Extraction of Lycopene from Gac Fruit (Momordica cochinchinensis Spreng) and Preparation of Nanolycopene

Vietnam Journal of Chemistry, International Edition, 55(6): 761-766, 2017 DOI: 10.15625/2525-2321.2017-00541 761 Extraction of Lycopene from Gac Fruit (Momordica cochinchinensis Spreng) and Preparation of Nanolycopene Ho Thi Oanh 1,2 , Hac Thi Nhung 1 , Nguyen Duc Tuyen 1 , Le Thi Kim Van 3 , Trinh Hien Trung 4 , Hoang Mai Ha 1,2* 1 Institute of Chemistry, Vietnam Academy of Science and Technology (VAST) 2 Graduate University of Science and Technology, VAST 3 National Institute of Medicinal Materials 4 School of Pharmacy, Haiphong University of Medicine and Pharmacy Received 11 October 2017; Accepted for publication 29 December 2017 Abstract In this work, we extracted high purity lycopene from dried Gac (Momordica Cochinchinensis) aril using organic solvents. We also succeeded in the preparation of nanolycopene. The effect of temperature on the drying of Gac aril and suitable solvents on the extraction of lycopene has been investigated. The results showed that the suitable drying temperature for Gac aril was 60-70 o C. The suitable solvents for lycopene extraction were dichloromethane or chloroform. The lycopene content in dried Gac aril is about 0.28-0.46 %. Nanolycopene was prepared successfully by freeze-dried method, with relatively small particles size of 40-60 nm. Nanolycopene is relatively stable in the inert environment in the presence of antioxidants such as butylated hydroxytoluene (BHT). Keywords. Momordica cochinchinensis Spreng, Gac Fruit, lycopene, nanolycopene, extraction. 1. INTRODUCTION Gac fruit (Momordica Cochinchinensis Spreng) is a special fruit of Southeast Asia, especially familiar to Vietnamese people due to its high nutritional value [1-3]. Gac aril is a thin aril surrounding a gac fruit seed, containing some carotenoids (lutein, beta- cryptoxanthin, zeaxanthin, alpha-carotene, beta- carotene, cis-lycopene, trans-lycopene, vitamin C, vitamin E, and some fatty acids (omega-3, omega- 6)) [4-5]. Among these carotenoids, lycopene has attracted much interest because of its antioxidant activity in vitro [6]. It is commontly extracted with organic solvents such as chloroform, toluene, petroleum ether, acetone, hexane and ethanol. Amount of lycopene in Gac aril ranged from: 0.380-0.408mg/g, respectively (Aoki et al., 2002; Vuong et al., 2006) [7-8]. While, Ishida et al. (2004) reported that lycopene concentration in gac aril was ranged from 1.546-3.053 mg/g [9]. Nhung et al. (2010) reported higher concentration of lycopene at 2.378-3.728mg/g [10]. According to research from the University of California, the lycopene content in Gac fruit was 70 times higher than lycopene content in tomatoes and much higher than that in other vegetables such as watermelon, papaya, red guava, red grapefruit or strawberries [8]. Lycopene is a nonpolar and acyclic carotenoid completely insoluble in water and only slightly soluble in vegetable oil. Thus, only a minor part of lycopene is absorbed by humans during consumption of raw vegetables and fruits. [5] Regardless of the promising results, the extensive use of lycopene has met only limited success, largely due to its instability, poor solubility, inefficient systemic delivery and low bioavailability. To overcome these physicochemical and pharmacokinetic limitations, the encapsulation of lycopene into nanostructure is a major challenge, and nanotechnology represents a powerful strategy [11]. In this study we selected the solvents and appropriate conditions to extract lycopene from gac fruit. Moreover, in order to improve the absorption of lycopene into the body, this active element was made in the form of nanoparticles by emulsification/solvent evaporation follow by freeze- dried method. VJC, 55(6), 2017 Hoang Mai Ha et al. 762 2. EXPERIMENTAL 2.1. Materials Materials used in this study are ripe Gac fruits collected in Bac Ninh, Bac Giang, Hai Duong and Nghe An province in the January of 2017. Chemical standards including Lycopene (> 90 %) were purchased from Sigma Aldrich. All solvents used in this study were freshly dried under the standard distillation method. All deuterated solvents were purchased from Cambridge Isotope Laboratory. 2.2. Extraction of Lycopene from Gac fruit The extraction process of lycopene from Gac fruit was shown in figure 1. The whole Gac fruit was scooped out and the red aril surrounding the seeds was completely separated. Gac aril was dried in the convection oven under drying temperature of 60-80 o C. Lycopene was extracted at least three times from the dried Gac aril using organic solvents such as dichloromethane (DCM), chloroform, tetrahydrofuran (THF), toluene, ethylacetate, petroleum ether, hexane and ethanol. The collected solution was concentrated under reduced pressure; then, ethanol was added slowly to precipitate out the lycopene. The solid was filtered, recrystallized in DCM/ethanol to afford a purple powder. 2.3. Preparation of nanolycopene Nanolycopene was prepared by emulsification/solvent evaporation method. Firstly, lycopene (10 mg), tween (10 mg), span (10 mg), BHT (1 mg) were dissolved in 3 mL DCM. The organic phase was added dropwise to 10 mL polyvinyl ancohol (PVA) solution (0.7% w/v) with an Ultra-Turrax (T18 IKA, Germany) homogenizer at 8400 rpm for 10 min. The nanosuspension was subjected to a Buchi freeze-dryer (Inlet temperature: 150 o C, outlet temperature: 60 o C, aspirator 100 %, pumb 46 %) to afford a deep red nanolycopene powder. 2.4. Instrumentations NMR spectra were recorded on a Varian AS400 (399.937 MHz for 1 H and 100.573 MHz for 13 C) spectrometer. The HPLC analysis was performed with LC-20A System (Shimadzu Co. ltd, Japan) equipped with a high pressure pump and UV-Vis detector. All scanning electron microscopy (SEM) images were obtained using a Jeol JSM-6500F scanning electron microscope. 3. RESULTS AND DISCUSSION 3.1. Drying of Gac aril Suitable temperature for the drying of Gac aril was investigated. Figure 2 showed a general tendency of the aril mass loss within 15 hours. At the drying temperatures from 60 to 80 o C, the moisture content was rapidly decreased within first 5 hours and slowly down in the following hours. After 15 hours of drying at 60-70 o C, the weight of dried Gac aril was fixed at 31.5%. This weight was remained event after drying up to 20 hours because the moisture was not significantly reduced further. By drying Gac aril at 80 o C, the moisture rapidly decreased in the first 5 hours. However, after 15 hours, the weight of Gac films was still remained at 33 %. It is because the high drying temperature led to the rapidly evaporation of water on the surface, resulted on the shrink of outer capillaries and cells that prevent the transfer of moisture from the inside to the surface. By drying Gac aril at high temperature (> 80 o C) on a long time, Gac aril lost their characteristic color and odor. In order to avoid the oxidation of the carotenoids by thermal air, the drying of Gac aril at 60-70 o C within 15 hours in the convection oven was suitable condition to obtain high quality dried aril for the extraction of lycopene. 3.2. Extraction of lycopene from dried Gac aril using organic solvents Lycopene was extracted from dried Gac aril using various organic solvents including DCM, chloroform, THF, toluene, ethylacetate, petroleum ether, hexane and ethanol. Because of the nonpolar and strong intermolecular interaction structure, lycopene showed a poor solubility in polar solvent such as alcohols. This molecule exhibited a highly solubility in DCM, chloroform, THF. Because of the low toxicity, DCM is a suitale solvent for the extraction of lycopene. The average content of lycopene extracted from dried Gac aril in different provinces was shown in table 1. The average lycopene content in dried Gac aril from Bac Giang and Bac Ninh province was surround 0.44-0.46 % while dried Gac aril from Nghe An showed lower content of 0.28 %. Table 1: The average lycopene content extracted from dried gac aril in different provinces. Sample Bac Giang Bac Ninh Nghe An Hai Duong Lycopene content (%) 0.46 0.44 0.28 0.37 VJC, 55(6), 2017 Extraction of Lycopene from Gac Fruit 763 Time (h) 0 2 4 6 8 10 12 14 16 G a c a ri l m a s s ( % ) 40 60 80 100 60oC 70oC 80oC Drying Extraction Purification (1) (2) (2) (3) (3) (4) (4) (5) (5) (6) (6) (7) -carotene Lycopene Figure 1: The extraction process of lycopene from dried Gac aril: (1)- Chloroform or DCM; (2)- Tetrahydrofuran (THF); (3)- Toluene; (4)- Ethylacetate; (5)- Petroleum ether; (6)- Hexane; (7)- Ethanol Figure 2: The tendency of the Gac aril mass loss The HPLC analysis was performed with LC- 20A System. Separation was conducted on Supelco C18 (250 mm, 4.6 mm ID, 5 µm) column. The solvent system was composed isocratic of MeOH:ACN: DCM (10:50:40). This eluent contained 0.05 % BHT as antioxidant agent. The flow rate was 1.3 ml/min. Injection volume was 10µl. The quantification of lycopene and beta carotene was performed at 472 nm. Identification of lycopene and beta carotene was carried out by comparisons of the HPLC retention time and absorption spectra in 472 nm of unknown peaks and chromatography with standards (figure 3). The HPLC result showed the purity of extracted lycopene was higher than 90 %. A B Lycopene -carotene Figure 3: Chromatogram of Gac aril extract developed with MeOH:ACN:DCM (10:50:40) system (A) and the standard line of lycopene quantification (B) The structure and purity of extracted lycopene VJC, 55(6), 2017 Hoang Mai Ha et al. 764 were analysed by 1 H-NMR and 13 C-NMR using CDCl3 (Fig. 4). Chemical shifts (in ppm) corresponding to 56 protons in the 1 H-NMR spectrum of Fig. 4A and to 40 carbons in the 13 C-NMR spectrum of Fig. 4B were assigned. The chemical shifts were reported in ppm; the CHCl3 (7.28 ppm for 1 H) and CDCl3 (77.02 ppm for 13 C) signals were used as the internal standard reference. 1 H-NMR (CHCl3 at 7.28 ppm): 6.68-6.20 (14H, m); 5.98 (2H, d, J = 10.0 Hz); 5.135 (2H, bs); 2.15 (8H, bs); 1.99 (12H, s); 1.84 (6H, s); 1.71 (6H, s); 1.64(6H, s). The value of the coupling constant of the signals centred at 5.98 ppm is assigned for a trans configuration around a partial double bond. 13 C-NMR (CDCl3): 139.50 (2C); 137.38 (2CH); 136.56 (2C); 136.18 (2C); 135.43 (2CH); 132.66 (2CH); 131.75 (2C); 130.10 (2CH); 125.76 (2CH); 124.82 (2CH); 123.97 (2CH); 40.25 (2CH2); 26.71 (2CH2); 25.71 (2CH3); 17.71 (2CH3); 16.97 (2CH3); 12.92 (2CH3); 12.81 (2CH3). A B Figure 4: 1 H-NMR (A) and 13 C-NMR (B) spectrum of purified lycopene in CDCl3 Table 2: Feeding composition of nanolycopene samples Sample Lycopene (mg) DCM (g) BHT (mg) Tween 80 (mg) Span 80 (mg) PVA (mg) H2O (mL) Dispersion in water Lycopene content (%)* S1 10 5 - 10 10 70 20 Well 9.9 S2 10 5 1 10 10 70 20 Well 9.8 * Lycopene content in nanolycopene powder analysed by HPLC. VJC, 55(6), 2017 Extraction of Lycopene from Gac Fruit 765 3.3. Preparation of Nano lycopene Nanolycopene was prepared by emulsification/solvent evaporation method using an Ultra-Turrax homogenizer. The feeding composition of sample S2 included 1 mg of BHT as anti- oxidation agent (table 2) while sample S1 was prepared without any anti-oxidation agent. The HPLC analysis showed that the lycopene content of S1 nanoparticles was 9.9 % while lycopene content of S2 nanoparticles was 9.8 %. Figure 5 showed SEM images of S1 and S2 samples. Similar morphological aspects of nanoparticles with distinct spherical shape were observed. The results indicated that the average particles size of both samples were about 40-60 nm, independently of the initial BHT amount loaded. Figure 6 showed the stability of sample S1 and S2 on the ambient and inert environment. By the presence of BHT, sample S2 was much more stable than sample S1. Otherwise, the degradation of lycopene was inhibited if these samples were storaged in innert environment. For example, in the case of sample S2, with the presence of 1% BHT, 92 % of lycopene was remained if the sample was storaged in nitrogene. S1 S2 Figure 5: SEM images of S1 and S2 samples 0 10 20 30 40 50 4 5 6 7 8 9 10 L y c o p e n e c o n te n t (% ) Time (days) S1 on air S1 on N 2 S2 on air S2 on N 2 Figure 6: The stability of lycopene nanoparticles 4. CONCLUSION We have successfully extracted lycopene (the purity > 90 %) from Gac fruit with the average lycopene content in dried Gac aril was surround 0.28-0.46 %. Nanolycopene was prepared by emulsification/solvent evaporation method. These particles showed spherical shape with average particles size of 40-60 nm. BHT was used as an antioxidation agent to improve the stability of nanolycopene. Acknowledgements. The author acknowledges the financial support from Institute of Chemistry (VHH.2017.2.07 grant). REFERENCES 1. Vo Van Chi. Tu Dien Cay Thuoc Viet Nam [A Dictionary of Medicinal Plants of Vietnam], Nha Xuat Ban Y Hoc, Ho Chi Minh City, (1997). 2. Do Tat Loi. Nhung Cay Thuoc va Vi Thuoc Viet Nam [Medicinal Plants and Drugs of Vietnam], Nha Xuat Ban Khoa Hoc va Ky Thuat, Hanoi (1991). 3. Pham Gia Tien, F. Kayama, F. Konishi, H.Tamemoto, K. Kasono, Nguyen Thi Kim Hung, et al. Inhibition of tumour growth and angiogenesis by water extract of Gac fruit (Momordica cochinchinensis Spreng), International Journal of Oncology, 26(4), 881-889 (2005). 4. Le Khac Lam Dien, Nguyen Phuoc Minh, Dong Thi Anh Dao. Investigation different pretreatment methods and ratio of carrier materials to maintain carotenoids in gac (Momordica cochinchinensis Spreng) powder in drying process, International Journal of scientific & technology research, 2(12), 360-371 (2013). VJC, 55(6), 2017 Hoang Mai Ha et al. 766 5. J. Kubola, N. Meeso, S. Siriamornpun. Lycopene and beta carotene concentration in aril oil of gac (Momordica cochinchinensis Spreng) as influenced by aril-drying process and solvents extraction, Food Research International, 50, 664-669 (2013). 6. H.C. Mai, V. Truong, F. Debaste Carotenoids purification from gac (Momordica cochinchinensis Spreng.) fruit oil, Journal of Food Engineering, 172, 2-8 (2016). 7. H. Aoki, Nguyen Thi Minh Kieu, N. Kuze, K. Tomisaka and Nguyen Van Chuyen, Carotenoid Pigments in GAC Fruit, Biosci. Biotechnol. Biochem., 66(11), 2479-2482 (2002). 8. Le Thuy Vuong, A. A. Franke, l. J. Custer, S. P. Murphy. Momordica cochinchinensis Spreng. (gac) fruit carotenoids reevaluated, Journal of Food Composition and Analysis, 19, 664–668 (2006). 9. B. K. Ishida, C. Turner, M. H. Chapman, T. A. Mckeon. Fatty acid and carotenoids composition of Gac (Momordica cochinchinensis Spreng) fruit, J. Agric. Food Chem., 52, 274-279 (2004). 10. Dang Thi Tuyet Nhung, Pham Ngoc Bung, Nguyen Thu Ha, Thai Khanh Phong. Changes in lycopene and beta carotene contents in aril and oil of gac fruit during storage, Food Chemistry, 121, 326–331 (2010). 11. S. O. Kim, T. V. A. Ha, Y. J. Choi, S. H. Ko. Optimization of Homogenization–Evaporation Process for Lycopene Nanoemulsion Production and Its Beverage Applications, Journal of Food Science, 79, 8 (2014). Corresponding author: Hoang Mai Ha Institute of Chemistry, Vietnam Academy of Science and Technology 18, Hoang Quoc Viet road, Cau Giay district, Hanoi, Viet Nam E-mail: hoangmaihand@ich.vast.vn Tel: 84-4-38361282, Fax: 84-4-38361283.