Evaluation of antioxidant activities from Thai herbs extracts to apply it in rice noodle (khanom chin).
The synthetic antioxidants, such as butylated hydroxyanisole (BHA) and butylated hydrohytoluene (BHT) are the most commonly used hazardous chemicals. These compounds have been applied in foods which containing fats and oils to prevent oxidative degradation. However, BHA found to be carcinogenic in laboratory animals and the lack of high BHT can cause bleeding inside and outside their babies.  Herb plants are usually used to flavor food. They are important and rich sources of natural antioxidants, [2-4] such as phenolics, flavonoids and terpenoids. [5,6] Serveral studies have reported the chemical characterization and quantification of the chemicals in this group, as well as their antioxidant, hypocholestero lemic, anti-cancer and antimicrobial properties. [7,8] Thus, they may use as natural food preservatives. It is found that herbs usually contain essential oils that showed antioxidative and antibacterial agents. It is possible, especially to extract essential oils from herbs, such as garlic, lemongrass, galangal and kaffir lime leaves to use in the process of preservation. Garlic has two main classes of antioxidant components, flavonoids  and sulfur-containing compounds such as dially sulfide, trisulfide, and allyl-cysteine. [10-14] It is the nature of the compounds against free radicals and compounds against microorganisms that affect health. It can reduce the cholesterol and sugar level and blood pressure. The therapeutic used as an antimimrobial, antifungal and antiviral activities. [15,16] Lemongrass contains the volatile oil called citral. It helps to reduce uric acid along with blood pressure, cholesterol, excess fats and other toxins. It has been used in treatment of candiurectic, emmenagogue, antiflatulence and antimicrobial agents. [17-20] Ginger shows antioxidant and anti-carcinogenic properties, 6-gingerol, a phenolic alkanone which found in ginger, has anti-bacterial, anti-inflammatory, and anti-tumor properties.  Kaffir lime leaves is an antioxidant with cancer-preventing properties, it shows high level of beta-carotene , free radical scavenging ability , antimicrobial activity , and anti-inflammatory activity.  Sweet basil is also a source of aroma compounds and essential oils containing biologically active constituents that possess insecticidal, nematicidal, fungistatic and antimicrobial properties. [25-28]
The aim of this research was to measure the total phenolic contents in 5 types of Thai herb (garlic, ginger, lemongrass, kaffir lime's leave and sweet basil) using Folin-Ciocalteu method. The antioxidant activity was determined by metal ion chelating. In addition, the radical scavenging capacity by DPPH assay was performed. Finally, the volatile oil from herb that showed the highest antioxidant property was then added to rice noodle (khanom chin) to measure the color stability and the lipid oxidation by thiobarbituric acid method (TBA).
Materials and methods
Plant Materials. The 5 types of Thai herb used in this study were garlic, ginger, lemongrass, kaffir lime's leave, sweet basil. All herbs were obtained from the local market, Mahasarakham province, Thailand, on 2010.
Reagents and Chemicals. All reagents include Folin-Ciocalteu reagent, 2, 2-diphenyl-1-picrylhydrazyl, gallic acid, sodium carbonate, vitamin E, butylated hydroxyanisole, and all solvents (HPLC grade) were purchased from Fluka (Switzerland).
Extraction of crude antioxidants. The fresh Thai herb samples (garlic, ginger, lemongrass, kaffir lime's leave and sweet basil.) were washed with distilled water, then chopped into small pieces and ground in a motar. The ground samples divided in two parts to prepare extraction and essential oil. The extraction, each of samples 100 g of sample was extracted with 95% ethanol (3x200 mL) or dichloromethane (3x200 mL) for 60 min with intermittent shaking at room temperature. The extracts were combined and filtered through a 0.45 [micro]m Nylon membrane filter. After that, the extracts were then slowly concentrated under reduced pressure, at temperature below 40[degrees]C, in a rotary evaporator to yield the crude extracts. The crude samples were stored at 4[degrees]C before determination of antioxidant activity. To prepare the essential oil, 100 g of each sample and 500mL of water were placed in a Clevenger type apparatus and isolated by hydrodistillation for 3 h. After that, the each extracts was filtered and dried over anhydrous solution sulphate, then they were stored at 4[degrees]C before used for the determination of antioxidant activity.
Total phenolic compounds. The total phenolic contents of Thai herbs were determined by spectrophotometric method using Folin-Ciocalteu's phenol reagent. The crude extracts (0.5 mL) were placed in a test tube and diluted to 5.0 mL with a glass of distilled water. Folin-Ciocalteu's phenol reagent (1.0 mL) was added, and the contents of the test tube were mixed thoroughly. After 3.0 min, 5 mL of 10% sodium carbonate solution was added, and the mixture was allowed to stand for 1 h with intermittent shaking. The absorbance of the blue color was measured in a Lamda 25 UV-VIS spectrophotometer (PerkinElmer, USA) at 750 nm. The concentration of total phenolic compounds was determined using the gallic acid equation (mg/mg extract) obtained from the standard gallic acid calibration curve. This experiment was carried out three times, and the results were averaged for the different fractions in Thai herb samples.
DPPH free radical-scavenging activity. The radical scavenging activity of crude samples were measured using the method of Yamaguchi et al.  The crude extracts and [alpha]-tocopherol (5-40 mg/mL) were added to 1.5 mL of 0.1 mM DPPH (2, 2-diphenyl-1-picrylhydrazyl) in ethanol. The mixture was shaken vigorously and was left to stand for 20 min at room temperature in the dark. The absorbance was measured in a Lamda 25 UV-VIS spectrophotometer (PerkinElmer, USA) at 517 nm. The control reaction contained all reagents except for the crude extracts. The radical scavenging effect was calculated by the following equation:
Scavenging effect (%) = [([A.sub.c]-[A.sub.s])/[A.sub.c]] x 100,
where [A.sub.c] is the absorbance of the control at 517 nm, and [A.sub.s] is the absorbance of the extract/standard at 517 nm. This experiment was repeated thrice, and the results were averaged for the different fractions in Thai herb samples.
Metal ion chelating activity. The chelating of ferrous ion was measured using the method of Dinis et al.  The crude extracts (5-25 mg/mL) were reacted with 0.05 mL of 2.0 mM Fe[Cl.sub.2]. The mixture was then added with 0.2 mL of 5.0 mM ferrozine.
After that, the reaction was shaken and incubated at room temperature for 10 min. The absorbance of the red color was measured in a Lamda 25 UV-VIS spectrophotometer (PerkinElmer, USA) at 562 nm. This experiment was carried out three times, and the results were averaged for the different fractions in Thai herb samples. The percentage of metal chelating activity was calculated by the following equation:
% Metal chelating activity = [([A.sub.c]-[A.sub.s])/[A.sub.c]] x 100,
where [A.sub.c] is the absorbance of the control at 562 nm, and [A.sub.s] is the absorbance of the extract/standard at 562 nm. This method was performed three times, and the results were averaged for the different fractions in Thai herb samples. EDTA was used as positive control
Preparation of rice noodle samples (khanom-chin) mixed with Thai herb.
Khanom-Chin is a fresh Thai rice noodle, which popular in Thailand. It made from fermented rice or rice starch. Commonly, khanom-chin is eaten with curry soup made from fish, chicken and pork. The result of this study is to apply the volatile oil from herb as the highest an antioxidant activity in hnanom-chin. The flow chart for khanom-chin mixed with volatile oil from herb is shown in Figure 1.
[FIGURE 1 OMITTED]
The color of Khanom-Chin mixed with volatile oil from herb was measured using Minolta colorimeter CR-300 (Minolta Camera Co., Osaka, Japan). Color was described as coordinates; lightness ([L.sup.*]), redness ([a.sup.*]) and yellowness ([b.sup.*]).
Thiobarbituric acid Method (TBA)
The lipid peroxidation was assay by thiobarbituric acid or TBA.  A 100 g of khanom-chin mixed with volatile oil from herb was add to 15% trichloroacetic acid and 0.375% thiobarbituric acid (TBA) in 0.2 N hydrochloric acid. The mixture was heated in a boiling water bath for 20 min, it was cooled immediately and centrifuged for 10 min. The absorbance was measured at 532 nm. This experiment was performed three times, and the results were averaged for the different fractions in the sample. The percentage of inhibition of lipid peroxidation was calculated using the following equation:
Inhibition (%) = [([A.sub.c] - [A.sub.s])/[A.sub.c]] x 100,
where [A.sub.c] is the absorbance of the control solution and [A.sub.s] is the absorbance in the presence of the extract.
Total phenolic compounds. The total phenolic content was measured by the Folin-Ciocalteu reagent method using gallic acid as the standard. A linear calibration curve of gallic acid resulted with a correlation coefficient of [R.sup.2] = 0.999 over the concentration range 20-180 [micro]g/mL. This linear equation was used to determine the total phenolic compounds in Thai herbs. The total phenolic contents of 5 herb extracts were performed by ethanol, dichloromethane and volatile oil. The average quantity of the total phenolic compounds found in crude extracts is shown in Table 1. The amount of total phenolics content of Thai herb extracts were in the range of 0.54-129.07mg GAE/g of extract. The results revealed that the total phenolic contents which were extracted by ethanol of all herbs gave higher in total phenolic contents than the other parts. For ethanolic extract, garlic showed the highest of total phenolic content (129.07 mg GAE/g of extract). The order of total phenolic content in Thai herb extract is garlic, sweet basil, kaffir lime's leave, ginger and lemongrass, respectively.
DPPH scavenging activity. In the DPPH radical-scavenging assay, Table 2 shows the concentrations of butylated hydroxyanisole (BHA) and the Thai herb extracts, at which the DPPH radicals were scavenged by 50 % ([IC.sub.50]), the lower the [IC.sub.50], the higher the antioxidant activity. Furthermore, ethanolic extract also showed the greatest capability in antioxidant activity which garlic indicated the highest potential in scavenging DPPH radicals ([IC.sub.50] = 0.32 mg/mL) and lowest in lemongrass ([IC.sub.50] = 0.70 mg/mL) (Table 2). The results in this experiment also showed that the ethanolic extract possessed the highest scavenging activity, followed by dichloromethane extract, and volatile oil.
Metal chelating activity. The metal chelating activity of Thai herb extracts was estimated by the ferrozine assay . The results indicate that the chelating ability increased with the increased concentration of all herb antioxidants in the range of 0-5 mg/mL and it was constant at concentration above 10.0 mg/mL as shown in Figure 2. The results in this experiment also showed that the ethanolic extract possessed the highest scavenging activity, followed by dichloromethane extract, and volatile oil. The order of percentages of metal chelating of all extraction was EDTA > garlic > sweet basil > kaffir lime's leave > ginger > lemongrass.
[FIGURE 2 OMITTED]
Color measurement. The color measurement of the application of volatile oil from garlic 1.0 mL mixed in khanom-chin was study by the [L.sup.*] (as the lightness), [a.sup.*] (as the redness) and [b.sup.*] (as the yellowness) during 5 days of storage at room temperature are shown in Figure 3. In all sample, the [L.sup.*] and [a.sup.*] were decrease while b* value was increase during storage. The khanom-chin containing volatile oil from garlic had lower [L.sup.*] value than the control (khanom-chin is not containing volatile oil from garlic) after 5 days of storage at room temperature. For [a.sup.*] and [b.sup.*,] the khanom-chin containing volatile oil from garlic had the least yellow and less red than the control after 5 days of storage at room temperature.
[FIGURE 3 OMITTED]
Lipid oxidation. The results of antioxidative assays by the TBA method carried out with the application of the khanom-chin containing volatile oil from garlic compared with that of the control are shown in Figure 4. In this study, the inhibition was amplified with the increased TBA value of all samples after 3 days, and it was constant at TBA value above 4 days. The khanom-chin contains volatile oil from garlic shown higher TBA values than the control.
[FIGURE 4 OMITTED]
The present research showed that Thai herb possessed a relatively strong antioxidant activity and total phenolic contents of the Thai herb samples. They are important and rich sources of antioxidative component such as flavonoids, phenolic compounds, carotenoids and vitamin C. The phenolic extracts by ethanol solvents extraction showed a higher amount of phenolic components than dichloromethane and hydrodistillation. It is suggested that ethanol is a polar, able to extract those of polar compounds such as polyphenol. The garlic extracts showed the higher amount of phenolic components than ginger, lemongrass, kaffir lime's leave and sweet basil respectively (Table 1). A group of researchers was reported that garlic is rich in [gamma]-glutamylcysteine and contained many other sulfur-containing compounds in it. [10-14] In the DPPH assay, radical scavenging activity in the phenolic extracts was related to the level of phenolic compounds. Garlic showed the higher level of DPPH radical scavenging activity. Therefore, we suggest that there are several different antioxidants in garlic. Although the DPPH assay is not specific to any particular antioxidant components, the possible mechanism of hydrogen donating suggests that the radical-scavenging effects of garlic extracts might due to the hydroxyl groups in the antioxidants of the extracts. The chelating activity against [Fe.sup.2+] was examined. The garlic extract showed the highest percentage of metal chelating, which was reduced the concentration of the catalyzing transition metal in lipid oxidation, which form [sigma]-bonds with a metal, are effective as secondary antioxidants because they reduce the redox potential . Study the application of the volatile oil from garlic as antioxidant in khanom-chin, the results from color measurement and oxidative stability by the TBA method should also be considered. The addition of antioxidants did not change [L.sup.*,] [a.sup.*] and b* value of samples. In the khanom-chin contained volatile oil from garlic, [L.sup.*] value decreased with increased storage time, and the lowest value of [L.sup.*] was obtained in the control sample. This result suggests that the fresh khanom-chin that contained volatile oil from garlic can absorbe water within the product, subsequently decreasing lightness values. For [a.sup.*] values decreased while [b.sup.*] values increased with increased storage time in greenness and yellowness, respectively. Because it was associated with the oxidation process by the presence of component pigments such as carotene in garlic extract.  Finally, oxidative stability is measured by TBA value. The khanom-chin containing volatile oil from garlic showed the highest inhibition of lipid oxidation. It is suggested that during the oxidation process, peroxide is gradually decomposed to lower molecular compounds.
The results presented in this study showed that Thai herbs had strong antioxidant activity. The application of volatile oil from garlic could be useful to control the development of rancidity and off flavours in the rice noodle (khanom-chin). Therefore, the Thai herb is a potential source of antioxidative phytochemicals and is a useful ingredient for nutraceutical or functional food products.
The authors wish to thanks to Department of Chemistry, Faculty of Science, Mahasarakham University and Center of Excellence for Innovation in Chemistry for a partial support of some chemicals. Financial support from Division of Research Facilitation and Dissemination, Mahasarakham University is acknowledged.
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Muntana Nakornriab (1) *, Nicom Nakornriab (2) and Audchara Sanekham (1)
(1) Department of Chemistry, Faculty of Science, Mahasarakham University, Mahasarakham 44150, Thailand.
(2) Department of Physic, Faculty of Science, Mahasarakham University, Mahasarakham 44000, Thailand
* Corresponding Author E-mail: email@example.com
Table 1: The content of total phenolics from Thai herb extracts compared with garlic acid (mg GAE/g of extract) Thai herb Total phenolic content (mg GAE/g of extract) Ethanolic extract Dichloromethane extract garlic 129.07 [+ or -] 0.19 94.25 [+ or -] 0.12 ginger 75.46 [+ or -] 0.14 62.51 [+ or -] 0.16 lemongrass 66.33 [+ or -] 0.27 55.86 [+ or -] 0.17 kaffir lime's 78.96 [+ or -] 0.25 69.48 [+ or -] 0.17 leave sweet 81.36 [+ or -] 0.21 70.32 [+ or -] 0.26 basil Thai herb Total phenolic content (mg GAE/g of extract) Volatile oil garlic 5.22 [+ or -] 0.22 ginger 1.06 [+ or -] 0.23 lemongrass 0.54 [+ or -] 0.18 kaffir lime's 1.19 [+ or -] 0.20 leave sweet 2.07 [+ or -] 0.22 basil Table 2: Antioxidant activity in Thai herb extracts by DPPH assay Thai herb (mg/mL) Ethanolic extract Dichloromethane extract garlic 0.32 [+ or -] 0.16 0.78 [+ or -] 0.20 ginger 0.63 [+ or -] 0.14 1.13 [+ or -] 0.21 lemongrass 0.70 [+ or -] 0.17 1.27 [+ or -] 0.19 kaffir lime's 0.59 [+ or -] 0.15 0.91 [+ or -] 0.18 leave sweet basil BHT 0.54 [+ or -] 0.16 0.86 [+ or -] 0.21 0.19 [+ or -] 0.15 0.19 [+ or -] 0.15 Thai herb (mg/mL) Volatile oil garlic 1.10 [+ or -] 0.14 ginger 1.75 [+ or -] 0.21 lemongrass 2.00 [+ or -] 0.22 kaffir lime's 1.63 [+ or -] 0.14 leave sweet basil BHT 1.46 [+ or -] 0.24 0.19 [+ or -] 0.15