SUMMARY Phytosterols in pumpkin seed oil extracted by organic solvent and supercritical CO2 Phytosterols in Cucurbitapepoconvar.

citrullina seed oil were analyzed by GCMS after extraction by the organic solvents, hexane and petroleum ether, and by supercritical carbon dioxide at 400 bar and 408C. Desmosterol, campesterol, stigmasterol, b-sitosterol, spinasterol, D7,22,25-stigmastatrienol, D7- stigmastenol, D7,25-stigmastadienol, and D7-avenasterol were identied mass spectrometrically via detection of the parent molecular ions and fragmentation patterns of corresponding trimethylsilyl derivatives. The used derivatizing agents N-methyl-N(trimethylsilyl)triuoroacetamide (MSTFA) and N,Obis(trimethylsilyl)triuoroacetamide (BSTFA) are equally effective for phytosterol content determination. As expected, the predominant phytosterols were D7-sterols that ranged from 91.0 to 94.2% of the total phytosterol content given as sum of free sterols and steryl esters. Comparison of extraction methods revealed that, although the oil yields obtained by extraction with hexane, petroleum ether and supercritical CO2 were 43.37, 44.65, and 36.17%, respectively, the total phytosterol content in supercritical CO2 extract (294 mg/100 g oil, for both MSTFA and BSTFA) was about 30% higher than in hexane and about 20% higher than in petroleum ether extract. Practical applications: Extraction with the application of uids in supercritical state opens a new approach to the production of edible vegetable oils. Many economical, health, and environmental aspects are interlaced in this modern approach. By applying the supercritical extraction, the selectivity of the extraction can be signicantly improved compared to traditional methods of extraction. Thus, depending on the operating conditions of supercritical CO2 extraction higher total phytosterol content can be achieved resulting in improved nutritional value of the pumpkin seed oil.

1. INTRODUCTION Previously, most types of vegetable oils are extracting by using non polar solvents such as hexane. However, the usage of organic solvent has its own disadvantage, which is the persistence of toxic solvent in the oil. Thus, supercritical extraction is used to produce oil free of such residues and with higher nutrition value. The common solvent of choice for production of such oil is supercritical carbon dioxide (CO2) due to its complete dissipation on exposure to atmospheric pressure. Besides that, CO2 also has low critical temperature (31.1 C) and pressure (73.8 bar), that makes it suitable extraction solvent for natural products, since there is no thermal degradation during the process. Oil yields obtained by this separation and by conventional organic solvent extraction are found similar in many studies. However, Temelli achieved higher yield by extraction with petroleum ether (38%) than by supercritical fluid extraction (21 25 %) flaxseed, as a result of extraction of undesirable compounds with the organic solvent. Pumpkin seed oil contains about 98 % of triglycerides and other minor component. Some of these minor components are valuable nutrients, as an example, phytosterols have multiple positive effects on human health. Phytosterols in plant material is usually determined by performing capillary GC-FID or GC-MS. All forms of phytosterols present in vegetable oils are: free alcohols (free sterols FS), steryl esters (SE), steryl glycosides (SG), and acylated steryl glycosides (ASG) can be analyzed by GC after the isolation from the oil by a combination of alkaline saponification and acid hydrolysis. Alkaline saponification alone is sufficient to cleave all of the conjugated phytosterols since plant oils usually contain primarily SE and little or no SG and ASG. Pumpkin seed oil is different from other oils as it contains more D7- than D5-sterols. Thus, acid hydrolysis is additionally not applicable as D7sterols decompose or isomerise after a short time. However, GC separation of phytosterols is possible without derivatization but, better resolution is achieved for their trimethylsilyl (TMS) derivatives. Thermal stability of unsaturated phytosterols, peak shape, and the sensitivity of the GC measurement are also improved by derivatization. Analysis of phytosterol silyl ethers (or acetate derivatives) by GC coupled with electron or chemical ionization MS with SIM mode provides the most effective resolution, identification, and quantitation.

Mandl reported the phytosterol content in Cucurbita pepo L. convar. citrullina I.GREB. Var. Styriaca I.GREB seed oil extracted with petroleum ether, while Neperal Nakic measured the phytosterol content in C. pepo L. seed oil obtained by the extraction with hexane. When petroleum ether is used, the phytosterol content in pumpkin seed oil is higher instead of hexane as extraction solvent. Moreover, Breinholder, who extracted oil from C. pepo L. convar. citrullina I. GREB. Var. styriaca I. GREB seed, and Phillips, who extracted oil from Cucurbita spp. (Shine Skin variety), both using a mixture of chloroform or methanol (2:1, v/v) as extraction solvent, also reported data concerning phytosterol content. For supercritical fluid extraction of oils from Cucurbita ficifolia and Cucurbita moschata seed extraction conditions, yields and fatty acid compositions of oils have been published; however, phytosterol contents were not determined. The present work is therefore aimed to compare the oil yields as well as total phytosterol contents, given as sum of FS and SE; in C. pepo L. seed extracts obtained by Soxhlet extraction with hexane and petroleum ether, and by supercritical CO2 extraction.The GCMS was used to determine the phytosterol composition. Besides that, the application of different derivatizing agents for phytosterols content determination was also examined.

2. METHODOLOGY

Plant material

Chemicals

Extraction of pumpkin seed oil

Standard solutions and calibration

Preparation of phyposterol TMS Ethers

Saponification of pumpkin seed oil

GC-MS analysis

LOD,LOQ, and method validation

Statistics

Plant material Pumpkin seed of C. pepoconvar.citrullina was obtained from the Parting Trade company. The moisture content of the raw material, determined gravimetrically by air drying at 1038C, was 0.0920 kg H2O/kg raw material. The seed was ground for 15 s in a laboratory mill. The fraction of particles having mean diameter of 0.25 mm (between 0.16 and 0.35 mm) that was 82% of the ground seed mass was subjected to extraction.

Chemicals Pro-analysis grade hexane and petroleum ether were purchased from Lach-Ner. 99.9% CO2 was supplied by Tehnogas .Analytical grade methanol, tert-butyl methyl ether and dichloromethane were obtained from JT Baker. Ethyl acetate was purchased from Carlo Erba. Anhydrous pyridine and anhydrous sodium sulfate were acquired from FlukaChemie AG. MSTFA and BSTFA were supplied by MachereyNagel. Silica gel, 75150 mm particle size, was purchased from Analtech, Newark USA. Standards of cholesterol, stigmasterol, b-sitosterol, and betulin were used.

Extraction of pumpkin seed oil Conventional Soxhlet extraction of about 15 g ground pumpkin seed was carried out using hexane or petroleum ether. Supercritical uid extraction of about 66 g ground pumpkin seed was carried out for 4 h at 400 bar and 408C under a constant CO2 ow rate of 0.44 kg/h.

Saponification of pumpkin seed oil A slightly modied procedure proposed by Mandl et al. [15] was applied. Prior to saponication, 5 mg/mL pyridine solutions of betulin that used as internal standard and cholesterol that used for recovery test were added to the oil sample that was further dissolved in dichloromethane and saponiedwith 1 mL KOH solution (20 g KOH in 88 mL methanol with 12 mL deionised water to limit the transestericationto methyl esters) for 45 min at 708C in a 10 mL screwcapped reaction vial. The end of the reaction was indicated by the clearing of the two-phase oilmethanol/water system. A glass column with glasswool frit at the bottom was dry-lled with anhydrous sodium sulfate as the lowest layer and silica gel as a second layer. The total saponiedoil mixture, adsorbed on silica gel, was packed on top to form the third layer. The unsaponiable fraction was eluted from the silica column with tertbutyl methyl ether/ethyl acetate mixture with the ratio 1:1, while the potassium salts of the fatty acids retained on the column. An aliquot of eluted unsaponiable fraction was further derivatized by silylation

Preparation of phytosterol TMS ethers The aliquot, transferred to a 2 mL vial, was evaporated to dryness with nitrogen. After addition of 50 mL of derivatizingagent (MSTFA or BSTFA)/dry pyridine mixture with the ratio 2:1, and 400 mL of tert-butyl methyl ether to the dry residue, silylation was run at 708C for 2 hour. One microliter of obtainedmixture was used for GCMS analysis.

Standard solutions and calibration Stock solutions of cholesterol, stigmasterol and b-sitosterol in pyridine were each used for preparing ve standard solutions that ranged in concentrations 6.64250 mg/mL. The solutions were made by transferring appropriate volumes of the stock solution and the constant volume of internal standard stock solution such as betulin to 2 mL screw cap vials. After addition 40 mL of MSTFA or BSTFA, the solutions were heated at 708C for 2 hour, diluted with appropriate amount of tert-butyl methyl ether and analyzed by GCMS.

GC-MS analysis The phytosterol content in extracts was determined by GCMS analysis, using a Thermo Finnigan Trace GC coupled with Finnigan Trace MS and furnished with Thermo Finnigan AS 2000 autosampler, under following conditions: TR-50MS capillary column (30 m, 0.25 mm ID, 0.25 mm thickness lm), 1 mL injection volume. He carrier gas at 1.5 mL/min, PTV injector with a splitless mode temperature raised from 55 to 2508C, oven temperature programmed as 708C (hold 1.5 min), 408C/min to 2458C (hold 1.5 min), 28C/min to 2808C (hold 10 min), transfer line temperature was 2908C, ion source temperature was held at 2208C. Mass spectra (EI, 70 eV) were recorded in the range between m/z 200600 with a scan time of 1 seconds. Spectrum acquisition was performed in the full scan mode, to conrm the retention times of phytosterol TMS ethers, and in the SIM mode for their quantitative analysis. The response factors were determined using sterol standard mixtures with betulin as internal standard. The NIST Mass SpectraLibrary was used to identify some sterols, while others were identied via detection of the parent molecular ions and fragmentation patterns of corresponding TMS derivatives.

LOD,LOQ, and method validation The limit of detection (LOD) and the limit of quantitation (LOQ) were obtained for a signal-to-noise ratio (S/N) of 3 and 10, respectively, using calibration plots for TMS derivatives of standard compounds in the range of 6.64 21.2 mg/mL (appropriate mass of sterol TMS ether diluted in tert-butyl methyl ether). The reliability of the method was veried by determination of accuracy and precision. The accuracy was determined by measuring the recovery; pumpkin seed extract samples were spiked with 300 mg cholesterol/100 g extracted pumpkin seed oil before the saponication. The precision was determined by means of replicate tests, each extracted pumpkin seed oil sample was prepared on three consecutive days and analyzed in triplicate.

Statistics Results were reported as means SD. Excalibur 1.3 software and SPSS 11.5 for Windows were employed to interpret the results. The inuence of different derivatizing agents on total phytosterol content determination was assessed by the paired samples t-test.

3. RESULTS AND DISCUSSION

3.1 Comparison Of Extract Yields Each particles fraction having a mean diameter of 0.25 mm was selected for extraction. In order to increase the extraction rate, the bigger particles were not used while in order to avoid compacting the extraction bed, the smaller particles were excluded because it will lead to increasing the internal mass transfer resistance and channeling inside the bed. The oil yields obtained by extraction of C were 43.37 1.63% and 44.65 1.97%. The extraction of supercritical CO2 that was carried out for 4 hour gives 36.17 0.68% of oil. The oil products obtained by pumpkin seed supercritical CO2 extraction have been reported, but due to the difference or lack of process conditions, they cannot be compared with the results presented in this work. The oil yields comparison obtained in this industry shows that the extraction with hexane and petroleum ether gives 16.6 and 19% respectively, higher yields than supercritical CO2 extraction, due to the different extraction rates of triglycerides. Organic solvents also extract free fatty acids, pigments, and phospholipids, the extracted oil require refining. Moreover, there was an obvious difference in extract color. The color of oils extracted with hexane and petroleum ether were browngreenish red, while supercritical CO2 extracted oil was an orange-yellow. The later oil was clearer, and can be explained by the lower selectivity of organic solvent extraction.

3.2 Qualitative GC-MS Analysis The TMS ethers of nine phytosterols such as desmosterol, campesterol, stigmasterol, -sitosterol, spinasterol, 7,22,25-stigmastatrienol, 7-stigmastenol, 7,25-stigmastadienol and 7-avenasterol, were identified (Figure 1) via detection of the parent molecular ions [M]+ and fragmentation patterns of corresponding TMS derivatives presented in Table 1. Stigmasterol and sitosterol TMS ethers mass spectra were confirmed with a certainty of 97-98% according to the NIST library . The mass spectra of the later three phytosterol TMS ethers recorded in this work are presented in Figure 2 and their confirmations are explained according to the fragmentation data given in Table 1 . For instance, desmosterol TMS ether was confirmed via its molecular ion (456), fragmentation ions derived from the lost of the side chain (345) and the side chain together with two hydrogen atoms (343) and other ions . In spite of spinasterol and 7,25-stigmastadienol TMS ethers having the same molecular masses and fragmentation patterns, they can be discerned because the later has molecular ion (484) less abundant than ion (469) which represents the lost of methyl group, while for former it is opposite . Additionally, stigmastadienol always has a longer GC retention time . 7,25-stigmastadienol and 7,22,25stigmastatrienol TMS ethers have the same fragmentation patterns but different mass spectra due to different number of double bonds, mass of their molecular are 484 and 482 respectively and fragmentation ions differ . Thus,on the basis of MS data (Table 1), peak 7 and 8 in Figure 1 were identified as overlapped peaks of 7stigmastenol and 7,25-stigmastadienol .

3.3 Validation Of The Method The correlation coefficients values confirmed the linearity of the calibration plots (Table 2). The accuracy of the average recovery added was 97.41% (n = 54). In order to determine the relative SD in was 1.95% indicates good repeatability. LOD and LOQ were found to be between 0.11- 0.19 g/mL (0.21- 0.36 mg/ 100 g oil) and 0.37- 0.65 g/mL (0.70- 1.2 mg/ 100 g oil), respectively.

Figure 1. Chromatograms of pumpkin seedoils extracted with petroleum ether (a) and supercritical carbon dioxide (b): (1) desmosterol, (2) campesterol, (3) stigmasterol, (4) b-sitosterol, (5) spinasterol, (6) D7,22,25-stigmastatrienol, (7, 8) D7stigmastenol and D7,25-stigmastadienol, (9) D7-avenasterol and (I.S.) betulin as internal standard.

Table 1. Relative retention times (RRt) and fragmentation ions with relative intensity (RI) used for identification of the phytosterol TMS ethers in pumpkin seed extracts
Main fragmentation ions, m/z (R) peak 1 sterol Desmosterol RRt
a) b) -c)

[M ] 456(7)

[M-15]

[M-90]

[M-105]

[M-129]

Others 372(6), 345(9), 343(30),253(28)

0.88

441(11)

366(11)

351(17)

327(20)

Campesterol

0.90

472(10)

457(5)

382(34)

367(17)

343(40)

315(2), 261(7), 255(6)

Stigmasterol

0.93

484(13)

469(2)

394(19)

379(7)

355(7)

255(25), 253(7), 213(11)

4 5

b-Sitosterol Spinasterol

1.00 1.02

486(11) 484(17)

471(4) 469(14)

396(46) 394(6)

381(21) 379(9)

357(48) -

255(3) 441(5), 345(16), 343(79), 329(7), 303(5), 255(73), 253(30), 213(31)

D7,22,25Stigmastatrienol

1.05

482(9)

467(9)

392(3)

377(6)

345(13), 343(59), 329(7),255(59), 253(22), 213(24)

D7-Stigmastenol

1.09

486(73)

471(19)

396(8)

381(24)

345(7),329(7),303 (5), 255 , 213(50)

d)

D7,25Stigmastadienol

1.10

484(17)

469(24)

394(7)

379(15)

345(8), 343(90), 329(5), 303(4), 255(43), 253(21), 213(46)

D7-Avenasterol

1.15

484(1)

469(5)

394(1)

379(4)

386(31), 371(7), 345(7), 343 , 329(5), 303(2), 296(6), 255(12), 253(27), 213(23)
d)

3.4 Phytosterol Content According to Table 3 shows that the results of phytosterol analysis of extracted pumpkin seed oils . They were quantified using a plot of a nearest eluted sterol with calibration plot due to absence of campesterol, desmosterol and 7-sterols solutions. The phytosterol contents obtained are within the range of values about 190-320 mg/100 g oil . Breinholder, who extracted C. pepo L. convar. citrullina I . GREB. var. styriaca I. GREB seed oil with mixture of chloroform or methanol (2:1, v/v), reported the presence of FS and SE in quantity of 189 mg/100 g oil, while the total phytosterol content was 215 mg/100 g oil, this also included 26 mg/100 g oil of SG. Philips, extracted from Cucurbita spp. (Shine Skin variety) oil with the same extraction mixture, yielding a FS, SE and SG content of 265 mg/100 g oil. Nederal-Nakic, extracted naked C.pepo L. seed with hexane and obtained a total phytosterol content of 317.2 mg/100 g oil. However, in our work the total phytosterol (FS and SE) content in C. pepo convar. citrullina seed oil extracted with hexane was about 28% lower; 225 mg/100 g oil, in case of derivatization with MSTFA and 228 mg/100 g oil, when derivatization was accomplished with BSTFA . The total phytosterol contents in oil extracted with petroleum ether were 244 mg/100 g oil and 246 mg/100 g oil when MSTFA and BSTFA were used,respectively.

From this study it show that the petroleum ether is better than hexane for phytosterol extraction, recovering 7.9-8.4% more phytosterols from seed. However, in the Table 3, the results indicate that the supercritical CO2 is even more selective for phytosterol extraction than petroleum ether, extracting 20.5 and 19.5% more phytosterols determined with MSTFA and BSTFA respectively. The difference in phytosterol extraction could be explained with different extraction kinetics phytosterols and triglycerides; using CO2 the sterols are extracted are much quicker than the triglycerides. In this way, under proper conditions, the lower yield of oil, however enriched in phytosterols, is obtained . The content of all individual phytosterols is higher in supercritical CO2 extracted oil . Besides that, the content of 7-sterols is gradually higher than content of 5-sterols. The paired sample test showed that the MSTFA and BSTFA, as derivatizing agents, are equally effective, since the differences in the measured phytosterol contents are statistically insignificant ( Table 3); the two-tail p values for hexane and petroleum ether extraction were 0.23 and 0.40 respectively and for supercritical; CO2 extraction p was 0.47.

Figure 2. Full scan mass spectra of desmosterol(1), spinasterol (5), and D7,22,25stigmastatrienol (6) TMS ethers, denoted according to the peak numbers on chromatograms presented in Fig. 1.

Compound

Correlation coefficient

Regression equation

LOD (g/mL) (mg/100 g oil)

LOQ (g/mL) (mg/100 g oil) 0.43 0.82

Cholesterol

0.9943

y = 0.4227x 1.4146

0.13

0.25

Stigmasterol

0.9957

y = 0.2455x + 0.7780

0.11

0.21

0.37

0.70

-Sitosterol

0.9958

y = 0.2106x+ 0.5441

0.19

0.36

0.65

1.2

Table 2. Calibration data for standard compounds (TMS derivatives of 5-sterols)

Sterol Hexane

Phytosterol content (mg/100 g oil) Petroleum ether Supercritical carbon dioxide MSTFA BSTFA MSTFA BSTFA 5.98 1.15 MSTFA 8.67 0.84 BSTFA 8.15 0.45 3.88 0.29 3.35 0.38 9.68 0.38 75.3 4.75 83.1 4.77

Desmosterol

4.41 0.52

4.37 0.43 5.88 0.38

Campesterol

2.71 0.38

2.38 0.21 2.86 0.42

2.49 0.39

3.51 0.38

Stigmasterol -Sitosterol

0.96 0.41

0.78 0.56 3.24 0.33

3.10 0.39

3.87 0.39

5.19 0.65

5.95 0.48 9.57 0.77

9.44 0.36

10.0 0.44

Spinasterol 7,22,25Stigmastatrie nol 7Stigmastenol +7,25stigmastadien ol 7Avenasterol 5-Sterols 7-Sterols Total

55.1 5.38

55.0 5.29 57.0 5.41

57.0 5.02

74.5 5.05

61.8 5.26

63.5 5.01 63.7 5.31

65.9 5.07

81.5 5.74

58.4 4.82

58.5 4.68 60.4 4.38

60.4 4.73

68.2 5.71

66.1 3.87

36.9 3.79

37.0 3.44 41.2 4.06

41.7 3.36

43.7 3.11

44.3 2.87 25.1 1.50

13.3 1.03

13.5 1.15 21.6 1.52

21.0 1.49

26.1 2.05

212 16.6 225 17.6

214 16.4 228 18.6

222 17.6 244 18.4

225 18.0 246 19.0

268 19.6 269 16.3 294 21.7 294 17.8

Table 3.Phytosterol contents (mg/100 g oil) in pumpkin seed oils extracted with hexane, petroleum ether, and supercritical carbon dioxide and derivatized with MSTFA and BSTFA

APPENDIX