Antioxidant Properties of Red Betel (Piper crocatum) Leaf Extract and its Compounds

Background: Piper crocatum, also known as red betel, is a potential herbal medicine. Aims: Current study was planned to determine the antioxidant activities of red betel (Piper crocatum Ruiz and Pav.) leaves extract (RBLE) as compared to eugenol and hydroxychavicol compounds. Methods: DPPH radical scavenging, H2O2 scavenging, ABTS reduction, and FRAP reduction assay were carried out. Results: In DPPH scavenging, RBLE showed an IC50 value of 3.98 μg/mL, eugenol of 2.98 μg/mL, and hydroxychavicol of 18.00 μg/mL. Meanwhile, H2O2 scavenging activity showed an IC50 value of RBLE, eugenol, and hydroxychavicol as 186.33 μg/mL, 97.36 μg/mL, and 41.06 μg/mL respectively. ABTS reduction assay showed an IC50 value of 38.43 μg/mL, 181 μg/mL, and 3.10 μg/mL for RBLE, eugenol, and hydroxychavicol respectively. The highest FRAP reduction activity was shown by Eugenol with a concentration of 50 μg/mL which was equal to 424.67 μM Fe (II)/μg. Conclusion: The RBLE and its compounds (eugenol and hydroxychavicol) have antioxidant activity as indicated by the results of the DPPH scavenging, H2O2 scavenging, ABTS reduction, and FRAP reduction assays. However, RBLE had the lowest antioxidant activity compared to other compounds. DOI:10.18311/jnr/2019/23633


Introduction
Molecules or fragment of molecules when they lose an electron in an atomic orbital make them as unstable free radicals. To attain stability, free radicals damage or react with neighboring molecules. External origin of free radicals are X-rays, ozone, cigarette smoke, industrial chemicals, ultraviolet light, and environmental pollutants 1,2 . Imparity between Reactive Oxygen Species (ROS) and the anti-oxidative defense systems cause oxidative stress that could lead to many diseases such as cancer, cardiovascular diseases, rheumatoid arthritis, and atherosclerosis 3 .
Piper betle leaves have antioxidant activity against 1,1-diphenyl-2-picrylhydrazyl (DPPH), hydroxyl (OH), anion superoxide (O 2 -) radicals, and lipid peroxidation assay. P. betle leaf extract was found to be a powerful trapper of OH radicals 4 . P. crocatum, also known as red betel of the family Piperaceae, has a more bitter taste, fragrant aroma, and is a better potential as herbal medicine than the regular betel. However, the volatile oil content and the antimicrobial activity were lowerer 4 . Red betel leaves (P. crocatum) extract and its compound namely, eugenol have antioxidant activities including DPPH scavenging activity, Super Oxide Dismutase (SOD) activity and anticancer activity Journal of Natural Remedies | ISSN: 2320-3358 http://www.informaticsjournals.com/index.php/jnr | Vol 19 (4) | October 2019 against cervical cancer 5 . Red betel has been reported to contain several active compounds such as flavonoids, steroids, tannins, saponins, alkaloids, polyphenolics, quinones, and essential oil 4,6 . Red betel also contains chavicol, chavibetol, carvacrol, caryophyllene, estragole, eugenol, and hydroxychavicol 5 . Hydroxychavicol and eugenol were antifungal compounds 7 . In addition, eugenol at lower concentration has been reported to have antioxidant and anti-inflammatory properties, but at higher concentration exhibits pro-oxidant properties 7 . Hydroxychavicol isolated from the aqueous extract of P. betle was the major phenolic component and has been suggested as antimutagenic, anticarcinogenic, antioxidant, anti-inflammatory, and chemopreventive agent 8 . The extract of red betel has been reported to be active against Colletotrichum gloeosporioides, Candida albicans, and Botryodiplodia theobromae 9 . Betel oil nowadays has been used as an antiseptic component in gels, balms, it is anti-inflammatory and also is a treatment for several diseases 10 . The aim of the study was to observe the antioxidant activity of the red betel (P. crocatum Ruiz and Pav.) leaf ethanol extract as compared to hydroxychavicol and eugenol.

Preparation of Extract
Red betel (P. crocatum Ruiz and Pav.) leaves were obtained from Pabuaran Cilendek Timur, Bogor. The plants were identified by Herbarium Bogoriense, Botanical Field Research Center for Biology-Indonesian Institute of Sciences, Bogor, Indonesia. Red betel leaves were dried using food dehydrator (Zhengzhou Wellknown) then mashed (160 g) and extracted by 500 mL of distilled ethanol 70% by maceration method. Ethanol filtrate was filtered in every 24 h and wastes were remacerated until there was colourless filtrate. The filtrate was concentrated in evaporator at 50 o C (Zhengzhou Well-known, RE-201D) to obtain the red betel leaves extract (RBLE) 11 -13 .

DPPH Scavenging
A total of 200 µL DPPH (Sigma Aldrich D9132) 0.077 mmol in methanol was added with 50 µL of samples including RBLE, hydroxychavicol (Chengdu Biopurify Phytochemical Ltd, BP3020), eugenol (Chengdu Biopurify Phytochemical Ltd, BP0569) with various concentration added on the 96-well microplate. The mixture was incubated for 30 min at room temperature, then the absorbance value was read at 517 nm wavelength using a micro plate reader (Multiskan™ GO Microplate Spectrophotometer, Thermo Scientific). For the sample, 200 µL of DPPH and 50 µL of sample was used, while for negative control 250 µL of DPPH was used, while for blanks, 250 µL of an absolute DMSO was used 11 -14 . Calculation of DPPH scavenging activity was done by using the following formula: DPPH scavenging activity (%) = (A-B)/A × 100 A: control absorbance B: sample absorbance

H 2 O 2 Scavenging
The scavenging of H 2 O 2 was measured based on the method described by Mukhopadhyay et al 15,16 . with slight modification. Each sample contained 60 µL of sample, 12 µL of ferrous ammonium sulphate (1mM, Sigma Aldrich 7783859), and 3 µL of H 2 O 2 (5mM, Merck 1.08597.1000). For the negative control, 12 µL of ferrous ammonium sulphate and 63 µL of DMSO were used, while for blanks, 60 µL of RBLE and 90 µL of DMSO was used.
After adding H 2 O 2, control, sample, and blank solutions was added into the 96-well plate and incubated for 5 min in a dark room at room temperature. Sample and control solutions were added with 75 µL of 1, 10-phenanthrolines and incubated again for 10 min in a dark room at room temperature. Absorbance value was measured at 510 nm wavelength. The percentage of scavenging activities was calculated using the formula: H 2 O 2 scavenging activity (%) = (A-B)/A X 100 A: control absorbance B: sample absorbance

Statistical Analysis
Statistical analysis was performed using SPSS software (version 20.0). Values are presented as Mean ± Standard Deviation. Significant differences between the groups were determined using the Analysis of variance (One Way ANOVA) followed by Tukey's HSD Post-hoc Test.

Results and Discussions
Red betel was extracted using 70% ethanol to extract active compounds that have antioxidant activity, such as flavonoids, polyphenols, alkaloids, and tannins. Ethanol is a polar solvent but less polar than water so it is more efficient in degrading cell walls in red betel leaf 17 .
DPPH is a free radical that belongs to the hydrogen radical group. DPPH is sensitive to light, oxygen, and pH. However, it is stable in a radical form so it may be quite an accurate measurement of antioxidant activity. DPPH free radical can capture hydrogen atoms from the component of antioxidant sample which are mixed and then react to their reduced form and are characterized by reducing intensity of purple DPPH solution with maximum uptake at 517 nm 11 -13 . When antioxidants interact with DPPH, they shift an electron or hydrogen atom (H + ) to DPPH to counteract its free radical character 18 . This process changes the color of the solution from purple to yellow. DPPH assay in this study shows that the radical-scavenging activities of the samples were in the order of hydroxychavicol < RBLE < eugenol. The differences between each concentration were significant ( Figure 1) and the IC 50 value of samples against DPPH free radical scavenging activity is shown in ABTS-reducing activity assay is for measuring the comparative potential of antioxidant to capture ABTS induced by reacting a strong oxidizing agent (potassium permanganate/potassium persulfate) with the ABTS salt. The long wave absorption spectrum was  used to measure decrease of blue-green colored ABTS radical solution by hydrogen-donating antioxidant. 13 The result of ABTS-reducing activity has been shown at Figure 3. All samples showed high activity at a concentration of 50 µg/mL. RBLE has the lowest ABTSreducing activity, indicated by the highest IC 50 value (38.43 µg/mL) compared to hydroxychavicol (3.10 µg/mL) and eugenol compounds (1.81 µg/mL). Based on the result, it can be indicated that RBLE had lower antioxidant activity compared to hydroxychavicol and eugenol (Table 3). ABTS-reducing assay is based on the ability of the antioxidants to quench the ABTS + radical cation. ABTS-reducing activities of samples are RBLE < hydroxychavicol < eugenol. IC 50 of each sample was 38.43 µg/mL (RBLE), 3.10 µg/mL (hydroxychavicol), and 1.18 µg/mL (eugenol). IC 50 of hydroxychavicol and eugenol was equivalent with 20.64 µM and 7.19 µM. According to Widowati et al 11 ., eugenol had IC 50 value of 1.56 µg/mL, equivalent with 9.54 µM.
The Ferric Reducing Antioxidant Power (FRAP) method is based on the decrease of a ferroin analog, the Fe 3+ complex of tripyridyltriazine (Fe(TPTZ) 3+ ) changes to the extremely blue colored Fe 2+ complex (Fe(TPTZ) 2+ ) by antioxidants in acidic medium. Antioxidant reduction of appropriate tripyridyltriazine Fe(III) complex produces absorbance of Fe(II) complex at 593nm 13 . Based on Figure 4, at the highest concentration, each sample had high activity

Conclusion
The red betel leaf extract (P. crocatum Ruiz and Pav.) and its compounds (eugenol and hydroxychavicol)