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Abstract • Introduction • Materials and Methods • Results and Discussions

Scavenging Effects of FRC001 China No.1 Tian Xian Liquid on Oxygen Free Radicals

Ba-Lu Zhao1 Ph.D., Wen-Juan Xin 1 Ph.D., Chung-Yu Kao2 MD, Hong Zhang3 MD. Ph.D.
1. Institute of Biophysics, Academia Sinica, Beijing
2. FRC Free Radical Biology and Medical Research Center, Taipei
3. Department of Pathology, Univewrsity of Linkoping, Sweden

ABSTRACT :
The transplantation sarcoma S180 and hepatic carcinoma of mouse are the most popular and important models in the screening of antineoplastic drugs. This paper studied the inhibition effects of FRC001 on these two models. The results showed that on FRC001 had inhibition effects, to some extent, sarcoma S180 and hepatic carcinoma. Among then, the inhibition rate of high, middle and low dose of FRC001 on S180 were 59.73%, 52.57%, 41.33% respectively which showed obvious dose-dependent effects; FRC001 also had some effects on hepatic carcinoma foci, and the inhibition rate was 47.81%; after the treatment of FRC001, the average weight decreased and the carcinoma foci shrinked apparently compared with the control group (P<0.05 or P<0.01). FRC001 is an antineoplastic using traditional Chinese medicine as the main ingredient.

Key Words : FRC001; Transplantation tumor; Sarcoma S180; Inhibition effect on hepatic carcinoma.

INTRODUCTION
Active oxygen free radicals can damage components of cell, even kill normal cells and cause aging and some very serious diseases, such as cancer and heart disease. Usually, the production and scavenging of the active oxygen free radicals are balanced in healthy human body. If there is imbalance in regular mechanism of some enzymes, such as superoxide dismutase (SOD) or catalase, there can be excessive amounts of active oxygen radicals generated in the body. It is possible that scavengers of active oxygen radicals might be beneficial for prevention of such diseases and for human health. So it is very important to search for effective scavengers of active oxygen radicals. In this paper, with ESR, BJL-chemiluminescence and other techniques, the scavenging effect of FRC001 on oxygen free radicals has been studied in aqueous enzyme, irradiation system and cell system. It was found that FRC001 could effectively scavenge the oxygen free radicals generated from PMN stimulated with PMA, xanthine/xanthine oxidase, irradiation riboflavin/EDTA and Fenton Reaction. It was also found that it could inhibit the conjugated dienes and TBARM formed during lipid peroxidation of linoleic acid and liposome respectively.

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BACKGROUND
Malignant tumor is a series of commonly encountered diseases which harm the human. The treatment of it is a difficult problem of today’s world medical science. Besides operation, radiotherapy, chemotherapy, the roles of TCM and the combination of Chinese and Western medicine are widely noted by medical circles. FC001 is prepared mainly using traditional Chinese drugs which can eliminate the pathogenic factor and support healthy energy. The supporting healthy energy includes invigorating Qi and enriching the blood, warming Yang and nourishing Yin; the eliminating the pathogenic factor includes promoting blood circulation to remove stasis, clearing away heat and toxic material and softening and resolving hard mass. These methods have better inhibition effect on tumors.

This paper studies the effects of FRC001 on transplantation carcinoma S180 and hepatic carcinoma of mice.

MATERIALS AND METHODS
Agents : DMPO (5.5-dimethyl-pyrroline-1-oxide) was purchased from Sigma Chem Co. and purified by active charcoal before use PMA (phorbol myristate acetate), linoleic acid, lipoxidase, SOD (6500 U/mg), xanthine/xanthine oxidase (2.125 U/ml) and luminol were purchased from Sigma Chem Co., PMA was dissolved in a little acetone and diluted with 50 mM phosphate buffer to a proper concentration before use. Other agents made in China are AR Levels. Measurement of SOD activity included in FRC001.

BJL-chemiluminescence measurement : The reaction of xanthine and xanthine oxidase can generate oxygen free radicals which give a luminol-dependent chemiluminescence. SOD can scavenge the chemiluminescence generated from this system, so the SOD activity can be measured with this system. The measurement system includes 0.2 mM luminol, 0.32 mM xanthine, and 0.09 U/ml xanthine oxidase. The chemiluminescence was measured with WDD-1 chemiluminescencemeter.

The scavenging effect of FRC001 on O2 is defined as :

E = ((ho-hx)/ho) X 100%

Here ho is the Chemiluminescence of xanthine/xanthine oxidase control system and hx is the chemiluminescence of xanthine/xanthine oxidase after addition of FRC001 solution.

The measurement system was the same as above except that different concentrations of FRC001 were added to the system. First, a standard curve of SOD activity was developed with this system. Then the scavenging curve of FRC001 on oxygen free radicals was measured in this syustem. By using both of these curves, the SOD activity included in FRC001 solution can be determined.

Scangenging effect of FRC001 on OH free radicals generated from Fenton reaction.

A solution of 50 mM DMPO 1% H2O2 and 100 µM Fe(II) as ferrous ammonium sulfate were mixed and transferred to a quartz capillary for ESR measurement. When the scavenging effect of FRC001 was measured, different concentrations of FRC001 solution were added to the system. The scavenging effect was calculated as above but here the height of the second peak was used for calculation. ESR conditions : All ESR spectra were recorded at Varian E-109 ESR spectrometer. The conditions are : microwave power 20 mW. X-band, 100 kHz modulation with amplitude 1G, central magnetic field 3250 G, scan width 200 G, time constant 0. 128 S, room temperature.

Scavenging effect of FRC001 on . O2 generated from irradiation of riboflavin/EDTA system.

A mixture containing 0.3 mM riboflavin, 5 mM EDTA and 0.1 M DMPO was transferred to a quartz capillary and put into the cavity of ESR spectrometer. After irradiation of the sample for 20 seconds with a xenon lamp (500 W, distance 70cm), the ESR spectra were recorded immediately. When the scavenging effect of FRC001 was measured, different concentrations of FRC001 were added to the system. The scavenging effect was calculated as above but here the height of the first peak was used for calculation. The ESR measurement condition was the same as above.

Measurement of scavenging effect of FRC001 on conjugated dienes generated from lipid peroxidation of linoleic acid by lipoxidase.

0.1 mM linoleic acid in PBS was mixed with 480 U/ml lipoxidase and measured at 232 nm with time. When inhibition effects of FRC001 on the generation of conjugated dienes were measured, different concentrations of FRC001 were added into the sytem. The scavenging effect was calculated as above, but here the h and hx was used by reaction rate of control and sample respectively.

Measurement rate of scavenging effect of FRC001 on TBA reacted materials (TBARM) generated from lipid peroxidation of liposome initiated by Fe2+.

Liposome made from lecithine (10mg/ml) was peroxidized by addition of Fe2+ (100 µM), then 95ºC mixed with 6.7 mg/ml TBA and 0.05 M HCL. The sample was incubated for 60 minutes at 95 C, then cooled to room temperature. The TBARM was extracted by butanol : methanol (85:15) and measured at 532 nm. When the inhibition effects of FRC001 were measured, different concentrations of FRC001 were added in the system. The scavenging effect was calculated as above.

Scavenging effect of FRC001 on oxygen free radicals generated from PMA stimulated PMN

Isolation of PMN : Fresh whole blood of healthy donor was purchased from Red Cross Blood Center of Beijing. PMN were separated from other cellular components by using 6% dextran sendimentation, hypersonic lysis of remained red cells and Ficoll density gradient centrifugation separation of mononuclear cells.

Production and measurement of active oxygen free radicals generated from PMN stimulated with PMA : In a typical experiment, a mixture containing 107 /ml PMN, 0.1 mM DETAPAC (diethylentriaminepentacetic acid) and 100 ng/ml PMA was incubated for 2 minutes at 37ºC, then 0.1 M luminal was added and mixed homogeneously before measurement with BJL-chemiluminescence. When the scavenging effect of FRC001 was measured, different concentrations of FRC001 were added into the system. The scavenging effect was calculated as above.

Scavenging effect of FRC001 on ONOO

Peroxynitrite synthesis : Peroxynitrite was synthesized in a quenched flow reactor [12]. Solutions of (i) 0.6 mol/L NaNO2 , and (ii) 0.6 mol/L HCL/0.7 mol/L H2O2 were pumped at 26 ml/min into a T-junction and mixed in a 3 mm diamter by 2.5 cm glass tube. The acid catalyzed reaction of nitrous acid with H2O2 to form peroxynitrous acid was quenched by pumping 15 mol/L NaOH at the same rate into a second T-junction at the end of the glass tube. Excess H2O2, was removed by passage over a 1 X5 cm column filled with 4g of granular MnO2. The suolution was frozen at –20 ºC for as long as a week. Peroxynitrite can oxidize luminol and give a very strong chemiluminescence. The scavenging effect of FRC001 on peroxynitrite was measured by chemiluminescence method and the scavenging effect was calculated as above.

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RESULTS AND DISCUSSION
Scavenging effects of FRC001 on oxygen free radicals generated from xanthine/xanthine oxidase system.
A standard curve of scanvenging effect of SOD on free radicals generated from xanthine/xanthine oxidase was shown in Figure 1.
SOD activity (U/ml)

Figure 1. The standard curve of scavenging effect of SOD on oxygen free radical generated from the reaction of xanthine/xanthine oxidase system.

The scavenging effect of FRC001 on oxygen free radicals generated from the reaction of xanthine/xanthine oxidase system was shown in Figure 2.

Figure 2. The scavenging effect of FRC001 on oxygen free radicals generated from xanthine/xanthine oxidase system.

From above two curves, the SOD ACTIVITY INCLUDED IN 1g FRC001 IS CALCULATED AND EQUAL TO 300.000 U/ml.

2.Scavenging effects of FRC001 on oxygen free radicals generated from PMN stimulated with PMA.

When PMN are stimulated or they are in phagocytes., there will be a respiratory burst and production of active oxygen free radicals. The active oxygen radicals produced in this process play an important role in microbicidal and tumorcidal processes and in protecting the healthy body from diseases. But if there are excess active oxygen radicals in the body, they will damage the components of cells and even kill the normal cells and cause aging and very serious disease, such as heart disease and cancer. Here it was used to examine effect of FRC001 on the oxygen free radicals. Figure 3 shows the scavenging effect of FRC001 on oxygen free radicals generated from PMA stimulated PMN measured by BJL-chemiluminescence. It was determined C50=0.62 mg/ml which was smaller than that of Vitamin C (C50=0.2 mg/ml) and bigger than that of Vitamin E.

Figure 3. Scavenging effect of FRC001 on oxygen free radicals generated from PMA stimulated PMN.

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3. Scavenging effect of FRC001 on · OH free radicals generated from Fenton’s Reaction
Fenton’s Reaction can generate · OH and has been used for examination of scavengers of . OH free radicals

H2O2 + Fe²+ -> · OH+OH- + Fe3+

Here it was used for examination of the scavenging effect of FRC001 on · OH free radicals. The ESR spectrum of DMPO-OH was shown in Figure 4b (aN=aH=14.9G). The scavenging effect on FRC001 on · OH was shown in Figure 5.

Figure 4. ESR spectra of DMPO spin trapped . O2 generated from irradiated riboflavin/EDTA system (a) and . OH free radicals generated from Fenton reaction (b).

4.The scavenging effect of FRC001 was shown in Figure 5 FRC001 can effectively scavenge the hydroxyl free radicals generated from Fenton Reaction but Vitamin C just has a little scavenging effect on the hydroxyl free radical Vitamin E only give a scavenging effect of 37.5% at the concentration of 5mg/ml.

Figure 5. Scavenging effect of FRC001 on · OH free radicals generated from Fenton reaction.

4.Scavenging effect of FRC001 on · O2 generated from irradiated riboflavin/EDTA system.
Irradiated riboflavin/EDTA has been used for generation of · O2- and examination of scavenger of · O2- in photo system. Here it was used for examining the scavenging effect of FRC001 on . O2-. The ESR spectrum of · O2- spin adducts DMPO-OOH generated from irradiated riboflavin was shown in Figure 4a (aN=14.3, aHß=11.3G,aHy =1.25G).

According to the definition of the scavenging effect, the curve of scavenging effect of FRC001 on O2- generated from irradiated riboflavin/EDTA system was shown in Fig 6. The concentration of FRC001 for 50% scavenging is about 17 mg/ml. It’s scavenging effect is smaller than that of Vitamin C (C50 = 0.0009 mg/ml) but stronger than that of Vitamin E.

Figure 6. Scavenging effects of FRC001 on . O2 generated from irradiated ribolfavin/EDTA system.

5.Inhibition effect of FRC001 on conjugated dienes generated from lipid peroxidation of Linoleic acid by lipoxidase.
Conjugated dienes were generated at the first step of lipid peroxidation, which has an absorbance at 233nm. The inhibition effect of FRC001 on the generation of conjugated dienes from lipid peroxidation of linoleic acid catalyzed by lipoxidase were shown in Figure 7. From the curves, it could be found that the inhibition effects were increased with the concentrations of FRC001 added in the system. When the concentrations of FRC001 was 0.35mg/ml, about 20% of conjuugated dienes was scavenged. When the concentration was increased, the absorbance at 233 nm would increase, which disturbed the determination at high concentration.

Figure 6. Inhibition effects of FRC001 on the generation of conjugated dienes from the lipid peroxidation of linoleic acid by lipoxidase.

6.Inhibition effects of FRC001 on TEA reacted materials (TBARM) generated from lipid peroxidation of liposome.
The inhibition effects of FRC001 on TBA reaction materials generated from lipid peroxidation of liposome were shown in Figure 7. From the curve, it can be found that the inhibition effect on TBARM was increased with the increase of concentration of FRC001. The concentration of inhibition for 50% is about 19 mg/ml. Its inhibitory effect on TBA reaction materials is smaller than that of Vitamin C (C50=1mg/ml) but larger than that of Vitamin E.

Figure 7. Inhibition effects of FRC001 on TBARM generated from lipid peroxidation of liposome initated by Fe2+.

7.Scavenging effect of FRC001 on peroxynitrite.

Nitric oxide has many biological functions, such as the endothelium-derived relaxing factor (EDRF), which can relax vascular smooth cell and inhibit platelet coagulation, and reverse messenger in neuron transmission. Biosynthesis of nitric oxide from L-arginine may be a pathway for the regulation of cell function and communication. Macrophages produce nitric oxide as part of their cytotoxic armamentarium. On the other hand, nitric oxide, which contains an unpaired electron, is a paramagnetic and active frree radical, it can react with ·O2 to form peroxynitrite anion (ONOO-). In alkaline solutions, ONOO- is stable but has a pKa of 6.6 at 0°C and decays rapidly once protonated, to give a species with hydroxyl radical-like and NO2 free radicals respectively, according to the following reaction.

O2 + NO -> ONOO- + H+ -> ONOOH -> · OH +NO2

Recently, it is proposed that nitric oxide reacts with . 02- in many pathological cases to yield cytotoxic species. The investigation of peroxynitrite has been given much attention. Its oxidation of sulfhydryls and membrane lipid, which cause cell toxicity and some diseases. Here the scavenging effect of FRC001 on ONOO- was measured and it was showed in Figure 8. It can be found that FRC001 could effectively scavenge ONOO- (C50 = 0.03 mg/ml). Its scavenging effect on peroxynitrite is smaller than that of Vitamin C (C50= 0.00003 mg/ml) but stronger than that of vitamin E.

FRC001 is a drug designed on the basis of the theory of scavenging free radicals. From above experiment results, it can be found that FRC001 can effectively scavenge the oxygen free radicals generated from PMN stimulated with PMA xanthine/xanthine oxidase, irradiation riboflavin/EDTA and Fenton’s Reaction. It was also found that it could inhibit the conjugated dienes and TBA reacted materials (TBARM) formed during lipid peroxidation of linoleic acid and liposome respectively and scavenge ONOO-. These results suggest that the therapy effect of FRC001 on diseases maybe pass through the pathway of scavenging toxic oxygen free radicals in human body.

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