Anti-aging Effects of Mangosteen Peel Extract and Its Phytochemical Compounds: Antioxidant Activity, Enzyme Inhibition and Molecular Docking Simulation

Skin aging is a complex natural process characterised by gradual diminishment of structural integrity and physiological imbalance of the skin tissue. Since the oxidative stress is tightly corelated to the skin aging process, the usage of antioxidant may serve as favourable strategies for slowing down the skin aging process. Mangosteen is an important fruit commodity and its extract had been extensively studied and revealing various biological activities. Present study aimed to assess the antioxidant and antiaging activity of mangosteen peel extract (MPE) and its phytochemical compounds. MPE and its compounds were subjected to ferric reducing antioxidant power (FRAP), hydroperoxide (H2O2) scavenging, anti-collagenase, anti-elastase, anti-hyaluronidase and anti-tyrosinase assay. MPE has the highest FRAP 116.31 ± 0.60 μM Fe(II) μg−1 extract, IC50 of MPE on H2O2 scavenging activity was 54.61 μg mL−1. MPE also has the highest anti elastase activity at IC50 7.40 μg mL−1. Alpha-mangostin showed potent anti-collagenase activity (IC50 9.75 μg mL−1). While gamma-mangostin showed potent anti-hyaluronidase (IC50 23.85 μg mL−1) and anti-tyrosinase (IC50 50.35 μg mL−1). MPE and its compounds were evaluated in vitro for antioxidant and antiaging activities. Current findings may provide scientific evidence for possible usage of mangosteen extract and its compounds as antioxidant and antiaging agent.


INTRODUCTION
Skin aging is a complex natural process characterised by a gradual diminishment of structural integrity and physiological imbalance of the skin tissue (Farage et al. 2013). The structural change caused by the degradation of extracellular matrix that underlies dermal tissue. There are various factors that affect the progression of skin aging which categorised into two groups, internal and external (Farage et al. 2008). Increased production of reactive oxygen species (ROS) is the main characteristic of cellular aging. Due to the nature of reactive oxygen species, radicals are highly reactive and can interact violently toward biological macromolecule. Skin fibroblast may undergo apoptosis when exposed directly toward radicals through activation of caspases pathway (Tanigawa et al. 2014). Not only affect cellular component of the skin, radical compound also holds an important role in the expression of protein related in degradation of extracellular matrix (Pittayapruek et al. 2016).
Since the oxidative stress is tightly corelated to the skin aging process, usage of antioxidant may serve as favourable strategies for slowing down the skin aging process (Masaki 2010). Previous study showed that the administration of plant extracts that are rich in antioxidant compounds can protect skin cell from cell death (Silverberg et al. 2011). Previously known plant extracts contain various phytochemical compounds that have various biological activities including antioxidants. Our previous studies showed strong antioxidant and antiaging activities in vitro in some plant extracts: White rice (Oryza sativa) (Widowati, Fauziah et al. 2016), jasmine flower (Jasmimum sambac) (Widowati et al. 2018), and rosella flower (Hibiscus sabdari a) ). This characteristic is the main reason underlies the plant utilisation as medicine. Mangosteen peel extract (MPE) had been extensively studied and revealing various biological activities (Widowati et al. 2014;Widowati, Fauziah et al. 2016, Gondokesumo et al. 2019Widowati et al. 2020). These activities were likely attributed by the phytochemicals in mangosteen peel. Mangosteen peel was already characterised both physically and chemically, revealed various xanthones, including alpha-mangostin, gamma-mangostin, garcinone C, garcinone D, garcinone E, gartanin and smeathxanthone-A (Widowati et al. 2014;Gondokesumo et al. 2018;2019). But to this date, antiaging benefit of mangosteen, especially its peel extract is very much unknown. To address this, we measured antioxidant properties and inhibitory activity against aging-related enzyme inhibition of MPE and its compounds. Present study describes the antioxidant and antiaging benefit of MPE.

Total Flavonoid Content
Total flavonoid content in MPE was measured using aluminum chloride colorimetric assay (Prahastuti et al. 2019

Peroxide Radical Scavenging Activity
Peroxide radical scavenging activity was measured using a method previously mentioned with slight modifications (Mukhopadhyay et al. 2016;Utami et al. 2017). The mixture was then transferred into 96-well plate and was incubated for 5 min at room temperature. Then 75 µL 1,10-phenanthroline was added to the mixture and incubated again for 10 min at room temperature. The absorbance was measured using a spectrophotometer at 510 nm using spectrophotometer [Thermo, 51119200, Waltham, MA, US].

Molecular Docking Simulation
The possible binding mode of phytochemicals found in mangosteen peel towards protein related in aging process was modelled using molecular docking. The 3-dimensional structure of matrix metalloproteinase (MMP1), nuclear export protein (NEP), and prophenoloxidase (PPO3) were obtained from RCSB protein data bank, with PDBID 2Y9X, 5JMY, 966C, respectively. The data was then prepared by removing crystallographic water and removing any co-crystallised ligand. Molecular docking simulation was performed using AutoDock Vina (Vina) using default configuration (Trott & Olson 2010). Best docked conformation that ranked by vina scoring was used in the visual analysis using UCSF Chimera (Pettersen et al. 2004). Intermolecular interaction of protein-ligand complex was inferred using Pose View accessible through Protein Plus web server (https://proteins.plus/) (Stierand & Rarey 2010).

Flavonoid Content in Mangosteen Peel Extract (MPE)
Flavonoid content of MPE was measured. Present study found flavonoid content from MPE was around 17.66 µg QE mg -1 extract (Table 1). Previous study found mature mangosteen peel contains 4.08 g QE/100 g (Pothitirat et al. 2009). Thus, current finding showed higher concentration than previously found.

Antioxidant Capacity Activity
Antioxidant work through reduction of radicals onto its neutral form. Thus, the reduction capacity of certain molecule attributed to its antioxidant activity (Baranowska et al. 2018). Antioxidant activity of MPE and its phytochemical was measured using colorimetric assay based on reducing capacity toward ferric ion.

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A compound with antioxidant activity will reduce Fe 3+ into Fe 2+ . These ions will absorb different colour when complexed with TPTZ. The higher Fe 2+ ion concentration indicating higher reducing capacity of a compound of interest has, thus describe its antioxidant activity. Present study found that MPE showed statistically very significant (P = 0.00) increase in reducing capacity as concentration rise compared to all other compounds in all concentrations. MPE was showed the highest antioxidant activity both in high (116.31 µg mL -1 ) and low (2.51 µg mL -1 ) concentration compared to its compounds alone (Table 2). Table 2 shows that MPE has a very good linearity which mean that this extract has very high antioxidant activity. Gamma-mangostin also showed good linearity in the data although the gradient is not quite high, that showed weak antioxidant activity. Garcinome C, garcinome D, and alpha-mangostin did not show good linearity and the gradient is very low (nearly 0) which can be robustly concluded that these compounds do not have antioxidant activity.

Hydroperoxide Scavenging Activity
The hydroperoxide scavenging assay was carried out in order to measure antioxidant activity of the compound of interest, especially its activity against peroxide radicals. The assay was done using a colorimetric assay based on radical reduction and formation of coloured complexes (Mukhopadhyay et al. 2016;Utami et al. 2017;Jusri et al. 2019). Differ from previous method, the level scavenging activity of the extract and natural compound was measured based on their reduction activity toward peroxide radicals. Reduced radical was unable to oxidise ferric ions and Fe (II) will react directly with phenanthroline, forming a complex that has a strong orange colour which then quantified by a spectrophotometer (Mukhopadhyay et al. 2016;133 Utami et al. 2017;Jusri et al. 2019). In present study peroxide scavenging of MPE and its compounds were measured (Fig. 1). MPE showed the highest scavenging activity based on its IC 50 value (54.61 µg mL -1 ) when compared to alpha-mangostin (120.52 µg mL -1 ), garcinone D (238.916 µg mL -1 ), garcinone C (437.64 µg mL -1 ) and gamma-mangostin (1051.45 µg mL -1 ) ( Table 3).

Elastase inhibitory Activity
In present study, anti-elastase of MPE and its compounds were measured (Fig. 2). MPE had the strongest inhibitory activity against elastase when compared with its known compound alone. Based on its IC 50 value the inhibitory activity was increase as follows: gamma-mangostin < garcinone D < alpha-mangostin < garcinone C < MPE (Table 4).

Hyaluronidase Inhibitory Activity
The hyaluronidase inhibitotry activity of MPE and its compounds were measured (Fig. 5). Based on IC 50 value alpha-mangostin had the strongest inhibitory activity against hyaluronidase when compared with another compound and MPE. The inhibitory activity was increase as follows: garcinone D < garcinone C < MPE < gamma-mangostin < alpha-mangostin (see Table 7).

Molecular Docking
Molecular docking was performed to model the possible binding conformation of compounds found in mangosteen torwards MMP1, NEP and PPO3. Molecular docking was validated by redocking of bound ligand (N-hydroxy-2-[4-(4-phenoxybenzenesulfonyl)-tetrahydro-pyran-4-yl]-acetamide, LBQ657, tropolone) to its respective protein. The RMSD of crystal conformation and docked conformation was less than the known cut off (> 2 Å, data not shown). Thus, the molecular docking method used was reasonable and continued with phytochemicals used in present study.
All compound was successfully docked to the receptor. The binding affinity was retrieved and compared to each other (Table 8). Alpha-mangostin has the highest binding affinity towards MMP1 (-8.9 kcal mol -1 ). Gamma-mangostin has the highest binding affinity towards NEP (-7.8 kcal mol -1 ). While gamma-mangostin has the bind strongly towards PPO3 (-6.8 kcal mol -1 ). None of the compounds exceeded binding affinity of bound ligand, except for gamma-mangostin towards PPO3.
The binding conformation with highest binding affinity was then visualised in Fig. 6. Visualisation analysis showed that all the potential compound was occupied the active site of the receptor, like the bound ligand.

DISCUSSION
The biological activities of plant extract often attributed to its phytochemical content. Plant especially its peel often characterised with enormous amount phytochemical compound. Mangosteen (Garciana mangostana L.) (Clusiaceae) peel previously known to be rich in polyphenolic compound belong to xanthone group. There were 40 xanthones present in the pericarp of the mangosteen fruit, the most abundant xanthones found are alpha-mangostin, beta-mangostin and gammamangostin (Chen et al. 2008;Pedraza-Chaverrí et al. 2009;Zarena & Sankar 2009). Mangostins (alpha, beta and gamma) are the most frequently studied. They have a unique chemical structure with a tricyclic aromatic system carrying isoprene, hydroxyl and methoxyl groups (Obolskiy et al. 2010). Phytochemical found in mangosteen peel includes garcinone, mangostin, isomangostin and garcimangosone (Obolskiy et al. 2010). These compounds have been reported to possess anti-oxidant, anti-proliferative, pro-apoptotic, anti-inflammatory and anti-carcinogenic activities in in vitro and in vivo studies 2020). Present study found MPE has higher flavonoid concentration than previously described. The phytochemical content of plant extract may different because of various factors, including seasonal or nutritional factors.
Aging is a natural process that affects various organs of the body and often characterised the build-up of ROS in cells (Davalli et al. 2016). ROS in normal circumstance holds an important role in various biological process, such as immune response, but through various stimulus or age, the homeostasis of radical was impaired. Increased cytoplasmic ROS is able to induce the synthesis of related to the degradation of extracellular matrix causing tissue structural diminishment that manifested as formation of wrinkles and sagging elasticity (Farage et al. 2008). Antioxidant phytochemical compound helps decrease ROS induced skin damages (Tanigawa et al. 2014). Not only relives the oxidative stress, administration phytochemical compound followed by the decreased collagen degrading enzyme activity (Tsai et al. 2014). Present study found that all compound of MPE have strong antioxidant potency based on its measured reductive capacity. Thus, MPE may alleviate skin cells damage caused by oxidative stress. MPE may also indirectly attenuates the activity of enzyme-related in the degradation of extracellular matrix.
Previous study shows that increasing ROS generation induces expression of various enzyme, including elastase. Human skin elastase is a serine protease responsible elastin degradation process. Chronic irradiation of UVB increased elastase activity significantly, causing the disintegration of elastin fibre in the dermal matrix and further manifested the loss of the skin elasticity. Previous study found Zingiber officinale (L.), extract inhibit the human skin fibroblast elastase activity (Imokawa & Ishida 2015). Another study measured the anti-elastase activity of 21 plant extracts (Thring et al. 2009). Present study shows MPE has a potent anti-elastase activity even when compared with its phytochemical alone. Present finding may serve as a mechanistic explanation of previous study, where oral administration of MPE was found increased skin elasticity (Ohno et al. 2015).
The tensile strength of the skin was provided by the collagen fibre present in dermal matrix. The synthesis of matrix metalloproteinase (MMPs), an enzyme involved in collagen hydrolysis, plays important role in the progression of skin aging (Pittayapruek et al. 2016). Since elevated MMPs synthesis was found to be correlated to the formation of skin changes, especially wrinkles. Several plant extracts can inhibit MMPs activity, especially white tea extract which contains the highest phenolic (Thring et al. 2009). In present study, alpha-mangostin has the highest inhibition activity towards MMPs. Thus, showing the potency of mangosteen phytochemicals for hampering the progression of wrinkles formation.
Hyaluronic acid (HA) is an example of glycosaminoglycans (GAGs) that plays an important role in maintaining skin the moisture of the skin (Stern & Maibach 2008). HA is a strong hydrophilic compound that can absorb 1000x of water volume it has (Baumann 2007). The degradation of HA related to the loss of skin moisture, as usually found on aging skin. In addition, GAGs are also served as peripheral proteins for other structural proteins such as collagen and elastin (Baumann 2007). Thus, the loss was also found to contribute to diminished dermal integrity. The result show gamma-mangostin inhibit hyaluronidase. This finding may explain how consumption of mangosteen peel extract helps to maintain skin moisture as can be found in previous study (Ohno et al. 2015).
Besides the structural changes, aging stimulus was able to induce darkening of the skin or often called hyperpigmentation (Ebanks et al. 2009). Skin pigmentation caused by the production of melanin pigment on the dermal melanocyte. This process is regulated by the rate-limiting enzyme tyrosinase, which serves as an important target for preventing pigmentation. Natural compound was already used commercially as anti-browning agent and skin whitening. Kojic acid works through the inhibition of tyrosinases functions since its share similar structure with its substrate (Chen et al. 1991). Current study found gammamangostin strongly inhibit tyrosinase activity. Thus gamma-mangostin may serve as potential skin whitening agent in further study.
To further investigate possible interaction between phytochemicals found in MPE towards enzyme related in aging process previously described, molecular docking was performed. Molecular docking of phytochemicals found in mangosteen peel predicted alpha-mangostin was a potential binder towards MMP1 while gamma-mangostin towards NEP and PPO3. Visual analysis showed that docked compound reside the active site cavity of the respective protein. Thus, present study proposed the possible binding conformation of alpha-mangostin towards MMP1 and gamma-mangostin towards NEP and PPO3.
Our results show that pure active compound of mangosteen such as alpha-mangostin, gamma-mangostin, garcinone C, and garcinone D had lower activity than MPE, even though they are the active compounds of MPE. These possibly caused by the reactivity of polyphenolic compounds that readily reacted with components of cell culture media to interact with H 2 O 2 , quinones, and semiquinones, and to up regulate the antioxidant defense(Gutierrez-Orozco & Faila 2013). Study done by  showed that alpha-mangostin readily degraded in differenty types of serum-free media (i.e., RPMI, DMEM, MEM and McCoy's 5A) when added in a dimethyl sulfoxide (DMSO) stock solution .

CONCLUSION
MPE and its compounds were evaluated in vitro for antioxidant and antiaging activities. Mangosteen peel extract showed potent antioxidant, anti-elastase and anti-collagenase activities, while gamma-mangostin showed potent anti-hyaluronidase and anti-tyrosinase. Current findings may provide scientific evidence for possible usage of mangosteen peel extract and its compounds as antiaging agent.