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Anti-oxidant activity and effect of Pinus morrisonicola Hay. on the survival of leukemia cell line U937.

Abstract

The free radical scavenging and anti-cancer activites of Pinus morrisonicola Hay. were studied using different parts of the pine, namely, needle, bark and cone. Results showed that pine needle water extract has the highest scavenging superoxide anion activity and the lowest I[C.sub.50] value in inhibiting superoxide anion formation; however, the bark water extract showed the best anti-lipid peroxidation activity. Additionally, needle water extract displayed the highest inhibition of leukemia cell line U937 growth. The results indicated that P. morrisonicola Hay. possesses potential chemopreventative and therapeutic properties.

[c] 2005 Elsevier GmbH. All rights reserved.

Keywords: Anti-oxidant activity; Lipid peroxidation; Cytotoxicity; Leukemia

Introduction

In recent years, considerable number of studies have indicated the important role of free radicals and reactive oxygen species (ROS) in the progress of many human diseases (Halliwell et al., 1993). ROS production in excess of cellular anti-oxidant capacity may result in damage to lipids, proteins, and DNA (Breimer, 1990; Cerutti, 1985). Oxidative damage to DNA is supposed to be potentially carcinogenic. Here the ROS play an important role in carcinogenesis, especially in the promotion stage (Breimer, 1990; Cerutti, 1985). Many studies have indicated that anti-oxidant nutrients and/or medicines have a protective role in human health (Ames, 1983; Weisburger, 1991). Therefore, anti-oxidants are considered effective inhibitors of carcinogenesis and pathogenetically associated with oxidative mechanisms.

Recently, the biological and pharmacological properties of Chinese herbs have begun to receive more attention in the scientific community and have become a very important research focal point. This is attributed to the fact that they contain phytochemicals, such as anti-oxidants, and other bioactive compounds. We have been successful in evaluating the anti-oxidant and hepatoprotective activities of several Chinese traditional medicinal herbs (Lin et al., 1993; Hsu et al., 1999) and Taiwanese folk medicines (Lin et al., 1995, 1996, 1997, 2000). Pinus morrisonicola Hay., also named Taiwan white pine or Taiwan Short-leaf Pine, belongs to gymnosperms and is one of Pinaceae sub. P. that grows at elevations of 300-2300 m in the Taiwanese mountains. Various curative effects of different parts of the pine have been recorded in ancient Chinese medical books. Many previous studies also indicate its physiological activities and therapeutic effects, including as a remedy for carcinoma. The aims of this study were to investigate whether different parts of this pine--precisely needle, bark and cone--from P. morrisonicola Hay, show anti-oxidant activity or cytoxicity against leukemia cell line U937.

Materials and methods

Preparation of extracts

Each different part of the plant sample (100g) was minced and decocted with 11 of boiling water for 2 h for three times. The decoction was filtered, collected and concentrated by evaporation and lyophilization. Yields of water extracts were 14.5, 12.0 and 4.9% (w/w) relative to starting material for pine needle, bark and cone, respectively.

Drugs and chemicals

Dimethyl sulfoxide (DMSO), ethylenediamine tetra-acetic acid (EDTA), cytochrome c, xanthine, xanthine oxidase, allopurinol, thiobarbituric acid (TBA), L(+)-ascorbic acid and Tris-HCl, sodium dodecyl sulfate were purchased from Sigma Chemical Co. (St. Louis, MO, USA). K[H.sub.2]P[O.sub.4] was acquired from Ferak Chemical Co. (Berlin, Germany). Trolox was obtained from Acros Organics Co. (NJ, USA). Fe[Cl.sub.2] was purchased from Wako pure chemical Industries Ltd. (Osaka, Japan). 3'-[1-[(phenylamino)-carbonyl]-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzenesulfonic acid hydrate, and the source of the XTT [sodium 3'-[1-(phenylamino)-carbony]-3,4-tetrazolium]-bis(4-methoxy-6-nitro) benzenesulfonic acid hydrate] kit was Roche Dignostics GmbH (Germany). RPMI-1640 medium, fetal calf serum (FCS), antibiotics (penicillin/streptomycin/amphotericin B) and L-glutamine were all procured from Gibco Co. (Grand Island, New York). Human leukemia cell line U937 was kindly provided by Dr. Chiang, Lien-Chai (Graduate Institute of Medicine, Kaohsiung Medical University).

Scavenging effect on superoxide anion (cytochrome c reduced method)

The cytochrome c reduced method was performed according to McCord and Fridovich (1969). Briefly, extracts were dissolved in distilled water and adjusted to appropriate concentration. The following reagents were added, in order: 50 [micro]l extracts or trolox, 400 [micro]l working solution (0.1 mM xanthine/0.1 mM cytochrome c/0.1 ml EDTA/50 mM K[H.sub.2]P[O.sub.4]), and 530 [micro]l [H.sub.2]O. After mixing well, 20 [micro]l xanthine oxidase (1 U/ml) was added. The adsorption at 550 nm was measured for 66s. Scavenging effects on superoxide anion were calculated as follows:

Inhibition rate (I)% = [([T.sub.abs] - [D.sub.abs])/[B.sub.abs]] X 100%,

where [T.sub.abs] is the absorption at 550 nm of the control group, [D.sub.abs] the absorption at 550 nm of the treated group and [B.sub.abs] the absorption at 550 nm of the blank group.

Inhibition effect on the formation of superoxide anion (xanthine oxidase inhibition test)

The xanthine oxidase inhibition test was conducted according to Chang et al. (1994). Extracts were dissolved in distilled water and adjusted to appropriate concentration. The following reagents were added, in order: 50 [micro]l extract or allopurinol, 400 [micro]l working solution (0.1 mM xanthine/[H.sub.2]O), and 530 [micro]l [H.sub.2]O. After mixing well, 20 [micro]l xanthine oxidase (1 U/ml) was added. The measurement range was within 66 s of absorption at 295 nm. The calculation of inhibition effects on the formation of superoxide anion was similar to the cytochrome c reduced method.

Fe[Cl.sub.2]-ascorbic acid-stimulated lipid peroxidation in rat liver homogenate (TBA method)

The effect of P. morrisoncola Hay. on rat liver homogenate induced with Fe[Cl.sub.2]-ascorbic acid and lipid peroxidation was determined by MDA-BA adduct according to the method described by Yoshiyuki et al. (1981). A mixture containing 0.5 ml of 4-week-old Wistar Albino strain male rat liver homogenate, 0.1 ml Tris-HCl buffer (pH 7.2), 0.05 ml 0.1 mM ascorbic acid, 0.05 ml 4 mM Fe[Cl.sub.2] and 0.05 ml various concentrations of crude drug extracts or trolox, was incubated for 1 h at 37[degrees]C. After incubation, 9 ml distilled water and 2 ml 0.6% TBA were added to 0.5 ml of the incubation solution and were shaken vigorously. The mixture was heated for 30 min in a boiling water bath. After cooling, 5 ml n-BuOH was added and the mixture was shaken vigorously again. The n-BuOH layer was separated by centrifugation at 1000g for 10 min and malonic dialdehyde (MDA) production was measured at 532 nm (Wong et al. 1987). The inhibition rate of lipid peroxidation was calculated as follows:

Inhibition rate (I)% = [([T.sub.abs] - [D.sub.abs])/([T.sub.abs] - [B.sub.abs])[B.sub.abs] X 100%,

where [T.sub.abs] is the absorption at 532 nm of the control group, [D.sub.abs] the absorption at 532 nm of the treated group and [B.sub.abs] the absorption at 532 nm of the blank group

Survival influence on leukemia cell line U937 (XTT-based colorimetric assay)

The XTT-based colorimetric assay was performed following the method described by Sundstrom and Nisson (1976). Briefly, human leukemia cell line U937 was cultured in RPMI-1640 medium (containing 10% FCS, 1% antibiotics, 1% L-glutamine) in a 5% C[O.sub.2] incubator at 37[degrees]C. Cell number was adjusted to 1.2 X [10.sup.5] cells/ml and 90 [micro]l were transferred to the wells in a 96-well tray. After adding, respectively, 10 [micro]l of various concentrations (100, 250 and 500 [micro]g/ml) of each extract to the above-mentioned 96-well tray and culturing in a 5% C[O.sub.2] incubator at 37[degrees]C for 72 h, 50 [micro]l of the mixture from 5 ml of XTT (labeling reagent) and 100 [micro]l PMS (electron coupling reagent) were added into every well. Culturing in a 5% C[O.sub.2] incubator at 37[degrees] for 4 h followed. The absorption was measured using an ELISA reader and the differences between O[D.sub.492] and O[D.sub.690] were calculated. The inhibition of survival rate was calculated as follows:

Inhibition rate (I)% = [100 - (O[D.sub.T])/(O[D.sub.C]) X 100] X 100%,

where O[D.sub.T] is the absorption of the treated group (added various concentration of extract) and O[D.sub.C] is the added of control group (not added extract)

Statistical analysis

Statistical analysis involved the use of the Statistical Analysis System software package. Data are indicated as the mean + s.e.m. (n = 3) and were determined statistically using one-way analysis of variance (ANOVA) procedures. Significant differences between the means of water extracts treated and positive control groups were determined using Duncan's multiple range test (Duncan, 1957).

Results

Scavenging effect of superoxide anion

Scavenging effects of superoxide anion by the water extracts of P. morrisonicola Hay. are shown in Table 1. The I[C.sub.50] values of various concentrations of all extracts were found to be significantly less than that of the trolox positive control, with values ranging from 0.08 to 0.61 mg/ml. Among the different parts of the pine tested, the needle extract was shown to have the lowest I[C.sub.50] value when compared with others, meaning that it has the strongest scavenging superoxide anion activity.

Inhibition effect on the formation of superoxide anion

Inhibitory effects of superoxide anion by the water extracts of P. morrisonicola Hay. are shown in Table 2. The I[C.sub.50] values of various concentrations of all extracts were found to be higher than allopurinol positive control, with values ranging from 3.15 to 9.39 mg/ml. Among the different components that were tested, bark extract showed the highest inhibition effect at concentration of 1 mg/ml; however, it had shown suspension phenomenon at 5 mg/ml and caused a shift when the absorption was detected at 295 nm. It was also difficult to calculate the I[C.sub.50] value.

Anti-oxidant activity on Fe[Cl.sub.2]-ascorbic acid induced lipid peroxidation

The rat liver homogenate was induced with Fe[Cl.sub.2]-ascorbic acid for nonenzymatic lipid peroxidation. The results of Fe[Cl.sub.2]-ascorbic acid-induced lipid-peroxidation are shown in Table 3. Water extracts at concentration of 0.5-10 mg/ml exhibited anti-lipid-peroxidation activities, with inhibition effects in the range of 13.1-91.2%, and the bark water extract had the lowest I[C.sub.50] value of 2.22 mg/ ml. Among these, bark water extract had the highest inhibitory effect at 5 mg/ml, still far lower than that of the positive control trolox.

Cytotoxicity on leukemia cell line U937

The XTT-based colorimetric peroxidation assay was performed to measure the inhibition of U937, in order to evaluate the anti-cancer activity of water extracts of different pine samples. Needle water extract showed a dose-dependent response and the highest inhibition of leukemia cell line U937 growth (Fig. 1). The inhibitory effect of bark and cone water extracts were similar to the positive control Catharanthus roseus at high concentrations.

[FIGURE 1 OMITTED]

Discussion

The Cytochrome c test is usually employed to evaluate whether drugs possess anti-oxidant activity; however, it is different from the xanthine/xanthine oxidase (X/XO) system method for direct inhibition of the superoxide anion. The pine needle water extract showed the highest free-radical-scavenging, superoxide-anion activity (Table 1) in our cytochrome c test. The X/XO system is used commonly as the generator of superoxide anions that may attack cell membrane. Inhibition of xanthine oxidase leads to a decrease in ROS generation in target cells. Xanthine-oxidase inhibitors are suggested to be scavengers of hydroxyl radicals and, therefore, be the anti-tumor promotion agents (Husain et al., 1987; Reiners et al., 1987). The needle extract showed the lowest I[C.sub.50] value in inhibiting superoxide formation (Table 2) in the X/XO system test. [Fe.sup.+2] is well-known to stimulate lipid peroxidation in liver microsomes and mitochondria (Yuda et al., 1991). Lipid peroxidation is long recognized as a potential mechanism of cell injury (Esterbauer et al., 1988; Vaca et al., 1992). The bark water extract showed the lowest I[C.sub.50] value in Fe[Cl.sub.2]-ascorbic-acid-induced lipid peroxidation in a rat liver homogenate in vitro (Table 3). From the results we consider that P. morrisonicola Hay. water extracts have free-radical-scavenging capacity and anti-lipid peroxidation effect. Plant flavonoids show a variety of biological effects in numerous mammalian cell systems in vitro and in vivo. Most flavonoids are effective in scavenging radical (Middleton and Kandaswami, 1994). Recently, much attention has been paid to their anti-oxidant and anti-tumor effects. Pine needle possesses an anti-tumor effect (Kong et al., 1995) and contains proanthocyanidins (Nyman, 1985). The different proportions of P. morrisonicola Hay. are shown in Fig. 2. Fang also indicated that P. morrisonicola Hay. contains various flavonoids and stilbenes (Fang et al., 1987). Needle water extract displayed the highest inhibition of leukemia cell line U937, even better than that of C. roseus (Fig. 1). Needle water extract showed anti-oxidant capability and cytotoxicity effects on leukemia cell line U937, possibly associated with its ability to quench superoxide anion and by different mechanisms connected with different extract antioxidants. We demonstrated here an inclination to T helper cell type I (Th1) and II (Th2) immune response under tube feeding P. morrisonicola Hay. water extract to mice. The preliminary data indicated that P. morrisonicola Hay. water extract stimulated Th1 and Th2 cell gene expression, implying that it is conducive to cellular and humoral immune response. In conclusion, these water extracts of P. morrisonicola Hay. containing certain anti-oxidant substances are capable of scavenging the superoxide radical, and furthermore, they show anti-tumor activity. The isolation, purification and mechanism of active components in P. morrisonicola Hay. are of interest for further investigation, which will be carried out in our future studies.

[FIGURE 2 OMITTED]

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T.-Y. Hsu (a), S.-C. Sheu (a), E.-T. Liaw (a), T.-C. Wang (b), C.-C. Lin (c,*)

(a) Department of Food Science and Technology, National Pingtung University of Seience and Technology, Neipu, Pingtung. Taiwan, ROC

(b) Fengshan Tropical Horticultural Experiment Station, TARI, Fengshan, Kaohsiung, Taiwan, ROC

(c) Graduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung, Taiwan, ROC

Received 8 January 2004; accepted 18 March 2004

*Corresponding author. Tel.: 886 7 3121101 2122; fax: 886 7 3135215.

E-mail address: aalin@ms24.hinet.net (C.-C. Lin).
Table 1. Scavenging effect on superoxide anion by the water extracts of
P. morrisonicola Hay. in the cytochrome c reduction system

 Scavenging
Extract Conc. (mg/ml) effect (%) I[C*.sub.50] value (mg/ml)

Needles 0.1 57.7 [+ or -] 3.4 0.08 [+ or -] 0.01 (d)**
 0.5 73.2 [+ or -] 0.0
 1.0 81.7 [+ or -] 5.1
Bark 0.1 49.3 [+ or -] 2.3 0.38 [+ or -] 0.00 (c)
 0.5 52.0 [+ or -] 0.0
 1.0 53.3 [+ or -] 2.3
Cones 0.1 13.5 [+ or -] 4.8 0.61 [+ or -] 0.02 (b)
 0.5 45.5 [+ or -] 4.9
 1.0 64.9 [+ or -] 4.3
Tx*** 1.40 [+ or -] 0.04 (a)

Data were presented as the means + s.e.m. for three replicates.
* I[C.sub.50] value was defined as the concentration of 50% superoxide
anion radical inhibition.
** Values within a column with different superscripts were significantly
different (p<0.05).
*** Tx: trolox, was adopted as positive control.

Table 2. Inhibitory effect on the formation of superoxide anion by the
water extracts of P. morrisonicola Hay. in the xanthine-xanthine oxidase
system

 Inhibitory
Extract Conc. (mg/ml) effects (%) I[C*.sub.50] value(mg/ml)

Needles 1.0 15.8 [+ or -] 3.5 3.15 [+ or -] 0.14 (b)**
 3.0 52.8 [+ or -] 2.6
 5.0 74.8 [+ or -] 0.7
Bark 0.5 10.0 [+ or -] 1.8 N. D.***
 1.0 18.9 [+ or -] 2.3
 5.0 N. D.
Cones 1.0 2.1 [+ or -] 0.4 9.39 [+ or -] 0.28 (a)
 5.0 22.6 [+ or -] 0.5
 10.0 53.4 [+ or -] 2.0
AL**** 0.02 [+ or -] 0.00 (c)

Data were presented as the means + s.e.m. for three replicates.
* I[C.sub.50] value was defined as the concentration of 50% superoxide
anion radical inhibition.
** Values within a column with different superscripts were significantly
different (p<0.05).
*** N.D. means non-detectable.
**** AL: allopurinol was adopted as positive control.

Table 3. Inhibitory effects of P. morrisonicola Hay. on Fe[Cl.sub.2]-
ascorbic acid induced lipid peroxidation in rat liver homogenate in
vitro

 Inhibitory
Extract Conc. (mg/ml) effects (%) I[C*.sub.50] value (mg/ml)

Needles 1.0 13.1 [+ or -] 3.1 4.66 [+ or -] 1.56 (b)**
 5.0 55.8 [+ or -] 5.2
 10.0 89.4 [+ or -] 9.5
Bark 0.5 17.5 [+ or -] 3.9 2.22 [+ or -] 0.60 (c)
 1.0 33.5 [+ or -] 5.7
 5.0 91.2 [+ or -] 8.3
Cones 1.0 17.6 [+ or -] 6.5 8.39 [+ or -] 0.72 (a)
 5.0 36.0 [+ or -] 9.4
 10.0 56.0 [+ or -] 0.3
Tx*** 0.21 [+ or -] 0.04 (d)

Data were presented as the means + s.e.m. for three replicates.
* I[C.sub.50] value was defined as the concentration of 50% superoxide
anion radical inhibition.
** Values within a column with different superscripts were significantly
different (p<0.05).
*** Tx: trolox was adopted as positive control.
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Author:Hsu, T.-Y.; Sheu, S.-C.; Liaw, E.-T.; Wang, T.-C.; Lin, C.-C.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Geographic Code:1USA
Date:Sep 1, 2005
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