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J. Agric. Food Chem. 2001, 49, 5620−5629
Phytosterols in Sea Buckthorn (Hippophae1 rhamnoides L.) Berries: Identification and Effects of Different Origins and Harvesting Times Baoru Yang,*,† Riina M. Karlsson,† Pentti H. Oksman,‡ and Heikki P. Kallio† Department of Biochemistry and Food Chemistry and Department of Chemistry, University of Turku, FIN-20014 Turku, Finland
Sterols in seeds, pulp/peel fractions, and whole berries of sea buckthorn (Hippophae¨ rhamnoides L.) samples belonging to two major subspecies (sinensis and rhamnoides) from Finland and China were analyzed as TMS derivatives by gas chromatography-mass spectrometry after saponification of the oils. The total sterol contents in the seeds, the fresh pulp/peel, and the whole berries were 1200-1800, 240-400, and 340-520 mg/kg, respectively. The corresponding values in the extracted oils were 12-23, 10-29, and 13-33 g/kg. Sitosterol constituted 57-76 and 61-83%, respectively, of the seed and pulp/peel sterols. The sterol content and composition showed little variation between subspecies and collection sites. Different harvesting dates showed significant effects on the levels of some sterols both in the seeds and in the pulp/peel. The sterol profiles obtained are useful for characterizing sea buckthorn and detecting adulterations of the valuable oils. The information provided by the present investigation is also important for further chemical investigation of sea buckthorn sterols and industrial utilization of the berries as a raw material of functional foods. Keywords: Berries; sea; buckthorn; harvesting time; seeds; sterols; Hippophae¨ rhamnoides INTRODUCTION
Sea buckthorn (Hippophae¨ rhamnoides L.) is a hardy bush with nutritious berries, naturally distributed mainly in Asia and Europe and also cultivated in North and South America (1, 2). During the past decade, the species has attracted intense global attention not only because of its role in soil and water conservation and reforestation of eroded areas but also because of the nutritional and medicinal values of its berries. The yellow-red, pearl-shaped berries are important sources of vitamins for the local people at the natural growth sites of the plant. The berries have a long history of application in Tibetan and Mongolian medicines and are also an ingredient for traditional Chinese medicines found in the Chinese Pharmacopoeia. A wide spectrum of physiological effects of the berries and berry products has been shown by many animal experiments and the long history of clinical and nutritional practice, especially in China and Russia (3, 4). These positive effects include lowering plasma cholesterol level, inhibiting platelet aggregation, regulating immune function, promoting the repair of injuries on skin and mucosa, and anticancer effects (5-12). Recently, interest in sea buckthorn has also developed in Europe, Japan, Canada, and the United States as more information on the chemical composition and physiological effects of the berries has become available. In association with research on sea buckthorn, the industrial cultivation and utilization of its berries as a raw material for functional food and natural medicines have started in these regions. Although many components, such as vitamin C, sugars, acids, and fatty acids, of the berrries of sea * Corresponding author (telephone 358-2-3336843; fax 3582-3336860; e-mail
[email protected]). † Department of Biochemistry and Food Chemistry. ‡ Department of Chemistry.
buckthorn have been extensively investigated, very little is known about their sterols. In the few existing publications, only single samples were analyzed, and completely different sterol profiles have been reported (13-19). A lack of original mass spectrometric data in most of these publications makes an objective evaluation of their identifications impossible. Thus, there is an urgent need for further investigation of sterols in sea buckthorn. In the present study, we analyzed the seeds, soft parts, and whole berries of two major subspecies of sea buckthorn, sp. rhamnoides in Finland and sp. sinensis in China, to obtain a general profile of sterols in sea buckthorn berries on the basis of a larger number of samples. This profile will serve as a basis for further detailed chemical investigation and nutritional evaluation of the berries. The aim of the present study was also to compare the difference between the two subspecies and to follow changes in the contents and compositions of sterols in the seeds, soft parts, and whole berries during the harvesting period. MATERIALS AND METHODS Berries. Wild berries of H. rhamnoides ssp. sinensis were picked in six different natural growth sites in China around mid-October 1997. The growth areas ranged between longitudes of 104° 40′ E and 114° 41′ E, latitudes of 35° 06′ N and 40° 03′ N, and altitudes of 1370 and 2800 m. Four samples of wild ssp. rhamnoides berries were picked on the shores of the Gulf of Bothnia in southwestern Finland in September 1999. The areas ranged between longitudes of 21° 04′ E and 24° 24 ′E, latitudes of 60° 45′ N and 64° 47′ N, and altitudes of 0 and 50 m. To follow the changes during the harvesting period, wild berries of subsp. sinensis were collected at their natural growth sites (Wenshui and Xixian, Shanxi Province, People’s Republic of China) from the end of August to the end of November 1998. All samples were loosely frozen in sealed plastic bags immediately after collection to avoid desiccation and extra moisture condensation and stored at -20 °C until analyzed.
10.1021/jf010813m CCC: $20.00 © 2001 American Chemical Society Published on Web 11/02/2001
Sea Buckthorn Sterols
J. Agric. Food Chem., Vol. 49, No. 11, 2001 5621
Table 1. Samples of Sea Buckthorn Berries from Different Subspecies and Collecting Times sample
location
r1 r2 r3 r4
Finland Siikajoki Vaasa Pyha¨maa Pyha¨maa
s1 s2 s3 s4 s5
China Xunyi, Shaanxi Youyu, Shanxi Datong, Qinghai Dingxi, Gansu Wenshui, Shanxi
s6
Xixian, Shanxi
collection date
seed content subspecies (% of fresh wt)
Sept 2, 1999 Sept 3, 1999 Sept 8., 1999 Sept 8, 1999
rhamnoides rhamnoides rhamnoides rhamnoides
Oct 12, 1997 Oct 15, 1997 Oct 13, 1997 Oct 17, 1997 Aug 30, 1998 Sept 15, 1998 Sept 30, 1998 Oct 15, 19 98 Oct 30, 1998 Nov 15, 1998 Nov 30, 1998 Aug 30, 1998 Sept 15, 1998 Sept 30, 1998 Oct 15, 1998 Oct 15, 1998 Nov 15, 1998 Nov 30, 1998
sinensis sinensis sinensis sinensis sinensis
sinensis
5.7 4.3 5.6 5.1 11.5 7.7 7.6 9.4 5.9 6.0 5.0 3.6 6.1 6.7 8.4 5.9 5.8 4.7 4.7 4.4 3.9 3.9
Detailed information of the samples is presented in Table 1. Lipid Extraction. Berries (400 g) of each sample were taken from a 5 kg lot using a sample partitioning procedure. Seeds were isolated from frozen berries by pressing the juice and rinsing the residue with distilled water. The press residue was air-dried at room temperature, and seeds were separated mechanically. For the whole berry analysis frozen berries were freeze-dried (Dura-Top bulk tray dryer, FTS System Inc., Stone Ridge, NY) to 15-30% of the original weight, depending on the berry composition. Samples (5 g) of seeds, freeze-dried whole berries, and pulp/ peel (seedless parts of the berries) were crushed in a mortar in liquid nitrogen and the lipids isolated using a methanol/ chloroform extraction procedure (20, 21). The sample was homogenized in methanol (50 mL) for 1 min in a blender, chloroform (100 mL) was added, and homogenization was continued for a further 2 min. The mixture was filtered and the solid residue resuspended in chloroform/methanol (2:1, v/v, 150 mL) and homogenized for 3 min. The mixture was filtered again and washed with fresh solvent (chloroform/methanol, 2:1, v/v, 150 mL). The combined filtrates were cleaned with a repeat procedure using a 0.88% potassium chloride water solution followed by a methanol/water (1:1, v/v) solution. The purified lipids were filtered before the solvent was removed on a rotary evaporator. Saponification of Lipids. After the addition of cholesterol palmitate (1.6 mg) as an internal standard, lipids (400 mg) were saponified by refluxing in 20 mL of a 1 M KOH ethanol/ water (8:2, v/v) solution for 1 h. The refluxed mixture was then transferred into a separatory funnel, and the reflux bottle was washed with 40 mL of Milli-Q water. The unsaponifiables in the combined solution were then extracted three times with 40 mL of diethyl ether. The ether phase was combined, washed three times with 40 mL of water, and dried with sodium sulfate overnight. Isolation of Sterols and Preparation of TMS Derivatives. The unsaponifiables dissolved in petroleum ether (bp ) 60-80 °C) were applied to a Silica Sep-Pak Cartridge (Waters Corp., Milford, MA) preconditioned with 5 mL of petroleum ether. After elution of hydrocarbons with 10 mL of petroleum ether and carotenoids with 10 mL of petroleum ether/ether (96:4, v/v), the tocopherols and tocotrienols were eluted with 10 mL of petroleum ether/ether (91:9, v/v) and sterols with 20 mL of petroleum ether/ether (1:1, v/v). The sterols were derivatized by incubation in a mixture of N,Obis(trimethylsilyl)trifluoroacetamide (BSTFA) and trimethylchlorosilane (TMCS) (2:1, v/v), at 60 °C for 2 h.
Identification of Sterols. The sterol TMS derivatives were analyzed by gas chromatography-mass spectrometry (GCMS). A VG Analytical 7070E double-focusing mass spectrometer (VG Analytical, Altrincham, U.K.) equipped with an Opus data system (Fisons Instruments, Manchester, U.K.) was connected to a Dani 3800HR 2Ch gas chromatograph (Dani S.p.A, Milan, Italy) without a restrictor capillary. A 30 m × 0.25 mm i.d., 0.25 µm film DB-1701 capillary column (J&W Scientific, Folsom, CA) was inserted directly into the ion source. The column temperature was held at 230 °C for 1 min, increased to 275 °C at 1 °C/min, and finally held at 275 °C for 30 min. Helium (1.5 mL/min) was used as a carrier gas. The injector temperature was 275 °C. The positive ion electron impact (EI) mass spectra were recorded at an ionization energy of 70 eV and a trap current of 100 µA with a source temperature of 180 °C. Perfluorokerosene (PFK) (Merck Art. 10145) was used as the reference compound. The magnetic sector scans were performed within the range of m/z 35-600 at a rate of 1 scan/s with an intermediate time of 0.5 s between the scans, an acceleration voltage of 6 kV, and a resolution of 1000. Reference compounds, campesterol (campest-5-en-3β-ol), sitosterol (stigmast-5-en-3β-ol), stigmastanol (5R-stigmastan3β-ol), R-amyrin (5R-urs-12-en-3β-ol), and β-amyrin (5R-olean12-en-3β-ol) were purchased from Sigma Chemical Co. (St. Louis, MO). Sterols were identified by comparing the mass spectra and retention times with those of reference compounds, or the mass spectra published in the literature. For those compounds for which neither standard compounds nor reference spectra were available, the chemical structures were postulated according to the general patterns of mass spectrometric fragmentation of different sterols. Quantatitive Analysis. The sterol TMS derivatives were analyzed with a Varian 3300 gas chromatograph equipped with a flame ionization detector (FID). The same column and GC parameters were used as in the GC-MS analysis. The FID temperature was 300 °C, and the split ratio of the injector was 1:40. The quantification of sterol compounds was carried out with a cholesterol internal standard and calculated by applying the detector reponse of sitosterol. The contents of sterols in seeds, pulp/peel, and whole berries were calculated according to the oil contents in these fractions and the contents of sterols in the oils. The repeatability of the analytical procedure was tested, and the relative standard deviation (SD) of five repeated analyses of a single sample was
