外文翻譯-- 不溶性地衣代謝產(chǎn)物的增溶作用在細(xì)胞系的生物學(xué)測(cè)試（英文）

1、European Journal of Pharmaceutical Sciences 24 (2005) 539–543Solubilization of poorly soluble lichen metabolites for biological testing on cell linesTh´ ord´ ?s Kristmundsd´ ottir a,?, Elsa J´ onsd

2、80; ottir a, Helga M. ¨ Ogmundsd´ ottir b,c, Krist´ ?n Ing´ olfsd´ ottir aa Faculty of Pharmacy, University of Iceland, Hagi, Hofsvallagata 53, Reykjavik IS-107, Iceland b Faculty of Medicine, Un

3、iversity of Iceland, Reykjavik, Iceland c Molecular and Cell Biology Research Laboratory, Icelandic Cancer Society, Reykjavik, IcelandReceived 8 July 2004; received in revised form 11 January 2005; accepted 14 January 20

4、05AbstractThe depside atranorin and depsidone fumarprotocetraric acid, isolated from the lichens Stereocaulon alpinum and Cetraria islandica, respectively, were chosen as prototypes for poorly soluble natural compounds i

5、n an effort to facilitate testing in pharmacological models. Solubilizing agents previously identified as being non-toxic towards a malignant leukemic (K-562) cell line and suitable for testing of anti- proliferative act

6、ivity of the dibenzofuran lichen metabolite (+)-usnic acid were used in solubilization studies of the depside and depsidone. Cyclodextrin derivatives were found to be most suitable for solubilizing the lichen compounds,

7、the greatest rise in solubility being witnessed for fumarprotocetraric acid, increasing almost 300-fold from 0.03 mg/ml in water to 8.98 mg/ml in 10% 2-hydroxypropyl-?-cyclodextrin (HP?CD). Subsequently, the lichen compo

8、unds, including (+)-usnic acid, were solubilized in 10% HP?CD and tested for effects on three malignant human cell lines; T-47D (breast), Panc-1 (pancreas) and PC-3 (prostate) in a standard proliferation assay. Atranorin

9、 and fumarpro- tocetraric acid did not exhibit anti-proliferative effects but usnic acid was active against all test cell lines with EC50 values of 4.3–8.2 ?g/ml. The non-toxic solubilizing agents used in this study coul

10、d prove useful for pharmacological testing of other poorly soluble natural products. © 2005 Elsevier B.V. All rights reserved.Keywords: Lichen metabolites; Solubility; Cell lines; Fumarprotocetraric acid; Atranorin;

11、 Usnic acid1. IntroductionSome secondary metabolites found in Icelandic lichens have shown promising anti-proliferative results in in vitro tests on malignant human cell lines ( ¨ Ogmundsd´ ottir et al., 1998;

12、Haraldsd´ ottir et al., 2004). Further testing of other can- didates of the same origin has however often been hampered by the poor solubility that many of these metabolites show in non-toxic solvents. Numerous ways

13、 are possible when try- ing to increase the solubility of poorly soluble substances, for example the use of co-solvents, surfacants and complex forming agents (Tinwalla et al., 1993; Jonkman-de Vries et al., 1996; Li et

14、al., 1999a, b). These methods must however? Corresponding author. Tel.: +354 525 4370; fax: +354 525 4071. E-mail address: thordisk@hi.is (T. Kristmundsd´ ottir).produce solvent systems that are non-toxic for the ce

15、lls in culture. In a previous study the lichen metabolite (+)-usnic acid (Fig. 1), a dibenzofuran derivative, was used as a pro- totype for a water-insoluble natural product with the aim to find a solvent that was both c

16、apable of solubilizing usnic acid and was free of direct activity against a test cell line. The di- rect effects of various solvents and complexants were tested on the human leukemia cell line K-562 in a standard prolif-

17、 eration assay. Most of the compounds proved toxic with the exception of the solvents propylene glycol and polyethylene glycol 400 (PEG 400) and the complexant 2-hydroxypropyl- ?-cyclodextrin (HP?CD). Anti-proliferative

18、activity of usnic acid could be demonstrated with an EC50 of 4.7 ?g/ml us- ing PEG 400 and 2-hydroxypropyl-?-cyclodextrin but only the latter gave satisfactory solubility. 2-Hydroxypropyl-?- cyclodextrin was thus identif

19、ied as a solubilizing agent that0928-0987/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.ejps.2005.01.011T. Kristmundsd´ ottir et al. / European Journal of Pharmaceutical Sciences

20、24 (2005) 539–543 541Cells were cultured under standard conditions in RPMI 1640 medium with l-glutamine and supplemented with 50 IU/ml penicillin and 50 ?g/ml streptomycin with 10% fetal calf serum (all from Gibco Life T

21、echnologies, Paisley, UK). For testing cells were placed in 96-well plates at 104 cells per well. One of the test substances was added in stepwise dilu- tions. [3H] Thymidine (Amersham Pharmacia Biotech, UK) was added at

22、 1 ?Ci per well after 24 h of culture and culture continued for further 6 h. The cells were then harvested on to glass fibre filters in a Packard C961960 FilterMaid cell harvester and then placed in MicroscintO scintilla

23、tion liquid. The radioactivity was counted in a TopCount scintillation counter (all from Packard Instruments, Connecticut, USA). The results are expressed as percentage of untreated control.3. Results3.1. Effect of solub

24、ilizing agents on the solubility of atranorinThe solubility of atranorin in water was non-detectable us- ing HPLC. An attempt was made to solubilize atranorin using the solvents and complexants previously found to be sui

25、table for testing on cell lines, i.e. propylene glycol, PEG 400 and HP?CD. The solubility of atranorin in pure PEG 400 was found to be 0.37 mg/ml and in propylene glycol 0.017 mg/ml but there was no measurable solubility

26、 of atranorin in 10% aqueous mixtures of these solvents. However, atranorin was found to be soluble in HP?CD. Fig. 2 shows that the solu- bility of atranorin in 10% HP?CD is very low at pH 6 but is considerably higher at

27、 pH 8.0, 0.0055 mg/ml. In spite of higher solubility of atranorin at pH 8.0 than the physiological pH 7.4 the latter was chosen for the cell experiments. As the solubility of atranorin in HP?CD was low it was attempted t

28、o use HP?CD to increase its solubility. The central cavity of the ?-cyclodextrin is larger than that of ?-cyclodextrin and might therefore be able to solubilize more of the lichen compound.Fig. 2. Effect of pH on the sol

29、ubility of atranorin in 10% ?-hydroxypropyl- ?-cyclodextrin. Each point represents the mean ± S.D. of three experiments.Fig. 3. Effect of solubilizer concentration on atranorin solubility at pH 7.4 in solutions of H

30、P?CD (?) and HP?CD (?). Each point represents the mean ± S.D. of three experiments.This, however, was not the case. Fig. 3 shows the effect of cyclodextrin concentration on atranorin solubility at pH 7.4. The solubi

31、lity increases as a function of solubilizer concen- tration but there is little difference in the results obtained for the two cyclodextrins with the HP?CD being slightly better at the highest concentration tested.3.2. E

32、ffect of solubilizing agents on the solubility of fumarprotocetraric acidThe solubility of fumarprotocetraric acid in water was found to be 0.0325 mg/ml. At pH 4 the solubility was 0.0069 mg/ml but increased with increas

33、ing pH and was 2.83 mg/ml at pH 7.4. In solutions of both PEG 400 and propylene glycol the solubility of fumarprotocetraric acid appeared to be better than in water alone, but due to decom- position of the compound in th

34、ese solvents as was apparent from HPLC measurements it was not possible to determine the solubility. The solubility of fumarprotocetraric acid was increased to 8.98 mg/ml in 10% HP?CD at pH 7.4. Decompo- sition was not s

35、een in the cyclodextrin solutions presumably because a part of the fumarprotocetraric acid molecule fits into the non-polar cyclodextrin cavity and is protected from the environment.3.3. Effect of the lichen compounds on

36、 thymidine uptake of cell linesThe three lichen compounds, usnic acid, atranorin and fu- marprotocetraric acid were solubilized in 10% solutions of HP?CD and tested at physiological pH (7.4) on the cell lines Panc-1, T-4

37、7D and PC-3. The EC50 for usnic acid against Panc-1 was 4.3 ?g/ml at a solvent concentration of 10 ?l/ml, which is comparable to the EC50 previously found against K-562 of 4.7 ?g/ml. The EC50 for usnic acid against T-47D

38、 was 2.9 ?g/ml but against PC-3 it was higher, at 8.2 ?g/ml. In calculating the EC50 account was taken of direct effects of the solvents. The solubilizing agent HP?CD had no signifi- cant effect on PC-3 and Panc-1 but T-