Home | About PM | Editorial board | Search | Ahead of print | Current Issue | Archives | Instructions | Subscribe | Advertise | Contact us |  Login 
Pharmacognosy Magazine
Search Article 
  
Advanced search 
 


 
  Table of Contents  
ORIGINAL ARTICLE
Year : 2022  |  Volume : 18  |  Issue : 79  |  Page : 699-706  

Antioxidant and anti-wrinkle effects of Orostachys japonicus extracts as anti-aging cosmetic agents


1 Department of Smart Foods and Drugs; Pharmaceutical Engineering, Inje University, Gimhae, Gyeongnam, Republic of Korea
2 Department of Smart Foods and Drugs; Biomedical Laboratory Science, Inje University; Phytoeco, Inc, Gimhae, Gyeongnam, Republic of Korea
3 Department of Smart Foods and Drugs; Biomedical Laboratory Science, Inje University, Gimhae, Gyeongnam, Republic of Korea

Date of Submission23-Sep-2021
Date of Decision06-Apr-2022
Date of Acceptance12-Jul-2022
Date of Web Publication19-Sep-2022

Correspondence Address:
Dong-Seok Lee
Department of Biomedical Laboratory Science, Inje University, Gimhae - 50834, Gyeongnam
Republic of Korea
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/pm.pm_497_21

Rights and Permissions
   Abstract 


Background: Orostachys japonicus has traditionally been used for antifever, anti-inflammation, and anticancer. O. japonicus has not yet been studied for cosmetic effects. Objectives: In this study, O. japonicus extracts were investigated to verify the possibility of them as anti-aging cosmetic agents. Materials and Methods: O. japonicus were used to prepare experimental extracts using 90% methanol (MeOH) or ethanol (EtOH) as solvent. Safety and efficacy tests applied in this work are as follows: Folin-Denis' method for total phenolic contents, liquid chromatography for component analysis, 3-[4,5-dimethyl thiazol-2-yl]-5-(3-carboxymethoxyphenyl) -2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay for cytotoxicity, 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and ferric reducing antioxidant power (FRAP) assay for antioxidant activity, biochemical inhibition assays of elastase activity, collagenase activity, matrix metalloproteinase-1 (MMP-1) and MMP-3 mRNA expression), and MMP-1 protein production for anti-wrinkle activity, and tyrosinase activity inhibition assay for whitening activity. Results: This research revealed that O. japonicus extracts contained various flavonoids, showed no adverse cytotoxicity up to 300 or 500 μg/mL in HS68 and B16F10, exhibited significant antioxidant activity, exerted remarkable anti-wrinkle activity, and represented useful whitening activity. Conclusion: This study suggested that O. japonicus extracts could be utilized to develop anti-aging cosmetic agents exerting useful whitening activity as well as excellent antioxidant and anti-wrinkle activities.

Keywords: Anti-aging, antioxidant, anti-wrinkle, MMP-1, MMP-3, Orostachys japonicus


How to cite this article:
Noh DY, Hyun JY, Kim D, Lee DS. Antioxidant and anti-wrinkle effects of Orostachys japonicus extracts as anti-aging cosmetic agents. Phcog Mag 2022;18:699-706

How to cite this URL:
Noh DY, Hyun JY, Kim D, Lee DS. Antioxidant and anti-wrinkle effects of Orostachys japonicus extracts as anti-aging cosmetic agents. Phcog Mag [serial online] 2022 [cited 2022 Sep 26];18:699-706. Available from: http://www.phcog.com/text.asp?2022/18/79/699/356401



SUMMARY

  • In this study, the antioxidant activity, anti-wrinkle activity, and whitening activity of Orostachys japonicus extract were comprehensively investigated. As a result of the study, cytotoxicity was not observed, and the antioxidant activity and anti-wrinkle activity were excellent. In addition, the whitening activity showed a significant level. Therefore, the extracts of O. japonicus are expected to be utilized as cosmetic materials for anti-aging.




Abbreviations used: OJ: Orostachys japonicus; MeOH: Methanol; EtOH: Ethanol; DPPH: 2,2-diphenyl-1-picrylhydrazyl; ABTS: 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid); FRAP: Ferric reducing antioxidant power; FALGPA: 2-furanacryloyl-Leu-Gly-Pro-Ala; MMP-1: Matrix metalloproteinase-1; UPLC Q-TOF MS: Ultra-high performance liquid chromatography with quadrupole time-of-flight mass spectrometry


   Introduction Top


The aging of the skin can be divided into intrinsic and extrinsic ones. Intrinsic skin aging is caused by the passage of time, whereas extrinsic aging is usually by exposure to sunlight.[1] When skin is exposed to UV, reactive oxygen species (ROS) is produced, which accelerates skin aging by increasing the expression of matrix metalloproteinases (MMPs), impairing the extracellular matrix (ECM) that keeps the three-dimensional structure of the skin.[2],[3] Antioxidants produced in the body can remove overproduced ROS byproducts.[4] However, the antioxidant effect is decreased when aging and overexposure to UV go along. Recently, natural antioxidants have been developed in foods, cosmetics, and pharmaceuticals to replace some synthetic antioxidants that are limited in their use due to the possibility of carcinogenicity.[5],[6]

Collagen and elastin belonging to the ECM protein are produced by fibroblasts and help maintain skin elasticity, flexibility, and tension.[7] In fibroblasts, ECM is reduced by UV, that is, collagen biosynthesis is lowered and elastin denaturation is elevated.[8] In addition, MMPs denature and degrade ECM such as collagen, elastin, proteoglycan, and fibronectin, thereby destroying the epidermal-skin boundary and accelerating dermis degradation, and finally inducing wrinkle formation.[9]

Melanin, a pigment biosynthesized from l-tyrosine in melanocytes, plays an important role in protecting skin cells by absorbing UV.[10],[11] Decreased melanogenesis results in sunburn, mottling, and gray hair due to skin damage by UV exposure.[12] The key enzyme in the biosynthesis of melanin is tyrosinase, which is involved in the synthesis of l-dopaquinone from l-tyrosine, consequently resulting in the production of pheomelanin and eumelanin.[13]

Recently, the anti-aging and antioxidant effects of plant extracts have been studied widely. Antioxidant activity was reported in extracts from Coffea arabica and Quercus robur.[14],[15] Elastase inhibition activity was shown in Callistemon lanceolatus, Morinda citrifolia, and Glycine max.[16],[17],[18] Whitening effect (tyrosinase inhibition) was reported in Rhodiola rosea, Tagetes erecta, and Cassia fistula.[19],[20],[21]

Orostachys japonicus, a medicinal herb is distributed in East Asia.[22] O. japonicus has been utilized in traditional medicine for antifever, anti-inflammation, hemostasis, and anticancer.[22],[23],[24] In previous studies this revealed hypolipidemic, hypoglycemic, antiulcerogenic, anti-inflammatory, anticancer, bone-protective, hepatoprotective, and immunomodulating effects.[23],[24],[25],[26],[27],[28],[29],[30],[31],[32],[33],[34],[35],[36],[37],[38]

Since functional cosmetic effects of O. japonicus have not been studied, we focused on the investigation of antioxidant, anti-wrinkle, and skin whitening effects of methanol (MeOH) or ethanol (EtOH) extracts from O. japonicus to identify their potential of them as anti-aging cosmetic agents.


   Materials and Methods Top


Materials

O. japonicus was obtained from greenhouse in Miryang, Gyeongnam, Korea. DMEM, TRIzol, and FBS were obtained from Invitrogen Life Technologies (Carlsbad, CA, USA). 3- [4,5-dimethyl thiazol-2-yl]-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)- 2H-tetrazolium (MTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 2, 4, 6-Tri (2-pyridyl)-s-triazine (TPTZ), trichloroacetic acid (TCA), dimethyl sulfoxide (DMSO), the oligopeptide 2-furanacryloyl-Leu-Gly-Pro-Ala (FALGPA), epigallocatechin gallate (EGCG), Folin-Denis' reagent, ferric chloride, potassium ferricyanide, potassium persulfate, ascorbic acid, elastase from porcine pancreas, N-succinyl-(ala) 3-p-nitroanilide, l-tyrosine, tyrosinase from mushroom, arbutin, and ursolic acid were obtained from Sigma Aldrich (St. Louis, MO, USA). Recombinant human tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ) from R&D Systems, Inc. (Minneapolis, MN, USA). TOPscriptTM cDNA synthesis kit and TOPrealTM qPCR PreMIX (SYBR Green, low ROX) from Enzynomoics, Inc. (Daejeon, Korea). AccuPower® PCR PreMix from Bioneer corp. (Daejeon, Korea). Human MMP-1 ELISA kit from Abcam (Cambridge, MA, USA). Ethics committee number: INJE 2020-03-006 Date of approval : April 20, 2020.

Preparation of O. japonicus extracts

O. japonicus was separately extracted at 40°C with two different solvents, 90% methanol or 90% ethanol for 24 h. Each extract was prepared using filter paper (Whatman, UK) and evaporated under a vacuum, and then freeze-dried for further study. The residue obtained from methanol and ethanol were named OJ_MeOH and OJ_EtOH, respectively.

Total phenolic contents assay

The phenolic contents were determined by Folin–Denis' reagent. O. japonicus extracts (200 μL) and Folin–Denis' reagent (200 μL) were mingled and reacted at room temperature for 3 min. Furthermore, 2 M sodium carbonate (400 μL) and distilled water (200 μL) were added additionally. After 30 min, 420 nm of absorbance was assayed with microplate spectrophotometer (Power wave XS2, BioTek, USA). Phenolic contents were represented as gallic acid equivalents (GAE) in mg/g of dried extracts.

UPLC Q-TOF MS analysis

The extracts were analyzed by ultra-high performance liquid chromatography with quadrupole time-of-flight mass spectrometry.

(UPLC Q-TOF MS). Extracts were analyzed using an ACQUITY UPLC H-class system (Waters, USA). Mass spectrometry data were gained on the range m/z 100–1200. An ACQUITY UPLC BEH C18 column (2.1 100 mm, 1.7 μm, Waters, USA) was applied. The solvent system consisted of A (0.1% formic acid in water, v/v) and B (0.1% formic acid in acetonitrile, v/v) with the increasing concentration elution applied. The flow rate of solvent was kept at 0.3 mL/min, and 35°C was chosen as the operating temperature.

Cytotoxicity assay

The B16F10 melanoma cells were provided from Korean cell line bank (Seoul, South Korea). HS68 human fibroblast cells were obtained from American Type Culture Collection (ATCC) (Manassas, VA, USA). DMEM containing 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, and 2 mM l-glutamine were used for both cells. The cells were grown at 37°C under the condition of 5% CO2. When the confluence of cells was above 70%, subculture was performed using 0.5 mM EDTA and 0.05% trypsin. The cytotoxicity of O. japonicus extracts was measured by MTS assay, which was conducted by modifying the protocol of Mosmann method.[38] All extracts were dissolved in DMSO. The cells were put on 96-well plate at the density of 5 × 104 and cultured for 24 h. Cell culture medium was removed and rinsed three times with phosphate-buffered saline (PBS). Media containing extracts (200 μL) were applied to cells, and after 24 h, 20 mg/mL of MTS was put to every well and they were kept for 2 h and measured using a microplate spectrophotometer at a wavelength of 450 nm.

Assay of antioxidant activity

Antioxidant activity of O. japonicus extracts was measured by DPPH free radical scavenging activity assay, ABTS free radical scavenging activity assay, and FRAP assay. DPPH scavenging activities needed 0.2 mM of DPPH in absolute ethanol.[39] DPPH (100 μL) was put into extracts (200 μL), and the mixtures were kept at 25°C. Then, 517 nm of absorbance was assessed by microplate spectrophotometer. l-ascorbic acid was chosen as a positive control.

ABTS scavenging activity assay was performed as follows: an equal volume of 2.45 mM potassium persulfate and 7 mM ABTS was mixed and left for 24 h in the dark to obtain the ABTS • + radical solution. It was mixed with 50% methanol to the absorbance of 0.700 (± 0.05) at 745 nm. ABTS scavenging activity was assessed by adding 20 μL of extract to 180 μL of ABTS solution. l-ascorbic acid was chosen as a positive control. Doses (μg/mL) of DPPH and ABTS at 50% scavenging were expressed as IC50 values.

FRAP assay was done in the following way: FRAP reagent was produced by adding 0.3 M of acetate buffer (pH 3.6) to 20 mM ferric chloride and 10 mM TPTZ at the ratio of 10: 1: 1 (v/v/v). The extracts (20 μL) were added to the FRAP reagent (180 μL), and the absorbance at the wavelength of 593 nm was assessed by a microplate spectrophotometer. FRAP values were represented as ascorbic acid equivalents antioxidant capacity (AEAC) in mg/g of dried extracts.

Assay of elastase and collagenase inhibition activities

Anti-wrinkle enzyme inhibition activities of O. japonicus extracts were determined by elastase and collagenase inhibition activities. Elastase inhibition activity was assessed by N-succinyl-(ala) 3-p-nitroanilide as a substrate. Briefly, 0.5 U/mL of elastase was prepared in 50 mM tris-HCl buffer (pH 8.6). The substrate was dissolved at 2 mM in tris-HCl buffer. The extract (20 μL) was mixed with the substrate (30 μL) and tris-HCl buffer (85 μL), and finally, elastase (15 μL) was applied in 96-well plate. The mixtures were left at 25°C, and 410 nm of the absorbance was assessed with the microplate spectrophotometer. The elastase inhibition activity was yielded using the following equation:

Elastase inbition activity (%) = [1 -(Exp.-Blank)/ Control] × 100

Exp: Absorbance of a sample containing elastase

Blank: Absorbance of a sample lacking elastase

Control: Absorbance of a solvent containing elastase

Collagenase inhibition activity was assayed using Clostridium histolyticum collagenase and FALGPA as an enzyme and a substrate. The assay was prepared in 0.05 M tricine buffer (0.001 M CaCl2 and 0.4 M NaCl, pH 7.5). Collagenase (1 U/mL) and FALGPA (2 mM) were dissolved in 0.05 M tricine assay buffer. The extracts (30 μL) were incubated with collagenase (10 μL) and tricine buffer (60 μL) at 37°C for 20 min. Then FALGPA (20 μL) was put in the mixture. EGCG was chosen as a positive control. Collagenase activity was assessed at 340 nm with a microplate spectrophotometer.

Assay of mRNA expressions of MMPs

Inhibition of mRNA expressions of MMPs by O. japonicus extracts in HS68 cells was assayed using RT-PCR and real-time RT-PCR. HS68 cells were put at about cells/well. Twenty-four hour later, 10 ng/mL TNF-α and O. japonicus extracts were added and cultured for 18 h. The total RNAs were obtained from HS68 cells by TRIzol reagent used in the standard procedure. cDNA was produced from 2 μg of total RNA using TOPscriptTM cDNA synthesis kit. RT-PCR was conducted using the AccuPower® PCR PreMix. The primer was synthesized in the following way: MMP-1: 5'-CTGAGGGTCAAGCAGACATC-3' (forward) and 5'-GCTAGGGTACATCAAAGCCC-3' (reverse); MMP-3: 5'-CACTCACTCACAGACCTGAC-3' (forward) and 5'-CCAGCTCGTACCTCATTTCC-3' (reverse); GAPDH: 5'-ATCATCAGCAATGCCTCCTG-3' (forward) and 5'-CCTGCTTCACCACCTTCTTG-3' (reverse). RT-PCR was conducted under the condition of 30 cycles of 95°C for 30 s, 57°C annealing for 30 s, and 72°C extension for 45 s. PCR samples were confirmed using 1.2% agarose gel.

Real-time RT-PCR was conducted using the TOPrealTM qPCR PreMIX (SYBR Green, low ROX). All quantitations were normalized to endogenous control, GAPDH.

Assay of inhibition of MMP-1 protein production

Inhibition of MMP-1 protein production by O. japonicus extracts was assayed using ELISA. HS68 cells were put at about 1 ×105 cells/well. Twenty-four hour later, 10 ng/mL TNF-α and O. japonicus extracts were added and incubated for 18 h. The level of MMP-1 production was assayed by Human MMP-1 ELISA kit.

Assay of tyrosinase inhibition activity

l-tyrosine and tyrosinase obtained from mushrooms were used as a substrate and an enzyme. In addition, 3 mM l-tyrosine (20 μL) and 0.1 M phosphate buffer (pH 6.8, 200 μL) were mixed with the extract (60 μL). Furthermore, 2,000 U/mL tyrosinase (20 μL) was put additionally and kept at 37°C. The absorbance of the mixtures was assessed by a spectrophotometer at the wavelength of 490 nm.

Statistical analysis

The data were presented as means ± standard deviation (SD). Statistical differences were determined with Student's t test using GraphPad Prism 5 (San Diego, CA, USA). P values <0.05 were regarded to be significant.


   Results Top


Total phenolic contents and cytotoxicity

The total phenolic contents of O. japonicus extracts expressed as GAE are exhibited in [Table 1]. The contents were 202.5 ± 4.9 mg GAE/g in OJ_MeOH and 206 ± 12.0 mg GAE/g in OJ_EtOH. The contents were similar in OJ_MeOH and OJ_EtOH.
Table 1: Total phenolic contents of O. japonicus extracts expressed as GAE

Click here to view


MTS assay was performed using B16F10 and HS68 cells to examine the cytotoxicity of O. japonicus extracts. When the extracts were applied at each dose ranging from 100 to 1,000 μg/mL, B16F10 cells showed minimum cytotoxicity up to 500 μg/mL and HS68 cells exhibited minimum cytotoxicity up to 300 μg/mL [Figure 1].
Figure 1: Cytotoxicity of OJ_MeOH and OJ_EtOH in (a) B16F10 cells, and (b) HS68 cells. The data measured by MTS assay were expressed as means ± SD (n = 3). ***P < 0.001 compared with control. OJ_MeOH, methanol extract of O. japonicus; OJ_EtOH, ethanol extract of O. japonicus

Click here to view


UPLC Q-TOF MS analysis

Bioactive compounds of OJ_MeOH and OJ_EtOH were identified by UPLC Q-TOF MS, and data were shown in [Figure 2] and [Figure 3], and [Table 2]. The compounds with their own structure, retention time, molecular weight formula were presented [Figure 3] and [Table 2]. This analysis identified eight peaks; rutoside, hyperoside, isoquercitrin, epicatechin gallate, astragalin, afzelin, quercetin, and kaempferol in OJ_MeOH [Figure 2]a and OJ_EtOH [Figure 2]b.
Figure 2: Base peak intensity chromatograms of (a) OJ_MeOH and (b) OJ_EtOH

Click here to view
Figure 3: Structures of compounds identified in UPLC Q-TOF MS analysis of OJ_MeOH and OJ_EtOH

Click here to view
Table 2: UPLC Q-TOF MS analysis of O. japonicus extracts

Click here to view


Antioxidant activity

Antioxidant activities of O. japonicus extracts measured by DPPH, ABTS, and FRAP assay were presented in [Table 3]. For the DPPH assay, IC50 values of OJ_MeOH, OJ_EtOH, and ascorbic acid were 16.6 ± 0.8, 23.7 ± 0.6, and 74.5 ± 3.4 μg/mL, respectively. Antioxidant effects of O. japonicus extracts were much better than ascorbic acid, which is widely used as a reference antioxidant agent. IC50 values of ABTS assay were 174 ± 13.9 (OJ_MeOH), 233.5 7 ± 20.1 (OJ_EtOH), and 64 ± 5.3 μg/mL (ascorbic acid), respectively. Compared with DPPH scavenging activity of O. japonicus extracts, ABTS activity was relatively low. FRAP values of OJ_MeOH and OJ_EtOH were 292.4 ± 50 and 166.7 ± 22.7 mg AEAC/g, respectively.
Table 3: Antioxidant activities of O. japonicus extracts measured by different methods

Click here to view


Elastase and collagenase inhibition activities

Anti-wrinkle enzyme inhibition activities of O. japonicus extracts were measured by elastase and collagenase inhibition activities. IC50 values of elastase inhibition activity of OJ_MeOH, OJ_EtOH, and EGCG were 73.2 ± 9.9, 42.6 ± 2.9, and 258 ± 33.9 μg/mL, respectively [Table 4], suggesting that OJ_MeOH and OJ_EtOH exhibited remarkable elastase inhibition activities. IC50 values of collagenase inhibition activity of OJ_MeOH, OJ_EtOH, and EGCG were 145.1 ± 4.2, 120.3 ± 6.7, and 774.5 ± 71.2 μg/mL, respectively [Table 4]. O. japonicus extracts showed distinct collagenase inhibition activities.
Table 4: Inhibition activity of O. japonicus extracts on elastase and collagenase

Click here to view


Expression of mRNA expressions of MMPs

RT-PCR and real-time RT-PCR were utilized to confirm the effect of O. japonicus extracts on the transcription level of MMP-1 and -3. When TNF-α was applied in HS68 human fibroblast cells, expression of MMP-1 and -3 increased. In RT-PCR, the expressions of MMP-1 and -3 induced by TNF-α were suppressed by adding OJ_MeOH and OJ_EtOH [Figure 4]. Real-time RT-PCR was also conducted to quantitatively assess MMP-1 and -3 transcription, which supported RT-PCR results obtained previously. When compared with the TNF-α treating control group, the inhibition rate of MMP-1 transcription by OJ_MeOH and OJ_EtOH was 97.6% and 94.4% at 300 μg/mL, respectively [Figure 5]a. In addition, the inhibition rate of MMP-3 expression by OJ_MeOH and OJ_EtOH was 95.3% and 89.1% at 300 μg/mL, respectively [Figure 5]b.
Figure 4: Measurement of effects of OJ_MeOH and OJ_EtOH on mRNA expressions of MMP-1 and MMP-3 using (a) RT-PCR, (b) MMP-1 using real-time PCR, and (c) MMP-3 using real-time PCR

Click here to view
Figure 5: Measurement of effects of OJ_MeOH and OJ_EtOH on mRNA expressions of (a) MMP-1 and (b) MMP-3 by real-time RT-PCR. GAPDH was used as internal standard. The results were expressed as means ± SD (n = 3). **P < 0.01, ***P < 0.001 compared with TNF-α-treated control

Click here to view


Inhibition of MMP-1 protein production

To confirm whether O. japonicus extracts lower the production level of MMP-1 provoked by TNF-α treatment, the production of MMP-1 was measured by ELISA. The reduction rate of MMP-1 production by OJ_MeOH and OJ_EtOH was conspicuously 97.9% and 97.2% at 300 μg/mL, respectively [Figure 6].
Figure 6: Measurement of effects of OJ_MeOH and OJ_EtOH on MMP-1 production in HS68 cells using ELISA. The results were expressed as means ± SD (n = 3). **P < 0.01, ***P < 0.001 compared with TNF-α-treated control

Click here to view


Tyrosinase inhibition activity

Tyrosinase inhibition activity was assayed to examine the skin whitening effect of O. japonicus extracts. IC50 values of OJ_MeOH and OJ_EtOH were 775.6 ± 95.5 and 1,470.1 ± 207.2 μg/mL, respectively [Table 5]. Tyrosinase inhibition activities of O. japonicus extracts were inferior to the positive control, arbutin [Figure 7].
Figure 7: Tyrosinase inhibition of OJ_MeOH, OJ_EtOH, and arbutin. The percent inhibitions were expressed as means ± SD (n = 3)

Click here to view
Table 5: Inhibition activity of O. japonicus extracts on tyrosinase

Click here to view



   Discussion Top


This study explored the antioxidant, anti-wrinkle, and skin whitening effects of O. japonicus MeOH and EtOH extracts. The total phenolic contents of OJ_MeOH and OJ_EtOH were similar. O. japonicus extracts are known to include phenolic compounds such as quercetin, kaempferol, myricetin, and epicatechin gallate.[29],[40],[41],[42] OJ_MeOH and OJ_EtOH were identified to contain rutoside, hyperoside, isoquercetrin, epicatechin gallate, astragalin, afzelin, quercetin, and kaempferol as exhibited in [Figure 2] and [Figure 3], and [Table 2]. The antioxidant effect is closely related to anti-aging cosmetic products. Free radical scavenging activities using DPPH and ABTS were performed to measure the antioxidant activities of O. japonicus extracts. Antioxidant activities were measured by different methods reflecting different antioxidant mechanisms.[15] DPPH is dissolved only in organic solvents, and hydrophilic samples are not suitable to determine antioxidant activity using this method.[43] ABTS free radical scavenging assay can measure both hydrophobic and hydrophilic samples.[44] FRAP assay is based on the reduction power of samples.[45] The antioxidant effect is generally correlated with antiwrinkle and whitening effects. Previous research has reported a linear correlation between phenolic contents and several antioxidant activities.[46],[47] Because OJ_MeOH and OJ_EtOH contained various phenolic compounds and showed potent antioxidant activities, O. japonicus extracts were considered to be utilized as functional cosmetic agents.

Elastase overproduced by stress induces degradation of elastin in the skin, and contributes to wrinkles and stretch marks.[48] Previous studies have reported that kaempferol, quercetin, and myricetin inhibited elastase activity.[42],[49] Several plants including Epilobium angustifolium, Tagetes erecta, Nelumbo nucifera, and Phyllanthus emblica were known to show high elastase inhibition activity.[20],[50],[51],[52] O. japonicus extracts containing many phenolic constituents such as quercetin, kaempferol, and other flavonoid glycosides exhibited remarkable elastase inhibition activity. Collagen maintains elasticity and tension on the skin because it accounts for about 90% of the dermis and 80% of the ECM. Collagen in the dermis consists of 80%–85% type I collagen, 15%–20% type III collagen, and fibronectin.[53] sMMPs are zinc-dependent endopeptidases that break down most ECM. These are classified based on substrate specificity and are grouped into collagenases, gelatinases, stromelysins, and others.[54] MMP-1 (collagenase-1) affects skin aging by initiating the breakage of the collagen cross-links.[55] As skin aging progresses, the level of MMP-1 increases, and moreover, excessive collagen breakdown in the dermis by MMP-1 is also involved in proinflammation among connective tissues.[56] Therefore, the balance of lowering MMP-1 and maintaining type I collagen plays a pivotal function in inhibiting wrinkle formation. MMP-3 (stromelysin-1) damages collagen types II, III, IV, V, and X, proteoglycans, laminin, elastin, fibronectin, and others. It can also extensively activate other MMPs such as MMP-1, -2, -7, -8, and -9.[57] Previous works have reported the inhibitory effects of MMP-1 and -3 in Pinus densiflora, Michelia alba, and Coffea arabica.[58],[59],[60] This work revealed that mRNA transcription of MMP-1 and -3 and the production of MMP-1 are suppressed in a concentration-dependent manner in human fibroblast HS68 cells treated with extracts from O. japonicus. IC50 values of collagenase inhibition activities of other plants reported were 9,550 μg/mL for Viburnum mullaha and 640 μg/mL for Terminalia arjuna.[61],[62] O. japonicus extracts, OJ_MeOH and OJ_EtOH, showed IC50 values of collagenase inhibition activity 145.1 ± 4.2 and 120.3 ± 6.7 μg/mL, respectively. From these results, O. japonicus extracts were believed to have remarkable anti-wrinkle activity.

Tyrosinase inhibition is an important mechanism in the pathway of inhibition of the melanin accumulation in the epidermis.[63],[64] OJ_MeOH showed moderate tyrosinase inhibition activity.

In this study, O. japonicus extracts were investigated to verify the possibility of them to be utilized as anti-aging cosmetic agents. For efficacy tests, antioxidant, anti-wrinkle, and whitening activities were examined using various biochemical assays. From three different antioxidant tests using DPPH, ABTS, and FRAP, O. japonicus extracts, OJ_MeOH and OJ_EtOH, showed effective DPPH scavenging activity compared with ascorbic acid. From substantial inhibitions of elastase and collagenase activities, MMP-1 and -3 mRNA expressions, and MMP-1 protein production by OJ_MeOH and OJ_EtOH, we have found that O. japonicus extracts exhibited remarkable anti-wrinkle activity. Whitening effect measured by tyrosinase inhibition activity was considered to be useful.


   Conclusion Top


O. japonicus extracts, OJ_MeOH and OJ_EtOH, exerted useful whitening activity as well as excellent antioxidant and anti-wrinkle activities, enhancing promising potential of them as anti-aging cosmetic agents.

Financial support and sponsorship

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MOE) (NRF-2017R1D1A1B03034570).

Conflicts of interest

There are no conflicts of interest.



 
   References Top

1.
Jenkins G. Molecular mechanisms of skin ageing. Mech Ageing Dev 2002;123:801-10.  Back to cited text no. 1
    
2.
Rittié L, Fisher GJ. UV-light-induced signal cascades and skin aging. Ageing Res Rev 2002;1:705-20.  Back to cited text no. 2
    
3.
Thring TSA, Hili P, Naughton DP. Anti-collagenase, anti-elastase and antioxidant activities of extracts from 21 plants. BMC Complement Altern Med 2009;9:27.  Back to cited text no. 3
    
4.
Saeed N, Khan MR, Shabbir M. Antioxidant activity, total phenolic and total flavonoid contents of whole plant extracts Torilis leptophylla L. BMC Complement Altern Med 2012;12:221.  Back to cited text no. 4
    
5.
Sasaki YF, Kawaguchi S, Kamaya A, Ohshita M, Kabasawa K, Iwama K, et al. The comet assay with 8 mouse organs: Results with 39 currently used food additives. Mutat Res 2002;519:103-19.  Back to cited text no. 5
    
6.
Aidi Wannes W, Mhamdi B, Sriti J, Ben Jemia M, Ouchikh O, Hamdaoui G, et al. Antioxidant activities of the essential oils and methanol extracts from myrtle (Myrtus communis var. italica L.) leaf, stem and flower. Food Chem Toxicol 2010;48:1362-70.  Back to cited text no. 6
    
7.
Sun Z, Park SY, Hwang E, Park B, Seo SA, Cho JG, et al. Dietary Foeniculum vulgare Mill extract attenuated UVB irradiation-induced skin photoaging by activating of Nrf2 and inhibiting MAPK pathways. Phytomedicine 2016;23:1273-84.  Back to cited text no. 7
    
8.
Fisher GJ, Quan T, Purohit T, Shao Y, Cho MK, He T, et al. Collagen fragmentation promotes oxidative stress and elevates matrix metalloproteinase-1 in fibroblasts in aged human skin. Am J Pathol 2009;174:101-14.  Back to cited text no. 8
    
9.
Jung SK, Lee KW, Kim HY, Oh MH, Byun S, Lim SH, et al. Myricetin suppresses UVB-induced wrinkle formation and MMP-9 expression by inhibiting Raf. Biochem Pharmacol 2010;79:1455-61.  Back to cited text no. 9
    
10.
Kong YH, Jo YO, Cho CW, Son D, Park S, Rho J, et al. Inhibitory effects of cinnamic acid on melanin biosynthesis in skin. Biol Pharm Bull 2008;31:946-8.  Back to cited text no. 10
    
11.
Nasr Bouzaiene NN, Chaabane F, Sassi A, Chekir-Ghedira L, Ghedira K. Effect of apigenin-7-glucoside, genkwanin and naringenin on tyrosinase activity and melanin synthesis in B16F10 melanoma cells. Life Sci 2016;144:80-5.  Back to cited text no. 11
    
12.
Tsuji-Naito K, Hatani T, Okada T, Tehara T. Modulating effects of a novel skin-lightening agent, alpha-lipoic acid derivative, on melanin production by the formation of DOPA conjugate products. Bioorg Med Chem 2007;15:1967-75.  Back to cited text no. 12
    
13.
Cooksey CJ, Garratt PJ, Land EJ, Ramsden CA, Riley PA. Tyrosinase kinetics: Failure of the auto-activation mechanism of monohydric phenol oxidation by rapid formation of a quinomethane intermediate. Biochem J 1998;333:685-91.  Back to cited text no. 13
    
14.
Henning SM, Niu Y, Lee NH, Thames GD, Minutti RR, Wang H, Go VL, Heber D. Bioavailability and antioxidant activity of tea flavanols after consumption of green tea, black tea, or a green tea extract supplement. Am J Clin Nutr 2004;80:1558-64.  Back to cited text no. 14
    
15.
Dudonné S, Vitrac X, Coutière P, Woillez M, Mérillon JM. Comparative study of antioxidant properties and total phenolic content of 30 plant extracts of industrial interest using DPPH, ABTS, FRAP, SOD, and ORAC assays. J Agric Food Chem 2009;57:1768-74.  Back to cited text no. 15
    
16.
Kim JH, Byun JC, Bandi AKR, Hyun CG, Lee NH. Compounds with elastase inhibition and free radical scavenging activities from Callistemon lanceolatus. J Med Plants Res 2009;3:914-20.  Back to cited text no. 16
    
17.
Masuda M, Murata K, Fukuhama A, Naruto S, Fujita T, Uwaya A, et al. Inhibitory effects of constituents of Morinda citrifolia seeds on elastase and tyrosinase. J Nat Med 2009;63:267-73.  Back to cited text no. 17
    
18.
Zhao R, Bruning E, Rossetti D, Starcher B, Seiberg M, Iotsova-Stone V. Extracts from Glycine max (soybean) induce elastin synthesis and inhibit elastase activity. Exp Dermatol 2009;18:883-6.  Back to cited text no. 18
    
19.
Chiang HM, Chien YC, Wu CH, Kuo YH, Wu WC, Pan YY, et al. Hydroalcoholic extract of Rhodiola rosea L. (Crassulaceae) and its hydrolysate inhibit melanogenesis in B16F0 cells by regulating the CREB/MITF/tyrosinase pathway. Food Chem Toxicol 2014;65:129-39.  Back to cited text no. 19
    
20.
Vallisuta O, Nukoolkarn V, Mitrevej A, Sarisuta N, Leelapornpisid P, Phrutivorapongkul A, et al. In vitro studies on the cytotoxicity, and elastase and tyrosinase inhibitory activities of marigold (Tagetes erecta L.) flower extracts. Exp Ther Med 2014;7:246-50.  Back to cited text no. 20
    
21.
Limtrakul P, Yodkeeree S, Thippraphan P, Punfa W, Srisomboon J. Anti-aging and tyrosinase inhibition effects of Cassia fistula flower butanolic extract. BMC Complement Altern Med 2016;16:497.  Back to cited text no. 21
    
22.
Jung HJ, Choi J, Nam JH, Park HJ. Antiulcerogenic effects of the flavonoid-rich fraction from the extract of Orostachys japonicus in mice. J Med Food 2007;10:702-6.  Back to cited text no. 22
    
23.
Lee HS, Ryu DS, Lee GS, Lee DS. Anti-inflammatory effects of dichloromethane fraction from Orostachys japonicus in RAW 264.7 cells: Suppression of NF-κB activation and MAPK signaling. J Ethnopharmacol 2012;140:271-6.  Back to cited text no. 23
    
24.
Lee HS, Lee GS, Kim SH, Kim HK, Suk DH, Lee DS. Anti-oxidizing effect of the dichloromethane and hexane fractions from Orostachys japonicus in LPS-stimulated RAW 264.7 cells via upregulation of Nrf2 expression and activation of MAPK signaling pathway. BMB Rep 2014;47:98-103.  Back to cited text no. 24
    
25.
Ryu DS, Baek GO, Kim EY, Kim KH, Lee DS. Effects of polysaccharides derived from Orostachys japonicus on induction of cell cycle arrest and apoptotic cell death in human colon cancer cells. BMB Rep 2010;43:750-5.  Back to cited text no. 25
    
26.
Lee SJ, Zhang GF, Sung NJ. Hypolipidemic and hypoglycemic effects of Orostachys japonicus A. Berger extracts in streptozotocin-induced diabetic rats. Nutr Res Pract 2011;5:301-7.  Back to cited text no. 26
    
27.
Ryu DS, Lee HS, Lee GS, Lee DS. Effects of the ethylacetate extract of Orostachys japonicus on induction of apoptosis through the p53-mediated signaling pathway in human gastric cancer cells. Biol Pharm Bull 2012;35:660-5.  Back to cited text no. 27
    
28.
Lee GS, Lee HS, Kim SH, Suk DH, Ryu DS, Lee DS. Anti-cancer activity of the ethylacetate fraction from Orostachys japonicus for modulation of the signaling pathway in HepG2 human hepatoma cells. Food Sci Biotechnol 2014;23:269-75.  Back to cited text no. 28
    
29.
Ryu DS, Lee HJ, Kwon JH, Lee DS. Anti-cancer effect of ethylacetate fraction from Orostachys japonicus on HT-29 human colon cancer cells by induction of apoptosis through caspase-dependent signaling pathway. Asian Pac J Trop Med 2018;11:330-5.  Back to cited text no. 29
  [Full text]  
30.
Kim JH, Nam GS, Kim SH, Ryu DS, Lee DS. Orostachys japonicus exerts antipancreatic cancer activity through induction of apoptosis and cell cycle arrest in PANC-1 cells. Food Sci Nutr 2019;7:3549-59.  Back to cited text no. 30
    
31.
Kwon JH, Kim JH, Ryu DS, Lee HJ, Lee DS. Anticancer effect of the ethyl acetate fraction from Orostachys japonicus on MDA-MB-231 human breast cancer cells through extensive induction of apoptosis, cell cycle arrest, and antimetastasis. Evid Based Complement Alternat Med 2019;2019:8951510.  Back to cited text no. 31
    
32.
Jeong JH, Ryu DS, Suk DH, Lee DS. Anti-inflammatory effects of ethanol extract from Orostachys japonicus on modulation of signal pathways in LPS-stimulated RAW 264.7 cells [BMB rep: 399-404] [BMB rep: 399-404]. BMB Rep 2011;44:399-404.  Back to cited text no. 32
    
33.
Shim KS, Ha H, Kim T, Lee CJ, Ma JY. Orostachys japonicus suppresses osteoclast differentiation by inhibiting NFATc1 expression. Am J Chin Med 2015;43:1013-30.  Back to cited text no. 33
    
34.
Hur JM, Park JC. Effects of the aerial parts of Orostachys japonicus and its bioactive component on hepatic alcohol-metabolizing enzyme system. J Med Food 2006;9:336-41.  Back to cited text no. 34
    
35.
Park JC, Han WD, Park JR, Choi SH, Choi JW. Changes in hepatic drug metabolizing enzymes and lipid peroxidation by methanol extract and major compound of Orostachys japonicus. J Ethnopharmacol 2005;102:313-8.  Back to cited text no. 35
    
36.
Lee HY, Park YM, Kim J, Oh HG, Kim KS, Kang HJ, et al. Orostachys japonicus A. Berger extracts induce immunity-enhancing effects on cyclophosphamide-treated immunosuppressed rats. BioMed Res Int 2019;2019:9461960.  Back to cited text no. 36
    
37.
Park HJ, Yang HJ, Kim KH, Kim SH. Aqueous extract of Orostachys japonicus A. Berger exErts immunostimulatory activity in RAW 264.7 macrophages. J Ethnopharmacol 2015;170:210-7.  Back to cited text no. 37
    
38.
Mosmann T. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J Immunol Methods 1983;65:55-63.  Back to cited text no. 38
    
39.
Kim T, Kim S, Kang WY, Baek H, Jeon HY, Kim BY, et al. Porcine amniotic fluid as possible antiwrinkle cosmetic agent. Korean J Chem Eng 2011;28:1839-43.  Back to cited text no. 39
    
40.
Lee JH, Lee SJ, Park S, Kim HK, Jeong WY, Choi JY, et al. Characterisation of flavonoids in Orostachys japonicus A. Berger using HPLC–MS/MS: Contribution to the overall antioxidant effect. Food Chem 2011;124:1627-33.  Back to cited text no. 40
    
41.
Im DS, Lee JM, Lee J, Shin HJ, Park SH, Kim K. Inhibition of collagenase and melanogenesis by ethanol extracts of Orostachys japonicus A. Berger: Possible involvement of Erk and Akt signaling pathways in melanoma cells. Acta Biochim Biophys Sin (Shanghai) 2017;49:945-53.  Back to cited text no. 41
    
42.
Wittenauer J, Mäckle S, Sußmann D, Schweiggert-Weisz U, Carle R. Inhibitory effects of polyphenols from grape pomace extract on collagenase and elastase activity. Fitoterapia 2015;101:179-87.  Back to cited text no. 42
    
43.
Arnao MB. Some methodological problems in the determination of antioxidant activity using chromogen radicals: A practical case. Trends Food Sci Technol 2000;11:419-21.  Back to cited text no. 43
    
44.
Cano A, Acosta M, Arnao MB. A method to measure antioxidant activity in organic media: Application to lipophilic vitamins. Redox Rep 2000;5:365-70.  Back to cited text no. 44
    
45.
Govindan P, Muthukrishnan S. Evaluation of total phenolic content and free radical scavenging activity of Boerhavia erecta. J Acute Med 2013;3:103-9.  Back to cited text no. 45
    
46.
Barros HRM, Ferreira TAPC, Genovese MI. Antioxidant capacity and mineral content of pulp and peel from commercial cultivars of citrus from Brazil. Food Chem 2012;134:1892-8.  Back to cited text no. 46
    
47.
Huang H, Sun Y, Lou S, Li H, Ye X. In vitro digestion combined with cellular assay to determine the antioxidant activity in Chinese bayberry (Myrica rubra Sieb. et Zucc.) fruits: A comparison with traditional methods. Food Chem 2014;146:363-70.  Back to cited text no. 47
    
48.
Suganuma K, Nakajima H, Ohtsuki M, Imokawa G. Astaxanthin attenuates the UVA-induced up-regulation of matrix-metalloproteinase-1 and skin fibroblast elastase in human dermal fibroblasts. J Dermatol Sci 2010;58:136-42.  Back to cited text no. 48
    
49.
Kanashiro A, Souza JG, Kabeya LM, Azzolini AECS, Lucisano-Valim YM. Elastase release by stimulated neutrophils inhibited by flavonoids: Importance of the Catechol Group. Z Naturforsch C J Biosci 2007;62:357-61.  Back to cited text no. 49
    
50.
Onar HC, Yusufoglu A, Turker G, Yanardag R. Elastase, tyrosinase and lipoxygenase inhibition and antioxidant activity of an aqueous extract from Epilobium angustifolium L. leaves. J Med Plants Res 2012;6:716-26.  Back to cited text no. 50
    
51.
Kim T, Kim HJ, Cho SK, Kang WY, Baek H, Jeon HY, et al. Nelumbo nucifera extracts as whitening and anti-wrinkle cosmetic agent. Korean J Chem Eng 2011;28:424-7.  Back to cited text no. 51
    
52.
Pientaweeratch S, Panapisal V, Tansirikongkol A. Antioxidant, anti-collagenase and anti-elastase activities of Phyllanthus emblica, Manilkara zapota and silymarin: An in vitro comparative study for anti-aging applications. Pharm Biol 2016;54:1865-72.  Back to cited text no. 52
    
53.
Kim DE, Hwang YS, Chang BY, Kim DS, Cho HK, Kim SY. Effects of the Syzygium aromaticum L. extract on antioxidation and inhibition of matrix metalloproteinase in human dermal fibroblast. Asian Pac J Trop Biomed 2019;9:53-9.  Back to cited text no. 53
  [Full text]  
54.
Hayami T, Kapila YL, Kapila S. Divergent upstream osteogenic events contribute to the differential modulation of MG63 cell osteoblast differentiation by MMP-1 (collagenase-1) and MMP-13 (collagenase-3). Matrix Biol 2011;30:281-9.  Back to cited text no. 54
    
55.
You GE, Jung BJ, Kim HR, Kim HG, Kim TR, Chung DK. Lactobacillus sakei lipoteichoic acid inhibits MMP-1 induced by UVA in normal dermal fibroblasts of human. J Microbiol Biotechnol 2013;23:1357-64.  Back to cited text no. 55
    
56.
Varani J, Perone P, Fligiel SEG, Fisher GJ, Voorhees JJ. Inhibition of Type I procollagen production in photodamage: Correlation between presence of high molecular weight collagen fragments and reduced procollagen synthesis. J Invest Dermatol 2002;119:122-9.  Back to cited text no. 56
    
57.
Munhoz FBA, Godoy-santos AL, Santos MCLG. MMP-3 polymorphism: Genetic marker in pathological processes (Review) [review]. Mol Med Rep 2010;3:735-40.  Back to cited text no. 57
    
58.
Chiang HM, Lin TJ, Chiu CY, Chang CW, Hsu KC, Fan PC, et al. Coffea arabica extract and its constituents prevent photoaging by suppressing MMPs expression and MAP kinase pathway. Food Chem Toxicol 2011;49:309-18.  Back to cited text no. 58
    
59.
Chiang HM, Chen HC, Lin TJ, Shih IC, Wen KC. Michelia alba extract attenuates UVB-induced expression of matrix metalloproteinases via MAP kinase pathway in human dermal fibroblasts. Food Chem Toxicol 2012;50:4260-9.  Back to cited text no. 59
    
60.
Jung HY, Shin JC, Park SM, Kim NR, Kwak W, Choi BH. Pinus densiflora extract protects human skin fibroblasts against UVB-induced photoaging by inhibiting the expression of MMPs and increasing type I procollagen expression. Toxicol Rep 2014;1:658-66.  Back to cited text no. 60
    
61.
Singh H, Lily MK, Dangwal K. Viburnum mullaha D. DON fruit (Indian cranberry): A potential source of polyphenol with rich antioxidant, anti-elastase, anti-collagenase, and anti-tyrosinase activities. Int J Food Prop 2017;20:1729-39.  Back to cited text no. 61
    
62.
Mathen C, Thergaonkar R, Teredesai M, Soman G, Peter S. Evaluation of anti-elastase and antioxidant activity in antiaging formulations containing terminalia extracts. Int J Herb Med 2014;2:95-9.  Back to cited text no. 62
    
63.
Chang TS. An updated review of tyrosinase inhibitors. Int J Mol Sci 2009;10:2440-75.  Back to cited text no. 63
    
64.
Schallreuter KU, Hasse S, Rokos H, Chavan B, Shalbaf M, Spencer JD, et al. Cholesterol regulates melanogenesis in human epidermal melanocytes and melanoma cells. Exp Dermatol 2009;18:680-8.  Back to cited text no. 64
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]



 

Top
   
 
  Search
 
    Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
    Access Statistics
    Email Alert *
    Add to My List *
* Registration required (free)  

 
  In this article
    Abstract
   Introduction
    Materials and Me...
   Results
   Discussion
   Conclusion
    References
    Article Figures
    Article Tables

 Article Access Statistics
    Viewed48    
    Printed0    
    Emailed0    
    PDF Downloaded17    
    Comments [Add]    

Recommend this journal