Biomedical Research

Abstract

According to previous studies, phytochemicals have many pharmacological activities. Chinese herbal remedies that have potential use in many diseases. Caffeic acid, p-coumaric acid, oleanolic acid and ursolic acid are bioactive compounds from plants. In the present study, the antioxidant properties of the caffeic acid, p-coumaric acid, oleanolic acid and ursolic acid were evaluated by using different in vitro antioxidant assays such as 1, 1-diphenyl-2-picryl-hydrazyl free radical (DPPH•) scavenging, metal chelating activities and cytochrome c reduced method. The result of this study showed that caffeic acid, p-coumaric acid, oleanolic acid and ursolic acid all have apparent antioxidant activity by these assays. Among these compounds, oleanolic acid was capable of inhibiting xanthine oxidase and showed a significant antioxidant effect. Thus, these compounds have potential to be used as natural antioxidants against oxidative deprivation for preventing carcinogenesis and other diseases.

Keywords

DPPH radical activity, Antioxidant activity.

Introduction

It has been indicated that oxygen molecules and most of the organisms present in our environment produce Reactive Oxygen Species (ROS) through the metabolism of oxygen to produce energy, and that these ROS are pathologic mediators in many diseases. Thus, many scientists have conducted extensive studies on the prevention or improvement of tissue damage resulting from active oxygen [1-6]. The main active oxygen species stored in cells are superoxide and peroxide, and the generation of excessive reactive oxygen species causes depletion of intracellular GSH (Glutathione), lipid peroxidation, changes in enzyme activity and DNA damage [7]. Therefore, if cells succumb to excessive exposure to reactive oxygen species, this exposure would induce large amounts of ROS damage and excessive lipid peroxidation in the organism body and finally result in cell apoptosis or necrosis [8,9]. While the mitochondria within a cell are the main sites for the generation of ROS, they mostly come from the mitochondrial electron transport chain. Some studies have further pointed out that the mitochondria play a key role in regulating the process of cell death and many mitochondrialrelated proteins dominate the apoptosis process.

Hence, any kind of substance with antioxidant effects, could remove ROS or prevent lipid peroxidation [10,11]. Such a substance might even affect the biochemical conversion of the mitochondria and cause potential changes in the mitochondrial membranes, which regulate cell death messages. This possibility has led many scientists to conduct extensive research toward finding ways to prevent, improve, and reduce tissue damage caused by ROS in order to achieve anti-aging effects and disease prevention. Antioxidant effects elicited by plant species have a full range of prospective applications in human health care. Therefore, attention has shifted to nonnutritive phytochemicals present in a plant-based diet. It is estimated that more than 1000 different phytochemicals possess chemopreventive properties [12,13]. A lot of antioxidants minimize actions of ROS production. In this study was aimed at evaluating their antioxidative activities of caffeic acid, p-coumaric acid, oleanolic acid and ursolic acid by DPPH radical activity, chelating activity and cytochrome c reduced method.

Materials and Methods

Chemicals

DMSO, DPPH, Vitamin C, EDTA, caffeic acid, p-coumaric acid, ursolic acid and oleanolic acid were purchased from Sigma chemical Co, USA.

DPPH radical activity

Because fats produce free radicals in the oxidation process and result in fat rancidity, common antioxidants provide hydrogen (hydrogen donor) to remove peroxyl radicals and thus inhibit the oxidation chain reaction. The present study used DPPH to assess the hydrogen donor ability of a given antioxidant. Since DPPH has a strong absorption at 517 nm in methanolic solution, as antioxidants restore it, the absorbance value is decreased and the spectrophotometric method could be used to measure the absorbance values at 517 nm. If the absorbance value decreases, then this means that the antioxidant has stronger hydrogen donor ability [14]. Radical scavenging activity (%)=(Control OD 517 nm-sample OD 562 nm))/ Control OD 562 nm × 100%.

Chelating activity of Fe²⁺ ions

The present study used the chelating activity of Fe2+ ions to determine the pro-oxidant effect of metal ions, which is often the main factor that causes lipid oxidation. Through redox cycle reactions, only a small amount of metal ions could effectively produce free radicals and accelerate lipid oxidation. From a variety of metal ions, Fe²⁺ is often the most influential pro-oxidant, which would promote lipid oxidation. Using the absorbance value of Fe²⁺ and ferrozine complexes at 562 nm, the chelating ability of test samples with Fe²⁺ ions could be measured. If the absorbance value decreases, this represents the chelating ability of test samples with Fe²⁺ ions. If this value is low, the test sample has a stronger chelating ability with Fe²⁺ ions. When the chelating activity of Fe²⁺ ions is expressed in percentages, the higher the chelating ability, then the stronger the antioxidant effects [15]. Chelating effects (%)=(1-(sample OD 562 nm-Control OD 562 nm))/Control OD 562 nm × 100%.

Cytochrome c reduced method

The cytochrome c reduction method was used to conduct an antioxidant study. Xanthine oxidase catalyses xanthine to produce uric acid and the maximum absorption occurs at a wavelength of 295 nm through uric acid. Then the spectrophotometric method could be used to determine the absorbance change of uric acid and could be used as an indicator for xanthine oxidase activity. At the same time, when xanthine oxidase catalyses xanthine to produce uric acid, it would be accompanied by superoxide (.O^2-) generation. If cytochrome c (the most important electron carrier in the mitochondrial electron transport chain) is used as a detection agent, the redox reaction between cytochrome c and .O^2- could result in a substantial increase in the amounts of reduced state cytochrome c (Ferro-cytochrome c). Due to the fact that reduced state cytochrome c has a specific absorption at 550 nm [16,17], the spectrophotometer could be used to detect absorbance change at 550 nm and the amount of reduced state cytochrome c could be determined and could be used indirectly to assess the ability of drugs removing superoxide [18]. Cytochrome c reduction (%)=(Control OD 550 nm-sample OD 550 nm))/Control OD 550 nm × 100%.

Statistical analysis

All data were analysed using the Statistical Package for the Social Sciences Ver 22 software (SPSS Inc., Chicago, IL).

Results

The antioxidation activity of isolated compounds

DPPH radical activity was used to evaluate an antioxidant's capacity for hydrogen donation, the determination of chelating activity of Fe²⁺ ions and cytochrome c reduction methods in antioxidant studies. The results showed that DPPH radical activity analysed antioxidant capacity based on free radical removal capability and the activity order was caffeic acid>pcoumaric acid>ursolic acid> oleanolic acid (Table 1) with IC50 values of 0.97, 6.65, 6.88, and 26.47 μM, respectively. The IC50 value of caffeic acid was less than the IC50 value of Vitamin C (1.94 μM) (Table 1). Moreover, when using chelating activity of Fe²⁺ ions to measure antioxidant ability, the order of free radical removal ability was caffeic acid>pcoumaric acid> oleanolic acid >ursolic acid (Table 2), but their IC₅₀ values were 655.3, 1234.1, 1860.6 and 9310 μM, respectively (Table 2). The order of cytochrome c inhibition ability of cytochrome c reduction method was oleanolic acid>ursolic acid>p-coumaric acid>caffeic acid (Table 3) and the IC₅₀ values were 10.3, 15.3, 51.5 and 88.4 μM, respectively. The IC₅₀ value of oleanolic acid is less than the IC₅₀ value of Vitamin C (14.09 μM) (Table 3). From the above experimental results, we discovered that while oleanolic acid, ursolic acid, p-coumaric acid and caffeic acid all have antioxidant effects and their main mechanisms of action were different. The antioxidant effect of caffeic acid was mainly used for the removal of peroxyl radicals by being a hydrogen donor for the inhibition of the oxidation chain reaction or for the chelating of Fe²⁺ ions.

Table 1. The IC₅₀ value of DPPH radical activity of caffeic acid, pcoumaric acid, ursolic acid, and oleanolic acid.

Table 2. The IC₅₀ value of chelating activity of Fe²⁺ ions of caffeic acid, p-coumaric acid, ursolic acid, and oleanolic acid.

Table 3. The IC₅₀ value of the cytochrome c reduction of caffeic acid, p-coumaric acid, ursolic acid, and oleanolic acid.

Discussion

Free radicals have potential of reacting with biological macromolecules and inducing tissue damage such as oxidative damage in lipids, proteins and DNA. Medical plants play an important role in maintaining the health and cure disease over the thousand years in Asia. Many phytochemicals including flavonoids, terpenoids, phenolic compounds, saponins, plant sterols, and curcumins have been identified and evaluated the pharmacological activities. Some of these phytochemicals have been found to be potent antioxidants and free radical scavengers with anti-cancer activities [19-21]. In order to further examine the antioxidant mechanism of compounds, different experiments were performed. This experiment includes the radical scavenger, the metal chelating mechanisms and reduction of cytochrome c. The scavenger mechanism (DPPH) is based on the reaction of the antioxidant directly with the hydroxyl radical. In metal chelation assay, these compounds may chelate the ferrous ions with hydroxyl groups.Oleanolic acid and ursolic acid are extracted from many plants [22]. Oleanolic acid and ursolic acid are triterpenoid compounds and widely used in food and folk medicine. Oleanolic acid and ursolic acid have antiinflammatory and antihyperlipidemic properties in vitro and in vivo study [22]. The hepatoprotection of two compounds may result from the inhibition of toxicant activation. The current antioxidant effect of oleanolic acid was present in the inhibition of xanthine oxidase. The two compounds displayed anti-cancer activities could result from antioxidant effect. The detail mechanism of pharmacological activities can be examined in the future. Caffeic acid (3, 4-dihydroxycinnamic acid) is phenolic compounds with hydroxycinnamic acids chemical structure. Based on recent researches, caffeic acid and p-coumaric inhibited LDL oxidation and quenche radicals [23,24]. Based on these results, we found that caffeic acid, p-coumaric acid, oleanolic acid and ursolic acid have antioxidation activity by different mechanism.

Conclusion

According to data obtained from the present study, caffeic acid, p-coumaric acid, oleanolic acid and ursolic acid was found to be an effective antioxidant in different in vitro assays. However, further studies are required to determine the exact pharmacological mechanisms involved in preventing carcinogenesis, diseases or food products.

Acknowledgement

The authors gratefully thank the financial support of this research from the National Science Council of the Republic of China (Taiwan) under grant NSC 97-2320-B-242 -005 -MY3.

References

1. Li J, Li F, Xu Y, Yang W, Qu L, Xiang Q, Liu C, Li D. Chemical composition and synergistic antioxidant activities of essential oils from Atractylodes macrocephala and Astragalus membranaceus. Nat Prod Commun 2013; 8: 1321-1324.
2. Kokoglu E, Belce A, Ozyurt E, Tepeler Z. Xanthine oxidase levels in human brain tumors. Cancer Lett 1990; 50: 179-1781.
3. Alok S, Jain SK, Verma A, Kumar M, Mahor A, Sabharwal M. Herbal antioxidant in clinical practice: a review. Asian Pac J Trop Biomed 2014; 4: 78-84.
4. Li WJ, Lin YC, Wu PF, Wen ZH, Liu PL, Chen CY, Wang HM. Biofunctional constituents from Liriodendron tulipifera with antioxidants and anti-melanogenic properties. Int J Mol Sci 2013; 14: 1698-1712.
5. Wu YB, Zheng LJ, Wu JG, Chen TQ, Yi J, Wu JZ. Antioxidant activities of extract and fractions from Receptaculum nelumbinis and related flavonol glycosides. Int J Mol Sci 2012; 13: 7163-7173.
6. Yeh YT, Huang JC, Kuo PL, Chen CY. Bioactive constituents from Michelia champaca. Nat Prod Commun 2011; 6: 1251-1252.
7. Lin CC, Yen MH, Lo TS, Lin JM. Evaluation of the hepatoprotective and antioxidant activity of Boehmeria nivea var. nivea and B. nivea var. tenacissima. J Ethnopharmacol 1998; 60: 9-17.
8. Khaitan D, Dwarakanath BS. Endogenous and induced oxidative stress in multi-cellular tumour spheroids: implications for improving tumour therapy. Ind J Biochem Biophys 2009; 46: 16-24.
9. Kim J, Seok YM, Jung KJ, Park KM. Reactive oxygen species/oxidative stress contributes to progression of kidney fibrosis following transient ischemic injury in mice. Am J Physiol Renal Physiol 2009; 297: 461-470.
10. Bors W, Saran M. Radical scavenging by flavonoid antioxidants. Free Radic Res Commun 1987; 2: 289-294.
11. Bulkley GB. The role of oxygen free radicals in human disease processes. Surgery 1983; 94: 407-411.
12. Surh YJ. Cancer chemoprevention with dietary phytochemicals. Nat Rev Cancer 2003; 3: 768-780.
13. Cheng KC, Hsueh MC, Chang HC, Lee AY, Wang HM. Antioxidants from the leaves of Cinnamomum kotoense. Nat Prod Commun 2010; 5: 911-912.
14. Almeida IF, Fernandes E, Lima JL, Valentão P, Andrade PB, Seabra RM, Costa PC, Bahia MF. Oxygen and nitrogen reactive species are effectively scavenged by Eucalyptus globulus leaf water extract. J Med Food 2009; 12: 175-183.
15. Lee J, Hwang KT, Heo MS, Lee JH, Park KY. Resistance of Lactobacillus plantarum KCTC 3099 from Kimchi to oxidative stress. J Med Food 2005; 8: 299-304.
16. McCord JM, Fridovich I. The reduction of cytochrome c by milk xanthine oxidase. J Biol Chem 1968; 243: 5753-5760.
17. McCord JM, Fridovich I. The reduction of cytochrome c by milk xanthine oxidase. J Biol Chem 1968; 243: 5753-5760.
18. Butler J, Koppenol WH, Margoliash E. Kinetics and mechanism of the reduction of ferricytochrome c by the superoxide anion. J Biol Chem 1982; 257: 10747-10750.
19. Choi H, Lim W, Kim JE, Kim I, Jeong J, Ko Y, Song J, You S, Kim D, Kim M, Kim BK, Kim O. Cell death and intracellular distribution of hematoporphyrin in a KB cell line. Photomed Laser Surg 2009; 27: 453-460.
20. Samuelsen AB. The traditional uses, chemical constituents and biological activities of Plantago major L. A review. J Ethnopharmacol 2000; 71: 1-21.
21. Lewis JG, Adams DO. Early inflammatory changes in the skin of SENCAR and C57BL/6 mice following exposure to 12-O-tetradecanoylphorbol-13-acetate. Carcinogenesis 1987; 8: 889-898.
22. Stankovic MS, Curcic MG, Zizic JB, Topuzovic MD, Solujic SR, Markovic SD.Teucrium plant species as natural sources of novel anticancer compounds: antiproliferative, proapoptotic and antioxidant properties. Int J Mol Sci 2011; 12: 4190-4205.
23. Liu J. Pharmacology of oleanolic acid and ursolic acid. J Ethnopharmacol 1995; 49: 57-68.
24. Meyer AS, Donovan JL, Pearson DA, Waterhouse AL, Frankel EN. Fruit hydroxycinnamic acids inhibit low density lipoprotein oxidation in vitro. J Agric Food Chem 1998; 46: 1783-1787.
25. Fukumoto LR, Mazza G. Assessing antioxidant and prooxidant activities of phenolic compounds. J Agr Food Chem 2004; 8: 3597-3604.