Study on the chemical composition, Biological activity and Physico-Chemical indicators of the weft-peel oil of Bac Giang oranges extracted by cold-pressing method

Abstract: The oil of Bac Giang orange weft peels were obtained by cold-pressing method and dried with Na2SO4. By using the GC-MS method, 33 components of the oil were estimated by comparing their retention times and molecular weights with the standards. In particular, there were 15 hydrocarbons such as monoterpenes: 53.51%, sesquiterpenes: 7.19% and 18 oxygenated components like aldehydes: 18.30%, alcohols: 15.28%, esters: 4.30%. Antioxidant activity of the oil of Bac Giang orange weft peels has been determined by using DPPH and it was (IC50 = 6.18 ± 0.02 μg/mL) lower than that of vitamin C (IC50 = 4.19 ± 0.01μg/mL). The results show that Pseudomonas aeruginosa is inhibited by the oil of Bac Giang orange weft peels with the minimum inhibitory concentration of about 4.15 g/mL, whereas Escherichia coli are about 6.25 g/mL. The density of oil was 0.84 (20oC), acid index was 0.66 (mg KOH/g) and ester index was 0.73 (mg KOH/g).

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JOURNAL OF SCIENCE OF HNUE DOI: 10.18173/2354-1059.2016-0056 Natural Sci. 2016, Vol. 61, No. 9, pp. 53-59 This paper is available online at 53 STUDY ON THE CHEMICAL COMPOSITION, BIOLOGICAL ACTIVITY AND PHYSICO-CHEMICAL INDICATORS OF THE WEFT-PEEL OIL OF BAC GIANG ORANGES EXTRACTED BY COLD-PRESSING METHOD Nguyen Van Loi Institute of Technology, Hanoi University of Industry Abstract: The oil of Bac Giang orange weft peels were obtained by cold-pressing method and dried with Na2SO4. By using the GC-MS method, 33 components of the oil were estimated by comparing their retention times and molecular weights with the standards. In particular, there were 15 hydrocarbons such as monoterpenes: 53.51%, sesquiterpenes: 7.19% and 18 oxygenated components like aldehydes: 18.30%, alcohols: 15.28%, esters: 4.30%. Antioxidant activity of the oil of Bac Giang orange weft peels has been determined by using DPPH and it was (IC50 = 6.18 ± 0.02 μg/mL) lower than that of vitamin C (IC50 = 4.19 ± 0.01μg/mL). The results show that Pseudomonas aeruginosa is inhibited by the oil of Bac Giang orange weft peels with the minimum inhibitory concentration of about 4.15 g/mL, whereas Escherichia coli are about 6.25 g/mL. The density of oil was 0.84 (20oC), acid index was 0.66 (mg KOH/g) and ester index was 0.73 (mg KOH/g). Keywords: The oil of Bac Giang orange weft peels, biological activity, cold - pressed, constituents, physico - chemical indicators. 1. Introduction The Bac Giang orange wefts (Citrus sinensis) are planted in mountainous area of Luc Ngan, Yen The district of Bac Giang province. Currently, Bac Giang orange wefts have only been being used in this zone, despite their peels accountìng for 20 - 25% of the whole fruit contain many valuable oil scent attractive compounds.The demand for orange peel oil used in food and cosmetics industries is increasing. In Vietnam, several research projects on the constituents and biological activity of pummelo, orange, lime and kumquat (except Bac Giang orange) peel oils were published [1]. Sawamura M et al., identified 64 constituents orange peel oil were identified by Swamura M et al. in China [2], 41 constituents in Japan by Moshonas M.G et al., Japan [3] respectively. Meanwhile, Nguyen Van Loi et al., identified five flavoring constituent signatures in Ham Yen orange peel oil [4]. Therefore, the aim of this study was to identify the chemical constituents, biological activity and physical-chemical indicators of Bac Giang orange weft peel oil. It may use as the basis for the further oil application in the industry food and cosmetics. Received September 26, 2016. Accepted November 25, 2016. Contact Nguyen Van Loi, e-mail address: loichebien@yahoo.com Nguyen Van Loi 54 2. Content 2.1. Experiment * Materials Scientific name of Bac Giang oranges weft, collected in Yen The district of Bac Giang province was identified as Citrus sinensis by Dr. Do Dinh Ca. The voucher specimen was placed at the Department of Biotechnology and Food Technology, Hanoi University of Industry. The oil of Bac Giang orange weft peel was obtained by cold-pressing method after being dried with Na2SO4. The tested bacterial strains (Pseudomonas aeruginosa and Escherichia coli) were obtained from the School of Biotechnology and Food Technology, Hanoi University of Science and Technology. * Methods - Oil extraction Bac Giang orange weft peels were pressed at temperatures from 5 to 10 °C. The mixture of oil and water was collected by centrifugation at a speed of 2500 rev/min within 5 minutes, and then separated into two layers: lighter oil emerged above; meanwhile, water remained at the bottom. Even after dewatering oil revenues, the remaining water wasdried with Na2SO4. - Gas chromatography mass spectrometry (GC-MS) The sample and standards were run parallel in the GC-MS experiment. Gas chromatography (GC) analysis was performed by using Agilent Technologies HP 6890 Plus Gas chromatograph system equipped a with FID (Flame Ionization Detector) and fitted with HP-5MS columns (30 m × 0.25 mm, film thickness 0.25 µm). The temperature was programmed as follows: the column temperature was programmed from 80 to 150 o C in 23.3 min at therate of 3 o C/min, and from 150 to 220 o C in 8.75 min at a rate of 8 o C/min. The injector temperature was set at 230 o C. The MS conditions were as follows: Ionization voltage was 70eV, transfering temperature was 250 o C. Carrier gas was helium used at a flowing rate of 0.5 mL/min, while split ratio of the injector was 1:5 [1, 5]. The MS fragmentation patterns were compared with known patterns of other oils and with those in the literature by using Wiley (Wiley 9th Version), NIST 08 Libraries (on ChemStation HP). The percentage of each component was calculated by the very peak one. - Determination of antioxidant activity using free radical scavenging activity [6-8] The free radical scavenging activity of the oil was measured by using 1, 1-diphenyl-2- picrylhydrazol (DPPH). Besides, a 0.5mM solution of DPPH in methanol and a 0.005M acetate buffer (pH 5.5) were prepared. An aliquot of 0.1 mL of the sample solution was added to the tube containing 2 mL of acetate buffer, 1.9 mL of methanol and 1 mL of DPPH solution. In the blank tube, DPPH was removed; in the control tube, 1 mL of DPPH containing 2 mL acetate buffer and 2 mL methanol was added. The mixture was shaken immediately after adding DPPH and allowed to stand at room temperature in the darkness. The decrease in absorbance at 517 nm was measured after each 30 minutes until the reaction curve reached plateau. Vitamin C was used as a positive control and its free radical scavenging activity was performed parallel in the same experiment. These experiments were run twice. The inhibitory percentage of DPPH was calculated as follows: Study on the chemical composition, biological activity and physico-chemical indicators... 55 Scavenging effect (%) = [(Ao – (A – Ab)) / Ao] x 100%. Wherein Ao is the value of absorbance of the control (Vitamin C) at the wave-length of 517 nm; A is the value of absorbance of the sample (the oil) at the wave-length of 517 nm; and Ab is the value of absorbance of the blank (without DPPH) at the wave-length of 517 nm. - Determination of anti-bacterial activity using agar diffusion method Anti-bacterial activity was roughly determined by agar diffusion method. A portion of 50 µl of the oil was put into each well of the 96-wells plate containing tested bacterial strains. The activity was roughly estimated by the diameter of the antibacterial round (mm), which was calculated by the formula D-d (mm), wherein D was the diameter of the antibacterial round (mm) and d was the hole diameter (mm) [8, 9]. - Determination of minimum inhibitory concentration (MIC) of the oil Minimum inhibitory concentration (MIC) of the oil is determined by using soluble method with TSB liquid medium, a control solution and a range of oil concentration test. Concentration range of the test is 4.15 μg/mL and 6.25 μg/mL. Pipette oil diluted to the concentrations of test suites, and certification solution to the prepared environmental one respectively. The mixed oil and the control solution are dissolved to the environment. Using a pipette of 20 μL to transfer bacteria for further in vitro culturing at 37 °C within 24 hour, the minimum inhibitory concentrations (MIC) in vitro was calculated with the lowest concentration of the oils could inhibit the growth of bacteria. Compared with a test tube before being adopted, carving in vitro demonstrated that oils at concentrations of bacteria are developing in vitro, and oils did not grow bacteria [9]. - Determination of physical-chemical properties of oil The density, angle of rotation, refraction index, acid index and ester index of the oil were determined by using ISO 8444: 2010, ISO 8446: 2010, ISO 8445: 2010, ISO 8450: 2010 and ISO 8451: 2010 on the following equipments: Average density, water bath, thermometer, analytical balance, flask 100 mL, copper pipe 5 mL, burette 2 mL and 25 mL, test tube 25 mL, voltage meter, respectively [10]. 2.2. Results and discussion * The components of the oil GC-MS of the sample was performed in order to determine roughly components of the oil. Based on the retention times and molecular weights of the sample and the standards (the GC profile was not shown here), 33 components and their percentages in the oil were evaluated and shown in Table 1. Table 1. The components of the oil of Bac Giang orange weft peels No. Components Retention time (min) Proportion (%) 1 α-pinene 2.269 4.07 2 β-pinene 2.492 2.16 3 sabinene 6.453 0.17 4 myrcene 11.394 7.96 Nguyen Van Loi 56 5 limonene 11.810 14.72 6 β-phellandrene 12.042 0.19 7 octanal 14.082 1.96 8 linalool 20.943 0.32 9 octanol 21.197 0.25 10 terpinen-4-ol 21.691 0.21 11 nonanal 22.632 0.22 12 citronellal 23.294 1.67 13 thujene 23.380 3.97 14 geranyl acetate 23.640 2.17 15 α-citronellol 24.171 1.93 16 α-terpineol 24.817 0.12 17 neryl acetate 25.083 2.13 18 β-citronellol 25.606 8.29 19 γ-terpinene 25.710 7.16 20 p-cymene 26.007 5.13 21 terpinolene 26.528 4.35 22 β-cubebene 27.422 7.04 23 decanal 27.818 5.22 24 dodecanal 27.966 4.02 25 decanol 28.257 2.15 26 trans-nerolidol 28.790 1.95 27 sabinene 29.655 3.57 28 geranial 36.001 5.12 29 α-phellandrene 36.638 0.06 30 α-farnesene 36.984 0.07 31 farnesol 37.712 0.06 32 δ-cadinene 42.951 0.08 33 β-sinensal 43.358 0.09 Total hydrocarbons 60.70 - Monoterpenes 53.51 - Sesquiterpenes 7.19 Study on the chemical composition, biological activity and physico-chemical indicators... 57 Total oxygenated 37.88 - Aldehydes 18.30 - Alcohols 15.28 - Esters 4.30 Total 98.58 Noted: (%) was calculated by the percentage of chromatographic peak area Figure 1. Gas chromatogram of the oil of Bac Giang orange weft peels Among them, fifteen components were hydrocarbons (such as monoterpenes: 53.51% and sesquiterpenes: 7.19%) and the rest were oxygenated ones (like aldehydes: 18.30%, alcohols: 15.28% and esters: 4.30%). The results provided an additional evidencethat the amounts of aldehydes and alcohols in the Bac Giang orange weft oil were higher than those of the oil from the Tuyen Quang ones [1]. Probably, the differences may be explainatorydue to the geographical conditions such as soil factors, climate, growing conditions and harvesting time [11]. * The biological activities of the oil of Bac Giang orange weft peels - The anti-bacterial activity of the oil of Bac Giang orange weft peels Tested micro-organisms used in this experiment were Escherichia coli and Pseudomonas aeruginosa. Diameters of anti-bacterial activity rounds of the oil against these bacteria were shown in Table 2. Table 2. The diameters of antibacterial activity rounds of the oil of Bac Giang orange weft peels No. Tested microorganisms Diameter of antibacterial round (mm) The minimum inhibitory concentration MIC (g/mL) 1 Escherichia coli 34 ± 2.5 6.25 2 Pseudomonas aeruginosa 38 ± 1.3 4.15 Results in Table 2 showed that the oil of Bac Giang orange weft peels possessed anti- bacterial activity against the two tested micro-organisms, showing that Pseudomonas aeruginosa Nguyen Van Loi 58 was inhibited by the oil of Bac Giang orange weft peels with the minimum inhibitory concentration of 4.15 g/mL, whereas Escherichia coli were 6.25 g/mL appropximately - The free radical scavenging activity DPPH of the oil of Bac Giang orange weft peels The free radical scavenging activity determined by DPPH of the oil of Bac Giang orange weft peel was carried out in laboratories of Hanoi University of Industry. Surveys were conducted in different concentrations and results were shown in Table 3. Table 3. The free radical scavenging activity determined by DPPH of the oil of Bac Giang orange weft peels No. Concentration (%) % DPHH of the oil of Bac Giang orange weft peel % DPHH of Vitamin C 1 0.055 22.05 90.02 2 0.045 15.65 65.95 3 0.035 12.18 50.32 4 0.025 8.62 34.73 5 0.015 4.98 20.64 As shown in Table 3, free radical scavenger ability of the Bac Giang orange weft oil is red- pods packaging (IC50 = 6.18 ± 0.02 μg/mL) lower than that of vitamin C (IC50 = 4.19 ± 0.01 μg/mL). * The physico-chemical indexes of the oil of Bac Giang orange weft peels The density, the angle of rotation, the refraction /acid/ and ester index of Bac Giang orange weft oil were shown in Table 4. Table 4. Physical-chemical indexes of the oil of Bac Giang orange weft peels No. Physical-chemical indexes Result 1 Density at 20 °C 0.84 2 Angle rotation α20D 86 o 32’ 3 Refractive indexn 20 D 1.46 4 Acid index (mg KOH/g) 0.66 5 Ester index (mg KOH/g) 0.73 According to the above Table 4, the density of the oil was 0.84, proving oils were lighter than water and floated on water reward. The oil of Bac Giang orange weft peels possessed a pleasant aroma and a mild spicy taste with a density of 0.84 (smaller than that of 0.9 as usual); and the refractive index of 1.46 (smaller than that of 1.47 as usual). This meant that the oil had a high rate of hydrocarbons. Study on the chemical composition, biological activity and physico-chemical indicators... 59 3. Conclusion By using the GC-MS method, 33 components in the oil were predicted by comparing their retention times and molecular weights with standards. In particular, there were 15 hydrocarbons such as monoterpenes: 53.51%, sesquiterpenes: 7.19% and 18 oxygenated components like aldehydes: 18.30%, alcohols: 15.28%, esters: 4.30%. Antioxidant activity of the oil of Bac Giang orange weft peels was determined by using DPPH and it was (IC50 = 6.18 ± 0.02 μg/mL) lower than that of vitamin C (IC50 = 4.19 ± 0.01 μg/mL). The results showed that Pseudomonas aeruginosa was inhibited by the oil of Bac Giang orange weft peel with the minimum inhibitory concentration of about 4.15 g/mL, whereas Escherichia coli was about 6.25 g/mL. Besides,the density of oil was 0.84 (20 o C), acid and ester index was 0.66 (mg KOH/g) 0.73 (mg KOH/g), respectively. REFERENCES [1] Nguyen Van Loi, Nguyen Thi Minh Tu, Hoang Dinh Hoa, 2011. Study mode extraction and chemical composition of determining some oil in Viet Nam citrus. Journal of Science and Technology 6A, 220-226. [2] Mariotti J.P, Tomi F, Casanova J, Castaand J and Bernardini A.F, 1997. 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