Effect of Gamma Irradiation on Angiotensin Converting Enzyme Inhibition, Antioxidant Activity, Total Phenolic Compound and Total Flavonoid of Peperomia pellucida Herbs Extract

Introduction: Peperomia pellucida L. Kunth has been reported to have some biological activities such as antihypertensive and antioxidants. Herbal materials susceptible to contamination during processing and storage which can shortens their shelf life. Gamma-irradiation has been used as a method for preservation. Methods: This research aimed to analyze the effect of gamma-irradiation on inhibition activity of angiotensin converting enzyme (ACE), antioxidant activity, total phenol content, total flavonoid, and thin layer chromatography profiles of P. pellucida L. Herbs extract. The extract was irradiated with 60Co gamma rays at 2.5; 5; 7.5; dan 10 kGy. Results: Irradiation up to 10 kGy did not change ACE inhibitory activity and TLC profile. No significant differences were noted in the inhibition activity of ACE and the type of chromatogram profiles between non-irradiated extract and irradiated extracts up to a dose of 10 kGy while total flavonoids showed a significant decrease. In addition, total phenolic content and antioxidant activity showed a significant increase of extracts were irradiated up to 5 kGy and decrease at dose 7,5 kGy and 10 kGy. Gamma-irradiation up to 10 kGy didn’t affect the activity of ACEinhibitor in-vitro while significant difference (P <0.05) of antioxidant activity, total flavonoids and phenolic content of the extract P. Pellucida. Conclusion: In conclusion, gamma irradiation can be used as a preservation method for ethanol extract P. Pellucida L. Kunth herbs.


INTRODUCTION
Herbs are prone to contamination by microorganisms and insect pests, that could occur either during preharvest processing or storage.This shortens their shelf life as well as in rare cases cause major illness. 1 Several reports have demonstrated that gamma irradiation method on herbal medicines is an effective method for decontamination, quarantine barriers in international trade and for improving shelf life due to its efficiency and high penetration. 2,3Gamma rays are a type of electromagnetic radiation which originates within the nucleus of a radioisotope.Irradiation facilities generally use the radionuclide cobalt-60 as the source of gamma rays.The energy threshold for inducing radioactivity in herbs is 5 MeV for gamma sources.Therefore, the energy of gamma rays from cobalt-60 (1.17 MeV and 1.33 MeV) is not sufficient to generate radioactive substances in medicinal plants. 3amma ray irradiation up to 10 kGy dose did not affect the biological activity of the methanol extract of Schizandra chinensis. 4Results of other studies have also known that exposure to gamma -irradiation up to a dose of 10 kGy in 70% ethanol extract of leaves of Nelumbo nucifera is not a significant effect on antioxidant activity and total polyphenol compounds. 5ikewise with Lee, Lee, Bai, Choi, Kim, and Chung (2013) who argued that there was no significant difference in tyrosinase inhibitory activity of the metha-nol extract Eremochloa ophiuroides Munro irradiated gamma rays up to a dose of 10 kGy. 6Peperomia pellucida is reported to have a wide range of biological activities.][9][10][11] The ethanolic extract of the plan has antihypertensive activity through inhibition of the angiotensin-converting enzyme (ACE) in vitro with EC 50 value of 7.17 mg/mL were compared with captopril (13.68 pg / mL). 11Irradiation can influence the levels of phytochemicals depending on the applied dose, moisture of material, exposure time, and the sensitivity of the phytochemicals towards irradiation and the nature of the raw material used.Therefore monitoring both qualitative and quantitative changes in bioactive phytochemicals after irradiation and storage is of importance. 1,12So this study aimed to analyze the effect of gamma-ray irradiation on the ethanol extract of P. Pellucida herbs in order to eliminate contamination, without causing a decrease in its activity as ACE inhibitors.

Materials
The aerial part of Peperomia pellucida L. Kunth.were collected from West Java, Indonesia.The species was identified and authenticated by the Indone-

Sample Preparation
Dried powder was sorted out to separate them from pollutants, blended, then were put through a 25 mesh sieve.

Water Determination
Determination of moisture content was done with toluene distillation method.

Gamma Irradiation
Ethanolic extract of the herbs P. pellucida L. Kunth was divided into two sets.One set was kept as a non-irradiated sample.The other set of samples, packed in a vial sealed with alumunium cap were irradiated in a gamma chamber with a cobalt-60 source to doses of 2.5, 5, 7.5 and 10 kGy at the dose rate of 6.01 kGy/h.Irradiated and non-irradiated samples were stored under ambient conditions.Irradiation was done at Atomic and Nuclear National Bureau, Jakarta.

Plant Extraction
The dried powder was extracted after irradiation process by reflux using 70% ethanol (1:10) for 45 minutes and the procedure was repeated 3 times.All filtrates were combined and concentrated using a rotary vacuum evaporator at 40°C, then dried using oven vacuum at 50 o C.

Free Radical-Scavenging Assay with DPPH
Various concentration of samples was prepared by dissolving the extract using methanol.About 1 mL of each concentration was put in a test tube, added 1 mL of 100 ppm DPPH solution and 2 mL of methanol pa.The mixture was vortexed for 20 seconds and incubated at 37 o C for 30 minutes.The absorbance of each concentration was measured with Uv-Vis spectrophotometer at 515 nm.Quercetin was used as the standard.

ACE Inhibitory Activity Assay
Both types of extract (irradiated and non-irradiated) were subjected to ACE-inhibitory assay using Dojindo ACE Kit-WST.The enzymatic reaction was initiated by the ACE and aminoacylase in the mixture containing 3HB-GGG (3-hydroxybutyrate glycyl glycylglycine) and the ACEinhibitor.The yield of 3HB was monitored indirectly through formazan concentration, which was measured at 450 nm after 10-minute reaction at 25°C.Testing procedures were run according to the manufacturer's instructions using a 96-well plate without modification, and the inhibition rate was calculated based on a comparison of the optical absorbance of samples treated wells (s), control wells (Ac), and blank wells (Ab).Absorbance was measured at 450 nm using the microplate reader.Captopril was used as the standard.

Determination of Total Phenolic Content
Total phenolic content was determined using the Folin-Ciocalteu colorimetric methods. 6Different concentration of gallic acid (30-80 ppm) and sample (3000 ppm) were dissolved in methanol.About 200 μL of each concentration was put into a test tube, and 1.5 mL Folin-Ciocalteu was added.All test tubes were incubated in a dark place at room temperature for 5 minutes.Then 1.5 mL 6% Na 2 CO 3 was added to each test tube and incubated for 105 minutes in a dark place at room temperature.The absorbance of each concentration was measured using UV-Vis spectrophotometer at 740 nm.

Determination of Total Flavonoid Content
Determination of total flavonoid content was done using aluminium chloride colorimetric methods. 7,8 arious concentration of quercetin (20 -120 ppm) and sample (3000 ppm) were dissolved in methanol.About 0.5 mL of each concentration was taken in a test tube, added 1.5 mL methanol, 0.1 mL 10% aluminium chloride, 0.1 mL sodium acetate (1M) and 2.8 mL distillate water into each test tube.Then, the test tubes were incubated at room temperature for 30 minutes.The absorbance of each concentration was measured by Uv-Vis Spectrophotometer at 434 nm.

Thin Layer Chromatography Profile
TLC profiles were performed using silica gel 60 F 254 and dichlormethane: methanol (92 : 8).The extracts were made at 10000 ppm and were spotted on the TLC plate for elusion.TLC was analyzed qualitatively under UV light of 254 nm and 366 nm, also by qualitative densitometry using TLC Scanner 3 with Camag Wincats software.

Statistical Analysis
Data was analyzed using one-way ANOVA and a significant difference was determined by the Tukey test (α = 0.05).Correlation between antioxidant activity, total phenolic compound, and total flavonoid compound was analyzed using Spearman test.

Water Content Determination
Experiment determination of water content of the 70% ethanol extract of P. pellucida Kunth herbs obtained with a value of 14.66%.Low water content can minimize the formation of free radicals OH* and H* from the radiolysis of water molecules in the samples irradiated gamma ray. 13tal Plate Count (TPC) Table 1 showed effect of gamma irradiation on TPC.TPC of the herb extract showed a decrease of microbial growth in all irradiated samples.Absorbed dose of dried herbs 1 kGy to eliminate insects, and 10 kGy to eliminate pathogenic microorganisms.Gamma irradiation can kill bacteria by destroying DNA of bacteria, therefore, obstructing bacteria division. 14

Extraction yields
Extraction yield of non-irradiated and irradiated samples ranged from 21.16 -24.86%.Ethanol was used because of its non-toxic characteristic and is permitted to be used by the department of health of the Republic of Indonesia.Free radical scavenging assay with DPPH Antioxidant activity is shown in EC 50 value as a percentage of inhibition of free radicals.During a test, the purple DPPH radical reduced by antioxidants / reducing compounds (antioxidants hydrogen donor) into pale yellow hydrazine.DPPH radical reactions and antioxidant occur where antioxidants (AH) is the donor molecule and A . is a product of free radicals. 15Table 2 showed effect of gamma irradiation on DPPH radical scavenging activity.
Based on non-parametric data analysis, it is known that there were no significant changes in the dose of 2.5 kGy and 5 kGy compared with non-irradiated samples, but at a dose of 7.5 kGy and 10 kGy significant changes marked by the increase in EC 50 compared with non-irradiated samples.

ACE Inhibitory Activity
ACE inhibitory activity of captopril at ppm was 34.42% and all ethanol extracts were measured at 100 ppm, giving results shown in Table 3.
The data obtained shows there were no significant differences between non-irradiated and radiation processed samples.Having an increasing the value of percent inhibition was possible due to the increased phenolic content could be attributed to the release of phenolic compounds from glycosidic components and the degradation of larger phenolic compounds into smaller ones by gamma irradiation.Having the lease decrease was the 10 kGy irradiated sample, was possible due to ionization effects of irradiation. 16Gamma irradiation may also generate formation of free radicals which induce breakage of chemical bonds , therefore, altering chemical structures or decomposition of phytochemicals which have an ACE inhibitory activity potential. 17,18tal Phenolic Content The increase in total phenolic content can be caused by the release of phenolic compounds from glycosidic components and degradation of phenolic compounds. 19Free phenolic levels may depend on the balance between the release of some phenolic bond to form free and free phenolic degradation by gamma irradiation.Total phenolic content in irradiated and non-irradiated samples can be seen in Table 4.

Total Flavonoid Content
Flavonoids are part of a group of phenolic compounds, which was mentioned earlier, a decrease of phenolic compounds due to gamma-ray irradiation caused by hydroxyl radicals formed from the interaction of gamma rays with the water contained in the sample resulting in hydroxylation reactions on the aromatic ring phenolic compounds and lead to termination of aromatic ring chain.The decrease levels of flavonoids was also allegedly caused by the loss of the aromatic ring of the phenolic compounds in the sample due to gamma irradiation. 20Table 5 showed total flavonoid content of samples after irradiation.

Thin Layer Chromatography (TLC) Profile
TLC profiles of all samples on UV 254 nm and 366 nm were similar.Under 254 nm, the non-irradiated sample gave 9 peaks which were also found in the irradiated samples.After spraying the TLC plate with 0.5% AlCl 3 , 10 spots had a change in color to yellowish and greenish (Figure 1). Figure 2 presented TLC densitogram of samples.The difference of amounts peaks and spotting faded from the non-irradiated and irradiated samples may indicate the formation of a different substance caused by irradiation, gamma ray irradiation generates free radicals which are can cause hydroxylation or degradation of components in the samples. 21Furthermore, decrease meant or increase meant of peak areas of the irradiated samples is thought to be a result of degradation of components into another component. 22Colour change of the spots' fluorescence after applying 0.5% AlCl 3 to greenish yellowish indicates the existence of flavonoids. 23

Correlation Analysis
Based on this correlation test showed that the total phenol compound or the total flavonoid compound was negatively correlated with EC 50 values, but positively correlated with antioxidant activity.Judging from the data results that the antioxidant activity and phenolic experienced a significant decrease in irradiation dose of 7.5 kGy and 10 kGy.As was mentioned earlier that the decrease is thought to be caused by the presence of radicals formed by the interaction of gamma -irradiation with the water contained in the sample.It can be concluded that the compounds that have the antioxidant activity in the sample are phenolic compound.

CONCLUSION
No significant differences were noted in the inhibition activity of ACE between non-irradiated extract and irradiated extracts.The type of chromatogram profiles in irradiated extracts was similar to those of non-irradiated extract.Total phenolic and total flavonoid content didn't change significantly compare to control at dose 5 kGy and 7.5 kGy.There's a correlation between antioxidant activity with total phenolic content but not with total flavonoid content.In conclusion, gamma ray irradiation can be used as a preservation method for ethanol extract of P. pellucida L. Kunth herbs.

Table 5 : Data from a total flavonoid compound of the five samples Sample Total Flavonoid mgQE/g Extract
* Figure 1: TLC profiles of the ethanolic extracts using Silica Gel 60 F 254 and dichloromethane: methanol (92:8) under UV light before (A) and after (B) spraying with AlCl 3 5%.