G.T. Maatooq et al. / Phytochemistry 71 (2010) 262–270
269
test compounds for four consecutive days (50 mg kgꢀ1, subcutane-
Acknowledgements
ously, suspended in corn oil). Normal group received corn oil). The
control group was treated with CCl4 (2 ml kgꢀ1, i.p., dissolved in
corn oil as single dose before the last dose of the test compounds).
Twenty-four hours after the last treatment, animals were anesthe-
tized by diethyl ether and blood was collected by puncture of the
retro-orbital plexus to determine the ALT and AST activities. Ani-
mals were then killed by cervical dislocation. Livers were dissected
and quickly frozen and kept for liver peroxidation analysis.
This work is a part of the research project ‘‘applications of bio-
technology in drug production, development and metabolic stud-
ies”, funded by Mansoura University Research Treasury Unit. The
authors gratefully acknowledge assistance from Prof. F.A. Badria
and Mr. A. Abbas, Department of Pharmacognosy, Faculty of Phar-
macy, Mansoura University, in carrying out the biological assays.
3.16. Hepatotoxicity evaluation
References
Arase, Y., Ikeda, K., Murashima, V., Chayama, K., Tsubota, A., Koida, I., Suzuki, Y.,
Saitoh, S., Kobayashi, M., Kumoda, H., 1997. The long term efficacy of
glycyrrhizin in chronic hepatitis C patients. Cancer 79, 1494–1500.
Baratta, M., Dorman, H., Deans, S., Figueiredo, A.C., Barroso, J., Ruberto, G., 1998.
Antimicrobial and antioxidant properties of some commercial essential oils.
Flav. Fragr. J. 13, 235–244.
Serum ALT and AST activities were measured using a spectro-
photometric diagnostic kit according to the manufacturer instruc-
tions. Liver peroxidation was measured by the formation of the
thiobarbituric acid reactive material malondialdehyde in 10% liver
homogenate, following a modified thiobarbituric acid reactive spe-
cies assay (Baratta et al., 1998).
Barran, J., Langford, D., Pitzele, B., 1974. Synthesis and biological activities of
substituted glycyrrhetic acids. J. Med. Chem. 17, 184–191.
Betts, R., Walters, D., Rosazza, J., 1974. Microbial transformation of antitumor
compounds. I. Conversion of acronycine to 9-hydroxyacronycine by
Cunninghamella echinulata. J. Med. Chem. 17, 599–602.
Bondet, V., Cuvelier, M., Berset, C., 2000. Behaviour of phenolic antioxidants in a
partitioned medium: focus on linoleic acid peroxidation induced by iron/
ascorbic acid system. J. Am. Oil Chem. Soc. (JAOCS) 77, 813–819.
3.17. Nitric oxide production in peritoneal rat macrophages
The assay was carried out according to methods described by
Ma and Kovanen (1995) and Yang et al. (2004). Peritoneal rat mac-
rophages (PMCs) were isolated by slow injection of 25 ml of 0.25%
trypsin-0.02% EDTA–Na2 into the rat abdominal cavity. The fluid
was removed from the peritoneal cavity under sterile conditions
after 15 min. Cellular components were isolated using low speed
(1500 rpm) centrifugation for 10 min at 4 °C. Pellets were then
washed briefly with phosphate buffered saline (PBS) and resus-
pended in RPMI-1640 medium supplemented with 10% (v/v) foetal
calf serum (FCS), penicillin (100 U/ml) and streptomycin (0.1 mg/
ml). Cells were placed into 25 cm2 tissue-culture flasks and incu-
bated overnight at 37 °C in a humidified 5% CO2 atmosphere. PMCs
were passaged by dissociating the confluent monolayer with 0.25%
trypsin-0.02% EDTA–Na2 and were reseeded in 96-well plates
(3 ꢁ 105 cells/well) and incubated in the same medium for 24 h.
Non-adherent material was removed the next day with two brief
washes using PBS. The adherent population was then incubated
at 37 °C, 5% CO2 in fresh culture medium for 2 h. The test com-
pounds dissolved in dimethyl formamide (DMF) were then added
Canonica, L., Ferrari, M., Jommi, G., Pagnoni, U., Pelizzoni, F., Ranzi, B., Maroni, S.,
Nencini, T., Salvatori, T., 1967. Ossidazioni microbiologiche di triterpenoidi.
Nota II. Acidi 15a-idrossi-glicerretico e 7b, 15a-diidrossi-glicerretico. Gazz.
Chim. Ital. 97, 1032–1051.
Chung, W., Lee, S.H., Kim, J., Sung, N., Hwang, B., Lee, S.Y., Yu, C., Lee, H., 2001. Effect
of the extracts from Glycyrrhiza uralensis Fisch. on the growth characteristics of
human cell lines: antitumor and immune activation activities. Cytotechnology
37, 55–64.
Fukuzawa, K., Seko, T., Minami, K., Terao, J., 1993. Dynamics of iron-ascorbate-
induced lipid peroxidation in charged and uncharged phospholipid vesicles.
Lipids 28, 497–503.
Ignatov, D., Prokof’ev, Yu., Ipatova, O., Timofeev, V., Medvedeva, N., Misharin, Yu.,
2003.
A simple method for preparation of 18b-glycyrrhetinic acid and its
derivatives. Russ. J. Bioorg. Chem. 29, 390–394.
Jeong, H., Kim, J., 2002. Induction of inducible nitric oxide synthase expression by
18b-glycyrrhetinic acid in macrophages. FEBS Lett. 513, 208–212.
Jeong, H., You, H., Park, S., Moon, A., Chung, Y., Kang, S., Chun, H., 2002.
Hepatoprotective effects of 18b-glycyrrhetinic acid on carbon tetrachloride-
induced liver injury: inhibition of cytochrome p450 2E1 expression. Pharmacol.
Res. 46, 221–227.
Kieslich, K., 1976. Microbial Transformations of Non-steroid Cyclic Compounds.
John Wiley and Sons, New York.
Kiso, Y., Tohkin, M., Hikino, H., Hattori, M., Sakamoto, T., Namba, T., 1984.
Mechanism of antihepatotoxic activity of glycyrrhizin. I. Effect of free radical
generation and lipid peroxidation. Planta Med. 50, 298–302.
at concentrations ranging from 1.5 to 50
lM. The concentration
Komoda, Y., 1984. Nuclear magnetic resonance of 18b-glycyrrhetinic acid and its
related compounds. Rep. Inst. Med. Dent. Engin. 18, 39–44 (in Japanese).
Luo, H., Huang, W., Zhang, Z., Wu, Q., Huang, M., Zhang, D., Yang, F., 2004. 18b-
glycyrrhetinic acid-induced apoptosis and relation with intracellular Ca2+
release in human breast carcinoma cells. Chi. Ger. J. Clin. Oncol. 3, 137–140.
Ma, H., Kovanen, P., 1995. Ig E-dependent generation of foam cells: an immune
mechanism involving degranulation of sensitized mast cells with resultant
uptake of LDL by macrophages. Arterioscl. Thromb. Vasc. Biol. 15, 811–819.
Mahato, S., Kundu, A., 1994. 13C NMR spectra of pentacyclic triterpenoids – a
compilation and some salient features. Phytochemistry 37, 1517–1575.
Mahato, S., Nandy, A., Roy, G., 1992. Triterpenoids. Phytochemistry 31, 2199–2249.
Nabekura, T., Yamaki, T., Ueno, K., Kitagawa, S., 2008. Inhibition of p-glycoprotein
of DMF did not exceed 0.1% v/v. CCl4 (10
l
M) was then added
and the plates incubated for 24 h. Nitric oxide was assayed in the
supernatants using Griess reagent. The assay depends on diazotiza-
tion of sulphanilamide with the produced nitrite in acid medium
and then formation of azo dye by coupling with N-(1-naphthyl-
ethylenediamine dihydrochloride, NEDA). The formed complex
has a bright reddish purple colour, which can be measured at
540 nm. Concentrations were determined by comparing absor-
bance of test solutions to that of a standard sodium nitrite solution
and multidrug resistance protein
Chemother. Pharmacol. 62, 867–873.
Ryu, S., Choi, S., Lee, S., Lee, C., No, Z., Ahn, J., 1994. Antitumor triterpenes from
medicinal plants. Arch. Pharmacol. Res. 17, 375–377.
1 by dietary phytochemicals. Cancer
(50 lM). Briefly, the reaction was carried out by mixing 100 ll of
the culture supernatants with 1 ml of 10 mM sulphanilamide con-
taining 1% orthophosphoric acid and standing for 5 min, followed
by adding 100
l
l of 1 mM NEDA, standing for another 5 min. The
SimeÓ, Y., Kroutil, W., Faber, K., 2007. Biocatalytic deracemization: dynamic
resolution, stereoinversion, enantioconvergent processes and cyclic
deracemization. In: Patel, R. (Ed.), Biocatalysis in the Pharmaceutical and
Biotechnology Industries. CRC Press, London, pp. 27–51 (Chapter 2).
Taylor, B., Alarcon, L., Billiar, T., 1998. Inducible nitric oxide synthase in liver:
regulation and function. Biokhimiya 63, 766–781.
Van Rossum, T., Vulto, A., De Man, R., Brouwer, J., Schlam, S., 1998. Review article:
glycyrrhizin as potential treatment for chronic hepatitis C. Ail. Pharmacol. Ther.
12, 199–205.
absorbance of the developed colour was measured at 540 nm.
Appropriate blanks were carried out for both sample and standard.
Concentration of NO was calculated using the following formula:
lM = Asample/Astandard ꢁ 50.
3.18. Statistical analysis
Williams, D., Fleming, I., 1997. Spectroscopic Methods in Organic Chemistry, fifth
ed. McGraw-Hill, Maidenhead, UK, pp. 98–99.
Xin, X., Liu, Y., Yo, M., Guo, H., Guo, D., 2006. Microbial transformation of
glycyrrhetinic acid by Mucor polymorphosporus. Planta Med. 72, 156–161.
Yamada, Y., Nakamura, A., Yamamoto, K., Kikuzaki, H., 1994. Transformation of
glycyrrhezic acid by Aspergillus spp. Biosci. Biotechnol. Biochem. 58, 436–437.
Differences among all groups were analyzed by one-way analy-
sis of variance (ANOVA) followed by Tukey–Kramer multiple com-
parisons test. N = 6.