The Antioxidant Activity Extract Leaves Avocado (Persea americana Mill) with Method Nitric Oxide (NO)

Antioksidan, Juni 2016

The Antioxidant Activity Extract Leaves Avocado (Persea americana Mill) with Method Nitric Oxide (NO) Donn R. Ricky1, Joshua H.L. Tobing2 Haryati Siahaan3 Jurusan Biologi, Fakultas Matematika dan Pengetahuan Alam, Universitas Advent Indonesia (UNAI), Jl. Kolonel Masturi no. 288, parongpong, Bandung Barat 40559, Indonesia

ABSTRACT This study was conducted to test the antioxidant activity of the extract of Avocado leaves (Persea americana Mill) using Nitrite Oxide, through the Ethanol extract, Acid and Dichloromethane fractions. Through laboratory testing the active compounds showed are: Alkaloids, Flavonoids, Phenolics, Tannins, Monoterpenes, Sesquiterpenes and Saponin. From 300 grams of simplicia the Ethanol yield of concentrated extract of crude drug is 6.06% or 18.20 grams. Using 1 gram of Ethanol the extract fractionation was carried out to obtain the Acid and dichloromethane fractions, the yield obtained is 60% or 0.6 gram of Acid fraction and 19% or 0.19 gram of Dichloromethane fraction. ANOVA results on Ethanol extract, Acid fraction and Dichloromethane fraction are significant with p value of 0.000 < α = 0.05. The Duncan's Multiple Range test showed that the Ethanol extract (50, 100 and 20 ppm), the Acid fraction (50, 20, 100, 500, 10, 200, and 1000 ppm), and Dichloromethane fraction (20, 50, 100, 10, and 200 ppm) are groups that contributed to the significance of the analysis on antioxidant activity. MANOVA test showed a significant antioxidant activity on the types and variations of extract/fractionation with p value of 0.000 < α = 0.05. Duncan multiple range test showed that Ethanol extract and Acid fraction contributed to the significance on the statistical analysis used. Keywords: Avocado leaves, Antioxidant, Nitrite Oxide

ABSTRAK Penelitian ini dilakukan untuk menguji aktivitas antioksidan daun alpukat ( Persea americana Mill) dengan metode Nitrat Oksida, melalui ekstrak Etanol, fraksi Asam, dan fraksi Diklorometan daun alpukat (Persea americana Mill). Pengujian senyawa aktif menunjukkan adanya Alkaloid, Flavonoid, Fenolat, Tanin, Monoterpen, Seskuiterpen dan Saponin. Rendemen ekstrak Etanol pekat dari simplisia daun alpukat yang diperoleh sebesar 6,06% (18,20 gram) dari 300 gram simplisia. Satu gram ekstrak Etanol digunakan untuk mendapatkan fraksi Asam dan fraksi Diklorometan. Rendemen fraksi Asam yang diperoleh adalah sebanyak 60% (0.60 gram) dan rendemen fraksi Diklorometan sebanyak 19% (0.19 gram). Hasil ANAVA pada ekstrak Etanol, fraksi Asam dan fraksi Diklorometan, menunjukkan p = 0,000 lebih kecil dari nilai α = 0,05. Hasil uji Jarak Berganda Duncan menunjukkan bahwa ekstrak Etanol (konsentrasi 50, 100 dan 20 ppm), fraksi Asam (konsentrasi 50, 20, 100, 500, 10, 200, dan 1000 ppm), dan fraksi Diklorometan (konsentrasi 20, 50, 100, 10, dan 200 ppm) adalah kelompok yang memiliki aktivitas antioksidan yang signifikan. Uji MANAVA menunjukkan adanya aktivitas antioksidan yang signifikan dari jenis dan variasi ekstrak/fraksinasi dengan nilai p = 0,000 lebih kecil dari nilai α = 0,05. Hasil uji berganda Duncan menunjukkan bahwa ekstrak Etanol dan fraksi Asam memberi signifikansi terhadap analisis statistik yang digunakan. Kata Kunci: Daun Alpukat, Antioksidan, Nitrat Oksida (NO)

FMIPA/ Biologi/ Universitas Advent Indonesia/ Bandung

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Antioksidan, Juni 2016 PREFACE Indonesia is a country that rich in diversity of plants, avocado is one of the plants that grow in the forest and can also grow in the yard of the house which of the many parts of this plant are used to treat diseases that people often suffered in the community. One of the benefits of the avocado plant is to treat diseases caused by an increase in free radicals that enter the body so that the body is unable to neutralize the increase in the concentration of free radicals. Free radicals are the molecules that in the outermost orbit have an unpaired electron, are highly unstable and extremely reactive so can damage the cell components such as DNA, lipids, proteins, and carbohydrates. Such damage can cause various biological disorders such as atherosclerosis, cancer, diabetes, and other degenerative diseases (Sie, 2013). Avocado (Persea americana Mill) is one of the plants that have benefits as a traditional medicine. Almost all parts of this plant have properties as a source of medicines. Avocado plant parts that have many properties are the leaves, although the family Lauraceae fruits also have high nutritional content. The leaves are used as a herbal medicine for renal and hypertensive disease, or in other words to have merit as an herbal remedy (Owalabi dkk, 2010). The results of the study Maryati et al (2007) say that the phytochemical screening extracts of leaves of avocado (Persea Americana Mill.) show that avocado leaves have some active compounds like flavonoids, tannins, quinone, saponin, and steroids / triterpenoid. Take a look from some pharmacological effects on the plant leaves Avocado, then the topic matter to be resolved in this study are: Leaf Extract Antioxidant Activity Test Avocado (Persea Americana Mill.) by the method of Nitric Oxide (NO). RESEARCH METHODS 1.

Tools and Materials

The tools used in this research is blender, jar, oven, funnel tube separator, shaker, digital scales, a measuring cup, stir bar, rotary vacuum evaporator, ultrasonic water bath (water bath), rotary shaker, spectrophotometer Boeco S- 26, plate evaporator and glassware commonly used in laboratories.

The materials used in this study is the avocado leaves, Griess reagent, sulfanilic acid, acetic acid 10%, Sulfanilamide 1% and the solution naphthyl-1 amin. Extract 0.75 mL, 0.25 mL of 50 mM Na-nitroprussida, 0.1 M phosphate buffer, NaH2PO4.2H2O, Na2HPO4.12H2O, NaCl, Ethanol 96%, distilled water, dichloromethane, chloroform, 2N HCl, Mayer reagents, reagent Dragendrof, FeCl3, Mg powder gelatin solution, Amil alcohol, ether, vanillin solution, concentrated H2SO4 and KOH 5%. 2.

Making of Simplicia The process of making simplicia done through several stages. These stages starting with gathering raw material such as two kilograms of avocado leaves. Avocado leaves washed with running water to clean dirt on the leaves, then drained. Then the leaves are dried for 3 days drying in the sun using the sack. This method is used so that the chemical constituents present in the leaves to stay awake, especially the compounds that are antioxidants such as flavonoids. The leaves are dried pulverized using a blender and then stored in a sealed container at room temperature and protected from exposure to sunlight. 3.

Screening of Active Compounds

Screening of active compounds was conducted to determine the content of classes of active compounds contained in avocado leaves. Compounds can be identified by the screening test using the treatment and the provision of certain reagents by searching eight groups active compounds in plants such as alkaloids, Felanot, tannins, flavonoids, monoterpenes and sesquiterpenes, Steroids and Triterpenoid, quinone and saponin. 4.

Maceration Macerate

and

Concentration

of

Maceration process is performed for 3 days. Simplicia of 300 gr Avocado leaves soaked into 1200 ml of 96% ethanol solvent. During the soaking process performed by using a shaker to obtain a homogeneous mixture. This is done to prevent saturation of the extract solution in order to obtain better. Every day, for three days, Ethanol solvent replacement. The filtrate obtained is collected and stored in containers that are protected from exposure to sunlight. 5.

Extract Concentration Extraction and concentration procedure extracts were performed in this study is to take 1200 ml


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Antioksidan, Juni 2016 maserat which has been obtained from maceration process is then evaporated using a vacuum rotary evaporator, so that the solvent evaporates and dissolved substances precipitate. By heating below the boiling point of the solvent, the compounds contained in the solvent is not damaged by high temperatures. 5.1

Making of Acid Faction and Dichloromethane Faction making of fraction of acid performed by weighing 1 gram of extract ethanol and dissolved in 50 mL of dichloromethane solvent that is inserted into the separator tube and added with HCl 5% as much as 15 mL and then stirred until homogeneous. The solution was then allowed to stand for 30 minutes until a separation and formation of layers of different positions, namely between acid fraction with dichloromethane fraction, the position is above the acid layer and dichloromethane layer position at the bottom. Layers acid fraction is then taken and separated from the layer of dichloromethane. Dichloromethane layer obtained was poured back into the tube dividers and added again with HCl 5% as much as 15 mL. This procedure is performed three times to obtain acid. 6.

Antioxidant Activity Testing Method with Nitric Oxide

Testing of antioxidant activity using the method of Nitric Oxide. Nitric Oxide provides absorption at a wavelength of 540 nm. In this study used several variations of the extract concentration of 10 ppm, 20 ppm, 50 ppm, 100 ppm, 200 ppm, 500 ppm and 1000 ppm. The procedure for obtaining the concentration variation is by weighing, in which each extract Ethanol, acid fraction, the fraction of dichloromethane each weighed as much as 0.15 grams. Then each extract and the fractions were dissolved in 150 ml of ethanol 96%. This solution is referred to as a stock solution. Further dilution starting from 10 ppm to 1000 ppm. Dilution to determine the concentration variations can be seen in Table 6.1 below. Table 6.1 Variations dilution Concentration Ethanol Extract, Acid faction and faction Dichloromethane

After dilution is done, each of the various concentrations of ethanol extract, fractions acid and dichloromethane fraction is divided into 5 tubes and each tube inserted by 2 mL Sodium Nitroprussida then added with 0.5 mL of phosphate buffer. Then added to a solution of 0.5 ml of the ethanol extract, fractions acid and dichloromethane fractions and then allowed to stand (incubation) for 3 hours. After incubation for 3 hours a solution of 0.5 mL of solution was transferred into a new test tube and then added 1 ml of sulfanilic acid and incubation for 5 minutes. After 5 minutes of added napthlyn-1-amine in 1 mL tube so that the total number is 10. After the addition of Nitric Oxide, the solution was incubated for 24 hours and protected from sunlight. Then the measured value of absorbance at a wavelength of 540 nm which is the maximum wavelength. The amount of antioxidant activity shown by the magnitude of percent inhibition was calculated using the following formula: (%) = (Abs. blank – Abs. sample) x 100% Abs. blank Tests were also conducted on each of the various concentrations of ethanol extract, fractions acid and dichloromethane fractions by time analysis 24 hours after the addition of Nitric Oxide and its absorbance values are calculated through a spectrophotometer at a wavelength of 540 nm. Then the documents used is 0.5 mL Ethanol 96% sodium nitroprussida as much as 2 mL and 0.5 mL of phosphate buffer is inserted into a test tube and incubated for 3 hours, after 3 hours of incubation of 0.5 mL were transferred into test tubes then add 1 ml of sulfanilic acid and incubation for 5 minutes, after 5 minutes of incubation and added napthlyn-1-amine in 1 ml and then incubating back for 30 minutes, then measurement of antioxidant activity, demonstrated by examination spectrophotometer on the value of its absorbance is 540 nm. 7.

Data Analysis In this study, the data obtained from the spectrophotometer form of analysis absorbance values using the Test of Statistical Analysis, and Test Linear Regression to determine the direction of the relationship between independent variables (concentration ppm) with the dependent variable (percent inhibition) and to predict the value of the dependent variable when the variable value independently increase or decrease. ANOVA (analysis of variants) was used to know the value of the average significant difference in the antioxidant activity of the


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Antioksidan, Juni 2016 extract concentration variations. If the ANOVA test results are significant value, then continued with Duncan's multiple range test analysis to determine the type and concentration extracts which have the highest antioxidant activity in avocado leaves after the addition of Nitric Oxide.

3. Antioxidant Activities of Ethanol Extracts

RESULTS AND DISCUSSION

From the testing of chemical compounds that have been performed, it is known that the plant leaves Avocados contain alkaloids, flavonoids, phenolics, tannins, monoterpenes, sesquiterpenes and saponin. But on the plant leaves Avocados there are compounds triterpenes and steroids.

3.2

Linear Regression Test

Results of Maceration and Fractionation

Can be seen in the following table Ethanol extract, fractions acid and dichloromethane fraction Avocado leaves in the form percent yield, namely Percentage of products that we produce compared to the raw materials processed and obtained by the formula: Extract weight

% Obtaining Extracts = Simplicia weight x 100%

percent Inhibition

2.

In Table 3.1 it was found that the highest rates antioxidant activity of the extract Ethanol as average percent inhibition, the Nitric Oxide as measured by the method Spectrophotometer Beoco S-26 after the addition of Nitric Oxide obtained at concentrations of 50 ppm with a mean value of 91, 56% and average lows in concentration of 500 ppm with a mean value of 86.56%. This means that the higher the value the higher the percent inhibition of antioxidant activity.

92 91 90 89 88 87 86 85

y = -0,0029x + 89,113 R² = 0,3067 rerata 24 jam Etanol

0

500

1000

1500

Extract Concentration (ppm)

Based on the calculation formula written above, the yield of ethanol extract that has been obtained is 6.06%, while the yield on acid fraction was 60% and the yield of dichloromethane fraction was 19%. The yield is calculated after the extract is concentrated by evaporation.

The results of figure 3.1 above, it was found that the ethanol extract of avocado leaves showed antioxidant activity in the presence of a positive increase in percent inhibition after mixed Nitric Oxide linearity equation Y = -0.0029 X + 89.113 which show non-linear regression with a correlation coefficient R2= 0.3067 (30.67%). This means that any increase in the concentration of ethanol extract will increase the percent inhibition of 30.67%.


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Antioksidan, Juni 2016

From table 3.2 above Ethanol extract regression results obtained with p = 0.000 smaller than the value of α = 0.05. Thus the concentration of ethanol extract of avocado leaves indicate that the regression correlation between increased concentration (ppm) with a mean percent increase in the value of the inhibitory activity of antioxidant extracts of avocado leaves Ethanol is significant. 3.3

4.

Antioxidant activity of Acid Fraction

Analysis of Variant (ANOVA) From Table 4.1 indicated that the highest average percent inhibition of the antioxidant activity of acid fraction after addition of Nitric Oxide is the concentration of 50 ppm with their mean value of 89.46% and the lowest average value of 1000 ppm with their mean value of 87.32%.

Ethanol extract after being mixed with Nitric Oxide shows the value of p = 0.000 which is smaller than the value of α = 0.05. These results indicate that the hypothesis "There is no antioxidant activity of ethanol extract of avocado leaves by using Nitric Oxide" rejected. This means that the avocado leaf extract obtained via Ethanol has significant antioxidant activity. 3.4

4.2

Linear Regression Test

Duncan's Multiple Range Test

From the table above is based on the results of ANOVA Duncan showed that the concentration of 50 ppm, 100 ppm and 20 ppm were one group differs significantly from kolompok concentration of 200 ppm, 1000 ppm, 10 ppm and 500 ppm. In other words, group concentration of 50 ppm, 100 ppm, and 20 ppm gives kesignifikansian the statistical analysis used.

From Figure 4.1 above shows that the acid fraction of avocado leaves showed antioxidant activity in the presence of a positive increase in percent inhibition after addition of linearity of Nitric Oxide equation Y = -0.0013 X + 88.622 which shows nonlinear regression with a correlation coefficient R2 = 0.2943 (29.43%). This means that the increase in the concentration of acid increases the fraction of the percent inhibition of 29.43% a low correlation.


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Antioksidan, Juni 2016 5. 1 Descriptive Data From table 4.2 above regression results obtained extract fraction Acid with p = 0.224 greater than the value of α = 0.05. Thus the concentration of acid fraction of avocado leaves indicate that the regression correlation between increased concentration (ppm) with a mean percent increase in the value of the resistor acid fraction of avocado leaves is not significant. 4.3 Analysis of Variant (ANOVA)

From Table 5.1 above shows that the highest average percent inhibition fraction of dichloromethane after addition of Nitric Oxide shows that the highest rates are at a concentration of 20 ppm with their mean value of 89.48% and the lowest average value of 1000 ppm with their mean value of 66.52%. 5.2 Linear Regression Test of Dichloromethane Faction

From table 4.3 ANOVA analysis results obtained with p = 0.000 which is smaller than the value of α = 0.05, so the hypothesis which states that "There is no antioxidant activity of the extract of avocado leaves acid fraction using the Nitric Oxide" rejected. This means that the avocado leaves, demonstrated by the fraction of the acid has antioxidant activity. 4.4

Figure 5.1 Percent Inhibition Antioxidant Activity Graph Dichloromethane Fraction Leaves Avocado

Duncan's Multiple Range Test

In table 4.4 can be seen the results of the statistical analysis of test Duncan's multiple range for the antioxidant activity of the fraction of the acid after the addition of Nitric Oxide shows that all kensentrasi fraction acids are in a group of 50 ppm, 20 ppm, 100 ppm, 500 ppm, 10 ppm, 200 ppm, and 1000 ppm. These results can be seen from the low correlation of the concentration of acid fraction of avocado leaves only 29.34%, although not statistically significant Anava is p = 0.000. 5. Antioxidant Activity Dichloromethane Fraction of Avocado Leaves.

From Figure 5.1 above shows that the avocado leaves dichloromethane fractions showed antioxidant activity with an increase in the percent inhibition of Nitric Oxide. Dichloromethane fraction of avocado leaves showed increased antioxidant activity with linearity equation Y = -0,022X + 90.473 which shows linear regression correlation coefficient R ^ 2 = 0.95 (95%). This means that an increase in the concentration of dichloromethane fraction increasing percent inhibition of 95% (high correlation level).

From the table 5.2 above it can be concluded regression results obtained dichloromethane extract fraction after addition of Nitric Oxide shows that the value of p = 0.000 is smaller than the value of α = 0.05.


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Antioksidan, Juni 2016 Thus the concentration of dichloromethane fraction of avocado leaves indicate that the regression correlation between the concentration (ppm) with a mean value of percent inhibition of avocado leaves dichloromethane fraction is significant. 5.3 Analysis of Variant (ANOVA)

From Table 5.3, ANOVA analysis results obtained with p = 0.000 which is smaller than the value of α = 0.05, so the hypothesis which states that "there is a fraction of the antioxidant activity dichloromethane avocado leaves by using Nitric Oxide" rejected. This means that the dichloromethane fraction of avocado leaves have antioxidant activity. 5.4 Duncan's Multiple Range Test

Table 5.4 shows the results of test analysis Duncan's multiple range for the antioxidant activity of the fraction of dichloromethane after addition of Nitric Oxide shows the concentration of 20 ppm, 50 ppm, 100 ppm, 10 ppm and 200 ppm are in the highest group with greatly differing antioxidant activity followed by group concentration of 500 ppm and 1000 ppm groups. This shows that the concentration of dichloromethane fraction at 20, 50, 1000, 10, and 200 ppm give the largest contribution to the rejection of the hypothesis or kesignifikansian level of statistical analysis used. 6. Comparison of Antioxidant Activity 6.1 Data Analysis By Statistics (Multivariate Analysis)

From table 6.1 above can be obtained that the value of p = 0.000 is smaller than the value of α = 0.05. These results suggest that "There is no difference in the antioxidant activity of the type and concentration variations avocado leaf extract using Nitric Oxide", was rejected. It can be concluded that the type extract / fractionation and varying concentrations of avocado leaves in this study are significant, therefore it is performed Duncan's multiple range test. 6.2 Duncan's Multiple Range Test Results in Group Extract

From the table above it can be concluded that the ethanol extract and fractionation acids contributed most to the rejection of the hypothesis or kesignifikansian statistical analysis is done. CONCLUSION 1. 1. From the analysis of variants for Ethanol extract that value of p = 0.000, less than the value α = 0.05, thus the hypothesis that states "There is no antioxidant activity of ethanol extract of avocado leaves by using Nitric Oxide" rejected. These results indicate that ethanol extract of leaves of avocado after the addition of Nitric Oxide has significant antioxidant activity with Nitric Oxide. Duncan's multiple range test results showed that the concentration of 50 ppm, 100 ppm, and 20 ppm is the highest group of Ethanol extracts that give kesignifikansian on the analysis used.


pg. 7

Antioksidan, Juni 2016 2. From the analysis of variants for the fraction of acid that the value of p = 0.000 less than the value α = 0.05, thus the hypothesis that states "There is no acid fraction of the antioxidant activity of avocado leaves by using Nitric Oxide" rejected. These results indicate that the acid fraction of avocado leaves after the addition of Nitric Oxide has no significant antioxidant activity by the method of Nitric Oxide. Duncan's multiple range test results showed that the concentration of 50 ppm, 20 ppm, 100 ppm, 500 ppm, 10 ppm, 200 ppm and 1000 ppm is the highest group of acid fraction which yielded significant results on the analysis used. 3. Dari hasil analisis Varian untuk fraksi Diklorometan bahwa nilai p = 0,000, lebih kecil dari nilai α = 0,05, dengan demikian maka hipotesis yang menyatakan “Tidak terdapat aktivitas antioksidan fraksi Diklorometan daun alpukat dengan menggunakan metode Nitrat Oksida” ditolak. Dari hasil analisis didapat bahwa fraksi Diklorometan Daun alpukat setelah penambahan Nitrat Oksida memiliki aktivitas antioksidan yang signifikan dengan metode Nitrat Oksida. Hasil uji jarak berganda Duncan menunjukkan bahwa konsentrasi 20 ppm, 50 ppm, 100 ppm, 10 ppm, dan 200 ppm adalah kelompok aktivitas antioksidan yang tertinggi dari fraksi Diklorometan yang memberi kesignifikansian pada analisis yang digunakan. 4. 4. From the analysis Manava that the value of p = 0.000 is smaller than the value of α = 0.05. These results suggest that "There is no difference in the antioxidant activity of the type and variety of avocado leaf extract concentration by the method of Nitric Oxide", was rejected. From Duncan's multiple range test results can be concluded that the group extracts and fractions Acid Ethanol is a type / fraction yielded significant results of statistical analysis used. BIBLIOGRAPHY Abraham, Ignatius., Rahul Joshi, Pushpa Pardasani dan R.T. Pardasani (2011). Recent Advances in 1,4Benzoquinone Chemistry. J. Bras.Chem. Soc., Vol. 22. No, 3, 385-421.

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The Antioxidant Activity Extract Leaves Avocado (Persea americana Mill) with Method Nitric Oxide (NO)

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