours and the solvent was, evaporated and dried under reduced pressure (yield =6.8% wiw) Preparation of culture medium with Daphne mucroncta extrac RPMI 1640 medium supplemented with 5% felal calf serum Gibco, Germany), 100 U/ml penicillin and 100 ig/ml streptomycin was used as culture medium. Dried extract was dissolved in this medium 10 the concentration of 20 mgiml and was mixed at 37°C for 20 min. This solution was centrifuged to remove insoluble ingredients, and the supernatant was passed through 0.22 yum fihers for sterilization. The solution was diluted with the medium and five concentrations (10, 50, 100, 400, 800, 11g/ml) were prepared Cell culture and the cytotoxicity assay: Cell lines including Vero, Hela, K562, SK. Br3, Ag.8, U937 and MDA-MB453 were maintained in medium. Cytotoxictly assay was carried out in 96-well tissue culture plates using an MTT proliferation kit (Gibco, Germany) Rriefly, vells were cultured in triplicate at a concentration of 10° cells/well in 100 jal culture medium containing various concenttations of D, mucronata extracts. Treated cultures were incubated in a CO: incubator (37 °C, 5% COs) far 24 to 72 hours After the incutation period, 10 ul of MTT (3: .S-Gimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide] in final concentration of 5 mgiml was added and then the plates were incubated for 4 hours in the incubator. The fomntion of the blue formazan was assessed by addition of 100 pi of solubilization solution, Plates were allowed to stand overnight in the incubator. ‘The absorbance of the samples was measured at 570 and 630 nm with an ELISA reader (Pharmacia, Sweden). The mean optical density (OD)=SD for each group of replicates was calculated. Inhibition percentage of cell proliferation was obiained as follows: Yelahibition= 100.{(est OD/nomreated OD) x 100}. "A concentration of 10 uM doxorubicin was used as positive control ‘without adding the extract Statistical analysis: Data wore analyzed_using SPSS software, one-way ANOVA and Duncan test. A p-value of £0.05 was considered sign2. Amirghofran, R. Miri, K. Javidnia, M. Davoodi 148 110 50 2:00 c1400 000 vere UST Aga MDA. skir Hele Figure 1: Exposure of tumor call fine to various goncentrations 119-800 gill. of hydrealcoholic extreet of Dephine mucronete in 9 48thour culture, Data represents the mean value of tiplicates with bars indicating SD (SD ranged 1-8). All values are significant, Results The cytotoxic activity of two hydroaleoholic and chloroformic extracts of D. mucronata was examined on seven different cell lines Results of the cytotoxic activity of hydroalcoholic extract are shown in Figure 1 and are as follows: U937: The extract showed a dose-dependent inhibitory etfect on the proliferation of this cell line after 24 hours (inhibition range, 24 56%). After 48 hours, the inhibitory effect was cominvously dose-dependent and inereased up to 60% at 800 ygiml. Finally this effect reached 75% in the 72-hour culture. 562: ‘The extract showed no marked cytotoxic activity on this cell line after 24 hours, Weak cytotoxic activity was observed on 48hour culture at all concentrations (Teimnitition range, 67-30%). This eytotosie activity reached up to the 38% in the 72-hour culture. MDA-MB- The exiraci showed significant cytotoxic activity inal concentrations afer 24 hours (Stinhibition 39%). The highest activity was at 50 ‘ l B10 B59 B1K0 Dato G80 vero UNS? KSE2 Ag8 MDA SKOr Hola Figure 2: Exposure of tumor cell line t9 various concentrations (10800 ugiml) 0! chloreformic extract ef Daphne mucronata in a 48-hour culture. Date represents the mean value of iplicates with bars indicating SO (SO ranges 1 8}, All values are significant usinl (73%). Alinost, the same results were obtained after 48 and 72 hours (inhibition 18-74%). Ag.8: Proliferation was inhibited in all concentrations. The highest percent inhibition after 48 hours was found at a concentration of 160 4.g'm of the exteect (55%). SK-Br-3: Anii-proliferative activity after 24 hours was found at all concentrations of the extrset (%inhibition range, 14 47%). This activity was strongly increased after 48 hours (inhibition range, 40-70%) and 72 hours (inhibition range, 45%-75%). The best result was obtained at 100 j.g/ml after 72 hours (Ginhibition, 75%) Hela; The extract showed no marked effect on the 24-hour culture of this cell line. Mild cytotoxic activity at 10-50 sup/ml of the extract (Sinhibition range, 26%-37 %) and stimulatory avtivity at concentrations more than 400 sig/ml alier 48 hours were observed. mild increase in Ue stimulatory activity was seen in the 72- hour culture (inhibition -48%) Vere: No cytotoxic activity but rather stimulatory effet was detected after 48 hours (inhibition range, 0% to -65%), Due to an overload of cells, te stimulatory acivity after9 Study of cytotoxic activity of Daphne mucrenata Royle extract 2 hours was not measurable. Treatwent of cell Tines with different concentrations of the chloroformic extract reduced the cell viability of MDA-MB-453, SK-Br-3 and U931 lines after 48 hours (inhibition range 20.33%, 5- 35% and 36-56%, respectively). Slight stimulatory activity was observed for the Vero cell line (inhibition, -10% to -18%), Other cell lines were almost not affected (binbibition < 7%) (Fig. 2). Doxonubicin as he positive control killed the cell lines (100% inhibition), Discussion The development of the effect of cytotoxic agents with therapeutic application remains an important need in clinical oncology. In recent years, the cytotoxicity and chemical constituents of different medicinal plants have bec evaluated. Dphoniun —fagettiforme (Araceace) commonly known as’ the “rodent tuber" in Malaysia is one of these plants, its hexane extract has shown cytotoxic activity against P388- murine leukemia cells." Albicla juilbrissin, another terbal plant tas also shown good inhibitory action against the KB cancer cell line in vitro" Aqueous extract, of Emblica officinalis has been found to be cytotoxic to L929 cells in culture in a dose dependent manner.” And the extract of Fuparornen perfoliatend tas shown potent cytotoxicity with BCs values (12-14 ugimL) comparable to a standard cytotoxic agent, chlorambucil.” The possible anti-proliferative effect of the hydroalchoholic and chloroformic extracts. of D. mucronata is the subject of this study. This effect was determined in four grouns af tumor cell Iines including 3 leukemic cell ines, 2 beast cancer, one cervix cancer and one non- malignant cell line, The most anti-protiferate activity of alcoholic extract of D. mucronata ‘was observed on breast cancer cell lines. This extract, ata concentration of 30 ug/ml, showed 73% and 34% inhibition in 24-hour culture of MDA-MB-453 and SK-Br-3 cell lines, respectively. The inhibitory effect of the extract on the later cell line increased « 70% after 48 hours which is indicative of the strong, anii-tunnor activity of the extcact om this type of cell line. This exiract also showed cytotoxic activity on leukemic cell lines. Among these lines, L937 and Ag 8 were more sensitive than K562. A 50% inhibition of cell proliferation duc 10 100 jgyil of the extract was observed in 24-hour culture of U937 and 48-hour culture of Ags cell lines whereas the highest % inhibition of K562 after 48 hours was 30%. In comparison 10 the hydrodlcoholie extract, the chlorofurmic extract showed a weaker cytotoxic activity and the effeci was only on MDA-MB-453, SK-Br-3 and U937 cell lines Taking into consieration that these fines were the most affected colls afier exposure. to hydroakotolic extract, itis concluded that dhe major components with cytotoxic propery in Damucronata might be the polar agents. In previous studies. the anti-leukemic activity of D. — genkawa, another species of thymelacaccous genus has been reported.” In thal study, Liou et al, showed tat the wo natural products isolated from D. gentawa including Genkwadaphnin and Yuanhuscine ‘were able to inhibit DNA and protein synthesis in P-338 loukemie ells. The major efects of these diterpene esters were the blockage of the elongation process and an interference with peptidyl transferase reaction.” Anti-leukemic activity has also heen reported for mezerein, a potent anti-tumor agent isolated from D. mecerewn.” Te is interesting 1 note dat mezesein structurally resembles phorbol esters which are tumor promoting agents.* In our study, despite the weak cytotoxic 2ctivity of Daplne extract on Hela cells at low concentrations, a stimulatory activity at concentrations. more than 400 ug/ml was observed. This result shows the difference in made of action of the extract on different cellZ. Amirghotran, R. Miri, K. Javidria, M. Davoodi lines. In addition, it is possible that the stimulaiory activity may in part be duet the presence of some similar weak tumor promoting or mitogenic agents in the extract. D. mucronata also. displays stimulatory activity on Vero, which is a nonmalignant Tibroblast-like cell. This finding suggests that thts plant deserves further stuly aS an ant neoplastic therapeutic agent, Indeed, further studies on D. mucronata extract and identification of its effective compounds will help 10 obtain more information with regard its anti-tumor activity Acknowledgment This study was supported by grant (No. 78- 047), jrom the Research Council of Shiraz University of Medical Sciences, Shirae, Iran. We are thankful to Dr. Gh. R. Amin for technical advice References 1 Safiness M, Perzato IM: Assay related to cancer drug discovery. Inv Hosttmann K eds: Metkods ta Platt Biochenutry Vol. 6. Academic Press, New Yor: 9ET-124 2. 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Barton K, Randall G, Sagone AL: The fests ofthe and onidative mieaolisn of human phvoe ell arni-taor agent mezerein om the eyttonie capacity Tevet Dig 1999,7:199-88 First International Congress of Anesthesiology And Perioperative Medicine snd Seventh franian Congress of Anesthesia and Critioal Cate Shim. fran, 2-5 Oct, 2002 Postal address PO Bow:71345/1997, Shiva, [ran ‘Vel: 987112337636, Fay: ~9871 [2307072 E-mail: Anescong@sums.2c.it Sponser: Shiraz University of Medical Sciences, Iranian Society Anesthesis and Critical Care Deadline for Abstract Submission: June, 21, 2002