F1000Research F1000Research 2046-1402 F1000 Research Limited London, UK 10.12688/f1000research.126762.2 Research Article Articles Antioxidant and phytometabolite profiles of ethanolic extract from the cascara pulp of Coffea arabica collected from Gayo Highland: A study for potential anti-photoaging agent

[version 2; peer review: 2 approved]

 Lestari Wahyu Conceptualization Data Curation Investigation Methodology Resources Validation Visualization Writing – Original Draft Preparation Writing – Review & Editing https://orcid.org/0000-0003-3119-7224 1 2 3 Hasballah Kartini Conceptualization Methodology Supervision Visualization Writing – Review & Editing https://orcid.org/0000-0002-1792-3990 a 4 Listiawan M. Yulianto Methodology Resources Supervision Visualization Writing – Review & Editing 5 Sofia Sofia Data Curation Methodology Resources Supervision Validation Visualization Writing – Review & Editing https://orcid.org/0000-0003-2466-6379 6 7 Department of Dermatology, Dr. Zainoel Abidin General Hospital, Banda Aceh, 24415, Indonesia Department of Dermatology, School of Medicine, Universitas Syiah Kuala, Banda Aceh, 23111, Indonesia Doctoral Program in Medical Science, School of Medicine, Universitas Syiah Kuala, Banda Aceh, 23111, Indonesia Department of Pharmacology, School of Medicine, Universitas Syiah Kuala, Banda Aceh, 23111, Indonesia Department of Dermatology, Faculty of Medicine,, Universitas Airlangga, Surabaya, 60131, Indonesia Department of Biochemistry, School of Medicine, Universitas Syiah Kuala, Banda Aceh, 23111, Indonesia Master of Public Health, School of Medicine, Universitas Syiah Kuala, Banda Aceh, 23111, Indonesia kartini.hasballah@unsyiah.ac.id

No competing interests were disclosed.

 18 9 2023 2023 12 12 6 7 2023 Copyright: © 2023 Lestari W et al. 2023 This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Background: As the most abundant coffee by-product, cascara pulp has been considered a good source of antioxidants which could be used to prevent photoaging. The aim of this study was to determine the phytometabolite profiles, antioxidant and photoaging properties of the ethanolic extract of Coffea arabica cascara pulp.

Methods: Ethanolic maceration was performed on the fine powder of C. arabica cascara pulp collected from Gayo Highland, Aceh Province, Indonesia. The filtrate obtained was evaluated for its 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity, total phenolic content (TPC), and total flavonoid content (TFC). The phytometabolite profiling was conducted qualitatively using reagents and quantitatively using gas chromatography–mass spectroscopy (GC-MS). The potential of the cascara pulp phytometabolites in inhibiting activator protein-1 (AP-1) was evaluated through molecular docking.

Results: The extract had TPC and TFC of 2.04 mg gallic acid equivalent/g extract and 91.81 mg quercetin equivalent/g extract, respectively. The half-maximal inhibitory concentration (IC 50) for the DPPH inhibition reached as low as 9.59 mg/L. Qualitative phytocompound screening revealed the presence of alkaloids, saponins, tannins, flavonoids, steroids, quinones, polyphenols, and triterpenoids. GC-MS revealed the extract containing 5-hydroxy-methylfurfural (22.31%); 2,5 dimethyl 4 hidroxy 3(2H) furanone (0.74%); and caffeine (21.07%), which could form interaction with AP-1 with binding energies of -172.8, -150.8, and -63.188 kJ/mol, respectively.

Conclusion: Ethanolic extract from C. arabica cascara pulp potentially have anti-photoaging properties which is worthy for further investigations in the future.

 AP-1 DPPH flavonoid in-silico natural product polyphenol The author(s) declared that no grants were involved in supporting this work. Amendments from Version 1

In this new version, we have revised our title from "photoaging agent" to "anti-photoaging agent". We have replaced all "hidroxy" to "hydroxy" in the entire article. We added more information about photoaging in Introduction section and we added the references for both methods of DPPH inhibition assay and total phenolic (TPC). In addition, we also have revised all the names of the compounds according to IUPAC.

 Introduction

Aging, a process that manifests in excessive wrinkle, cutis laxa, degraded collagen and hyperpigmentation, occurs naturally. Other than endogenous factors, the process could be accelerated by exogenous factors. 1 Exogenous aging is caused by the surrounding environment that could enhance the oxidative stress in skin tissue including but not limited to cigarette smoke, air pollution, and UV exposure. 1 , 2 The chronic exposure of solar radiation could also contribute to the development of skin cancer. 3 As an addition, maintaining youthful appearance has been the goal of many individual recently which create multibillion-dollar industry of anti-aging products. 4

UV radiation from the sun contains three spectra components including UV-A (320-400 nm), UV-B (280-320 nm), and UV-C (100-280 nm). 5 UV rays from the sun are known to accelerate the aging process of the skin by inducing oxidative stress that inhibits collagen synthesis. 6 Photoaging occurs at the molecular level by UV-induced matrix metalloproteinases (MMPs), which degrade skin collagen. UV stimulates growth factor and cytokine receptors on keratinocytes and fibroblasts, resulting afterward activates mitogen-activated protein (MAP) kinase pathways, leading to downstream signal transduction for the pathophysiology of photoaging in human skin. 7 An increase in transcription factors including activator protein-1 (AP-1) and nuclear factor kappa B (NF-κB) is also produced according to the effects of oxidative stress. Gene regulation in response to cell proliferation and differentiation depends on regulation by the transcription factor AP-1. 8 – 10 Clinical indications of photoaging are based on age, gender, particularly skin, and ethnic and the clinical signs include wrinkles, sagging, roughness, and telangiectasia. 11 – 13 The main effect that chronic UV radiation on the skin is the reduction of the skin's extracellular matrix by collagen degradation and is considered the main cause of the appearance of wrinkles in photoaged skin. 5 – 14 Therefore, searching for anti-photoaging has been a crucial research topic in the field of dermatology.

Natural products have been studied for their ability in providing photoprotection, hence preventing photoaging. 4 , 15 These natural products usually have high antioxidant activities which could suppress the excessive UV radiation-induced reactive oxygen species (ROS) generation. 16 Coffea arabica and its by-products have been reported multiple times containing abundant antioxidants. 17 , 18 Cascara pulp is one of the widely studied coffee by-products which have been utilized in food and beverage products. 19 – 21 Previously, cascara pulp has been extracted using water, methanol, or even supercritical CO 2. 22 , 23 However, the use of ethanol solvent that could attract polar compounds and several non-polar compounds is scarcely reported. 24 The present study tried to close the gap by determining the phytometabolite profile of the ethanolic extract of C. arabica cascara pulp along with its antioxidant properties. Furthermore, the potential of the cascara pulp extract as photoaging agent has been evaluated in-silico.

 Methods Materials

Materials used in this study included methanol, ethanol 96%, ascorbic acid, gallic acid, quercetin, potassium acetate, and sodium carbonate. Reagents for phytochemical screening included AlCl 3 powder, Mg powder, Liebermann-Burchard reagent, Folin–Ciocalteu reagent, Mayer reagent, Dragendorf reagent, and Wagner reagent. All materials and reagents were analytical grade and procured from Merck, Selangor, Malaysia. Other reagents were procured from specific companies as described in each section.

 Extraction of cascara pulp

The cascara of C. arabica was collected from Gayo Highland, Aceh Province, Indonesia. Cascara pulp was crushed into small pieces to produce simplicia powder and air-dried. The maceration was performed on the dried simplicia powder using ethanol 96% (Merck, Selangor, Malaysia) with ratio of 1:10 for 7 days. Thereafter, the filtrate was collected and dried in a rotary evaporator (45°C) (N-1300VW, EYELA, Tokyo, Japan).

 DPPH inhibition assay

Antioxidant activity of the cascara pulp extract was evaluated based on 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay as described previously 25 with some modifications. DPPH was procured from Merck, Selangor, Malaysia. Briefly, 1 mg extract was dissolved up to 10 mL using ethanol 96% to produce 100 mg/L extract solution, which was further dissolved into 2–10 mg/L. DPPH solution was prepared by adding its powder (7.9 mg) into 50 mL methanol (Merck, Selangor, Malaysia). One mL of DPPH 0.4 mM was added into priorly-dissolved extract, homogenized, and incubated at 37°C for 30 minutes. The absorbance was measured at 517 nm on UV-Vis spectrophotometer (Uvmini-1250, Shimadzu, Kyoto, Japan). Ascorbic acid (Merck, Selangor, Malaysia) was used as the control.

 Determination of total phenolic (TPC) and flavonoid contents (TFC)

The total phenolic (TPC) was determined using Folin–Ciocalteu method as described previously. 26 , 27 Briefly, the cascara pulp extract (0.2 mL) was added into 15.8 mL distilled water which had been pre-added with 1 mL Folin–Ciocalteu reagent (Merck, Selangor, Malaysia). The mixture was shaken rigorously and rested for 8 minutes before added with 15 mL sodium carbonate 10% (Merck, Selangor, Malaysia) and subsequently incubated at room temperature for 2 h. The absorbance was measured at 765 nm using an UV-Vis spectrophotometer (Uvmini-1250, Shimadzu, Kyoto, Japan). The value in concentration was obtained from the calibration curve constructed using gallic acid (gallic acid equivalent (GAE)/g extract) and assigned as total phenolic content (TPC). Gallic acid was purchased from Merck, Selangor, Malaysia.

The total flavonoid content (TFC) was determined using a method as described previously, 15 its value was derived from absorbance at 450 nm of a mixture consisting of potassium acetate (0.2 mL) (Merck, Selangor, Malaysia), AlCl 3 (0.2 mL), and distilled water (5.6 mL) which was incubated for 30 minutes at room temperature (all from Merck, Selangor, Malaysia). TFC was calculated through a calibration curve based on quercetin concentrations (quercetin equivalent (QE)/g extract). Both TPC and TFC measurements followed the procedure reported previously. 26

Qualitative phytochemical screening

Phytochemical screenings followed the procedure reported previously. 28 , 29 Flavonoids were detected from a reaction between the cascara pulp extract (0.5 in 10 mL methanol) and Mg powder (0.5 mg) in the presence of HCl 0.5 M. As for saponin contents, the extract (0.5) was shaken to form a stable foam after dissolved in 10 mL boiling water. Three reagents, namely Mayer, Dragendorf, and Wagner (all from Merck, Selangor, Malaysia), were used to identify alkaloid contents. Tannins were identified by reacting the extract (0.5 g in 10 mL distilled water) with FeCl 3 10%. Quinones was observed through the appearance of red color following the drop-wising of sulfuric acid (1–2 drops) into the extract (0.5 g in 10 mL methanol). As for the steroids and triterpenoids, their presence was indicated by color change following the a few drops addition of reagent Liebermann-Burchard (Merck, Selangor, Malaysia) to the cascara pulp extract (1 g in 10 mL n-hexane).

 Gas chromatography–mass spectroscopy (GC–MS) analysis

The cascara pulp extract was analyzed for its phytometabolites based on gas chromatography–mass spectrometry (GC-MS) (Shimadzu QP2010, Kyoto, Japan). The analysis was carried out with column temperature of 40°C (10°C/minute), flown with He gas. The retention was made 3 minutes (30°C/minute) until the temperature reached 299°C with total running time of 29.63 minutes through splitless injector (280°C; 4.34 psi) with total current of 8.4 mL/m in a 30 m-long Rti-1MS column. Data from mass spectrometer was compared with the database from National Institute of Standards and Technology (NIST).

 Molecular docking

To perform the molecular docking, 3D molecular structure was downloaded from PubChem for the following compounds: caffeine (ID: 2519); 5-hydroxymethylfurfural (ID: 237332); and 2,5-dimethyl-4-hydroxy-3(2H)-furanone (ID: 538757). As for the protein, activator protein-1 (AP-1; ID: 5vpf), its structure was downloaded from RSCB Protein Data Bank. Preparation on the protein and active sites prediction was carried out on Molegro Virtual Docker 5. The grid box position was X: -16.5A; Y: 55.64A; Z: -32.78A. The ligand-protein interaction and its visualization were performed on PyMol and Discovery Studio v.21.1.1, respectively.

 Results Antioxidant properties

Antioxidant properties of C. arabica cascara pulp were investigated based on the TPC, TFC, and DPPH assay. The TPC and TFC values of the cascara pulp extract were presented in Table 1. The calibration curves obtained from the measurement of gallic acid and quercetin had R 2 values of 0.9284 and 0.9868, respectively. When the extract was reacted with Folin–Ciocalteu reagent, the UV-Vis absorbance at 765 nm was found to be 0.055 a.u. Meanwhile, the absorbance of 1.545 a.u. was obtained following the reaction between the reaction with AlCl 3 and CH 3COOK. The foregoing absorbance gave the TPC and TFC of the cascara pulp extract as much as 2.04 mg GAE/g extract and 91.81 mg QE/g extract, respectively.

Antioxidant capacity of the ethanolic extract from the cascara pulp based on total phenolic content and total flavonoid content.
Phenolic Flavonoid
Calibration equation y = 0.0143x – 0.0258 y = 0.0166x + 0.0209
R 2 0.9284 0.9868
Absorbance 0.055 a.u. 1.545 a.u.
Total content 2.04 mg GAE/g extract 91.81 mg QE/g extract

GAE: gallic acid equivalent; QE: quercetin equivalent.

DPPH inhibition percentages yielded by the extract with concentrations ranged from 2 to 10 mg/L have been presented ( Figure 1). When the extract concentrations were at 2, 4, 6, 8, and 10 mg/L, the DPPH inhibition percentages reached 25, 29.41, 33.82, 42.65, and 54.41%, respectively. Based on these values, the linear equation with a slope and intercept of 3.603 and 15.44, respectively (R 2 = 0.933). As for the ascorbic acid (control), at the same concentration range, the DPPH inhibitions were 41.18–60.29%. The IC 50 value of the cascara pulp extract in scavenging free radicals DPPH was 9.59 mg/L, which could be considered highly active as an antioxidant. However, the value still fell short by almost 2-fold when compared with the ascorbic acid (5 mg/L).

DPPH inhibition by cascaara pulp extract and cascorbid acid (control). Qualitative phytometabolite screening

Results of the qualitative phytometabolites screening of C. arabica cascara pulp have been presented in Table 2. Positive results were indicated by the Dragendorf and Wagner tests, though the phytometabolites were not sensitive to Mayer test. Members of saponin, steroid, quinone, and tannin families were also found positive in the extract. Flavonoids and polyphenols, widely known antioxidant compounds, were shown to be contained in the extract. The presence of triterpenoids was not observable by the qualitative screening.

Qualitative screening results of the phytometabolites contained in the extract.
Phytometabolite Screening result
Alkaloids
Dragendorf +
Mayer -
Wagner +
Saponins +
Tannins +
Flavonoids +
Steroids +
Quinones +
Polyphenols +
Triterpenoids -

(-) and (+) indicate the presence and absence of the phytometabolites, respectively.

 Phytometabolite profile based on GC–MS

After the initial screening, GC–MS analysis was carried out to identify the phytoconstituents in the extract. The chromatogram depicting peaks from each phytometabolite detected has been presented in Figure 2. Details of the recorded compounds along with the quantitative information (peak area which could be associated to the metabolite contents) have been presented in Table 3. As many as 30 phytocompounds were identified in this present study. Compounds 5-hydroximethylfurfural; caffeine; n-hexadecanoic acid; cis-vaccenic acid; and 2(1H)-naphthalenone, 3,5,6,7,8,8a-hexahydro-4, 8a-dimethyl-6-(1-methylethenyl) topped the phytometabolite abundance rank in the extract with peak areas of 22.31, 21.07, 12.85, 11.94, and 5.26%, respectively. However, only 5-hydroxy-methylfurfural; 2,5-dimethyl-4-hydroxy 3(2H)-furanone; and caffeine (peak area = 22.31, 0.74, and 21.07%, respectively) were considered most bioactive based on previous research. 30 – 32 The aforementioned phytocompounds were then selected for molecular docking study against AP-1.

Chromatogram of cascara pulp extract. Phytometabolites of the ethanolic extract as identified by the GC–MS.
Retention time (min) Phytometabolite Peak area (%)
3.145 Formic acid 0.54
3.978 Acetic acid (CAS) ethylic acid 0.60
4.070 Propanal, 2-oxo- (CAS) pyruvaldehyde 2.08
6.755 Propanoic acid, 2-oxo-, methyl ester (CAS) methyl pyruvate 1.04
8.065 Ethane, 1,1′,1″-[methylidynetris (oxy)]tris-(CAS) triethyl formate 1.03
8.184 2-Furancarboxaldehyde (CAS) furfural 1.07
10.905 2-Propanone, 1-(acetyloxy)- 0.47
11.126 2-Furanmethanol 0.66
14.010 Isopropenylacetic acid 4.85
14.515 5-Methyl furfural 0.41
14.659 2-Furancarboxaldehyde, 5-methyl-(CAS) 5-methyl-2-furfural 0.28
17.409 Orcinol 0.37
17.903 2,5-Dimethyl-4-hydroxy-3(2H)-furanone 0.74
19.729 2,3-Dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one 1.03
21.377 5-Hydroximethylfurfural 22.31
21.825 5-Acetoxymethyl-2-furaldehyde 0.38
22.341 2-Propoxy-succinic acid, dimethyl ester 0.59
25.149 Phenol, 3,5-bis(1,1-dimethylethyl)- 0.40
25.849 9-Octadecenoic acid (Z)- (CAS) oleic acid 0.37
26.117 1H-Cycloprop [e]vazulen-4-ol, decahydro-1,1 0.93
27.458 2,6,10,14-Hexadecatetraen-1-ol, 3,7,11,15-tetramethyl, acetate (E,E,E) 1.52
27.675 2(1H) Naphthalenone, 3,5,6,7,8,8a-hexahydro-4, 8a-dimethyl-6-(1-methylethenyl) 5.26
28.005 Hydrazinecarboxamide, 2-(2-methylcyclohexylidene) 3.93
28.285 Tetradecanoic acid 0.92
29.277 Caffeine 21.07
29.735 Corymbolone 0.51
30.701 n-Hexadecanoic acid 12.85
32.435 Cis-Vaccenic acid 11.94
35.463 Hexadecanoic acid, 2-hydroxy-1-(hydroxym 0.91
37.002 9-Octadecenoic acid (Z)-, 2,3-dihydroxypropyl ester (CAS) 1-monoolein 0.95
<italic toggle="yes">In-silico</italic> interactions between the identified phytometabolites and AP-1

The summaries of molecular docking results were presented in Table 4. Highest binding energy was exhibited by 5-hydroxy-methylfurfural (-172.8 kJ/mol) attributed to six hydrogen bonds and five Van der Waals interactions. The second most potential AP-1 inhibitor was 2,5-dimethyl-4-hydroxy-3(2H)-furanone with a binding energy of -150.8 kJ/mol involving four H bonds and three non-polar interactions. Caffeine appeared to have the lowest affinity with AP-1, where the binding energy only reached -63.188 kJ/mol despite the seven H bond, six alkyl, and four pi-alkyl interactions. All the three compounds formed interactions with AP-1 through ARG176, ARG177, and LYS289. While both 5-hydroxy-methylfurfural and 2,5-dimethyl-4-hydroxy-3(2H)-furanone interacted with ARG173, only caffeine that did not. The 3D representations of the foregoing interactions have been presented in Figure 3.

Molecular docking results of the interaction between the phytometabolites and AP-1.
Ligand Binding energy (kJ/mol) Interaction Distance (Å) Interaction types
5-Hydroxy-methylfurfural -172.8 A:ARG176:HH11 - :10:O3 2.64 Conventional H bond
A:ARG173:HA - :10:O3 2.42 Carbon H bond
A:ARG173:HA:B - :10:O3 2.44 Carbon H bond
A:ARG176:HD1 - :10:O1 2.64 Carbon H bond
A:ARG176:HD1 - :10:O3 2.47 Carbon H bond
A:ARG177:HA - :10:O2 2.45 Carbon H bond
B:LYS289:NZ - :10 4.25 Pi-cation
:10 - A:ARG173 5.13 Pi-alkyl
:10 - A:ARG176 3.71 Pi-alkyl
:10 - A:ARG177 4.91 Pi-alkyl
:10 - B:LYS289 4.62 Pi-alkyl
2,5-Dimethyl-4-hydroxy-3(2H)-furanone -150.8 B:ARG286:HE - :10:O3 2.23 Conventional H bond
B:LYS289:HZ1 - :10:O4 2.54 Conventional H bond
B:LYS289:HZ2 - :10:O4 2.39 Conventional H bond
A:ARG176:HD1 - :10:O2 2.63 Carbon H bond
:10:C5 - A:ARG173 4.15 Alkyl
:10:C5 - B:LYS289 4.67 Alkyl
:10:C6 - A:ARG173 4.35 Alkyl
Caffeine -63.188 A:ARG177:HH11 - :10:O2 2.24021 Conventional H bond
B:ARG292:HH11 - :10:O1 2.37319 Conventional H bond
A:ARG177:HD2 - :10:O2 2.61266 Carbon H bond
:10:H1 - B:LYS289:O 2.98414 Carbon H bond
:10:H5 - B:LYS289:O 2.40609 Carbon H bond
:10:H6 - :10:O1 2.83378 Carbon H bond
:10:H7 - :10:O1 2.83398 Carbon H bond
A:ARG176:C,O;ARG177:N - :10 5.3792 Amide-pi stacked
A:ARG176:C,O;ARG177:N - :10 4.6741 Amide-pi stacked
:10:C6 - A:ARG176 4.19428 Alkyl
:10:C6 - A:ARG177 3.96796 Alkyl
:10:C6 - B:LYS289 4.9672 Alkyl
:10:C7 - B:LYS289 4.63265 Alkyl
:10:C7 - B:ARG292 3.32205 Alkyl
:10:C7 - B:ILE293 5.2454 Alkyl
:10 - A:ARG176 4.67272 Pi-Alkyl
:10 - A:ARG177 5.06042 Pi-Alkyl
:10 - B:LYS289 4.24254 Pi-Alkyl
:10 - B:LYS289 3.70068 Pi-Alkyl
3D (left and middle) and 2D (right) conformations of the interactions between the phytometabolites and AP-1. Discussion

In this present study, the antioxidant activity of C. arabica cascara pulp could be considered high since the IC 50 value of DPPH inhibition reached below 100 mg/L. 26 The antioxidant properties of the cascara pulp extract are further supported by the TPC and TFC of 2.04 mg GAE/g extract and 91.81 mg QE/g extract, respectively. C. arabica has previously been reported to possess high antioxidant activities. A study in India revealed that 200 mg/L C. arabica seed extract could yield >90% DPPH inhibition. 18 The authors attributed the finding with the donation of hydrogen from hydroxyl groups of the bioactive metabolites which could stop the oxidation process by DPPH via stable end products formation. 18 Other studies also observed the strong antioxidant activity of C. arabica along with its high TPC. 33 , 34 In our previous systematic review, we found there are at least 13 studies confirming the antioxidant activities of C. arabica by-products. 17 IC 50 value obtained from the DPPH assays of arabica coffee husk using supercritical fluid extraction was >0.25 mg/L. 23 Arabica coffee pulp extracted with distilled water was reported to contained TPC as high as 0.28 mg GAE/g extract. 35 Hence, our ethanolic extract from C. arabica cascara pulp had higher DPPH scavenging activity and TPC value than that of previously studied by-products.

UV radiation could induce aging process (photoaging) by promoting reactive oxygen species (ROS) production which could result in excessive oxidative stress and inflammation in skin. Indeed, ROS is naturally produced in mitochondria resulted from the aerobic metabolism, particularly in electron transport chain, which contributes to slow endogenous aging. 36 However, there are studies who suggest that ROS production could be enhanced by UV light exposure. Activities of keratinocytes and fibroblasts in producing ROS are positively correlated with the UV radiation intensity. 37 , 38 The increase of ROS release following the solar light exposure could also attributed to the downregulation of ROS neutralizing enzymes viz glutathione reductase and peroxidase. 1 In addition, ROS could mediate several signaling pathways to induce skin damage. 16 , 37 , 39 A terpenoid isolated from an ethyl acetate fraction of coffee silverskin was reported to be capable of inhibiting ROS production and potential as photoaging agent. 22 Taken altogether, the cascara pulp obtained herein is potential in preventing photoaging attributed to its high antioxidant activity that could suppress the oxidative stress in skin tissue following solar radiation.

ROS induced from the UV exposure could initiate the cascading reaction of mitogen-activated protein kinases (MAPKs), consisting of extracellular C-Jun N-terminal kinase (JNKs), signal-regulated kinase (ERK), and p38MAPK. 40 The activation of JNK and p38MAPK pathways promotes activator protein-1 (AP-1) and cyclooxygenase-2 (COX-2). 40 This reaction cascade contributes to the enhanced levels of interleukin (IL)-8, IL-10, prostaglandin G2, and vascular endothelial factor which are responsible for inflammatory reaction, differentiation, immunosuppression, proliferation, as well as angiogenesis. 1 Moreover, AP-1 is responsible to aging process by both inhibiting collagen synthesis via direct binding with procollagen promoter and degrading collagen via interaction with matrix metalloproteinase 1 (MMP1). 41 , 42 Based on the foregoing explanations, researchers have used AP-1 as the target for anti-photoaging agents. 43 – 45 Hence, we had performed molecular docking studies to observe the potential of the phytometabolites from the cascara pulp extract to interact with AP-1.

In this present study, two predominant compounds (5-hydroxy-methylfurfural and caffeine) and one minor compound (2,5-dimethyl-4-hydroxy-3(2H)-furanone) were shown to have strong affinity with AP-1 through molecular docking studies. From the highest to the lowest, the phytometabolites could be ranked as follows: 5-hydroxy-methylfurfural > 2,5-dimethyl-4-hydroxy-3(2H)-furanone > caffeine. Both 5-hydroxy-methylfurfural and 2,5-dimethyl-4-hydroxy-3(2H)-furanone are common food additives, while caffeine is a common compound in coffee products. Similarly, a study found that caffeine was among the isolates obtained from antioxidant assay-guided procedure on Robusta coffee seeds, yet its bioactivity is relatively minimum. 46 In our previous in-silico screening using gallic acid, malonic acid, gallic acid, and decanoic acid from water:ethanol combination extract of C. arabia cascara pulp, ARG173, ARG176, ARG177, and LYS289 were also found as common binding sites. 47 The highest binding energy found in that previous study was -242.24 kJ/mol. 47 Though the highest binding energy in this present study is -172.8 kJ/mol, obtained from 5-hydroxy-methylfurfural, the value is relatively high and could be considered as an effective binding. 48 , 49 Moreover, the antioxidant properties of the cascara pulp herein could contribute to other preventive mechanisms of photoaging. Nonetheless, it is worth noting that in-silico study alone is not sufficient to conclude the AP-1 inhibition by the cascara pulp extract, where in vitro study is a must. 24

Conclusions

The ethanolic extract from the cascara pulp of C. arabica is highly active as an antioxidant agent. Phytocompound profiling evidences the antioxidant metabolite richness of the cascara pulp extract which could act as anti-photoaging agent. In-silico studies reveal the potential of strong interaction between the bioactive phytometabolites with AP-1. Further research with in vitro or in vivo design is warranted to confirm the anti-photoaging activities of the phytometabolites contained in the C. arabica cascara.

 Data availability Underlying data

PubChem: Molecular structure for caffeine. 2519, https://identifiers.org/pubchem.compound:2519. 50

PubChem: Molecular structure for 5-hydroxymethylfurfural. 237332, https://identifiers.org/pubchem.compound:237332. 51

PubChem: Molecular structure for 2,5-dimethyl-4-hydroxy-3(2H)-furanone. 538757, https://identifiers.org/pubchem.compound:538757. 52

PubChem: Molecular structure for activator protein-1. https://doi.org/10.2210/pdb5VPF/pdb. 53

Figshare: ‘Antioxidant and phytometabolite profiles of ethanolic extract from the cascara pulp of Coffea arabica collected from Gayo Highland: A study for potential photoaging agent’, https://doi.org/10.6084/m9.figshare.21219323. 54

This project contains the following underlying data:

DPPH Inhibition Assay.docx

Determination of Total Phenolic Content.docx

Determination of Total Flavonoid Content.docx

Calibration Curve_Total flavonoid.png

Calibration Curve_Total Phenolic.png

DPPH inhibition by cascaara pulp extract and cascorbid acid (control).jpg

Figure 2. Chromatogram of cascara pulp extract.jpg

3D (left and middle) and 2D (right) conformations of the interactions between the phytometabolites and AP-1.jpg

Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).

 Acknowledgements

We would like to thank Badan Pengembangan Sumber Daya Manusia (BPSDM) Aceh, Indonesia.

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Purnama A Mardina V Puspita K : Molecular docking of two cytotoxic compounds from Calotropis gigantea leaves against therapeutic molecular target of pancreatic cancer. Narra. J. 2021;1(2):e37. 10.52225/narraj.v1i2.37 Purnama A Rizki D Qanita I : Molecular docking investigation of calotropone as a potential natural therapeutic agent against pancreatic cancer. J. Adv. Pharm. Technol. Res. 2022;13(1):44–49. 35223440 10.4103/japtr.japtr_143_21 National Center for Biotechnology Information: PubChem Compound Summary for CID 2519, Caffeine.[Data].2022. Reference Source National Center for Biotechnology Information: PubChem Compound Summary for CID 237332, 5-Hydroxymethylfurfural.[Data].2022. Reference Source National Center for Biotechnology Information: PubChem Compound Summary for CID 538757, 2,4-Dihydroxy-2,5-dimethyl-3(2H)-furanone.[Data].2022. Reference Source Yin Z Rudenko G Machius M : Transcription factor FosB/JunD bZIP domain in complex with cognate DNA, type-II crystal, Protein Data Bank.[Data].2019. 10.2210/pdb5VPF/pdb Hasballah K : Antioxidant and phytometabolite profiles of ethanolic extract from the cascara pulp of Coffea arabica collected from Gayo Highland: A study for potential photoaging agent. figshare.[Dataset]. Journal Contribution. 2022. 10.6084/m9.figshare.21219323.v2 10.5256/f1000research.152714.r207137 Reviewer response for version 2 Atanassova Maria 1 Referee https://orcid.org/0000-0002-6188-7990 Nutritional Scientific Consulting, Chemical Engineering, University of Chemical Technology and Metalurgy, Sofia, Bulgaria

Competing interests: No competing interests were disclosed.

 26 9 2023 Copyright: © 2023 Atanassova M 2023 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation approve

I have read the manuscript carefully and agree with the corrections the authors have made following my recommendations. I think your manuscript can be accepted for indexing.

Is the work clearly and accurately presented and does it cite the current literature?

Partly

If applicable, is the statistical analysis and its interpretation appropriate?

I cannot comment. A qualified statistician is required.

Are all the source data underlying the results available to ensure full reproducibility?

Yes

Is the study design appropriate and is the work technically sound?

Yes

Are the conclusions drawn adequately supported by the results?

Yes

Are sufficient details of methods and analysis provided to allow replication by others?

Yes

Reviewer Expertise:

My research area is Total Phenols and Total Flavonoids, Bioactive Compounds and Antioxidant Activity of Extracts from Different Natural Plants

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

 10.5256/f1000research.139202.r171576 Reviewer response for version 1 Atanassova Maria 1 Referee https://orcid.org/0000-0002-6188-7990 Nutritional Scientific Consulting, Chemical Engineering, University of Chemical Technology and Metalurgy, Sofia, Bulgaria

Competing interests: No competing interests were disclosed.

 9 5 2023 Copyright: © 2023 Atanassova M 2023 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation approve-with-reservations

The manuscript "Antioxidant and phytometabolite profiles of ethanolic extract from the cascara pulp of Coffea arabica collected from Gayo Highland: A study for potential photoaging agent" with authors Wahyu Lest, Kartini Hasballah, M. Yulianto Listiawan, Sofia Sofia is interesting and original. I think it would be of interest not only to readers of F1000Research but also to other researchers. I think that the task that the authors have set themselves is very serious.

In the Introduction part, the authors have cited 10 sources from the last 3-5 years, out of a total of 14. This shows that the authors have worked very seriously on their goal to prove the seriousness of the research they have done. I would like to say that I think the Introduction is too short and I ask the authors to add more sources.

As for the Methods and Materials section, the methods are generally known, well described, and could be replicated by any researcher. It struck me that two of the methods have no sources noted. I ask the authors to cite where they got the methods because according to the journal's requirements, this omission amounts to plagiarism. These are the methods: DPPH inhibition assay; Determination of total phenolic (TPC). The authors may have modified them in their use but they should also note this.

Results are presented accurately and clearly. They are supported by relevant tables and figures. Accordingly, the results obtained by the authors enable any reader of this manuscript to get a clear idea. In my opinion, the authors should note Total Phenolics and Total Flavonoids in the table. The phenols and flavonoids for which the authors present the results are not one specific compound and for that they use the corresponding equivalent. For example, if the authors had used another equivalent such as catechin or rutin to prove the flavonoids, I think they would have obtained different results. Another thing that I would like the authors to pay attention to is the formula of potassium acetate CH 3CO 2K, in my opinion, it should be written according to IUPAC CH 3COOK. I also ask the authors to write the formulas of the compounds in the text according to the IUPAC requirements, for example: 2,5-Dimethyl-4-hydroxy-3(2H)-furanone.

In the Discussion section, I think the authors presented their results accurately and clearly and supported them with relevant citations. In my opinion, the conclusion supports the results obtained by the authors.

Is the work clearly and accurately presented and does it cite the current literature?

Partly

If applicable, is the statistical analysis and its interpretation appropriate?

I cannot comment. A qualified statistician is required.

Are all the source data underlying the results available to ensure full reproducibility?

Yes

Is the study design appropriate and is the work technically sound?

Yes

Are the conclusions drawn adequately supported by the results?

Yes

Are sufficient details of methods and analysis provided to allow replication by others?

Yes

Reviewer Expertise:

My research area is Total Phenols and Total Flavonoids, Bioactive Compounds and Antioxidant Activity of Extracts from Different Natural Plants

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

Hasballah Kartini Faculty of Medicine, Syiah Kuala University, Indonesia

Competing interests: There's no conflict of interest.

 13 6 2023

We thank and appreciate the reviewer comment regarding our manuscript and we are humbled that our efforts have been well conveyed. We thank and appreciate the reviewer comment about our intention regarding the sources of our manuscript. We’d like to agree about the introduction part and would like to add more sources as soon as possible. Again, we’d like to thank the reviewer regarding her veracity as well as her precision. We apologize about our lack of source, we’d like to correct it soon enough. We’d like to thank the reviewer about the comment regarding our result and we’re content that our purpose have been well received. We’d also appreciate the advice regarding the phenolic and flavonoid matter. About the formula, yes, we’re completely agree that we should write it according to the IUPAC requirement. We’d like to correct it later. We thank and appreciate the reviewer comment regarding the discussion and conclusion section. We really do appreciate the reviewer for taking the time and for giving us her insights in order to make our manuscript better.

 Hasballah Kartini Universitas Syiah Kuala, Indonesia

Competing interests: I have no conflict of interest

 6 7 2023

Dear Reviewer,

Thank you for reviewing our article and proving the comments and suggestions. Based on your comments and suggestions we have revised our article:

We have added more information about photoaging in Introduction section with nine additional references.

We have added the references for both methods of DPPH inhibition assay and total phenolic (TPC).

The total of the total phenolics and total flavonoids have been provided in the Table 1.

We have revised all the names of the compounds according to IUPAC. We would like to thank you for pick some of them.

 Best regards,

Authors

 10.5256/f1000research.139202.r165756 Reviewer response for version 1 Salim Emil 1 Referee https://orcid.org/0000-0002-4667-6351 Department of Pharmacology, Faculty of Pharmacy, Universitas Sumatera Utara, Medan, North Sumatra, Indonesia

Competing interests: No competing interests were disclosed.

 23 3 2023 Copyright: © 2023 Salim E 2023 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation approve

The authors studied the antioxidant properties of ethanolic extract from the cascara pulp of  Coffea arabica collected from Gayo Highland. Using the DPPH method, they found that the extract has strong antioxidant activity. Moreover, they studied the phytochemical constituent of the extract using phytochemical screening methods using reagents and found that the extract contained saponin, steroid, quinone, tannin, flavonoids and polyphenols. Using GCMC, they found some bioactive compounds exist in the extract, such as 5-hydroxy-methylfurfural, 2,5 dimethyl 4 hydroxy 3(2H) furanone and Caffeine. By performing in silico study, they showed that these compounds have interaction with AP-1, a target for anti-photoaging agents.

The study's background, methods, results, discussion, and conclusion have been presented clearly. However, in the title, I would like to suggest that it should be "anti-photoaging agent" rather than "photoaging agent".

I spotted a tiny error in the sentence: The second most potential AP-1 inhibitor was and 2,5 dimethyl 4 hidroxy 3(2H) furanone with a binding energy of -150.8 kJ/mol involving four H bonds and three non-polar interactions. I think the word "and" should be deleted. Also in some sentences, "hidroxy" should be written as "hydroxy".

Is the work clearly and accurately presented and does it cite the current literature?

Yes

If applicable, is the statistical analysis and its interpretation appropriate?

Not applicable

Are all the source data underlying the results available to ensure full reproducibility?

Yes

Is the study design appropriate and is the work technically sound?

Yes

Are the conclusions drawn adequately supported by the results?

Yes

Are sufficient details of methods and analysis provided to allow replication by others?

Yes

Reviewer Expertise:

Pharmacology and Immunology

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

Hasballah Kartini Faculty of Medicine, Syiah Kuala University, Indonesia

Competing interests: There's no conflict of interest.

 13 6 2023

We thank and appreciate the reviewer comment regarding our manuscript and we are humbled that our efforts have been well conveyed. We thank and appreciate the reviewer comment about our intention regarding the sources of our manuscript. We’d like to agree about the introduction part and would like to add more sources as soon as possible. Again, we’d like to thank the reviewer regarding her veracity as well as her precision. We apologize about our lack of source, we’d like to correct it soon enough. We’d like to thank the reviewer about the comment regarding our result and we’re content that our purpose have been well received. We’d also appreciate the advice regarding the phenolic and flavonoid matter. About the formula, yes, we’re completely agree that we should write it according to the IUPAC requirement. We’d like to correct it later. We thank and appreciate the reviewer comment regarding the discussion and conclusion section. We really do appreciate the reviewer for taking the time and for giving us her insights in order to make our manuscript better.

 Hasballah Kartini Universitas Syiah Kuala, Indonesia

Competing interests: I do not have conflict of interest.

 5 7 2023

Dear Reviewer,

We would like thank you for your suggestions and corrections. We have revised our title from "photoaging agent" to "anti-photoaging agent". We also have replaced all "hidroxy" to "hydroxy" in the entire article.

Authors