Pumpkin (Cucurbita maxima) seed oil: chemical composition, antioxidant activities and its authentication analysis

Email: abdulrohmanugm@gmail.com eISSN: 2550-2166 / © 2019 The Authors. Published by Rynnye Lyan Resources Food Research 4 (3) : 578 584 (June 2020) Journal homepage: http://www.myfoodresearch.com M IN I R E V IE W Pumpkin (Cucurbita maxima) seed oil: chemical composition, antioxidant activities and its authentication analysis Rohman, A. and Irnawati Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Universitas Gadjah Mada, Yogyakarta Institute of Halal Industry and Systems, Universitas Gadjah Mada, Yogyakarta 55281 Indonesia Faculty of Pharmacy, Universitas Halu Oleo Kendari, Southeast Sulawesi 93232 Indonesia


Introduction
Pumpkin with the scientific name of Cucurbita maxima belongs to Cucurbitaceae family which widely grows in tropical regions and has a relatively high economic importance around the world. The food industry has exploited pumpkin for the production of juices, purees, jams and alcoholic beverages (Jiao et al., 2014). Meanwhile, pumpkin seed refers to the edible seed of a pumpkin, containing some bioactive compounds frequently used as herbal medicines and functional foods. Pumpkin seeds are also commonly used in culinary practices mainly in Southeast Asian countries. In Indonesia, pumpkin is one of the popular vegetables consumed and considered as functional food due to the extraordinary rich sources of bioactive compounds having beneficial health effects (Montesano et al., 2018). In addition, pumpkin seed oil (PSO) has gained great attention in fats and oils industry not only as edible oil but also as a potential nutraceutical (Rezig et al., 2012). The world production of pumpkins was 27 million tonnes, and China led the total production accounting of 29%.
PSO has been reported to contain phytosterols, phenolic compounds, antioxidants, tocopherols, and small levels of carotenoids responsible to some biological activities which are beneficial to human health (Cuco et al., 2019) including prevention of gastric, breast, colorectal and lung cancers (Elfiky et al., 2012), retardation of hypertension progression, antihypertension (Zuhair et al., 2000), prevention of prostate disease, mitigation of hypercholesterolemia and arthritis, alleviation of diabetes mellitus by enhancing hypoglycemic activity, reduction of bladder and urethral pressure (Fruhwirth et al., 2003a;Fu et al., 2006), improving bladder compliance and urinary disorder in human overactive bladder (Nishimura et al., 2014), and offering good antioxidant sources Naziri et al., 2016).
Due to the beneficial effects of PSO to human health, several studies also focused on the chemical characterization including triacylglycerol compositions, fatty acids (FA) composition, tocopherols, sterols, and phenolic acids and correlated these components with those biological activities (Romano et al., 2014). In the market of fats and oils industry, PSO may have high price value compared with other vegetable oils such as palm and corn oils and may have lower price value than, eISSN: 2550-2166 © 2019 The Authors. Published by Rynnye Lyan Resources for example, olive oil (Rohman et al., 2015), as a consequence, PSO could be adulterated with other oils. This review article highlighted some extraction methods, physico-chemical characterization of PSO, antioxidant activities, and the authentication of PSO.

Methodology
To accomplish this review article, numerous reputable databases such as Web of Science, PubMed and Scopus containing review and original articles related to the covered topics were identified and downloaded. The keywords explored during literature searching consisted of physico-chemical properties + pumpkin seed oil or antioxidant activities + pumpkin seed oil + in vitro or in vivo or biological activities + pumpkin seed oil.

Extraction of pumpkin seed oil
Numerous different methods have been employed for the extraction of PSO from the pumpkin seeds in order to obtain PSO with high yield and high content of bioactive compounds. These methods include the extraction by organic solvents, cold pressing (Rezig et al., 2018), mechanical pressing (Rabrenović et al., 2014), supercritical fluid extraction employing liquid CO 2 (Durante et al., 2014), aqueous enzymatic extraction assisted by micro-wave (Jiao et al., 2014), ultrasoundassisted extraction, and microwave-assisted extraction (Kouba et al., 2016). However, the extraction with an organic solvent and mechanical pressing are the most common methods applied for the commercial production of vegetable oils.
Supercritical CO 2 using the experimental design of composite rotatable design has been used for extraction of PSO. Some parameters affecting extraction efficiency (maximum yield) and physicochemical properties were optimized which included pressure, temperature, and time. The maximum extraction yield obtained was 30.7% using optimized conditions of pressure, temperature, and time of 32,140 kPa, 68.1 o C and 94.6 mins, respectively. These three variables exhibited quadratic effects and all parameters affected the extraction yield significantly (Mitra et al., 2009). Rezig et al. (2018) have compared two different extraction methods, namely solvent extraction with extracting solvents of pentane, hexane, and the mixture of chloroform: methanol (3:1, v/v) and cold pressing. These extraction techniques affected the oil stability and antioxidant activities. PSO extracted by mechanical extraction exhibited the best stability and highest tocopherol levels than other PSOs. PSO extracted by mechanical pressing also had the highest values in total carotenoids, total phenolic compounds (TPC), βcarotene, quercetin, squalene, fecosterol, stigmasterol and antioxidant activities DPPH using radical scavenging, ABTS radical scavenging, and reducing power. PSOs were extracted using n-hexane as the extracting solvent in the Soxhlet apparatus by thermal cycles at 76°C for 4 hrs (Can-Cauich et al., 2019). These antioxidant activities correlated with the levels of tocopherol in which PSO has α-tocopherol of 23.68±2.08 mg/kg of oil and γ-tocopherol of 12.35±1.10 mg/kg of oil. Hernández-Santos et al. (2016) have evaluated the effects of amplitude and time of ultrasound-assisted extraction (UAE) during PSO extraction. Using experimental design, the amplitude of 62.50% and a time of 5 mins offered a higher extraction yield than others. The extraction yield increased with the increased amplitude because the larger ultrasonic wave amplitude could enhance the cell disruption which led thus the extraction efficiency increased (Perez-Serradilla et al., 2007).
Compared to organic solvent extraction, aqueous enzymatic extraction technology is considered as safe, cheap, and environmentally-friendly. Jiao et al. (2014) performed PSO's extraction based on microwaveassisted aqueous enzymatic comprising of cellulose, pectinase and proteinase (w/w/w). Some extraction conditions namely extraction temperature, enzyme concentration (%, wt/wt), time (min) and power (W) were optimized using response surface methodology (RSM). Using optimum condition (extraction temperature of 44 o C and enzyme concentration of 1.40% (wt/wt), extraction time of 66 min and irradiation power of 419 W), the highest oil yield of 64.17% was achieved. PSO extracted using microwave-assisted aqueous enzymatic extraction showed higher oxidative stability than that extracted using Soxhlet extraction with hexane as extracting solvents.
The production processes of PSO also affected the chemical properties as studied by Nederal et al. (2012). Three different extraction procedures namely pressing of roasted pumpkin seed paste, pressing of unroasted ground pumpkin seeds, and pressing of unroasted ground pumpkin seeds while cooling the press two seed varieties (husked and naked) were evaluated and the results showed that PSO produced higher total phenol content, higher initial peroxide value and better oxidative stability while cold-pressed oils had higher tocopherol content. But, FA composition and TAG profiles were not affected significantly by these extraction processes. The chemometrics of principal component analysis (PCA), based on the score plot of eISSN: 2550-2166 © 2019 The Authors. Published by Rynnye Lyan Resources first principle component (PC1) and second principle component (PC2), using fatty acid and triglyceride compositions as variables could classify PSOs from two varieties and three production processes.

Chemical characterization of pumpkin seed oil
Determination of physico-chemical properties of PSO was needed to characterize which could be used further for the authentication purposes. The chemical composition and PSOs properties depend on several factors including varieties (cultivars) of pumpkin and region of origins. The fatty acid (FA) composition of PSO was significantly different among various cultivars of PSO (Stevenson et al., 2007). Table 1 compiles the physicochemical characterization of pumpkin seed oil which include some constants specific for PSO. Habib et al. (2015) have reported that the physical properties of PSO were as follows: specific gravity of 0.9412 (at 31°C ), the iodine value of 114.33 gI 2 /g PSO, saponification value of 193.73 mg KOH/g PSO, acid value of 0.516 mg KOH/g PSO and percentage of free fatty acid of 0.2646%.
PSO is known to have nutritional values because it contained some vitamins needed by the human diet (Nishimura et al., 2014), as shown in Table 2. Rezig et al. (2012) have reported that the major FAs in PSO were oleic, linoleic, and palmitic acids. In addition, PSO also contained δ-tocopherol, β-sitosterol, and syringic acid was the predominant phenolic acid present in PSO (Table 3). In addition, the chemical composition of PSO in terms of fatty acid composition exhibited that PSO contained 40.58% oleic acid, 27.06% stearic acid, 17.39% palmitic acid and 14.97% linoleic acid. Siano et al. (2016) have noted that the polyunsaturated FA (PUFA), monounsaturated FA (MUFA) and saturated FA (SFA) contents in PSO cultivated in southern Italy were of 48.14%, 25.54%, and 25.20%, respectively. The high degree of unsaturated FA makes PSO suitable to be used as valuable drying agent, while low value of free FA contents indicates the suitability of PSO as edible oils. Potočnik et al. (2018) have investigated the antioxidant activity of PSO due to roasting temperature using the capability to scavenge 2,2-diphenyl-1picrylhydrazil (DPPH) radicals. PSO used was obtained from the extraction of pumpkin seed Gleisdorf variety and the Rustikal hybrid variety produced in Slovenia. The results showed that antiradical activity values were 31.4-70.6% and 19.3-47.7% in the Gleisdorf and Rustikal samples, respectively at the same concentrations of PSO. This activity was correlated with the levels of polyphenol and tocopherol contents. Fruhwirth et al. (2003b) also suggested that 59% polar phenolics and 41% tocopherols contributed to the antioxidant capacities of PSO. The antioxidant activities of PSO from 12 cultivars have been also investigated . Using Trolox equivalent antioxidant capacity (TEAC), the values of 0.443 to 1.220 µM Trolox/g, the highest value was observed in PSO cultivar Karowita (1.220 TEAC), and the lowest value was obtained in PSO cultivars of Danka and Pyza, each with TEAC value of 0.443 and 0.446 TEAC, respectively.

Antioxidant activities
PSO obtained from microwave-assisted aqueous enzymatic extraction (MAAEEO) and that extracted by Soxhlet extraction (SEO) has been evaluated for its antioxidant activity using two different antioxidant methods, namely DPPH radical scavenging activity and β-carotene/linoleic acid bleaching test (Jiao et al., 2014). PSO obtained from MAAEEO exhibited higher antioxidant activity based on DPPH radical scavenging assay with IC 50 of 123.93±1.85 mg/mL than that obtained from SEO with IC 50 of 150.38 ± 1.07 mg/mL. In addition, using β-carotene/linoleic acid bleaching method, the lipid peroxidation inhibitory activity of PSO obtained by MAAEEO was 152.84±2.34 mg/mL, significantly higher (p<0.05) than that obtained from SEO with lipid peroxidation inhibitory activity of 183.26±3.79 mg/mL. Therefore, PSO obtained by MAAEEO showed higher antioxidant activities than that obtained using SEO either in aqueous (DPPH assay) or lipid-based system (β-carotene/linoleic acid bleaching) which can be explained by higher contents of antioxidant compounds. PSO obtained from MAAEEO exhibited higher PUFA accounting of 57.65% than that in SEO of 53.90%. Besides, MAAEEO method increased the release of tocopherols and phenolics compounds from PSO, thus contributing to the higher antioxidant activities than SEO. Several (Zhang et al., 2010;Latif and Anwar, 2011;Rezig et al., 2012). Bardaa et al. (2016) also reported that PSO had high antioxidant activity using β-carotene/linoleic acid bleaching. PSO revealed lipid peroxidation inhibition than butylhydroxytoluene (BHT) as positive control. PSO exhibited important antioxidant activities due to tocopherols contained.
The antioxidant activity using DPPH radical scavenging activity of PSO from three cultivars of Miranda, Golosemianaja, and Herakles grew in Lithuania and its correlation with phenolics content has been investigated by Kulaitienė et al. (2018). The highest antiradical activity was found in cultivar Miranda accounting of 3.28 μmol/g, followed by cultivar Golosemianaja (2.49 μmol/g) and Herakles (1.64 μmol/ g). DPPH antiradical activity of studied PSO increased with the phenolic content, allowing for establishing the linear correlation between DPPH-radical scavenging activity and total phenolic content with correlation coefficient (r) of 0.823 (p<0.05). The high levels of phenolic compounds and tocopherols have been correlated to its potent antioxidant capacities in vivo, as indicated the increased levels hepatic enzymatic activities of superoxide dismutase, catalase and glutathione peroxidase in vivo and decreased level of malonaldehyde (Bora, 2018).

Authentication of pumpkin seed oil
Some methods have been proposed, developed and used for the authentication of PSO which include molecular spectroscopy (ultraviolet-visible, nearinfrared, mid-infrared spectroscopy), chromatographic based technique (gas and liquid chromatography), carbon isotope analysis of the individual fatty acids by the use of gas chromatography-combustion-isotope ratio mass spectrometry (GC/C/IRMS) (Spangenberg, 2001).

Chromatography methods
Gas chromatography has been used for the analysis of Δ7,25stigmastadienol,and Δ7,22, in PSO for authentication purpose (Mandl et al., 1999). The column used was fused silica capillary column of HP 35 (30 m length and 0.25 mm i.d.), coated with a 0.25 mm layer of crosslinked 65% dimethyl-35% diphenyl polysiloxane. The temperatures of injector and detector were 300 o C. The oven temperature was isothermally set at 280 o C using helium as a carrier gas with a flow rate of 1.1 mL/min. PSO has different profiles of Δ7phytosterols from sunflower oil, used as the adulterant model in this study, even at low concentrations (2% PSO mixed with sunflower oil). HPLC using detector refractive index combined with chemometrics of principal component analysis has been used for authentication of PSO. The stereospecific triacylglycerol (TAG) composition data was used for the differentiation of PSO and 29 TAGs were analyzed. Among these, 25 TAGs were identified using TAGs standard and 4 TAGs were could not be identified marked as NAS1, NAS2, NAS3, and NAS4. Based on TAGs composition, PCA could classify PSO from different region of origin as group I comprising of TAGs of LnLnLn, LLnLn, NAS1, LLLn, OLnLn; group II LLL, OLLn, PLLn, OLL, OOLn; group III, PLL, LnLS, POLn, LOO, SLL; group IV, PLO, PLP, OOO, NAS2, SOL; group V, POO, SPL, POP, NAS3, SOO; and group VI, SLS, POS, NAS4, PPS (Butinar et al., 2010).
FTIR spectroscopy using attenuated total reflectance as sampling handling technique in combination with linear discriminant analysis (LDA) has been employed for discrimination of PSO from different species namely Cucurbita maxima, C. pepo, and C. moschata. The different cultivars of pumpkin seeds may exhibit significant differences in PSO composition terms of fatty acid compositions of linoleic, oleic, and stearic acids and tocopherols composition. LDA could discriminate PSO eISSN: 2550-2166 © 2019 The Authors. Published by Rynnye Lyan Resources from different species and the main wavenumbers contributing for such discrimination using LDA model was 2952-2900 cm -1 corresponding to CH 2 , symmetrical stretching, 1418-1361 cm -1 (-C-H in CH 3 bending), 1147-1128 cm -1 (C-O stretching), 887-804 cm -1 (-CH 2 , wagging), and 665-639 cm -1 (O-H, bending out of plane) (Saucedo-Hernandez et al., 2011).
FTIR spectroscopy coupled with chemometrics and fatty acid composition as determined by gas chromatography-flame ionization detector was reported to analyze PSO as an adulterant in olive oil (Rohman et al., 2015). The absorbance values at combined wavenumbers of 3020-2995 and 1070-900 cm −1 were used as variables during quantification of PSO using partial least square (PLS) model with coefficient of determination (R 2 ) obtained of >0.99. The calibration and validation errors were relatively low, i.e. 0.166 and 1.32% (vol/vol), respectively. The chemometrics of Discriminant analysis could discriminate PSO in olive oil and pure olive oil. In addition, using fatty acid composition change, the presence of PSO in olive oil could be investigated by decreased levels of oleic with the increasing concentration of PSO with R 2 of 0.946. In addition, the level of linoleic acid was increased with the increasing level of PSO with R 2 of 0.978.

Conclusion
Pumpkin seed oil (PSO) is valuable oil having functional oil properties and exhibits several biological activities, mainly antioxidant due to high contents of phenolic compounds and tocopherols. The high price of PSO in the market make it to be adulterated with lower price oils, and some analytical methods have been successfully used for authentication of PSO. Besides, the characteristic properties of PSO including physicochemical constants and fatty acid compositions can assist the authentication of PSO from any adulteration practice.