Antioxidants in Thai Herb, Vegetable and Fruit Inhibit Hemolysis and Heinz Body Formation in Human Erythrocytes

This book focuses on the numerous applications of oxidative stress theory in effects of environmental factors on biological systems. The topics reviewed cover induction of oxidative stress by physical, chemical, and biological factors in humans, animals, plants and fungi. The physical factors include temperature, light and exercise. Chemical induction is related to metal ions and pesticides, whereas the biological one highlights host-pathogen interaction and stress effects on secretory systems. Antioxidants, represented by a large range of individual compounds and their mixtures of natural origin and those chemically synthesized to prevent or fix negative effects of reactive species are also described in the book. This volume will be a useful source of information on induction and effects of oxidative stress on living organisms for graduate and postgraduate students, researchers, physicians, and environmentalists.

flavanones in citrus fruits. The group of flavanol catechins is derived from green tea, red grapes, red wine and chocolate (Beecher, 2003). The antioxidant activities of phenolics are mainly due to their redox properties which allow them to act as reducing agents, hydrogen donors, and singlet oxygen quenchers. In addition they have a metal chelation potential (Halliwell and Gutteridge, 2001). Flavonoids may suppress LDL oxidation and inflammatory progression in the artery wall (Hertog et al, 1995;Peluso, 2006). It was found that catechins promote many biological functions, including prevention of cardiovascular diseases (Langley-Evans, 2001) and cancer (Kohlmeier et al, 1997;Steinmetz & Potter, 1991). Polyphenolic phytochemicals such as epigallocatechin from tea, the flavonoids quercetin and genistein from onions and soya, curcumin in curry spice and resveratrol from red grapes are diet constituents with high efficacy in preclinical carcinogenesis of colorectum, breast and prostate (Thomasset et al., 2006;Lambert et al., 2005;Surh, 2003). Low cerebrovascular disease was associated with high intake of the flavonol kaempferol and of the flavonones naringenin and hesperitin and there was a trend of reduction in type 2 diabetes was associated with higher quercetin and myricetin intake (Knekt et al., 2002). The other reported a strong inverse association between the sum of quercetin, myricetin, luteolin, and apigenin intaked and stroke (Keli et al.,1996) and low risk of lung cancer at high flavonoids intake (Knekt et al.,1997). Black rice anthocyanins reduced oxidative damage (Sangkitikomol et al., 2010a), anti-inflammation (Wang et al., 2007), enhanced LDLreceptor (Sangkitikomol et al., 2010b) and promoted cardiovascular health status (Ling et al., 2002;Wang et al., 2007). Aging is the major risk factor for neurodegenerative diseases such as Parkinson's diseases and Alzheimer's. Polyphenolic compounds could affect on cells not only due to their antioxidant activities but also due to their modulation of different pathways including signaling cascades, antiapoptotic processes and the synthesis of the amyloid β peptide (Ramassamy, 2006).
CytochromeP450 isoenzymes (CYP450s) are major enzymes in phase I of biotransformation system which involved in the metabolism of various endogenous chemicals such as; fatty acids, steroids, hormones, bile acids, eicosanoids and exogenous chemicals such as; xenobiotics, carcinogens, mutagens and environmental pollutants. A major function of CYP450s is catalyzed the reactions then conversion these nonpolar compounds into polar metabolites which can be conjugated by phase II enzymes. The oxygen activation by CYP450s' catalytic function generate the reactive oxygen species (ROS) ( Guengerich, 2008). Flavonoids may be beneficial for health protection by reducing oxidative damage and minimizing toxicity by regulating mRNA CYP450s expression for suitable production or inhibition of CYP450s isoenzymes, thereby maintaining xenobiotic biotransformation balance. Flavonoids from St. John's wort can selectively inhibit CYP1 enzymes may be useful as chemoprotective agents in prostate cancer (Chaudhary & Willett, 2006). Inhibition of PAH-induced carcinogenesis using cancer chemoprevention; methoxylated flavones and stilbene resveratrol, could effectively inhibit the benzo[a]pyrene-DNA binding and CYP 1A induction which were the early step in molecular levels of cencer prevention (Tsuji & Walle, 2007). Emerging evidence indicates that transcriptional activation of the antioxidant response element (ARE) plays a crucial role in modulating oxidative stress and providing cytoprotection against prooxidant stimuli (Nguyen et al., 2003). Several chemopreventive agents, such as curcumin, caffeic acid phenethyl ester, rectinoic acid, (-)-epigallocatechin-3gallate and (-)-epicatechin-3-gallate from tea, directed to protect DNA and other important cellular molecules by inducing the synthesis of phase II detoxifying genes and antioxidant genes via the Nrf2-ARE signaling pathway. Thereby enhancing those genes transcription and stimulating carcinogen detoxification/inactivation. The Nrf2 (nuclear transcription factor erythroid 2p45 (NF-E2)-related factor 2), is a regulator of genes encoding antioxidants and phase II enzymes such as glutathione S-transferase, NAD(P)H:quinine oxidoreductase 1, UDP-glucuronosyl-transferase, -glutamate cysteine ligase, and hemeoxygenase-1. The Nrf2 is known to mediate detoxification and/or to exert antioxidant functions thereby protecting cells from genotoxic damage (Lee & Surh, 2005;Zhang & Gordon, 2004;Hayes & McMahon, 2001). NF-κB regulated genes such as inducible nitric oxide synthase (iNOS) and cyclooxgenase-2 (COX-2) which are the inflammatory mediators and may promote cancinogenesis (Greten & Karin, 2004;Surh, 2003). Nrf2 encodes for antioxidant and general cytoprotection genes, while NF-κB regulates the expression of proinflammatory genes. A variety of antiinflammatory or anticarcinogenic phytochemicals suppress NF-κB signalling and activate the Nrf2-ARE pathway (Bellezza et al., 2010) There is no evidence that different types of tea, herbal tea, fruit and vegetable currently available in the markets of Thailand have any significant antioxidant contents. The aim of this study was to carry out a survey of relative levels of total antioxidant activities by TEAC assay (Re et al., 1999) with reference to their total phenolic contents (Singleton et al., 1999). In order to find the natural sources of antioxidants, thereafter some of them were selected to study the inhibition effect on hemolysis (Sangkitikomol et al.,2010a) as a marker of lipid peroxidation, induced by 2, 2´-azobis (2-amidinopropane) hydrochloride and the inhibition effect on Heinz body formation, as a marker of protein oxidation, induced by Nacethylphenylhydrazine (Sangkitikomol et al., 2001;2010a) by using normal fresh human red blood cells.

Preparation of plant extracts
Different types of Thai plants were purchased from Thai-herb shops, health food shops, and local markets. These samples were crushed into small pieces and some were ready-made, they were 152 samples of fruit, vegetable and herb, 33 brands of tea. A hundred milligram of dry material was extracted with 2 x 10 ml of solvent using ultrasonic bath for 5 minutes sonication, centrifuged at 3000 rpm for 10 minutes and the combined extracts were kept in deep freezer at -80°C until used. Using 2 kinds of solvent for extraction, one is 80 % aqueous methanol and the other is deionized reverse osmosis water. The plant water-extracts were used to determine lipid and protein oxidation in human red blood cells, and the other were used to analyze the antioxidant activity. Fourteen samples of ready-to-drink beverages were purchased from supper markets and directly used for analysis.

Preparation of fresh whole blood
Human blood samples were obtained from The National Blood Centre, Thai Red Cross Society, Bangkok, Thailand. Fresh blood was collected in heparinised tubes and centrifuged at 252 × g for 3 min. Plasma was carefully removed by aspiration in order to obtain a hematocrit of approximately 50% (packed red blood cells; RBCs) for hemolysis test and Heinz body formation. The blood was stored at 4°C and used within 3 h.

Determination of antioxidants in plant extracts
The total antioxidant determination and total phenolic contents were performed on the Shimadzu spectrophotometer model UV160A (Tokyo, Japan). All determinations were carried out at least three times of the standards and samples. And the correlation analysis had been done by using program Microsolf Excel 2007.

Trolox Equivalent Antioxidant Capacity (TEAC) Assay
Total antioxidant activity is measured by TEAC assay of Re et al. (1999). It is a screening of lipophilic and hydrophilic antioxidants activity by decolorization assay. The radical monocation of 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS•) is generated by oxidation of ABTS with potassium persulfate. Then the radical ABTS• is reduced in the presence of hydrogen-donating antioxidants from the plant extracts. The percentage inhibition of absorbance at 734 nm is calculated and plotted as a function of concentration of antioxidants and of Trolox for the standards. The results were expressed as Trolox equivalent in mM per kilogram of dry weight (TE mM/kg.dw).

Folin cioculteau phenol assay (Folin assay)
Modified Folin Cioculteau Phenol assay (Singleton et al., 1999) is used to determine reducing properties of phenolics contents. Briefly, 500 μl of samples or standards was mixed with 500 μl of 10% Folin reagent, let stand for 20 min, added 10 mM Na 2 CO 3 350 μl, let stand for 20 min for the solution turned blue color and then measured the absorbance at 750 nm by using gallic acid as the standards. The results were expressed as Gallic acid equivalent (GE) mM/kg.dw.

Determination of AAPH-Induced oxidative damage of erythrocyte membrane
The erythrocyte susceptibility to oxidative destruction was evaluated in vitro by subjecting the cells to oxidative stress. Since a peroxyl radical initiator, AAPH, has been proved to cause an oxidation of cell membrane proteins and lipids resulting in lysis of red blood cells (RBCs), oxidative hemolysis induced by AAPH (Sangkitikomol et al.,2010a) was used as a tool in the present study. Briefly, positive control was added 0.100 ml of fresh whole blood into pre-incubated medium [1.00 ml of phosphate buffer saline (PBS) pH 7.4], then incubated at 37 o C for 5 min. One ml of PBS solution with 200 mM AAPH was added to the whole blood suspension. The reaction mixture was shaken gently at 37 o C under aerobic condition for three and a half hours. Reaction mixture (0.100 ml) was withdrawn to 1.5 ml of ice cold PBS at 120, 150, 180 and 210 minutes after added AAPH and centrifuged at 1006 × g for 10 min. The extent of hemolysis was determined by measuring the absorbance of hemolysate at 540 nm. Negative control was done the same as positive control without AAPH presented. And the plant extracts was done the same as positive control excepted only using the extract 0.5 ml mixed with 0.5 ml of double strength of PBS in stead of 1.00 ml pre-incubated medium. Similarly, 0.100 ml of reaction mixture of negative control was treated with 1.5 ml of distilled water to yield complete hemolysis. Percentage hemolysis was calculated according to the equation. % hemolysis = absorbance of the sample aliquot divided by absorbance of the complete hemolysis x 100. Data was represented as the time required to achieve 50 % hemolysis (T 50 min).

Determination of APHZ-Induced Heinz body formation in cytosol of erythrocytes
APHZ was used as a free radical initiator inside RBCs to oxidize proteins mostly hemoglobin based on the modification of protocol described previously (Sangkitikomol et al. 2001). Using Heinz body formation was a marker of protein oxidation for testing the antioxidants properties of plant extracts. Briefly, positive control was added 0.1 ml of fresh whole blood into 2.0 ml of reaction medium [contained 1.0 g/L of APHZ, 2.0 g/L of glucose in phosphate buffer (1.3 parts of 1/15 M of KH 2 PO 4 and 8.7 parts of 1/15 M of Na 2 HPO 4 , pH 7.4)] and incubated for 2 hours at 37 o C under aerobic condition. Heinz bodies are precipitates of oxidized or denatured hemoglobin that adhere strongly to the red blood cell membrane. Negative control was done without APHZ added. And the plant extracts were done the same as positive control excepted only using the plant extract 1.0 ml mixed with 1.0 ml of double strength of reaction medium. Staining Heinz bodies in RBC with 3 drops of crystal violet solution (10 g/L in 0.73 % of normal saline) and 3 drops of RBC from reaction medium for 5 minutes at room temperature. Made blood smear on glass slides and counted RBC with Heinz body inside/1,000 of RBC using light microscope. The results were reported in % inhibition of Heinz body formation.

Results
Antioxidant activities of 152 herbs and 33 brands of tea using TEAC assay varied considerably with the types of plants and the types of tea.The range of antioxidant activities and total phenolic contents were several hundred-fold. The plants which are very good sources of antioxidants are Quercus infectoria, Areca catechu Linn., Terminalia spp., Phyllanthus emblica Linn., Punica granutum Linn., Eugenia caryophyllus (Table 1) and tea (Camellia sinensis) ( Table 2). Correlation analysis (R) between the antioxidant activity measurements of plantextracts and tea-extracts with the measured total phenol concentration were calculated to be 0.988 and 0.902, respectively (Figure 1-2). Antioxidant activities varied considerably with the types of tea (Table 2). In the TEAC assay tea-extracts with the highest to the lowest activity were green tea, oolong tea and black tea, respectively. The range of antioxidant activity was 15.6-fold for the TEAC analysis and 6.6-fold for Folin assay. Fourteen samples of ready-todrink beverages were found small amount of polyphenolics and antioxidant contents (Table  3). Selected 30 samples with high level of total antioxidant activities, within the range 141-9490 mM TE/kg.dw., were used to study the inhibition effect of lipid peroxidation and protein oxidation. The results showed that 13 out of 30 samples showed inhibition effect on Heinz body formation and the other had no effect may be caused by antioxidant contents in the extracts was not enough to inhibit Heinz body formation inside red blood cells. All www.intechopen.com samples showed prolong T 50 min. of hemolysis assay, and most of them had very strong inhibition effect which showed the T 50 min. were longer than 3 and a half hours. Time required to get T 50 min. of control positive was between 120-150 min. (Table 4).   Zingiber officinale Roscoe (young) 37 + 3 60 + 4 All determinations were carried out at least three times .
*Polyphenolics are expressed as the mean + SD in mM of catechin equivalents per kilogram of dry weights.
**Total antioxidant activities are expressed as the mean + SD in mM of Trolox equivalents per kilogram of dry weights. All determinations were carried out at least three times .
*Polyphenolics are expressed as the mean + SD in mM of catechin equivalents per kilogram dry weight.
**Total antioxidant activities are expressed as the mean + SD in mM of Trolox equivalents per kilogram dry weight.

Conclusion
For our knowledge this current work is the first report of phenolic contents and total antioxidant activities of Thai herb. The value of antioxidant activities of plant polyphenols showed varied widely with the various types of herb. The range of antioxidant activities and total phenolic contents were several hundred-fold for herbal extracts and 15.6-fold using TEAC analysis and 6.5-fold using Folin assay for 33 brands of tea. Nevertheless there was a very good relationship between the results from the TEAC assay which can measure the antioxidants activities and polyphenols in herbal extracts (r = 0.988) and in tea extracts (r = 0.902). It is suggested that the process of oxidation to make black tea and oolong tea may cause decreasing their antioxidant activities, and the correlation between the antioxidant activities and total phenolic contents of tea extracts was lower than that of herbal extracts.
Tea is one of the most popular and widely consumed as daily beverage in the world. At present, tea has become an important agricultural product that more than 40 countries in the world, especially Thailand, grow tea trees within Asian countries producing 90 % of the world output. All tea trees have their origin directly or indirectly in China and come from the same plant, Camellia sinensis. The composition of tea varies with the age of the leaf (plunking position), climate, horticultural practices and the process of storage (Lin et al., 1996(Lin et al., , 1998. The different types of tea result from variation in processing of the leaves after they are harvested. The difference in processing results in different types of polyphenolics profiles between oxidation and no oxidation tea. There are different methods in manufacturing tea that give green tea (no oxidation) contains catechins. (Lin et al, 1998) black tea (fully oxidation tea) contains the polymeric compounds, thearubigins and theaflavins, and oolong tea (semi-oxidation tea) contains a mixture of the monomeric polyphenols and higher molecular weight theaflavins (Graham, 1992). Several epidemiological studies have shown beneficial effects of tea in cancer, cardiovascular, and neurological diseases (Zaveri, 2006).
Selected 30 types of herb with various polyphenol levels to study antioxidant activities by the modified methods which were developed by the author. For the test of AAPH induced hemolysis, most studies used isolated erythrocyte suspension for the simplicity of the system and the data interpretation. However fresh whole blood was employed as the ex vivo sample for this study to ensure the erythrocytes were tested in the least modified state. To study the effect of herbal extracts against oxidative damage to lipids and proteins in human erythrocytes, free radical initiators, AAPH and APHZ, were employed for inducing oxidative stress. AAPH was source of free radical formation capable of inducing oxidation of lipid and protein structurally located on erythrocyte membranes (Chaudhuri et al., 2007). The results showed that erythrocytes were more resistant to AAPH-induced oxidation when herbal extracts were added, most of them had very strong inhibition effect. It was showed that the T 50 mins were longer than 3 and a half hours. Plant polyphenols are membrane-active antioxidant agents (Saija et al., 1995) and are the cell metabolism regulators by modulating the fluidity of lipid bilayer, since they have been demonstrated to control cell signal pathways by targeting receptors on the cell surface or by intercalating the lipid bilayer of membranes (Tarahovsky et al., 2008). Polyphenols interact with the surface of bilayer through hydrogen bonding have been shown to reduce the accessibility of oxidants, thus protecting the structure and function of membranes (Oteiza et al., 2005).
APHZ was a source of free radical formation inside cytosol of erythrocytes leading to induction of proteins mostly hemoglobin and lipid bilayer of membranes oxidation (Sangkitikomol et al., 2001). Phenylhydrazine in the presence of oxidase or peroxidase , it reacts with oxyhemoglobin to form phenylhydrazine radicals. Phenylhydrazine slowly oxidizes in aqueous solution to form O 2 -• and H 2 O 2 and the end products of the reaction are benzene and N 2 . Phenylhydrazine radical is the most damaging agent that can denature hemoglobin molecules. The oxidation of reactive sulfhydryl (S-H) groups creates disulfide bonds that may change the conformation of globin chains, resulting in precipitation of the hemoglobin molecules called Heinz bodies, then follows by membrane lipids oxidation and causing hemolysis (Winterbourn, 1985). Bioavailability differs greatly from one polyphenol to the other; therefore the most abundant polyphenols in plants are not leading to the highest concentrations of active metabolites in target cells. Gallic acid and isoflavones are the most absorbed polyphenols, followed by catechins, flavanones, and quercetin glucosides. The least absorbed polyphenols are proanthocyanidins, galloylated tea catechins, and anthocyanins (Manach et al., 2005). The results showed that some herbal extracts could inhibit Heinz body formation inside erythrocytes, but the other could not. This finding suggested that the process of proteins and lipids oxidation was still taking place as a result of insufficiency of absorbed polyphenols inside erythrocytes to inhibit oxidation.
The advantage of using erythrocytes (human living cells) as the models for screening test of antioxidant properties of herbal extracts, because it is simple and low cost to perform the analysis and the given data could be extrapolated to happen in human body.

Acknowledgment
This work was supported by Faculty of Allied Health Sciences, Sciences, Chulalongkorn University, Bangkok.