1. 124 American Scientific Research Journal for Engineering, Technology, and Sciences (ASRJETS) ISSN (Print) 2313-4410, ISSN (Online) 2313-4402 © Global Society of Scientific Research and Researchers http://asrjetsjournal.org/ Potential ABTS Reducing and Anti-collagenase Activity of Scutellarein and Apigenin Feliciaa, Ermi Girsangb* , Ali Napiah Nasutionc, I Nyoman Ehrich Liesterd aFaculty of Medicine, Universitas Prima Indonesia, Medan, North Sumatera, Indonesia bDepartment of Public Health, Faculty of Medicine, Universitas Prima Indonesia, Medan, North Sumatera, Indonesia cDepartment of Tropical Medicine, Faculty of Medicine, Universitas Prima Indonesia, Medan, North Sumatera, Indonesia dDepartment of Physiology, Faculty of Medicine, Universitas Prima Indonesia, Medan, North Sumatera, Indonesia bEmail: ermiunpri@yahoo.co.id Abstract In developed and developing countries, the aging population growth rapidly and the consumption of natural product which rich antioxidant increasing. The aim of this study was to investigate the ABTS reducing activity and anti-collagenase activity of scutellarein and apigenin. This study used ABTS reducing activity for antioxidant assay and collagenase inhibitory activity for antiaging assay. Scutellarein (16.77 μg/ml) has higher antioxidant activity through ABTS reducing activity compared with apigenin (80.19 μg/ml). Scutellarein (99.20 μg/ml) has higher antiaging activity through collagenase inhibitory activity compared with apigenin (211.67 μg/ml). Scutellarein has very strong antioxidant activity through ABTS reduction activity and strong antiaging property particularly as collagenase inhibitor. Apigenin has strong antioxidant activity through ABTS reduction activity and weak antiaging property particularly as collagenase inhibitor. Keywords: Scutellarein; apigenin; ABTS; anti-collagenase; Antioxidant; antiaging. ------------------------------------------------------------------------ * Corresponding author. http://asrjetsjournal.org/ American Scientific Research Journal for Engineering, Technology, and Sciences (ASRJETS) (2019) Volume 57, No 1, pp 124-130 125 1. Introduction In the 20th century, the aging population is growing rapidly both in developed and developing countries [1]. The skin aging process is developed from a combination of intrinsic aging (age-related) and extrinsic aging (environmental factors) [2]. Benchmarks and wrinkles are the combination from ultraviolet (UV) light and poor lifestyle to accelerate the skin depression [2,3]. Ultraviolet radiation (UVR) like UVB can penetrate to the epidermis, destructive the skin function and keratinocyte development [4]. UVA can induce ROS that caused DNA damage [5]. Mitogen-activated protein kinase (MAPK) pathway activated by ROS and increasing MMP production that degrades collagen [6]. In this 21st century, antioxidant intake is increasing [7]. Fruits, vegetables, whole grains, and herbs are rich of antioxidants like vitamin C, vitamin E, carotenoids and phenolics [8]. Antioxidant donates hydrogen atom or proton to free radical so that the oxidation process can be slowed down [9]. Bioactive compounds in the natural sources have been used widely in cosmeceuticals substance to prevent skin aging [10]. The antiaging activity plays a role in stimulating collagen synthesis useful for improving skin appearance by restoring skin firmness and elasticity [11]. Scutellarein, apigenin, linalool, ursolic acid, estragole are rich in Ocimum basilicum L. leaf, they have applied for tropical cream because of their antiaging and antioxidant activity [12,13,14]. Arterbery and his colleagues [15] reported that apigenin use in cosmetical products contributed to improving skin firmness and skin health. Gu and his colleagues [16] reported that scutellarein has antioxidant activity particularly as DPPH scavenging activity. The aim of this study was to investigate the potential of scutellarein and apigenin as antioxidant sources particularly as ABTS reducing activity and antiaging sources particularly as the collagenase inhibitor. 2. Experimental Section Materials Materials used in this study are ABTS (Sigma A1888), Potassium persulfate, Distilled water, PBS 1x, Dimethylsulfoxide (DMSO), Scutellarein, Apigenin, N-[3-(2-Furyl)acryloyl]-leu-gly-Pro-Ala (FALGPA) (Sigma F5135), Collagenase from Clostridium histolyticum (Sigma C8051), Tricine, Calcium chloride, Sodium Chloride and Hydrochloric acid solution. Instrumentation Instruments used in this study are Multiscan Go Reader, Micropipette ((1-10 µl, 50- 200 µl, 100-1000 µl), Tips (1-10 µl, 50- 200 µl, 100-1000 µl), 96 well plate, Falcon tube (15 ml and 50 ml), Analytical Balance, Eppendorf tube 1.5 ml, Vortex, pH meter, Beaker glass, Spatula, and Incubator. 2.1 ABTS Reducing Activity Assay Mix the ABTS 14 mM and potassium persulfate 4.9 mM with a volume ratio 1:1 for 12-16 hours in dark room at room temperature to produce ABTS solution. Add PBS 1X to the mixture until the solution’s absorbance was 0,7 ± 0,02 at 745 nm wavelength. In brief, 2 µL various level of Scutellarein and Apigenin added to each well, then add 198 µL ABTS solution into the well. The absorbance was measured at 745 nm wavelength after the American Scientific Research Journal for Engineering, Technology, and Sciences (ASRJETS) (2019) Volume 57, No 1, pp 124-130 126 plate incubated for 6 minutes at 37°C [17]. % 𝑺𝒄𝒂𝒗𝒆𝒏𝒈𝒊𝒏𝒈 = 𝒄 − 𝒔 𝒄 𝒙 𝟏𝟎𝟎% c: control absorbance (without sample), s: sample absorbance 2.2 Collagenase Inhibitory Activity Assay The mixture consists of 30 µL sample (0.78-50 µg/mL), 10 µL collagenase from Clostridium histolyticum (0.1 mg/mL) and 60 µL Tricine buffer (50 mM Tricine, 10 mM calcium chloride, 400 mM sodium chloride, pH 7.5) incubated for 20 minutes at 37°C. After incubated for 20 minutes, add 20 µL FALGPA substrate (1 mM) into the mixture. Absorbance was measured at 335 nm wavelength [17]. % 𝐼𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 = 𝑐 − 𝑠 𝑐 𝑥 100% c: control absorbance (without sample) s: sample absorbance 3. Result and Discussion 3.1 ABTS Reducing Activity Assay From table 1 showed that at 50.00 µg/mL, the ABTS reducing activity of scutellarein was 99.02 ± 0.06, at 25.00 µg/mL was 66.78 ± 2.99, at 12.50 µg/mL was 39.53 ± 1.88, at 6.25 µg/mL was 34.57 ± 4.94, at 3.13 µg/mL was 31.36 ± 2.90, at 1.56 was 25.42 ± 2.56. at 50.00 µg/mL, the ABTS reducing activity of apigenin was 30.88 ± 0.42, at 25.00 µg/mL was 16.93 ± 0.14, at 12.50 µg/mL was 10.42 ± 0.72, at 6.25 µg/mL was 4.26 ± 0.51, at 3.13 µg/mL was 2.47 ± 0.25, at 1.56 was 0.55 ± 0.09. Table 1 indicated that an increase in the concentration level was directly proportional to the increase of the ABTS reducing activity. Table 1: ABTS reducing activity of scutellarein and apigenin Final concentration (µg/ml) Mean of ABTS reducing activity (%) Scutellarein Apigenin 50.00 99.02 ± 0.06e 30.88 ±0.42e 25.00 66.78 ± 2.99d 16.93 ± 0.14d 12.50 39.53 ± 1.88c 10.42 ± 0.72c 6.25 34.57 ± 4.94b,c 4.26 ± 0.51b 3.13 31.36 ± 2.90a,b 2.47 ± 0.25a,b 1.56 25.42 ± 2.56a 0.55 ± 0.09a American Scientific Research Journal for Engineering, Technology, and Sciences (ASRJETS) (2019) Volume 57, No 1, pp 124-130 127 Data were presented as mean ± standard deviation. Different small letters in the same column are significant at P < 0.05 (Tukey HSD post hoc test). From table 2 showed the IC50 value of ABTS reducing activity from scutellarein (16.77 ± 0.15 µg/mL) was lower than apigenin (80.19 ± 0.99 µg/mL). The IC50 is the concentration of scutellarein and apigenin needed to reduce half of ABTS. Table 2 indicated that scutellarein needed 16.77 ± 0.15 µg/mL and apigenin needed 80.19 ± 0.99 µg/mL to reduce ABTS activity. Based on the result indicated that scutellarein had ABTS reducing activity higher than apigenin. Table 2: IC50 value of ABTS reducing activity of scutellarein and apigenin Sample Equation R2 IC50 (µg/mL) IC50 (µg/mL) Scutellarein (1st repetition) Y = 1.5219x + 24.292 0.98 16.89 16.77 ± 0.15 Scutellarein (2nd repetition) Y = 1.5369x + 24.474 0.97 16.61 Scutellarein (3rd repetition) Y = 1.4843x + 25.038 0.99 16.82 Scutellarein (mean) Y = 1.5144x + 24.601 0.98 16.77 Apigenin (1st repetition) Y = 0.6104x + 1.0496 0.98 80.19 80.19 ± 0.99 Apigenin (2nd repetition) Y = 0.6108x + 0.7413 0.99 80.65 Apigenin (3rd repetition) Y = 0.6269x + 0.6366 0.99 78.74 Apigenin (mean) Y = 0.616x + 0.8091 0.99 80.19 The IC50 was used to classified antioxidant activity of scutellarein and apigenin. If the IC50 less than 50 µg/mL is very strong antioxidant, 50-100 µg/mL is a strong antioxidant, 101-150 µg/mL is a moderate antioxidant, over than 150 µg/mL is a weak antioxidant [3]. Li and his colleagues [18] reported that the IC50 value of scutellarein was 18.3 ± 1.2 µM, which indicated that scutellarein has very strong antioxidant activity. Martinez and his colleagues [19] reported that the IC50 value of apigenin was 379.7 µg/mL, which indicated that apigenin isolated from Alchornea coelophylla pax & k. Hoffm. leaf extract has weak antioxidant activity. From the study, scutellarein has antioxidant activity through ABTS reducing activity higher than apigenin. Apigenin plays a major role in protecting cells from the breakdown of DNA double-stranded caused by oxidative stress [20]. Scutellarein plays a role in transferred electron to quench the free radical and reduced the activity [18]. 3.2 Collagenase inhibitory activity assay From table 3 showed that at 250 µg/mL, the collagenase inhibitory activity of scutellarein was 92.83 ± 0.37, at 125 µg/mL was 60.63 ± 1.47, at 62.5 µg/mL was 50.57 ± 1.92, 31.25 µg/mL was 26.78 ± 0.54, at 15.625 was 20.29 ± 1.00, at 7.8125 was 17.18 ± 0.18 At 250 µg/mL, the collagenase inhibitory activity of apigenin was 54.05 ± 7.98, at 125 µg/mL was 42.71± 0.31, at 62.5 µg/mL was 33.41 ± 0.50, 31.25 µg/mL was 29.48 ± 1.83, at 15.625 was 25.12 ± 1.17, at 7.8125 was 21.10 ± 0.06. Table 3 indicated that an increase in the concentration level was directly proportional to the increase of the collagenase inhibitory activity. American Scientific Research Journal for Engineering, Technology, and Sciences (ASRJETS) (2019) Volume 57, No 1, pp 124-130 128 Table 3: Anti-collagenase activity of scutellarein and apigenin Final concentration (ug/ml) Mean of collagenase inhibitory activity (%) Scutellarein Apigenin 250 92.83±0.37f 54.05±7.98d 125 60.63±1.47e 42.71±0.31c 62.5 50.57±1.92d 33.41±0.50b 31.25 26.78±0.54c 29.48±1.83ab 15.625 20.29±1.00b 25.12±1.17ab 7.8125 17.18±0.18a 21.10±0.06a Data were presented as mean ± standard deviation. Different small letters in the same column are significant at P < 0.05 (Tukey HSD post hoc test). From table 4 showed the IC50 value of collagenase inhibitory activity from scutellarein (99.20 ± 0.94 µg/mL) was lower apigenin (211.67 ± 46.81 µg/mL). the IC50 is the concentration of scutellarein and apigenin needed to inhibit half of collagenase. Table 4 indicated that scutellarein needed 99.20 ± 0.94 µg/mL and apigenin needed 211.67 ± 46.81 µg/mL to inhibit collagenase activity. Based on the result indicated that scutellarein had collagenase inhibitory activity higher than apigenin. Table 4: IC50 value of anti-collagenase from scutellarein and apigenin Sample Equation R2 IC50 (µg/mL) IC50 (µg/mL) Scutellarein (1st repetition) Y = 0.3061x + 19.46 0.96 99.77 99.20 ± 0.94 Scutellarein (2nd repetition) Y = 0.309x + 19.685 0.94 98.11 Scutellarein (3rd repetition) Y = 0.3091x + 19.179 0.94 99.71 Scutellarein (mean) Y = 0.3081x + 19.441 0.95 99.19 Apigenin (1st repetition) Y = 0.1536x + 22.724 0.98 177.58 211.67 ± 46.81 Apigenin (2nd repetition) Y = 0.0921x + 25.59 0.80 265.04 Apigenin (3rd repetition) Y = 0.1407x + 22.93 0.98 192.40 Apigenin (mean) Y = 0.1288x + 23.748 0.95 203.82 The IC50 was used to classified anti-collagenase activity of scutellarein and apigenin. If the IC50 less than 50 µg/mL is very strong anti-collagenase, 50-100 µg/mL is a strong anti-collagenase, 101-150 µg/mL is a moderate anti-collagenase, over than 150 µg/mL is a weak anti-collagenase [3] Kristina and his colleagues [21] reported that the IC50 value of lycopene was 85.09 ± 1.81 µg/mL, which indicated that lycopene has strong anti- collagenase activity. Widowati and his colleagues [22] reported that the IC50 value of myricetin was 11.65 ± 2.08 µg/mL, which indicated that myricetin has very strong antioxidant. From our finding, that bioactive compounds did not always have strong anti-collagenase activity. According to Arterbery and his colleagues [23] in tropical products apigenin improve firmness, elasticity, maintain hydration, decrease MMP-1 and MMP-2 production. The bioactive compound can boost the collagen synthesis and reduce the collagenase activity American Scientific Research Journal for Engineering, Technology, and Sciences (ASRJETS) (2019) Volume 57, No 1, pp 124-130 129 through the collagen breakdown [21]. Clinical application of the present study is to investigate the antiaging effect of scutellarein in topical product compared with apigenin. 4. Conclusions Scutellarein has very strong antioxidant activity through ABTS reduction activity and strong antiaging activity particularly as collagenase inhibitor, apigenin has strong antioxidant activity through ABTS reduction activity and weak antiaging property particularly as collagenase inhibitor. Scutellarein has antioxidant activity through ABTS reduction activity and antiaging activity through collagenase inhibitory activity better than apigenin. Acknowledgements The author would say thanks to Dr. Ermi Girsang, SKM., M.Kes. and dr. Ali Napiah Nasution, M.KT as mentor, Dr. dr. 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