Novel α-Mangostin Derivatives from Mangosteen (Garcinia mangostana L.) Peel Extract with Antioxidant and Anticancer Potential

Institute of Research and Development, Duy Tan University, Danang 550000, Vietnam Faculty of Environmental and Chemical Engineering, Duy Tan University, Danang 550000, Vietnam Department of Genetics, University of Science, Ho Chi Minh City, Vietnam Vietnam National University, Ho Chi Minh City, Vietnam Faculty of Chemical & Food Technology, University of Technology and Education,+u Duc, Ho Chi Minh City 700000, Vietnam Faculty of Chemical Engineering, Industrial University of Ho Chi Minh City, 12 Nguyen Van Bao, Ho Chi Minh 700000, Vietnam Faculty of Technology, Van Lang University, Ho Chi Minh City, Vietnam NTTHi-Tech Institute, Nguyen Tat+anh University, 300ANguyen Tat+anh,Ward 13, District 4, Ho ChiMinh City, Vietnam Center for Advanced Chemistry, Institute of Research and Development, Duy Tan University, Da Nang 550000, Vietnam Department of Organic Chemistry, Faculty of Chemical Engineering, Ho Chi Minh City University of Technology (HCMUT), 268 Ly +uong Kiet Street District 10, Ho Chi Minh City, Vietnam


Introduction
Nowadays, the study of natural phytochemical substances from plants has continuously increased due to their effectiveness in preventing and treating various human diseases [1][2][3][4]. ese natural substances are an excellent alternative for therapeutic due to abundant resources, low cost, high biocompatibility, nontoxicity to the human body, and high biological and pharmacological activity. Various parts of plants, including roots, stems, leaves, fruits, flowers, and seeds, can be used as sources for the extraction of natural bioactive compounds [5][6][7][8][9]. Many studies have recently focused on the utilization of inexpensive or waste sources from the consumption and food industry for the production of valuable natural substances [2,[10][11][12]. Additionally, peels of several fruits, e.g., pomegranate, citrus, longan, sapodilla, dragon fruit, banana, and apple, have been reported to be rich in bioactive components (vitamins, flavonoids, and phenolic compounds), which promote high free radical scavenging activity [13][14][15]. Antioxidants scavenge the oxidants or free radicals created by multiple degenerative and disease processes, e.g., diabetes, cancer, and cardiovascular disorders [12,16,17]. erefore, finding, extraction, and semisynthesis of these active ingredients from traditional medicine play an important role in discovering the new active ingredients in antioxidant, healing, and cancer treatment processes [4,9,18,19].
Mangosteen (Garcinia mangostana L.) is a tropical fruit that can be found in South East Asia, such as Vietnam, Indonesia, Malaysia, and ailand. is unique fruit is used both in medicine and in cosmetics. e soft and juicy white part inside mangosteen is mainly consumed fresh as dessert. In contrast, the mangosteen peels have been utilized extensively in traditional medicine for treating several illnesses, including skin infection, dysentery, trauma, abdominal pain, and wound infection, as well as cancer treatment [20][21][22]. e mangosteen peels contain a variety of biologically active compounds (e.g., xanthones, isoflavones, tannins, flavonoids, alpha-, beta-, and gamma-mangostin), exhibiting various biological and medicinal effects such as antioxidant, anti-inflammatory, antimicrobial, and anticancer effects [9,[23][24][25][26][27][28].
Among these isolated phytochemicals, α-MG-(1,3,6-trihydroxy-7-methoxy-2,8-bis(3-methyl-2-butenyl)-9H-xanthen-9-one)-is one of the most powerful natural antioxidants, and it has recently received great attention to the production of antioxidant and anticancer compounds [29,30]. Several studies have described the synthesis and medicinal chemistry of α-MG derivatives, and evaluation of their therapeutic activity has been reported [31][32][33][34][35]. A diversity of novel xanthone analogs based on α-MG was synthesized and tested as anticancer agents by cytotoxic activity using human cancer cell lines [36,37]. e structure-activity relationship study suggested that the modification of the phenol groups on C3 and C6 of α-MG played a critical role in inhibiting cancer cell lines [38]. It was also established that the modification at the C4 position of α-MG increased the anticancer activity and drug-like properties [39]. Besides, the compound having the Cl − group at the C4 position of α-MG showed strong potency and improved solubility many times over α-MG [35].
In this study, α-MG was extracted and isolated from mangosteen peels. To improve the antioxidant and anticancer activities of α-MG, a series of new compounds were designed and synthesized from the pristine α-MG by simultaneously changing the substituents OH group on the C-3 and C-6 of α-MG by F − , Cl − , and Br − of benzoyl chloride. α-MG derivatives structures were determined using MS, 1 H-NMR, 13 C-NMR, and HPLC. e antioxidant activity of α-MG and its derivatives were investigated by the DPPH method. e effects of cytotoxicity of the synthesized compounds were studied on four cancer cell lines (HeLa, MCF-7, NCI-H460, and HepG2).

High-Performance Liquid Chromatography (HPLC).
HPLC was conducted at RT on the Agilent 1260 Infinity HPLC system equipped with a DAD-detector using the C-18 analytical column (250 mm × 4.6 mm, 5 µm) and a 20 μL injection volume with a flow rate of 1 mL/min [42]. e standard stock solutions of α-MG and α-MG derivatives were set by dissolving accurately weighed compounds in methanol to make a concentration of 1 mg/mL. All samples were held at 4°C and brought to room temperature before use. e mobile phase consisted of 0.1% solution of phosphoric acid in water (solution A) and methanol (solution B). Solution gradient mode was as follows: (i) at 0 minutes: 30% of solution A, 70% of solution B; (ii) at 15 minutes: 100% of solution B; and (iii) at 20 minutes: 100% of solution B.
e structure of α-MG and its derivatives were determined by MS, 13 C-NMR, and 1 H-NMR spectra were determined on MicroOTOF-Q 10187 mass spectrometer (Brucker, Germany) and 500 Ultrashield NMR Spectrometer (Brucker, Germany).

Extraction of α-MG.
Fresh mangosteen fruits were obtained from the Mekong river delta in Vietnam. e fullyripe fruits (dark purple peel) were selected for the study. e pieces of mangosteen peels were dried in a hot air oven at 45 ± 0.5°C and then ground into a fine powder. Typically, 500 g of powdered mangosteen peel was soaked into 1 L of ethyl acetate at room temperature (25°C) for 96 h. e solid fraction was separated from the extract by filtration; TLC preliminarily determined the presence of α-MG in the extract. e filtered mangosteen peel powder was further soaked into ethyl acetate solvent 2 times for 96 h to extract the remaining α-MG. Next step, the obtained extract was mixed with the silica gel and put into the evaporating flask to evaporate ethyl acetate resulting in α-MG powder. e condensed extract was partitioned with n-hexane to remove indeterminate compounds that were soluble in n-hexane. en, the ratio of ethyl acetate was gradually added to the mixture, so that the E : H ratio increased from 1 : 99 to 30 : 70. e mixture containing α-MG was isolated by CC. e processes of mixing the silica gel, evaporation, and isolation were repeated until obtaining pure α-MG (tested by TLC and HPLC as well as NMR spectra). e yield percentage was calculated from the weight ratio of α-MG and the dry raw material [43].  (25 C). e reaction was tracked by TLC to determine the change of the initial substance (α-MG) and the newly formed substances after 48 or 60 or 55 h, respectively. e light-yellow liquid of the formed compounds (compounds 1, 2, and 3) was isolated by CC using a solvent mixture of E : H � 20 : 80, respectively.

Synthesis of α-MG Derivative from 3-Chlorobenzoyl
Chloride (Compound 6). 100 mg of α-MG (0.2 mM) previously dissolved in 5 mL CH 2 Cl 2 was reacted with 3-chlorobenzoyl chloride (0.07 mL, 0.6 mM) in the presence of 0.10 mL of trimethylamine at room temperature for 50 h. e formation of a new compound was followed by TLC. e yellow powder of a new compound (compound 6) was purified by silica gel CC with the mobile phase of E : H � 20 : 80.

Antioxidant Activity Evaluation.
e antioxidant activity of α-MG and its derivatives were investigated by using 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay [44]. e scavenging behavior tests the antioxidant potential to capture the DPPH radical by donating a hydrogen atom. By antioxidants quenching the DPPH radical, the DPPH solution changes its color to light yellow from deep violet, and the absorbance decreases at 517 nm. Ascorbic acid was used as the reference for evaluating the antioxidant activity of α-MG and its derivatives. e concentration of the working DPPH solution was 6 mM prepared by dissolving DPPH in methanol, and the concentration of the sample solutions was varied at 0.1, 0.5, and 1.0 mg/mL. 100 μL of DPPH (6 mM) was dropped into 2.8 mL methanol, followed by adding 100 μL of every sample solution. e resultant solution was shaken at RT for 30 min before the absorption was measured at 517 nm on a Cary 60 UV-Vis spectrophotometer (Agilent, USA). e scavenging activity of the synthesized compounds was calculated using Q (%) is the percentage of the scavenging activity of the compound, A is the absorbance in the presence of the tested compound, and A o is the absorbance without sample.
DMSO was dissolved in the camptothecin and synthesized compounds at the concentrations of 2, 4, 6, 8, and 10 µg/mL. e camptothecin, an anticancer compound, was used as a positive control. e control group has only been handled under similar conditions with DMSO.

In Vitro Cytotoxicity of α-MG Derivatives.
e 4 kinds of tested cells were seeded in 96-well microtiter plates at a density of 7.5 × 10 3 cells per well (NCI-H460) or 10 × 10 3 cells per well (MCF-7, HeLa, and Hep G2) for 24 hours before incubation about 48 h with various concentrations of the compounds. When the incubation ends, cells were fixed with cold trichloroacetic acid 50% (w/v) for 1.5 h; then, plates were washed 5 times with H 2 O. e plate was stained with 0.2% (w/v) sulforhodamine B for 20 minutes [45,46]. e protein-bound dye was solubilized in 10 mM Tris base solution after five washes with 1% acetic acid. e absorbance was measured at a wavelength of 492 nm/620 nm with the microplate reader (Synergy HT, BioTek, Vermont, USA). e growth inhibition percentage (Inh %) was determined by in which ODc and ODt are the values of the optical density of the control sample and test sample, respectively. A drug concentration is needed for inhibition of 50% (IC 50 ), which was calculated by GraphPad Prism ver 5.

Results and Discussion
e previous studies have reported several semisynthetic amphiphilic α-MG derivatives as potential compounds for in vitro bactericidal activity on Gram-positive bacteria (Figure 1). α-MG, ß-MG, and c-MG can kill bacteria quickly and avoid the phenomenon of drug resistance (Figure 1(a)) [49,50]. Besides, the synthetic and medicinal chemistry of α-MG derivatives for the activity of anticancer was recorded. Xanthone analogs based on α-MG were carried out and estimated as an anticancer compound by an in vitro cell test. A variety of compounds have shown promising activity on cancerous cell lines (compounds i, ii, and iii in Figure 1(b)).
ese studies suggest that C-3 and C-6 phenol groups are critical to inhibiting cancer cell lines and increase the activity of drug-like properties [51][52][53].

Extraction, Isolation of α-MG, and Chemical
Reactions. α-MG yields obtained from 500 g of dried mangosteen peel corresponded to 0.56% (2.813 g) and moisture content of α-MG of 19.94 ± 0.06%. After isolation by CC technology with the solvent mixture of E : H, the α-MG yield was 1.321 g. e extractable performance was 0.26%. e following are the yield, analytical data, and spectra data for each compound:   Figure S3) and (Table S2) Figure S3) and (Table S4) Figure S3) and (Table S5) Figure S3) and (Table S6) Figure S3) and (Table S7) e synthetic procedure to obtain the α-MG derivatives compounds is shown in Figure 2. e natural compound, α-MG, reacted with 2-fluorobenzoyl chloride, 3-fluorobenzoyl chloride, and 4-fluorobenzoyl chloride in the presence of trimethylamine using dichloromethane solvent to obtain compounds 1, 2, and 3, respectively. Subsequently, α-MG was treated with 2-bromobenzoyl chloride, 4-bromobenzoyl chloride with the solvent of dichloromethane, and the presence of trimethylamine to form α-MG derivatives of 4 and 5. Next, also α-MG reacted with 3-chlorobenzoyl chloride under the same conditions to provide compound 6. e chemical structures were determined by 1 H-NMR, 13 C-NMR, MS, and HPLC. A relevant synthesis and analytical data were provided in the experimental section and supporting information.

Evaluation of Antioxidant Activity.
e free radical scavenging potential of α-MG and its derivatives at different concentrations were also detected by the DPPH assay [54]. Compounds 1, 2, and 6 showed a weak antioxidant activity even at high concentrations of 1 mg/mL, which are lower than those of α-MG. e antioxidant activity of compounds 1, 2, and 6 at 1.0 mg/mL was 7.1%, 3.8%, and 5.9%, respectively, and lower than that of α-MG (8%). In the case of compound 3, the antioxidant activity increased slightly to 12.2% at a concentration of 1.0 mg/mL. For compound 5, the antioxidant activity at a concentration of 0.1, 0.5, and 1 mg/ mL was 6.2, 12.7, and 13.6%, respectively, which were higher than those of α-MG. Significantly, the antioxidant activity of compound 4 climbed up to 18.2, 43.3, and 68.7% at 0.1, 0.5, and 1.0 mg/mL, respectively, and these values were far beyond those of α-MG and remaining derivatives, as shown in Figure 3.
In this study, the data indicated that α-MG and its derivatives could be free radical inhibitors, which may be assigned to hydroxyl groups at C-3 and C-6. e phenolic compounds may attribute these antioxidant functions to their hydrogen-donating capacity. e free radicals induce autoxidation of unsaturated lipids. Antioxidants inhibit the free oxidation radical chain reaction in transferring hydrogen from the phenolic hydroxyl groups, creating a stable end compound. e difference in the antioxidant activity of α-MG derivatives could be due to different positions of Cl − , F − , and Br − onto the chemical structure of benzoyl, as reported by previous studies on the relationship between chemical structures and DPPH antioxidant activity [55].

In Vitro Cytotoxicity against Cancerous Cell Lines.
Based on the outstanding results of antioxidant activity, compound 4 was selected for in vitro cytotoxicity against human cancer cell lines. To investigate the anticancer activity of compound 4, the cytotoxicity assay was performed in MCF-7, NCI-H460, HeLa, and Hep G2 cancerous cells for 48 h. As shown in Figure 4, 19.1, 20.6, and 29.3% of NCI-H460 cells die after being treated by compound 4 with a corresponding concentration of 2, 4, and 6 µg/mL. is result indicated the high sensitivity of NCI-H460 cancerous cells toward compound 4, even at low concentrations. Besides, compound 4 showed lower cytotoxicity for HeLa and MCF-7 cell lines than for NCI-H460. At low concentrations (2, 4, and 6 µg/mL), compound 4 exhibited insignificant cytotoxicity against Hep G2 cancerous cells.
However, almost cell of Hep G2 was killed by compound 4 at the concentration of 10 µg/mL. Meanwhile, the lower cytotoxic percentage for the remaining cancerous cell lines was signified at 10 µg/mL. e cytotoxicity for MCF-7, HeLa, and NCI-H460 corresponded to the values of 68.5, 77.4, and 88.5%. ese results indicated that compound 4 could notably kill four cancerous cell lines at a concentration of 10 µg/ mL. And this result is significantly higher than that of α-MG as shown in Figure S5.
To measure the potency of compound 4 in inhibiting four cancerous cell lines, the half-maximal inhibitory concentration (IC 50 ) was evaluated. As shown in Figure 5

Relationship between Structure and Anticancer Activity of α-MG Derivatives.
To explore the relationship between structure and activity more thoroughly of α-MG and its derivatives, we collected and compared the structure and cytotoxicity of α-MG derivatives with the previous studies as shown in Table 1   According to Ly et al., the same cytotoxic activity was for both cowanin and cowanol despite having different side chains at C-2. is demonstrated that the substitution of a methyl group at C-13 via a hydroxymethyl group did not impact the cytotoxicity. Besides, since both cowanin and cowanol are less active than α-mangostin, the length of the alkyl side chains at C-2 or C-8 is significant as well [52].  Table 1). e bromide substitution (compound 3e) was created, taking into account the vacant sites of α-MG C-4 and C-5 positions. 2k compound with mild antiproliferation activity against all the tested cancer cell lines is less toxic to five cell lines compared to α-MG. e finding revealed that cyclization at C-12 and OH of α-MG is more sensitive to some cancer cells studied in vitro than normal cells and thus has good selectivity [59]. Besides, the biological effects of halogen were worth studying. e bromide substitution (compound 3e) was created, taking into account the vacant sites of α-MG C-4 and C-5 positions. e effect of halogenation on 3e's selective potency is weak. e halogenated product exhibited better cytotoxicity; compound 3e in SMMC-7721 cell lines is up to three times more cytotoxic than α-MG with an IC 50 value of 3.98 μM [59]. In the case of the isopropyl substitution compounds at C-6 to form isopropyl mangostin (IPM) and dimethyl substitution compounds at OH groups of C-3 and C-6 to generate Di-O-methyl mangostin (DMM), the cytotoxicity is not good. e IC 50 in cervical cancer for IPM and DMM was 34.84 and 15.57, respectively, as discussed by Kirthanashri et al. [60].
In this work, α-MG showed cytotoxic activity, manifested by the decrease of cell viability in four cancer cell lines at high concentrations. e IC 50 values of α-MG in MCF-7, NCI-H460, HeLa, and Hep G2 were corresponding to 48.47, 47.72, 60.61, and 65.48 μM, respectively. Significantly, the 2bromobenzoyl chloride substitution at C-3 and C-6 of α-MG (compound 4) indicated remarkable cytotoxicity. In detail,

Conclusions
In summary, we have successfully extracted pure α-MG from mangosteen peels for the synthesis of α-MG derivatives. e esterification reaction at positions of C-3 and C-6 of α-MG proved that the hydroxyl group (-OH) at C-3 and C-6 plays an essential role for antioxidant and anticancer agents. Compound 4 exhibited the highest free radical scavenging DPPH activity (68.7% at the concentration of 1 mg/mL) compared to that of α-MG and remaining derivatives. e result of the in vitro cytotoxicity study for the synthesized derivatives against four cancer cell lines MCF-7, NCI-H460, HeLa, and Hep G2 was significantly improved. Notably, compound 4 showed a significant enhancement of the antioxidant activity and the cytotoxicity toward MCF-7, NCI-H460, HeLa, and Hep G2 cell lines in comparison with pristine α-MG at low concentration. In particular, the IC 50 values of compound 4 exhibited five to six times lower than those of α-MG for all tested cancerous cell lines after 48 h. Our results provide new opportunities for further explorations of α-MG derivatives for antioxidants and promises as drugs in cancer therapy. is result is the platform for a deeper research of the α-MG for bioapplication.

Data Availability
Data supporting the results of our study can be found in supporting information.

Conflicts of Interest
e authors declare that there are no conflicts of interest regarding the publication of this article.