Chemical composition and in vitroevaluation of the cytotoxic and antioxidant activities of supercritical carbon dioxide extracts of pitaya (dragon fruit) peel
© Luo et al.; licensee Chemistry Central Ltd. 2014
Received: 15 December 2013
Accepted: 30 December 2013
Published: 3 January 2014
Hylocereus polyrhizus and Hylocereus undatus are two varieties of the commonly called pitaya fruits, and pitaya fruits have gained popularity in many countries all over the world. However, studies on chemical composition and the nutritional quality of pitaya flesh peel are limited.
Extracts of pitaya (H. polyrhizus and H. undatus) peel were extracted by supercritical carbon dioxide extraction, and analyzed by gas chromatography–mass spectrometry analysis. Their cytotoxic and antioxidant activities were investigated. The main components of H. polyrhizus extract were β-amyrin (15.87%), α-amyrin (13.90%), octacosane (12.2%), γ-sitosterol (9.35%), octadecane (6.27%), 1-tetracosanol (5.19%), stigmast-4-en-3-one (4.65%), and campesterol (4.16%), whereas H. undatus were β-amyrin (23.39%), γ-sitosterol (19.32%), and octadecane (9.25%), heptacosane (5.52%), campesterol (5.27%), nonacosane (5.02%), and trichloroacetic acid, hexadecyl ester (5.21%). Both of the two extracts possessed good cytotoxic activities against PC3, Bcap-37, and MGC-803 cells (IC50 values ranging from 0.61 to 0.73 mg/mL), and the activities of their main components were also studied. Furthermore, these extracts also presented some radical scavenging activities, with IC50 values of 0.83 and 0.91 mg/mL, respectively.
This paper provides evidence for studying the chemical composition of supercritical carbon dioxide extracts of pitaya peel and their biological activity.
Pitaya is often called “dragon fruit” following its bright red skin with green overlapping fins covering the fruit, which has gained popularity in many countries all over the world . Three varieties that have been commercialized are Hylocereus polyrhizus, which has red-skinned fruit with red flesh, Hylocereus undatus (Red pitaya), which has red-skinned fruit with white flesh, and Hylocereus megalanthus (Yellow pitaya), which has yellow-skinned fruit with white flesh . They belong to the vine cacti from the subfamily Cactoideae of the tribe Cacteae, and are native to the tropical forest regions of Mexico and Central and South America . H. polyrhizus and H. undatus have recently drawn much attention from growers worldwide, because of their powerful antioxidative activity [4–6]. Betanin, phyllocactin, hylocerenin, and betacyanin with 5–O-glycosides or 6-O-glycosides have been discovered in many species of the Cactaceae family [7, 8]. Furthermore, these types of compounds are responsible for many pharmacological activities such as antitumor, antioxidant, and anti-inflammatory actions. The objectives of this study were to evaluate the nutritional quality of pitaya flesh peel and study whether the peel of pitaya, the waste product from juice manufacture, could be utilized as a potential alternative for various sources of nutrients or antioxidants to improve human health.
The aim of the present study was, as a first step, to determine the chemical composition of supercritical carbon dioxide extracts of the peel of pitaya (H. polyrhizus and H. undatus) by gas chromatography–mass spectrometry (GC-MS) analysis. In a second step, we examined the in vitro cytotoxic and antioxidant activities of the two extracts by MTT and DPPH assays, respectively. We compared their activities with the activity of the major component of each extract sample. To our knowledge, there are no published reports on the chemical compositions, cytotoxic and antioxidant activities of supercritical carbon dioxide extracts of the pitaya (H. polyrhizus and H. undatus) peel.
Results and discussions
Chemical composition of supercritical carbon dioxide extract
Chemical composition of supercritical carbon dioxide extracts of pitaya peel
(Z, Z)-9, 12-Octadecadienoic acid
Trichloroacetic acid, hexadecyl ester
6-Tetradecanesulfonic acid, butyl ester
1,2-Benzenedicarboxylic acid, mono (2-ethylhexyl) ester
Phthalic acid, 6-ethyloct-3-yl 2-ethylhexyl ester
A total of 24 components in H. polyrhizus extract, representing 90.66% of the total composition, were identified, of which 29.77% were triterpenoids and 16.46% were steroids. Its extract was characterized by a high content of β-amyrin (15.87%), α-amyrin (13.90%), octacosane (12.2%), γ-sitosterol (9.35%), octadecane (6.27%), 1-tetracosanol (5.19%), stigmast-4-en-3-one (4.65%), and campesterol (4.16%).
The predominant constituents of H. undatus extract were β-amyrin (23.39%), γ-sitosterol (19.32%), and octadecane (9.25%), which formed approximately a half of the extract. Heptacosane (5.52%), campesterol (5.27%), nonacosane (5.02%), and trichloroacetic acid, hexadecyl ester (5.21%) were also present at significant concentration. A total of 19 components were identified, comprising 92.82% of the total extract. Moreover, its extract was also dominated by triterpenoids (23.39%) and steroids (19.32%).
In conclusion, both of H. polyrhizus and H. undatus contained mostly triterpenoids and steroids. In contrast, the content of triterpenoids in supercritical carbon dioxide extract of H. polyrhizus was higher than that of H. undatus, whereas the extract of H. undatus had higher content of steroids. It would also be worth pointing out that the constituents of the two extracts are normally influenced by several factors such as geographical, climatic, seasonal and experimental conditions.
Effect of steroids and triterpenoids from supercritical carbon dioxide extracts of H. polyrhizus and H. undatus against cell viability of different cancer cell lines
73.2 ± 1.02
78.4 ± 0.93
51.9 ± 0.87
74.4 ± 0.65
58.2 ± 0.44
43.8 ± 0.63
65.4 ± 1.13
79.3 ± 0.49
56.9 ± 0.81
1.09 ± 0.18
1.34 ± 0.30
0.83 ± 0.22
It can be seen from the IC50 values that β-amyrin, β-sitosterol, and stigmast-4-en-3-one suppressed proliferation of the above three cancer cell lines in different extents (IC50 values of 43.8-79.3 μM). These compounds showed similar inhibition activity against PC3 and MGC-803 cells, while the proliferation inhibition of MGC-803 cells was superior to other kinds of cancer cells. However, α-amyrin displayed weak activities against the three cells. These finding indicated that β-amyrin, β-sitosterol, and stigmast-4-en-3-one may be responsible for the activities of the two extracts.
Antioxidants terminate these chain reactions by removing free radical intermediates, and inhibit other oxidation reactions, and they do this by being oxidized themselves [17–19]. High phenolic content were usually correlated with high radical scavenging activity . Choo et al. found that H. polyrhizus and H. undatus had great antioxidant properties, because of high content of polyphenols . Moreover, polyphenols can be extracted by supercritical carbon dioxide extraction . Hence, antioxidant activities of the pitaya peel extracts were most probably due to the presence of polyphenols, which have the hydrogen-donor ability to scavenge the free radicals. However, the polyphenols were not detected by GC-MS. Studies of the content of polyphenols in the extracts are currently underway.
In summary, the composition of supercritical carbon dioxide extracts of pitaya (H. polyrhizus and H. undatus) peel has been analyzed by GC-MS, and their cytotoxic and antioxidant activity were investigated. The predominant constituents of H. polyrhizus extract were β-amyrin (15.87%), α-amyrin (13.90%), octacosane (12.2%), γ-sitosterol (9.35%), whereas H. undatus were β-amyrin (23.39%), γ-sitosterol (19.32%), and octadecane (9.25%). The two extracts showed a wild range of cytotoxic activities against PC3, Bcap-37, and MGC-803 cells, and it was found that β-amyrin, β-sitosterol, and stigmast-4-en-3-one, the main components, were responsible for their activities. In addition, they had some DPPH radical scavenging activities, with IC50 values of 0.83 and 0.91 mg/mL, respectively.
There is a trend to find cytotoxic and antioxidant materials from natural products in the modern medical industry. The above results show that supercritical carbon dioxide extracts of pitaya (H. polyrhizus and H. undatus) peel could be a potential source of compounds with cytotoxic and antioxidant activities and the results provide a reference point for further research on the chemical components of supercritical carbon dioxide extracts of pitaya peel as well as for their utilization.
Materials and methods
General procedures and reagents
The melting points of the products were determined using an XT-4 binocular microscope (Beijing Tech Instrument Co. Ltd., Beijing, China). 13C NMR were recorded using a JEOL-ECX500 spectrometer at 22°C, with tetramethylsilane as the internal standard and CDCl3 as the solvent. Column chromatography was performed using silica gel (200–300 meshes) (Qingdao Marine Chemistry Co., Qingdao, China) and Sephadex LH-20 (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). Sodium dodecyl sulfate (SDS) were purchased from Beijing Dingguo CO., Ltd; 2,2-diphenlyl-1-picrylhydrazyl (DPPH) and vitamin C (Vc) were purchased from Aladdin Reagent Inc; 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and DMSO were purchased from Roche Molecular Biochemicals (1465–007); Adriamycin (ADM) was purchased from Zhejiang Hisun Pharmaceutical Co., Ltd; α-amyrin, β-sitosterol, and stigmast-4-en-3-one had been prepared in previous work [22, 23]. β-amyrin was isolated from supercritical carbon dioxide extract of H. undatus peel, and its purification process and NMR data are presented in Additional file 1. All other chemicals were of analytical reagent grade and used without further purification.
Fresh peel of pitaya (H. polyrhizus and H. undatus) were collected from Guiyang, Guizhou province in China, in July 2013. Voucher specimens were deposited at Guizhou Fruit Institute, Guiyang, China.
Supercritical carbon dioxide extraction
About 250 g of dried peel of pitaya (H. polyrhizus and H. undatus) were cut into pieces and submitted to extraction. A CO2 flow rate of 30 L/h and an extraction period of 60 min were used. The extraction was performed under a pressure of 30 MPa and at a temperature of 40°C. The two extracts obtained by supercritical carbon dioxide extraction assay were pale yellowish. These extracts were dried over anhydrous Na2SO4 and placed at a low temperature in the refrigerator until analysis.
Gas chromatography-mass spectroscopy (GC-MS) analysis
A gas chromatographic-mass spectral analysis was performed on the extracts using an Agilent 6890 GC with Agilent 5973 mass selective detector (EI-MS, electron energy = 70 eV, scan range = 10-550 amu), and a fused silica capillary column (HP-5 ms, 30 m × 0.25 mm) coated with 5% phenyl methyl siloxane (0.25 μm phase thickness). The carrier gas was helium (99.999%) with a flow rate of 1.0 mL/min. The injector temperature was 250°C, and the oven temperature was programmed to 50°C for 2 min, and then increased to 290°C at a rate of 5°C/min. The interface temperature was 280°C. A 1% (w/v) solution of each sample in dichloromethane CH2Cl2 was prepared, and 1 μL was injected using a split injection technique with split ratio 20:1. The components were identified by comparison of their mass spectra with those of the NIST 5 mass spectra library.
Cell lines and culture
PC3, Bcap-37, and MGC-803 cell lines were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The entire cancer cell lines were maintained in the RPMI 1640 medium. They were supplemented with 10% heat-inactivated fetal bovine serum (FBS). All cell lines were maintained at 37°C in a humidified 5% carbon dioxide and 95% air incubator.
DPPH free radical scavenging assay
All statistical analyses were performed using SPSS 10.0, and the data were analyzed using one-way ANOVA. The mean separations were performed using the least significant difference method. Each experiment was performed in triplicate, and all experiments were run thrice and yielded similar results. Measurements from all the replicates were combined, and the treatment effects were analyzed.
The authors wish to thank the Scientific Research of Guizhou (No.20126006) for the financial support.
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