Will Grapefruit Essential Oil Affect Birth Control?
Molecules. 2020 January; 25(1): 217.
Chemic Composition, Antimicrobial, Antioxidant, and Antiproliferative Backdrop of Grapefruit Essential Oil Prepared by Molecular Distillation
Raffaele Capasso, Bookish Editor
Received 2019 December 3; Accepted 2020 Jan 3.
Abstract
Grapefruit essential oil has been proven to take wide range of bioactivities. However, bioactivity of its molecular distillate has not been well studied. In this report, a light stage oil was obtained by molecular distillation from cold-pressed grapefruit essential oil and GC-MS was used to identify its chemical composition. The antimicrobial activity of the light phase oil was tested by filter paper diffusion method, and the anticancer activity was determined by the Cell Counting Kit-8 (CCK-8) analysis. Twenty-4 components were detected with a full relative content of 99.74%, including 97.48% of terpenes and 1.66% of oxygenated terpenes. The light phase oil had the best antimicrobial effect on Bacillus subtilis, followed by Escherichia coli, Staphylococcus aureus and Salmonellaty phimurium. DPPH and ABTS assays demonstrated that the light phase oil had good antioxidant action. The CCK-viii assay of cell proliferation showed that the light phase oil had a good inhibitory issue on the proliferation of HepG2 liver cancer cells and HCT116 colon cancer cells.
Keywords: grapefruit essential oil, molecular distillation, GC-MS, antimicrobial, antioxidant, anticancer activity
one. Introduction
With the frequent occurrence of food safety issues and the toxicity of synthetic chemicals, the need for safe and natural alternatives is growing. Plant extracts have been used since ancient times, and now the focus is on their role in health promotion and their treatment and prevention properties for various diseases. In the past few decades, plant essential oils (EOs) accept attracted a lot of interest due to their prophylactic and pharmacological properties including bacteriostatic, costless radical scavenging, anti-inflammatory, and inhibitory effects on cancerous tumor cell proliferation [ane,ii,iii,4]. Citrus Eos are the chief aromatic by-products of the juice extraction industry and are widely used in nutrient, cosmetics and pharmaceutical manufacture [5,half dozen,seven]. The annual global product of citrus EO is approximately xvi,000 tons, and the cost is about $14,000/ton on the international market. Thus, citrus EO is of bully demand and is one of the more than promising market prospects [8]. Grapefruit (Citrus paradisi Macf.), 1 of the world′s largest product citrus families [nine], is famous for its taste and nutritional value. Grapefruit EO is extracted from grapefruit peel and has been used for a long time equally a valuable ingredient for its characteristic odour in flavor and fragrance [10,xi]. Similar to near citrus EOs, its major components are terpenes and terpene oxides. Terpene oxides include alcohols, ethers, aldehydes, ketones, and esters [12,13,14], which are the main source of the scent, whereas terpenes contribute less to the olfactory property. In spite of extensive studies on the aroma features of grapefruit EO, in recent years, more and more researchers have become interested in exploring their biological and pharmacological activities. Grapefruit EO has been reported to have a wide range of bioactivities. Information technology was shown to inhibit the growth of food-borne spoilage bacteria and pathogenic strains [15,sixteen,17]. Okunowo et al. (2013) found that grapefruit EO obtained from the skin by hydrodistillation exerted inhibitory effects confronting bacteria and fungi, andmay be further developed for the treatment of certain diseases [eighteen]. Grapefruit EO has shown antioxidant activeness, which was of import for food preservation and disease prevention [19,20]. Ahmed et al. (2019) reported that grapefruit EO extracted by hydrodistillation had antioxidant activeness by using DPPH and FRAP assays [21]. Grapefruit peel extracts have been shown to decrease the HL-threescore cell viability in a concentration-dependent mode [22]. In fact, grapefruit extracts (a mixture of EO and other nonvolatile phytochemicals) could also inhibit the growth and proliferation of cancer cells such equally neuroblastomas, leukemias, and prostate and lung cancer lines [23,24,25]. Cuthrell et al. (2006) reviewed the anticancer activities of phytochemicals establish in grapefruit [26].
Most grapefruit EO samples used for bioactivity studies were made by cold-pressing, steam distillation, or hydrodistillation methods. Cold-pressing is the predominant method to excerpt about citrus peel EOs, including grapefruit EOs. In commercial practice, grapefruitis candy to obtain juice and other past-products. EO is one of the main grapefruit by-products. Large-scale grapefruit EO is mainly prepared by a cold-pressing method based on John Bean FoodTech (JBT) juice extractors and its technology, which is used by 75% of the globe's citrus juice production [27]. The juice and EO are extracted separately and simultaneously. The EO was extracted by mechanical rupturing of the oil sacs in the flavedo, expressing the oil as an aqueous emulsion from which information technology is separated by centrifuging. The EO recovery is a physical separation process and no heat is practical throughout the whole extraction procedure. The performance temperature is much lower than in the distillation process. Thus, the EOs will have characteristics that are closer to those of the essence nowadays in the grapefruit matrix. Large scale production, low cost and the olfactory property characteristic remaining are the big advantages of the common cold-pressing method. However, common cold-pressed grapefruit oil contains waxes, pesticide residues, coumarins, carotenoids and other nonvolatile components [28,29,30], some of them also accept expert bioactivities that may cause bias in bioactivity research of EO. César et al. (2009) constitute that furanocoumarins isolated from grapefruit peel oil showed potent in vitro inhibitory activity confronting abdominal cytochrome P450 3A4, an enzyme involved in "grapefruit/drug" interactions in humans [28]. Steam distillation or hydrodistillation was carried out at relatively high temperature which may crusade degradation of some thermal sensitive molecules [18,31]. To avoid such bug, and notice a new way to use the commercial available cold-pressed grapefruit EO in biochemistry and pharmachutical fields, we used a molecular distillation method to prepare grapefruit oil samples for our bioactivity tests.
Molecular distillation is a special liquid–liquid separation technology under high vacuum, which is employed every bit a separation process in the nutrient industry [32,33]. Molecular distillation tin can divide the EO mixture into ii different phases according to the free path of different molecules at low temperature. Molecular distillation is prominent with the advantages of low temperature treatment and high vacuum application, which is very suitable for thermolabile compounds and is used for concentrating and refining EOs [34,35]. At present, there are seldom reports on the antibacterial and anticancer activity of Eos obtained by molecular distillation. In this study, the cold-pressed grapefruit EO was processed by molecular distillation applied science and the low-cal phase essential oil (LPEO) was nerveless. Its constituents were identified by GC-MS. The activities of LEPO were tested on microorganisms and malignant proliferating cells (HCT116 colon cancer cells and HepG2 liver cancer cells) were tested. We expect that this piece of work can stimulate the development of new agents for nutrient preservation and chemo-preventive anti-cancer treatments.
2. Results and Word
two.1. Chemic Limerick of the Light Phase Grapefruit Essential Oil
The chemical limerick of the grapefruit calorie-free phase essential oil (LPEO)was analyzed by GC-MS. The total ion chromatogram (TIC)of LPEO is shown in Effigy 1. The relative content of each component was calculated past the pinnacle expanse normalization method. The components were identified according to retention index and the NIST mass spectral library.
Total ion chromatogram of grapefruit light phase essential oil (LPEO).
Every bit shown in Table 1, 20-four compounds, accounting for 99.74% of the full oil were identified. Monoterpenes were the major components, accounting for 96.93% of the total oil. Limonene (93.33%) was the predominant component of monoterpenes, followed by β-myrcene (2.xvi%), α-pinene (0.76%), and sabinene (0.60%). Monoterpene oxide (1.62%) included carvone (0.41%), cis-limonene oxide (0.43%), and trans-limonene oxide (0.33%). The sesquiterpene (0.55%) included caryophyllene (0.xx%), β-cubebene (0.xiv%), α-copaene (0.13%), etc. Caryophyllene oxide (0.04%) was the only sesquiterpene oxide detected. In addition, iii linear aldehydes: Octanal (0.36%), decanal (0.19%), and nonanal (0.05%) were found in LPEO. Pino et al. (1999) reported the chemical limerick of grapefruit EO prepared by steam distillation from solids and effluents produced during commercial oil extraction [31]. The limonene content (70.nine%) in steam-distilled oil was much less than LPEO (93.33%); however, the content of myrcene (13.6%) and α-pinene (iii.8%) was much higher than LPEO (myrcene 2.16% andα-pinene 0.76%). Also, Okunowo et al. (2013) reported the components of grapefruit EO obtained by hydrodistillation [18]. The content of limonene (75.07%)was airtight to that of steam-distilled oil. Cold-pressed grapefruit oil was shown to have a limonene content of 93.47%, nevertheless, the corrected limonene content became 85.60% when nonvolatiles were excluded [36]. The composition of distilled samples of grapefruit EO still vary from each other according to genetic differences, soil type, maturity stages, weather types and culturing conditions etc [37].
Table 1
Chemical composition of grapefruit light phase essential oil (LPEO) by GC-MS.
| No. | RIa | Compounds | Composition (%) |
|---|---|---|---|
| i | 938 | α-Pinene | 0.76 |
| 2 | 956 | Camphene | 0.01 |
| 3 | 977 | Sabinene | 0.60 |
| iv | 985 | β-Pinene | 0.05 |
| five | 992 | β-Myrcene | 2.xvi |
| half-dozen | 1007 | Octanal | 0.36 |
| vii | 1049 | Limonene | 93.33 |
| 8 | 1053 | β-Ocimene | 0.02 |
| 9 | 1103 | Linalool | 0.12 |
| x | 1108 | Nonanal | 0.05 |
| eleven | 1127 | trans-p-Mentha-ii,8-dien-ane-ol | 0.xvi |
| 12 | 1137 | cis-Limonene oxide | 0.43 |
| 13 | 1141 | trans-Limonene oxide | 0.33 |
| 14 | 1155 | Citronellal | 0.04 |
| 15 | 1199 | α-Terpineol | 0.13 |
| sixteen | 1208 | Decanal | 0.19 |
| 17 | 1251 | Carvone | 0.41 |
| 18 | 1377 | α-Copaene | 0.13 |
| xix | 1388 | β-Cubebene | 0.xiv |
| twenty | 1421 | Caryophyllene | 0.20 |
| 21 | 1457 | Humulene | 0.03 |
| 22 | 1482 | Germacrene D | 0.01 |
| 23 | 1519 | δ-cadinene | 0.04 |
| 24 | 1566 | Caryophyllene oxide | 0.04 |
| Full | 99.74 | ||
| Monoterpene hydrocarbons | 96.93 | ||
| Oxygenated monoterpenoids | i.62 | ||
| Sesquiterpene hydrocarbons | 0.55 | ||
| Oxygenated sesquiterpenes | 0.04 | ||
| others | 0.sixty | ||
2.2. Antimicrobial Activity
Grapefruit EOs prepared by common cold-pressing or hydrodistillation using a Clevenger-type appliance have shown a wide spectrum of antimicrobial activity in vitro [18,37]. Nonetheless, antimicrobial activity of grapefruit EO prepared by molecular distillation has non been well studied. Nosotros tested LPEO on v microorganisms and the results obtained are shown in Table 2. The filter paper diffusion method was used to test the antibacterial activity of LPEO against different leaner, and the activeness of LPEO was evaluated according to the diameter of the inhibition zone and the minimum inhibitory concentration (MIC) values. LPEO exhibited strong antibacterial furnishings on the 4 bacteria tested. From the calibration of the inhibition zone, LPEO had the strongest inhibitory effect on B. subtilis with a maximum diameter of 35.59 mm, followed by E. coli, Due south. aureus, and S. typhimurium. LPEO had no inhibitory activity against P. aeruginosa with an inhibition zone of 8.57 mm. P. aeruginosa belongs with Eastward. coli and Due south. typhimurium to the group of Gram-negative leaner merely exhibits quite a different response to LPEO. This phenomenon may be partly due to its relatively low outer membrane permeability. LPEO molecules enter the periplasm by improvidence through the channels of nonspecific porins in the outer membrane, and this pathway in P. aeruginosa is 10- to 100-fold less efficient than that in Due east. coli [38]. Regarding MIC values, LPEO showed the best antimicrobial activity against Bacillus subtilis with a MIC value of 0.78 µL/mL. Based on the inhibition zone and MIC values, the club of sensitivity of the different bacteria was: B. subtilis > E. coli > South. aureus > S. typhimurium > P. aeruginosa. Since common cold-pressed grapefruit oil (Citrus paradisi Macf.) has been evaluated as "more often than not recognized as safe" (GRAS) by the Skilful Panel of the Flavor and Extract Manufacturers Association (FEMA) [39], and LPEO, a distillate from common cold-pressed EO, showed strong sensitivity to nigh tested microorganisms, it appeared to be suitable to food applications. These results demonstrate that molecular distillation technology can provide a grapefruit EO fraction with adept antimicrobial activity.
Table 2
The antimicrobial activity of grapefruit lite phase essential oil (LPEO).
| Bacterial Strain | Diameter of Inhibition Zone (mm) | MIC (µL /mL) |
|---|---|---|
| Bacillus subtilis (G+) | 35.59 ± 1.06 a | 0.78 |
| Staphylococcus aureus (G+) | 24.34 ± 0.52 c | half dozen.25 |
| Escherichia coli (G-) | 26.86 ± 0.17 b | 6.25 |
| Salmonella typhimurium (G-) | 21.70 ± 0.21 d | 12.50 |
| Pseudomonas aeruginosa (G-) | 8.57 ± 0.13 e | 25.00 |
Uysal et al. (2011) evaluated the antibacterial activities of grapefruit Eos from solvent-free microwave extraction (SFME) and hydrodistillation (HD) by the disc-diffusion method [17]. The Eos obtained from SFME and Hard disk showed the highest activeness against S. aureus with inhibition zones of 53 and 41 mm, respectively, higher than LPEO (24.34 mm). The action against E. coli (30 mm and 28 mm) was close to our result (26.86 mm). Both of their samples and LPEO showed no obvious activity against P. aeruginosa. LPEO showed meliorate activity against S. typhimurium (21.lxx mm) than their samples (15 mm and 13 mm). Although a lot of plant EOs accept shown antimicrobial activity, the reason of this capacity is not well known. It could be provoked by the major components of the EOs or due to a synergistic effect amid the major components and the modest ones. Different preparation methods yield EO samples with differences in chemical composition and relative content, and cause differences in antimicrobial activity.
2.three. Antioxidant Activity
A lot of EOs accept been reported to scavenge the free radicals that cause damage to the trunk and reduce the risk of many diseases originating from oxidative stress. In order to measure the result of LPEO and make up one's mind its potential awarding in nutrient, cosmetic or pharmaceutical industries, we evaluated its antioxidant activeness using two dissimilar assays: The two,2-diphenyl-1-picrylhydrazyl (DPPH)and 2,two′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acrid) radical (ABTS) assays. Butylated hydroxytoluene (BHT) was used as positive control. The IC fifty values of BHT in DPPH and ABTS were 0.03 mg/mL and 0.01 mg/mL, which was consistent with the literature [40]. The DPPH and ABTS activities of LPEO were obtained with IC l values of 22.06 ± 0.92 mg/mL and xv.72 ± 0.32 mg/mL, respectively. LPEO had better antioxidant activity than cold-pressed grapefruit EO in the DPPH assay (EC 50 > xl mg/mL) and hydrodistilled grapefruit EO in the ABTS analysis (EC 50 = 27.v mg/mL) [35]. Compared with cold-pressed orange oil, LPEO had much lower antioxidant activity in the DPPH assay (IC 50 = 3.01 ± 0.20 mg/mL) and ameliorate activity in the ABTS assay (IC 50 = 23.25 ± 0.84 mg/mL) [41].
2.4. Antiproliferative Activity of LPEO in HepG2 and HCT116 Cancer Cells
The effects of dissimilar concentrations of LPEO on the proliferation of HepG2 liver cancer and HCT116 colon cancer cells were tested past the Cell Counting Kit-8 (CCK-8) method [42,43]. The results are shown in Figure ii. The viability rate of both jail cell types decreased with increasing LPEO concentration. When the concentration of LPEO was less than 0.1 μL/mL, no obvious change of viability of HepG2 cells was observed. However, when the concentration of LPEO was higher than 0.1 μL/mL, the viability of HepG2 cells significantly decreased; at the LPEO concentration of 0.3 μL/mL, the viability was 7.four%only. LPEO too had a practiced inhibitory effect on the growth of HCT116 colon cancer cells. At the concentration of0.05 μL/mL or college, the viability of HCT116 cells significantly decreased. It was as depression equally 7.43% when the concentration of LPEO was 0.5 μL/mL. GraphPad Prism™ (Version 5.00) software (GraphPad Software, San Diego, CA, USA) was used to calculate IC l values. IC 50 value of HepG2 and HCT116 was 0.24 and 0.xx μL/mL, respectively. These results indicate that LPEO has a significant inhibitory effect on the proliferation of HepG2 hepatoma cells and HCT116 colon cancer cells in vitro.
Effects on the viability of cancer cellsHepG2 and HCT116 as a part of LPEO concentration. Significant decreases in prison cell viability of cancer cells are seen at increasing LPEO concentrations compared to untreated controls (control group was set to 100%). **—Very significant at p < 0.01, ***—Highly significant at p < 0.001.
Sun et al. (2002) studied antiproliferative activity of grapefruit fruit extract on the growth of HepG2 human liver cancer cells in vitro [22]. The excerpt showed antiproliferative activeness in a dose-dependent manner with the median effective dose (EC l) value of 130.09 mg/mL. Nevertheless, they did not place the specific phytochemicals which were responsible for antiproliferative activity. Manassero et al. (2013) studied the antiproliferative activity of cold-pressed EO from mandarin peel and its principal component limonene [44]. Mandarin EO and limonene exhibited IC fifty of 0.063 μL/mL and 0.150 μL/mL confronting HepG2 cells, respectively. The much higher activity of mandarin EO than LPEO (0.24 μL/mL) may attributed to other loftier potent phytochemicals in cold-pressed EO. We have reported antiproliferative activity of the 'Gannanzao' orange EO (GOEO) prepared past hydrodistillation, which exhibited IC l of 0.29 μL/mL and 0.35 μL/mL against HepG2 cells and HCT116 colon cancer cells, respectively [43]. LPEO showed a slightly college activity than GOEO, which may exist attributed to its college limonene content (LPEO 93.33%, GOEO 88.07%).
The discussion about anticancer activity of some EO components has been made by Mukhtar et al. [45]. Our report preliminarily tested the inhibitory effect of LPEO on the proliferation of HepG2 liver cancer cells and HCT116 colon cancer cells. The anticancer activity of LPEO and its components on cancer cells and their mode of action deserve further written report.
3. Materials and Methods
iii.1. Materials
Cold-pressed Marsh white grapefruit (Citrus paradisi Macf., Lakeland, FL, USA) EO was purchased from Ungerer Limited. ii,two-diphenyl-i-picrylhydrazyl (DPPH) was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan), 2,two′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), northward-alkanes(C8–C20)were purchased from Sigma-Aldrich (St. Louis, MO, United states). Butylated hydroxytoluene (BHT) was purchased from Macklin, Shanghai, People's republic of china. The following microorganisms were purchased from Beijing, Prc General Microbiological Culture Collection Center (CGMCC): Escherichia coli (ATCC25922), Staphylococcus aureus (ATCC25923), Bacillus subtilis (ATCC6633), Salmonella typhimurium (ATCC14028), and Pseudomonas aeruginosa (ATCC9207).
3.2. Preparation of Grapefruit Light Phase EO Sample
Grapefruit low-cal stage EO(LPEO) was obtained past molecular distillation from cold-pressed grapefruit EO (Citrus paradisi Macf., Lakeland, FL, USA) using a wiped-moving picture molecular distillation apparatus (Pope 2 Inch Laboratory Scale Wiped-Moving-picture show Molecular Still & Evaporator, Pope Scientific Inc., Saukville, WI, U.s.). The evaporation temperature and operation pressure were 55 °C and 6.0 Torr, respectively. Cold-pressed grapefruit EO was fed at room temperature and the feeding charge per unit was three.0 mL/min. The rotational speed of the roller wiper (Pope Scientific Inc., Saukville, WI, U.s.) was 325 rpm, and the condenser temperature was 0 °C. The last grapefruit EO sample (LPEO) was obtained from the low-cal phase outlet with the yield of 86%.
3.3. GC-MS Analyses
The constituents of LPEO were analyzed using an Agilent 7890B gas chromatograph coupled with an Agilent mass spectrometer detector (Agilent Technologies, Santa Clara, CA, U.s.a.). The GC was equipped with a HP-5 column (30.00 m × 0.25 mm × 0.25 µm). Mass spectra were obtained by electron ionization (EI) at 70 eV. The injector and detector were operated at 250 °C and 300 °C, respectively. The temperature program was 80 °C for iv min, and then increased at 5 °C/min to 250 °C and held abiding for 10 min. The constituents were identified by comparing their mass spectra with the National Institute of Standards and Technology (NIST, version 2010, U.South. Department of Commerce, Gaithersburg, Physician, Us) data reference. The retention indices (RI) of the constituents were determined by adding a C8–C20 n-alkanes mixture to the essential oil before injecting in the GC-MS equipment and analyzing it nether the same weather described above.
3.4. Antimicrobial Activeness Assays
3.four.ane. Microbial Growth Conditions
The microbial strains were maintained in food agar media at 37 °C. Subsequently, one colony from each culture was inoculated in liquid medium for xviii–24 h with shaking (200 rpm) to obtain freshly cultured microbial suspensions (>10eight CFU mL−1) for test.
iii.4.2. Determination of Bore of the Inhibition Zone
LPEO was tested on five microbial strains, using filter paper improvidence method [46]. Briefly, a suspension of the tested microorganism (10six CFUmL−1) was spread on the solid media plates. The newspaper discs (Whatman No. 1 filter paper, half-dozen mm bore) were impregnated with 20 μL LPEO and placed on the inoculated agar. The plates inoculated with bacterial strains were incubated for 24 h at 37 °C. After incubation, diameter of the inhibition zone was measured in millimeters. Each test was performed in triplicates on at least iii split up experiments.
iii.four.3. Determination of Minimum Inhibitory Concentration (MIC)
MIC values of LPEO confronting microorganisms were determined by disc-diffusion method [46,47] Sterile filter paper discs were placed on the surface of Petri dishes and impregnated with twenty µL of EO at unlike concentrations (100.00, 50.00, 25.00, 12.50, vi.25, three.125, 1.56, 0.78, 0.39, and 0.195 mg/mL) in dimethyl sulfoxide (DMSO). DMSO alone was used as negative command. After staying at iv °C for 2 h, all Petri dishes were incubated at 37 °C for 24 h. All determinations were performed in triplicates. The minimum inhibitory concentration (MIC) values were adamant as the lowest concentration of EOs that inhibited visible growth of the tested microorganism.
three.five. Complimentary Radical-Scavenging Capacity
3.5.1. DPPH Radical-Scavenging Assay
The free radical-scavenging activeness of LPEO was measured using the stable radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) analysis [48]. DPPH was dissolved in ethanol at concentration of 0.ane mmol L−1. The absorbance of 2.7 mL DPPH solution and 0.three mL ethanol was measured every bit the negative control. A different concentration of the sample solution in ethanol (0.3 mL) was pipetted into a cuvette with 2.seven mL DPPH solution. The resultant solution was incubated for 30 min at room temperature in the nighttime, and so monitored at 517 nm. The DPPH scavenging activity was expressed according to the following equation:
DPPH scavenging activity (%) = (AC − AS)/AC × 100
(1)
where AC is the absorbance of the negative control, and AS is the absorbance containing 0.3 mL sample and 2.seven mL DPPH solution. All samples were analyzed in triplicates, and the results are expressed every bit the hateful ± standard deviation. The scavenging activity was expressed as the 50% inhibitory concentration (IC fifty), which was defined as the sample concentration necessary to inhibit DPPH radical activity by l% after incubation.
iii.five.ii. ABTS Radical-Scavenging Analysis
This method was performed equally described by Teles et al. [49], based on the capacity of LPEO to inhibit the 2,2′-azinobis (3-ethylbenzthiazoline-half-dozen-sulfonic acid) radical (ABTS). 20-five mL of ABTS (7 mM) were added to 440 μL of potassium persulfate (K2S2O8, 140 mM), and the solution was kept in darkness for 12 h at room temperature in order to form the radical. An accurate volume of the solution was diluted in ethanol until an absorbance of 0.lxx at 734 nm. Once the radical was formed, 2 mL of ABTS solution were mixed with 100μL of LPEO and the absorbance measured at734 nm. ABTS scavenging effect was calculated using the following equation:
ABTS scavenging activity (%) = (AC − AS)/AC × 100
(2)
where ASouthward is the absorbance of the solution when the sample has been added and AC is the absorbance of the ABTS solution as control. The IC l was calculated from the graph of scavenging percentage against LPEO concentration. The results are expressed as the mean ± standard deviation.
3.vi. Cancer Cell Civilisation
HCT116 colon cancer cells and HepG2 liver cancer cells were purchased from Library of Typical Culture of Chinese Academy of Sciences (Shanghai, People's republic of china). HCT116 cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Hyclone, UT, Usa), supplemented with x% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Hyclone, UT, USA). HepG2 cells were cultured in MEM containing 10% FBS and 1% penicillin/streptomycin (Hyclone, UT, USA). The above-mentioned cells were maintained in 25 cm2 prison cell culture flasks in a humidified atmosphere containing 5% CO2 at 37 °C. Cells were fed until ninety% confluence and the confluent cells were washed twice with phosphate buffered saline (PBS), treated with 0.25% trypsin (Invitrogen, MA, USA) for near 1 min, and incubated at 37 °C. When the cells were contracted and rounded under the microscope, FBS (Hyclone, UT, Us) containing medium was added, centrifuged at 200× g for 3 min, and subcultured at a split ratio of 1:iii.
3.seven. Antiproliferative Activity Exam of LPEO
The jail cell proliferation inhibition rate of LPEO was evaluated by CCK-8 assay [42,43]. LPEO (50 μL) was added to the medium and mixed well. The mixture was diluted in DMSO to ready solutions at a concentration of 0.v, 0.4, 0.3, 0.ii, 0.1, 0.05, and 0.0 μL/mL, respectively. The cells were placed into 96-well plates (3 × x3 cells/well). After 24 h, 100 μL of LPEO at different concentrations was added and continued to incubate for 48 h at 37 °C in a COtwo incubator, after which the medium in the 96-well plate was tending. A 100 μL of CCK-viii examination solution (DojinDo, Tokyo, Japan) was added and incubated for ii h at 37 °C. The optical density (OD) for each well was measured at 450 nm using a microplate reader (BioTek, Winooski, VT, USA). The cell viability rate at different concentrations of LPEO treatment was calculated according to the formula:
Viability rate (%) = (ODsample − ODblank)/(ODcommand − ODblank) × 100%
(three)
iii.8. Statistical Analysis
The mean and standard deviation of 3 experiments were determined. Statistical analyses of the differences between mean values obtained for experimental groups were calculated using IBM SPSS Statistics 23.0. (IBM Corp. Released 2015. IBM SPSS Statistics for Windows, Version 23.0. Armonk, NY, United states). p values < 0.05 were regarded every bit significant, p values < 0.01 as very significant and p values < 0.001 equally highly significant.
4. Conclusions
Essential oils are valuable institute extracts used in food, medicine and complementary treatment strategies [41]. The benign role of grapefruit EO has been widely reported. However, the bioactivities of grapefruit EO prepared past molecular distillation has non been well studied. Molecular distillation is a very useful technique to separate thermally-sensitive EOs. In our study, molecular distillation was used to remove undesired components from the cold-pressed grapefruit EO to provide light phase EO (LPEO). The chemical limerick and antimicrobial action of LPEO were studied. LPEO showed a wide spectrum of antimicrobial activity against some Gram-positive and Gram-negative microorganisms, with MIC values ranging from 0.78 to 12.50 µL/mL. LPEO might be used as a novel antimicrobial agent in the nutrient manufacture. The antioxidant activity of LPEO by DPPH and ABTS was obtained with IC 50 values of 22.06 ± 0.92 mg/mL and fifteen.72 ± 0.32 mg/mL, respectively. An in vitro exam showed a dose-dependent antiproliferative activity of LPEO on HepG2 and HCT116 cancer cells. Thus, LPEO may potentially be used as a new complementary anticancer agent. However, this still needs further studies.
Author Contributions
J.C. and B.Z. contributed to the conception and design of the written report and data assay; W.D., K.Fifty., J.Southward., and J.C. collected the found material and performed distillation and GC-MS analysis; K.L. and S.C. carried out bioactivity test; Westward.D. and J.C. wrote the manuscript. All authors take read and agreed to the published version of the manuscript.
Funding
This work was supported by Foundation of Jiangxi Province Educational Committee (KJLD14079), Natural Science Foundation of Jiangxi Province (20141BBG70002), and Innovation Team Plan of Jiangxi Province (20142BCB24007).
Conflicts of Interest
The authors declare no conflict of interest.
Footnotes
Sample Availability: Sample of the compound LPEO is available from the authors.
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Will Grapefruit Essential Oil Affect Birth Control?,
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