Bioactive Compounds of Palm Fatty Acid Distillate ( PFAD ) from Several Palm Oil Refineries

This research studied the characteristics of Palm Fatty Acids Distillates (PFADs) from several palm oil refineries. It was aimed to know the potency of PFAD as bioactive compounds source, including vitamin E (mainly tocotrienols), phytosterols, squalene and possibly co-enzyme Q10 and polycosanol. Sampling was conducted at 6 palm oil refineries. The results showed that PFAD was dominated by free fatty acids of 85-95% with low oxidation level indicated by peroxide value of 1-10 meq/kg and anisidin value of 6-31. Bioactive compounds found were vitamin E 60-200 ppm, phytosterols 400-7500 ppm and squalene 400-2800 ppm, meanwhile polycosanol and coenzyme Q10 were not found. Vitamin E was dominated by tocotrienols and γ tocotrienol was the major vitamin E, followed by α and δ tocotrienols. Phytosterols in PFADs from several palm oil refineries had variety in quantity and composition. Generally it was dominated by β sitosterol, followed by stigmasterol and campesterol

According to Liu et al. (2008), about 5-57% of tocopherols and tocotrienols is removed from CPO during deodorization and some of them is accumulated in PFAD that results PFAD with 0.7-1.0% of vitamin E. Goh and Gee (1985) indicated that PFAD hydrocarbon was squalene as main components and minor component of n-alkane (C 12 H 26 -C 36 H 74 ).PFAD had higher quantity of squalene (up to 1.03%) than other vegetable oils (Posada et al., 2007).Squalene has the ability to maintain healthy skin due to its antioxidant activity and protect skin from detrimental environmental effects (Nandi et al., 2008), as well as to reduce blood cholesterol level and anticancer (Loganathan et al., 2009).Newmark (1997) revealed that squalene had antitumor activity due to strong inhibiton of HMGCoA reductase that limit farnesyl pyrophosphate availability for oncogene prenylization thus inhibit oncogene relocation in cell membrane.
Co-enzyme Q10, one of palm oil bioactive compounds, has similar structure to vitamin E and K but its antioxidant activity is ten fold higher than vitamin E. It is also known that co-enzyme Q10 has cardio-protective effect and anticancer (Loganathan et al., 2009).Other hydrocarbon of PFAD is supposed to be polycosanol.Polycosanol is a bioactive wax (Awika and Rooney, 2004) that capable to reduce blood cholesterol level by inhibition of cholesterol synthesis (Jain et al., 2007) and reducing HMG-CoA reductase activity (Somaiya et al., 2010).
So far, variational characteristics of PFAD as a byproduct of palm oil refineries have not been yet reported.Palm oil refineries might have different processing conditions that imply to different characteristics of PFAD, especially bioactive compounds.Therefore, this study was objected to know the characteristics of PFAD from several palm oil refineries.
Characterization of PFAD: PFAD from 6 palm oil refineries were analyzed for oxidation level indicated by peroxide value (Hills and Thiel, 1946) and anisidine value (ISO, 2006), as well as free fatty acid content (AOCS, 1990).Bioactive compounds were analyzed as followed: PFAD saponification: About 10 g of PFAD was put into 250 mL erlenmeyer with rubber seal and ethanol 88.3 mL, 0.5 g ascorbic acid and 5 mL KOH solution (50% w/v) were added.The mixture was stirred and heated at temperature of 65°C for 32 min on waterbath shaker.The reaction mixture was added by hexane 150 mL and distilled water 200 mL and then shaked slowly.The mixturer was let to separate on separating funnel for ±1 h at ambient temperature.Aqueous lower layer contained saponifiable matters meanwhile upper hexane layer contained unsaponifiable matters.Hexane layer was evaporated by vacuum evaporator to obtain solvent free unsaponifiable fraction for further analysis.
Phytosterols analysis: Phytosterols analysis was followed method of Khatoon et al. (2010).About 1 mg of unsaponifiable fraction of PFAD was added by ethanol 1 mL and subsequently filtered.20 µL of sample solution was injected into high performance liquid chromatography (Shimadzu LC20AT) with C18 PPODS column 250×4.6 mm and uv vis detector (Shimadzu SPD20A) at wavelength of 206 nm.Mobile phase was methanol: water (99:1 v: v) with flow rate of 1 mL/min.Identification and quantification was conducted by using phytosterols (stigmasterol, β sitosterol, campesterol) standard that injected into HPLC separately.
Vitamin E analysis: This procedure was according to Ball (1988).About 1 mg of unsaponifiable fraction of PFAD was added by ethanol 1 mL and then filtered.20 µL of sample solution was injected into high performance liquid chromatography (Shimadzu LC20AT) with C18 PPODS column 250×4.6 mm and uv vis detector (Shimadzu SPD20A) at wavelength of 295 nm.Mobile phase was methanol: water (95:5 v: v) with flow rate of 1 mL/min.Identification and quantification was conducted by using α tocopherol, α tocotrienol, β tocotrienol, δ tocotrienol and γ tocotrienol standards that injected into HPLC separately.
Polycosanol and squalene analysis: This analysis was using GC MS with derivatization method according to Mendez et al. (2003) by using MSTFA.About 10 mg of unsaponifiable fraction of PFAD was put into reaction tube and added by MSTFA 10 µL.The mixture was heated at 60°C for 15 min.External standard of 1docosanol was used for quantification that prepared by diluting 1 mg of this standard with MSTFA 100 µL and the mixture was heated at 60°C for 15 min.
1 µL of derivatized sample was injected into GC MS (GCMS-QP2010S Shimadzu) (with column (AGILENT DB-1) 30 m, i.d.0.25 mm, helium as carrier gas and ionization EI 70 Ev.GC operational condition was column oven temperature of 80°C, injector temperature of 310°C, pressure of 16.5 kPa, total flow rate of 80.0 mL/min, column flow rate of 0.50 mL/min, linear velocity of 26.1 cm/s, suooorting gas rate of 3.0 ml/min and split ratio of 153.0.Initial temperature was 80°C and holded for 5 min.
Temperature was increased with rate of 10°C/min to the temperature of 305°C and holded for 25 min.MS analysis was conducted at ion source temperature of 250°C, interface temperature of 310°C, solvent cut time of 4.80 min, scan speed of 1250, start m/z 28.0 and end m/z 600.0.
Co-enzyme Q10 analysis: Co-enzyme Q10 analysis was refered to method of Mahendra et al. (2011).About 1 mg of unsaponifiable fraction of PFAD was diluted with acetone 1 mL.20 µL of sample solution was injected into (Shimadzu LC20AT) with C8 column 250×4.6 mm and uv vis detector (Shimadzu SPD20A) with mobile phase acetonitrile: isopropanol (86:14 v:v).Analysis was performed in isocratic condition at column temperature of 50ºC, flow rate of 1.0 mL/min and using uv vis detector (Shimadzu SPD20A) at wave length of 210 nm.

RESULTS AND DISCUSSION
PFAD chemical characteristics: PFADs from several palm oil refineries had slightly different oxidation level and free fatty acid content (Table 1) in the range of 85-93%.Free fatty acids level was in accordance to PORIM standard for PFAD that free fatty acid content should be minimum 70% (Affandi, 1994).Different deodorization process applied in palm oil refineries might lead to different free fatty acid content of PFAD, but we had no data of deodorization and other refining process from these 6 palm oil refineries.Ceriani and Mirelles (2004) showed that different deodorization temperature and pressure in canola, wheat and soybean oil processing had different deodorizer distillate composition indicated by different concentration of Triglycerides (TG), Diglycerides (DG), Monoglycerides (MG) and Free Fatty Acids (FFA), as well as bioactive compounds such as squalene, δ tocopherol and β sitosterol.Ceriani and Mirelles (2004) revealed that different temperature (240-260°C) and pressure (1-6 mmHg) during continuous deodorization of soybean oil produced Soybean Oil Deodorizer Distillate (SODD) with free fatty acids content of 8-65%.Increasing deodorization temperature caused increasing non free fatty acids components such as TG, DG, tocopherol and β sitosterol, thus decreasing free fatty acids content.Conversely, increasing deodorization pressure led to decreasing TG, DG, tocopherol and β sitosterol thus increased free fatty acid content.Therefore, variation in free fatty acids content of PFADs from several palm oil refineries possibly related to variation in deodorization process parameters as well as characteristics of CPO as input materials.
Oxidation level of PFADs also varied that indicated by peroxide value as indicator of primary oxidation products and anisidine value as indicator of secondary oxidation products.Deodorization is aimed to remove odoreous volatile compounds including products of secondary oxidation.Data in Table 1 showed that oxidation level of PFADs was quite low with peroxide value of 1-9 meq/kg and anisidine value of 6-31.Benites et al. (2009) showed that a deodorizer distillate of soybean oil had peroxide value of 7.5 meq/kg and TBA value of 16.0 mg MDA/kg.Oxidation products are volatile and vaporize during deodorization.Perhaps, only small amount of oxidation products were condensed during distillation for free fatty acids recovery.Deodorizer distillate was the product of low temperature condensation of vaporized components in CPO.High condensation temperature components might not capable to change into liquid and was not recovered in PFAD.Thus, low oxidation level of PFADs was due to not all of oxidation products condensed.
Data in Table 1 showed that unsaponifiable matters of PFADs was 0.5-4% and saponiafiable matters was 96-99%, and PORIM standard required minimum saponifibale matters of 95% (Affandi, 1994).Variation of usaponifiable matters of PFADs was possibly due to variation in processing conditions such as degumming, bleaching and deodorization, as well as raw material variability.Unsaponifiable matters of PFAD were supposed to contain bioactive compounds.
PFADs from several palm oil refineries had variation in composition and concentration of bioactive compounds.Ceriani and Mirelles (2004) showed that deodorizer distillate of soybean oil contained squalene, tocopherol and β sitosterol; wheat oil contained tocopherol; and canola oil contained β sitosterol and tocopherol.Benites et al. (2009) also showed that deodorizer distillate of soybean oil contained tocopherol.Meanwhile, Khatoon et al. (2010) showed that deodorizer distillate of soybean oil contained phytosterol.Bondioli et al. (1993) revealed that deodorizer distillate of olive oil contained squalene.Furthermore, Winkle-Moser and Vaughn (2009) indicated that deodorizer distillers of dried grain oils contained phytosterol, steryl ferulat, tocopherol, tocotrienol and carotenoid.
Co-enzyme Q10 or ubiquinone also was not detected in PFADs.Co-enzyme Q10 was found in palm oil in low concentration of 10-80 ppm and this compound had 10 fold higher antioxidant activity compared to vitamin E, involved in ATP or energy synthesis, acted as anticancer and had heart protective effect (Loganathan et al., 2009).Perhaps, due to its low concentration in palm oil and low volatility, co-enzyme Q10 was not detected in PFAD.

Vitamin E composition:
The superiority of PFADs over other deodorizer distillates was the presence of tocotrienols as predominat vitamin E. Tocotrienols were found in high concentration in palm oil and rice bran (Theriault et al., 1999), meanwhile other vitamin E sources such as coconut, soybean, cacao, barley, wheat germ, sunflower, groundnut, walnut, sesame and olive oils only contained tocopherols (Heinonen and Piironen, 1991).Tocotrienols have been reported to have more advantages over tocopherols due to their higher capability in inhibiting microsome and liver mitochondria peroxidation as well as dioleoylphosphatidylcholine liposomes oxidation (Serbinova and Packer, 1994;Packer et al., 2001).Deodorizer distillate of soybean oil only contained tocopherols meanwhile tocotrienols were not found (Wan et al., 2008).
Palm oil contained several vitamin E isomers, i.e., α tocopherol, α tocotrienol, γ tocopherol, γ tocotrienol and δ tocotrienol (Ng et al., 2004) with vitamin E concentration of 600-1000 ppm.Puah et al. (2004)  reported that vitamin E composition of palm oil was α tocopherol (20%), α tocotrienol (22%), γ tocotrienol (46%) and δ tocotrienol (12%).Data in Table 3 showed that tocotrienols were predominant vitamin E in PFADs from several palm oil refineries with proportion of 50-92% based on total vitamin E. This study revealed that tocotrienols concentration of PFADs from several palm oil refineries varied due to the influence of palm fruit characteristics, CPO extraction and handling, as well as refining process.The predominant vitamin E of PFADs was γ tocotrienol.The result of this study was in accordance to that reported by Puah et al. (2004) that vitamin E in palm oil was dominated by γ tocotrienol.Generally, tocotrienols concentration in PFADs was in order of γ>δ>α tocotrienol.

Phytosterols composition:
Phytosterols were predominat bioactive compounds of PFADs (Table 2).Khatoon et al. (2010) reported that phytosterols and vitamin E was found in unsaponifiable fraction of soybean oil deodorizer distillate and free fatty acids were the major component.Data in Table 4 showed that β sitosterol was predominat phytosterols in PFADs from several palm oil refineries.The composition of each phytosterols compound (β sitosterol, campesterol and stigmasterol) varied among PFADs.Campesterol was detected in all PFADs meanwhile stigmasterol was not detected in PFAD from palm oil refinery 6. Loganathan et al. (2009) reported that phytosterols of palm oil comprised of campesterol (13%), β sitosterol (60%) and stigmasterol (24%).Phytosterols had ability to reduce blood cholesterol level, anti thrombotic and anticancer (Awad and Fink, 2000;Piiron et al., 2000, Ostlund et al., 2002).Phytosterols from PFADs could be extracted to have phytosterol enrich fraction that could be used as food supplement for related diseases prevention.
It was interesting that palm oil refineries 1 and 5 were from one company but different location and showed different phytosterol composition.Although we did not have data about refining conditions especially deodorization, we supposed that both palm oil refineries applied similar processing parameters.The variability of phytosterols composition was supposed due to variability in CPO characteristics.Both palm oil refineries used same CPO source but different distance from palm milling industry to refineries.

CONCLUSION
PFADs from several palm oil refineries had variability in characterisctis.PFAD was dominated by free fatty acids with low level of primary and secondary oxidation products.Bioactive compounds of PFADs were phytosterols, vitamin E and squalene with variational concentration and composition.Generally, vitamin E was dominated by γ tocotrienol meanwhile predominat phytosterol was β sitosterol.The common concentration of tocotrienol was in order of γ>α>δ tocotrienol, meanwhile phytosterol was in order of β sitosterol>stigmasterol>campesterol.

Table 1 :
PFAD characteristics from several palm oil refineries

Table 3 :
Vitamin E profile of PFADs from several palm oil refineries