Indo. J. Phar. Scie. Tech. Vol. 2, No. 1, 22-32 (2015). http://dx.doi.org/10.15416/ijpst.v2i1.7807
Deden I. Dinata1, Dadih Supriadi1, Garnadi Djafar1, Syerliana1, Wahyu Wijayanti1, Shelvy E. Suherman2
1Sekolah Tinggi Farmasi Bandung, Bandung, West Java, Indonesia
2Faculty of Pharmacy, Universitas Padjadjaran, Sumedang, West Java, Indonesia
Korespondensi: email@example.com (Deden I. Dinata)
Kata Kunci: Antioksidan, daun kemangi, DPPH, granul
Globalization, urbanization, economic transition, and industrialization change the lifestyle of people. It can increase the heart disease incident. Consume unhealthy foods for examples fatty foods, baked or roasted and preserved tend to increase the risk of heart disease.1
Although the fatty foods consumption in Indonesia are generally at a lower level, but the cases of coronary heart disease is increase and has become one of the leading causes of death.2 A study of the Indonesian diet showed that the main source of fat in Indonesian are fried foods (80 90%).3 Frying is a thermal-chemical process that produces the characteristic fried food with a golden brown color, texture and flavor cracker desired appearance, so that fried food is very popular.4
The oil that used for frying heated continuously at high temperatures.5 The oil will chemycally changes, the common reactions are polymerization, oxidation, hydrolysis, and produce both volatile and nonvolatile compounds.5 ̶6 Most of volatile compounds will be evaporated and partly through a chemical reaction and absorption in fried food products.7
Food products absorb the oil during the frying and cooling.8 The oil that is absorbed during frying process is about 14 30% of total weight of the final fried food product.7 The oil is absorbed in the food that can degrade the quality of food products. Frying causes the decline in the quality of frying oil and the quality of the fried products.9
The heated oil or fat used for frying on the other. The unsaturated fatty acids are unstable and the double bonds can easily transform into saturated fatty acids or trans-fatty acids that harmful to health.10 The more the number of double bond then the more established the trans-fatty acids, especially if the oil is used repeatedly more than three times. In addition to structural changes, it will also form other compounds that are toxic.11
A decrease in quality of the cooking oil is causing the shelf life of the product is different from one frying process with the previous frying process.12 Therefore, quality of cooking oil needs to be analyzed before being used again to produce a product with a shelf life that has been defined.12,13 The quality of cooking oil is also related with security products.12
One effective way to prevent damage to the oil and fried products is the use of antioxidants. Synthetic antioxidants that usually use are tetra-butyl hidroquinone (TBHQ), butylated hydroxyanisole (BHA), and butylated hydroxytoluene (BHT). It is very effective for preventing oil or fat oxidation.14 In high concentrations, TBHQ can cause cancer.15 High concentrations and prolonged use of BHA and BHT can induce tumor. BHA can induce tumors in stomach of test animal while BHT can induce liver tumors in test animals.16
Many studies have showed the benefits of plants nutritious as antioxidants, such as to reduce the risk of heart disease, cancer, cataracts and other degenerative diseases due process. It makes nature antioxidants, much in demand in the world.17 Currently the use of basil (Ocimum americanum) is still not optimal. One of the benefits of basil is as natural antioxidants, and it was supported by research data that showed the antioxidant activity of ethanol extract of basil leaves, namely its ability to capture free radicals DPPH with IC50 value of 80.55 ppm.
Based on the research, granulation of ethanol extract of basil leaves determined its effect in preventing free radicals in fried foods. Research of Yoon and Choe showed that several parameters such as polar components, oxidation of free fatty acids (FFA), dienoat conjugate acid increased in each repetition frying for 60 times the period of frying.18 Determination of free fatty acids or commonly called the FFA is crucial to the quality of the oil.19 Because of the acid number is used to measure the amount of free fatty acids contained in the oil.20 The larger of acid number mean that free fatty acid content is higher.20 It can be derived from poor processing or hydrolysis because the hydrolysis can take place with the addition of heat.21
On the other hand, today has developed an instrument called Fourier Transform Infra Red or FTIR spectroscopy. This instrument has advantages of being able to detect the components of a sample of food quickly and cheaply because it does not need reagents preparation. This instrument has also been applied to food widely. This is an important method in quality control and monitoring processes in food industry because of its cheap, good works, and its use is more easily compared with other methods.21 This technique also offers a rapid and nondestructive for quantitative and qualitative analysis.22 Therefore, FTIR spectroscopy used for detecting cooking oil damage quickly and accurately.
This study begins with collecting basil leaf obtained from plantations in Lembang, Bandung, West Java. The procedure of this study is divided into three stages, namely the first is preparation of the extract, the second is testing the antioxidant activity of extract, the third is preparation for making extract granule, and the last is test the quality of fried foods after adding extract granul.
In the extract preparation phase, basil previously determined in Bandungense Herbarium, Department of Biology, School of Biological Sciences and Technology ITB to know the truth of the plants then dried basil leaves using an oven at a temperature of 35 oC and then reduced the size by using blender to obtain crude drug powder leaves basil for characteristic test include macroscopic examination, total ash determination, determination of the content of acid insoluble ash, water soluble ash content determination, water soluble ash assay, assay of soluble extract ethanol, water content determination, determination of drying shrinkage.23 24 The next stage crude drug basil leaves was macerated with 95% ethanol then repeated 3 times each for 3 hours. The liquid extract was filtered and concentrated using rotary evaporator. The rendemen was calculated by:
Concentrated extract x 100%
Weight of crude drugs
Phytochemical screening performed that crude drugs and ethanol extracts of basil leaves covering examinate tannins, quinone, steroid and terpenoids, alkaloids, and flavonoids. Besides the monitoring of polyphenols and flavonoids class using Thin Layer Chromatography (TLC). The mobile phase varied according to the gradient polarity. Stain reagent was FeCl3 10% in methanol for detect polyphenol compounds.25 AlCl3 in methanol was stain reagent for flavonoid compounds.26
Antioxidant activity test for ethanol extract of basil leaf was conducted using 2,2-diphenyl-1-picrylhydrazyl or DPPH carried further by measuring the extent of the reduction reaction against DPPH free radicals can take place.26 Measurement was made by measuring the absorbance of each sample using spectrophotometer that has been treated with a standard solution of 1 mM DPPH at λ 515 nm. The standar was ascorbic acid solution.27
The ethanol extract of leaves were granulated using 5% PVP, aerosil 1%, 89% lactose as a filler, the concentration of the extract used was 5%. Extract mixed with aerosil little by little until homogeneous then added PVP little by little and lactose to ethanol sufficiently homogeneous then spray until evenly mixed and can be clenched. Fist mass then sieved with 14 mesh, dried the granules by aired it. The quality of granules were evaluated includes water content test, flow rate, corner breaks and compressibility, and polyphenol and flavonoid compounds were monitored using TLC method same as that of the extract.28 Testing the ability of ethanol extract of leaves of basil granules in preventing radical in fried foods is done by adding the granules at a concentration of 0.1%, 0.05% and 0.025% of the total weight 100 g and then fried in oil 100 mL at 220 oC. Negative control was made without adding granules of ethanol extract of leaves of basil. The free fatty acid (FFA) value of the samples and the control were analyzed by use titrimetry and instrumental analysis using FTIR.29,30 The data were processed using a computer program. After that, parametric statistical test and one way ANOVA were performed followed by LSD test to determine the significance of differences in the levels of free fatty acids from four samples groups.
Sample Absorbance Spectrum Analysis with FTIR Spectroscopy was conducted to determine the spectral profile of cooking oil using FTIR spectroscopy. The FTIR used a model of IR-Prestige 21 production Shimadzu Corporation, Japan. Cooking oil samples to be measured was dripped into a KBr disk and flattened. After that, both the KBr disk were held with each other to form a sandwich KBr. Afterwards, cooking oil samples can be measured by using the wave number 400–4000 cm-1 at resolution of 1.9. Before start the next measurement, the disk KBr should be cleaned using pure n-hexane (p.a) and wiped with a lens tissue until completely clean.31
Free fatty acids testing were held with titration method. Samples were stirred and then weighed as much as 5 g and put in a erlenmeyer that have been weight before and mixed 50 mL of alcohol and heated at temperature 50 75 oC then adding 3 drops of phenolphtalein indicator to form slightly colored solution. Samples was titrated with 0.1 N NaOH. Write the NaOH volume that used to neutralize and calculate the levels of Free Fatty Acid (FFA) with the formula of calculation.32
Table 1 Characterization of Crude Basil
Table 2 Phytochemical Screening of Crude Basil
Basil plant was determinated in SITH ITB based on literature indicates the plant was Ocimum americanum Lin. The basil leaf then dried to form crude drugs. The results of crude drug calculate. The crude drug weight was 10.54% from the weight of basil leaf. Macroscopic examination for basil leaf crude drug showed that the shape is oval bone pinnate leaf, the color is green, and the aroma is typical of basil. The crude drugs characterized and phytochemical screening was held. The result showed in Table 1 and 2.
Flavonoids and phenols in ethanol extract of basil were monitored by TLC. RF value of ethanol extract of leaves of basil use non-polar mobile fase n-hexane:ethyl acetate (7:3) was 0.775. The spotted was sprayed by spotting stain reagent AlCl3 5% in methanol for flavonoids and FeCl3 10% in methanol for phenols. Flavonoids (AlCl3 5% in methanol) showed yellow spot (+) which was confirmed by observed under 254 nm UV flourescent light spotting and polyphenols (FeCl3 10% in methanol) showed a black spot (+).25
The antioxidant activity test of ethanol extracts basil using DPPH method showed IC50 values of the ethanol extract of leaf of basil was 80.55 ppm. The DPPH radical damping ability to extract ethanol mainly occurs in the secondary metabolites of polyphenols by TLC identified gave Rf value as 0.77. It was closely related to the structure of the metabolite.
The results of fried food quality test, the data analysis of free fatty acid that had undergone heating using one way ANOVA test with a value of 0.000<0.05, which indicates that there is a difference between the increase in free fatty acid groups of the samples after frying in other words, the unequal treatment of the sample affects the levels of fatty acids free on samples after fryings (Figure 1).
To find out more sample groups have the greatest effect in inhibiting the increase in free fatty acid LSD Post Hoc analysis.
The results of the data analysis of free fatty acid levels between the control sample (-) with three other samples 0.000<0.05 means that there was a difference between free fatty acid levels in the control sample (-) with three other samples or in other words the addition of ethanol extract granules basil leaves 0.1%, 0.05% and 0.025% effect on the levels of free fatty acids in the sample after being fried so that the increase in free fatty acid levels were not as high as in the control sample (-) (Figure 1).
Basil plant determination of plants in SITH ITB based literature indicates basil used was Ocimum americanum Lin. The basil leaf then dried to form crude drugs. The results of crude drug calculate. The crude drug weight was 10.54% from the weight of basil leaf. This shows the water content and weight of basil leaf stalks take a very large portion of which was approximately 90%.
Flavonoids and phenols in ethanol extract of basil was monitored by TLC. RF value of ethanol extract of leaves of basil use non-polar mobile fase n-hexane:ethyl acetate (7:3) was 0.775. The spotted was sprayed by spotting stain reagent AlCl3 5% in methanol for flavonoids and FeCl3 10% in methanol for phenols. Flavonoids (AlCl3 5% in methanol) showed yellow spot (+) which was confirmed by observed under 254 nm UV flourescent light spotting and polyphenols (FeCl3 10% in methanol) showed a black spot (+).25
Antioxidant activity test of basil leaf (Ocimum americanum) ethanol extract was done by a radical reduction reaction of 2,2-diphenyl-1-picrylhydrazyl or DPPH.
Figure 1 Levels of Free Fatty Acid (% FFA) in Sample after Frying
Figure 2 Mechanism of DPPH Acceptors33
Free radicals were known as a major factor in the biological damage, and it used to evaluate the activity of DPPH free radical reduction of a natural antioxidant. The DPPH free radical was a molecule with the color purple can be transformed into a stable compound with yellow by reaction with an antioxidant, wherein antioxidant gave one electron on DPPH resulting in reduction in free radical DPPH.34 DPPH test was a simple method test for small amount of antioxidant molecules because reaction could be observed visually by TLC, or also its intensity could be analyzed by simple spectrophotometric.35 Structure and reaction DPPH antioxidant were showed in Figure 2.
Unpaired electrons on DPPH gives a strong absorption, maximum at λ=517 nm and purple. Suppression of free radicals by antioxidant occur when unpaired electrons become paired with a hydrogen donor, thus forming a more stable DPPH.16 Reduction of free radical activity is usually expressed as percent inhibition of DPPH, but can also be expressed as the concentration that causes a loss of 50% DPPH activity (IC50). IC50 values are deemed to be good measure of the efficiency of antioxidant compounds or pure extract.18 Said to be a very strong antioxidant activity when the IC50 values less than 50 ppm, strong if IC50 values between 50–100 ppm, medium when the IC50 values between 100–150 ppm, and weak when IC50 between 151 ̶200 ppm.36
IC50 values of the ethanol extract of leaves of basil at 80.55 ppm. This means that in 80.55 ppm concentration of ethanol extract of basil leaves could reduce DPPH radical activity by 50% and classified as a strong antioxidant. DPPH radical damping ability to extract ethanol mainly occurs in the secondary metabolites of polyphenols by TLC identified gave Rf value as 0.77. It was closely related to the structure of the metabolit.
In polyphenol compounds, antioxidant activity is closely related to the structure of the side chain and also substitution on the aromatic ring.37 Its ability to react with DPPH free radicals can affect the order of antioxidant strength. Reduction of free radical activity of polyphenolic compounds believed to be influenced by the number and position of the phenolic hydrogen in the molecule.37 Thus the higher antioxidant activity will be generated on the amount of phenolic compounds having hydroxyl groups more at the core of flavonoids.37
The phenolic compounds have the ability to donate hydrogen, the antioxidant activity of phenolic compounds can be produced in the neutralization reaction of free radicals which initiate the oxidation process or the termination of radical chain reactions that occur.23
Antioxidant properties of flavonoids derived from the ability to transfer an electron to the free radical compounds (Figure 3).37,38 Antioxidant also forming complexes with metal (Figure 4).39 Both mechanisms that make flavonoids have several effects like suppress tissue damage by free radicals, inhibit lipid peroxidation and inhibit the activity of some enzymes.40
From the research results as well as the processing and analysis of data that has been done, it was evident that the increase in free fatty acid levels due to frying. The results of data analysis of free fatty acid that had undergone heating using one way ANOVA statistic test with a value of (significance 0.000<0.05), which indicates that there was a difference between the increase in free fatty acid groups of the samples after frying in other words, the unequal treatment of the sample affects the levels of fatty acids free on samples after fryings.
Figure 3 The Process Suppression of Free Radicals by Flavonoids37
Figure 4 Formation of Metal Complexes in Flavanoid39
Free fatty acids were the result of an overhaul that occurs in fatty acid that caused a reaction in the oil complex. This is consistent with the statement that the hydrolysis reaction that occurs in the oil will result in damage to the oil because there is a number of water in the oil and cause the formation of free fatty acids and some glycerol.11
To find out more sample groups have the greatest effect in inhibiting the increase in free fatty acid LSD Post Hoc analysis. The results of the data analysis of free fatty acid levels between the control sample (-) with three other samples 0.000<0.05 means that there was a difference between free fatty acid levels in the control sample (-) with three other samples or in other words the addition of ethanol extract granules basil leaves 0.1%, 0.05% and 0.025% effect on the levels of free fatty acids in the sample after being fried so that the increase in free fatty acid levels were not as high as in the control sample (-).
The addition of primary antioxidants (AH) (basil leaf ethanol extract containing polyphenolic compounds) with fatty acid low concentration may inhibit or prevent the autooxidation reaction of fats and oils.15 The expansion may hinder the oxidation reaction stages, initiation and propagation. Radicals, antioxidants (A*) which is formed in the reaction is relatively stable and does not have enough energy react with other lipid molecules form a new fatty acid radicals.41 The results of data analysis of free fatty acid levels between samples with addition granule of basil leaf ethanol extract 0.1% with the other two samples (0.05% and 0.025%). LSD Post Hoc test, significance value of 0.000<0.05 could be concluded that there are differences between the free fatty acid content of the samples after addition granule of basil leaf ethanol extract 0.1% with two other samples and the samples after the addition of granule extracts ethanol basil 0.05% with the sample after the addition of ethanol extract granule 0.025% basil leaves. So it could be concluded that the antioxidant ability of granules of basil leaf ethanol extract was 0.025%>0.05%>0.1% or in other words, the addition granules of basil leaf ethanol extract 0.025% has more effective antioxidant capabilities than in the addition granule of basil leaf ethanol extract 0.1% and 0.05%. It is caused by the adding granules with greater concentration of additives containing greater that could be expected to reduce the ability of ethanol extract of leaves of basil in inhibiting the increase in free fatty acids besides the more residue generated during frying which then could lead to increased levels of fatty acids be free.
The results of the data analysis of free fatty acid levels between samples with the addition of granules basil leaf ethanol 0.1% with the other two samples (0.05% and 0.025%). LSD Post Hoc test, significance value of 0.000<0.05 could be concluded that there are differences between the free fatty acid content of the samples after addition granule of basil leaf ethanol extract 0.1% with two other samples and the samples after the addition of granule extracts ethanol basil 0.05% with the sample after the addition of ethanol extract granule 0.025% basil leaves. So it could be concluded that the antioxidant ability of granules of basil leaf ethanol extract was 0.025%>0.05%>0.1% or in other words, the addition granules of basil leaf ethanol extract 0.025% has more effective antioxidant capabilities than in the addition granule of basil leaf ethanol extract 0.1% and 0.05%. It is caused by the adding granules with greater concentration of additives containing greater that could be expected to reduce the ability of ethanol extract of leaves of basil in inhibiting the increase in free fatty acids besides the more residue generated during frying which then could lead to increased levels of fatty acids be free.
This Research funded by Young Lecturer Competitive (Hibah Dosen Pemula) Scheme by Directorat General of Higher Education Republik of Indonesia by the year 2013 coordinated by Lembaga Penelitian dan Pengabdian kepada Masyarakat (LPPM) STFB.
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Figure 5 FTIR Result of Basil Leaf Extract 0.1%
Figure 6 FTIR Result of Negative Control the Sample was New Oil
Figure 7 FTIR Result of New Oil Sample