J. Verbr. Lebensm. (2011) 6:333–338 DOI 10.1007/s00003-010-0648-1
Journal fu¨r Verbraucherschutz und Lebensmittelsicherheit Journal of Consumer Protection and Food Safety
GENERELLE ASPEKTE
Bioactive lipids and fatty acids profile of Cistanche phelypaea Mohamed Fawzy Ramadan • Hefnawy Taha Mansour Hefnawy Ayman Mohamed Gomaa
•
Received: 12 July 2010 / Accepted: 22 July 2010 / Published online: 10 August 2010 Ó Springer Basel AG 2010
Abstract Oil extracted from the wild plant of Cistanche phelypaea was analyzed for its fatty acid, sterol, hydrocarbon and tocopherol contents. Total lipids (TL) content was 10 g/kg (on dry weight basis). The majority of fatty acids were of the unsaturated type (50.4 % of total fatty acids), while the saturated (mainly palmitic acid) were about 43.2 % of the total fatty acids. Oleic acid was the dominating fatty acid followed by palmitic and linoleic acids. High amounts of sterols were found in the oil with the main component b-sitosterol. Other phytosterols (e.g. stigmasterol, D7-avenasterol and D5-avenasterol) were present at approximately equal amounts (6–9 % of total sterols). The main identified hydrocarbon compounds were C21, C26 and C32 constituting about 61.2 % of total hydrocarbons. Small amounts of C12, C18 and C22, however, were also detected. Tocopherol levels were high in the oil (3.36 g/kg oil), wherein b-tocopherol was the main component followed by a-isomer. Both tocopherol components comprised more than 87 % of total vitamin E content in the oil. Furthermore, c- and d-tocopherols were detected in small amounts in the oil accounting for 14–16 % of the total vitamin E content. Information provided by the present work will be of importance for food applications and chemotaxonomy of Cistanche phelypaea.
Dr. M. F. Ramadan (&) Dr. H. T. M. Hefnawy A. M. Gomaa Biochemistry Department, Faculty of Agriculture, Zagazig University, 44511 Zagazig, Egypt e-mail:
[email protected]
Keywords Cistanche phelypaea Oil Fatty acids Sterols Hydrocarbons Tocopherols
1 Introduction The genus Cistanche that belongs to the family Orobanchaceae includes 16 species. They form an attractive group of phanerogamic root parasites. The occurrence of the genus is restricted to certain arid and semi arid regions of Africa, Asia and the Mediterranean area including parts of Southern Europe. The family Orobanchaceae represents two genera Cistanche and Orobanche. The genus Cistanche is represented in Egypt by three species namely C. phelypaea (Fig. 1), C. tubulosa and C. violacea according to Tackholm (1974). Many edible and medicinal uses are attributed to this genus. The whole plant of Cistanche tubulosa is used medically in Pakistan as a remedy for diarrhea and sores (Kobayashi et al. 1987). Shoots and stems of Cistanche species can be used for food applications and as a tonic in the traditional Chinese medicine for the deficiency of the kidney. Moreover, it is used for cold sensation in the lions and knees, female sterility, and constipation due to dryness of the bowel in the senile (Mabberley 1997; Namba 1994). The genus Orobanche (Orobanchaceae) comprises about 100 species of haloparasitic plants. Beack-Mannagetta (1930) proposed a subgeneric classification of the genus in four sections: Gymnocaulis Nutt., Myzorrhiza (Phil.) Beck., Trionychen Wallr. and Osproleon Wallr. The latter is known nowadays as sect. Orobanche
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2 Materials and methods 2.1 Materials The whole wild plants of Cistanche phelypaea were collected from South Sinai (Egypt) during April 2008. The plants were kindly authenticated by Prof. Dr. ElHadidy, Faculty of Science, Cairo University, Egypt. Herbarium specimens were kept at the Department of Agricultural Biochemistry, Faculty of Agriculture, Zagazig University. Standards used for sterols characterization, cholesterol, b-sitosterol, stigmasterol, lanosterol, ergosterol, campesterol, D5-avenasterol and D7-avenasterol were purchased from Supelco (Bellefonte, PA, USA). Standards used for the characterization of vitamin E (a-, b-, c- and d-tocopherol) were purchased from Merck (Darmstadt, Germany). All reagents and chemicals used were of the highest purity available. 2.2 Methods 2.2.1 Extraction of total lipids (TL) Fig. 1 The spectacular plant is a member of the broomrape (Orobancheaceae) family but has a lupin like shape, five lobed two lipped flowers and can grow half a meter tall
according to the rules of the International Code of Botanical Nomenclature (Greuter 1988). Many biological activities were reported for some species of this family including antimicrobial, anti-inflammatory activities (Endo et al. 1982), antispasmodic, smooth muscle relaxant (El-Shabrawy et al. 1989) and cardioactivity (Pennacchio et al. 1969). Furthermore, Orobanche is eaten, like asparagus, as mentioned recently by Rubiales et al. (1999). The plant contains a number of constituents which include phenylethanoid glycosides (sometimes called phenylpropanoids), iridoids (Xiong et al. 1996) and polysaccharides (Naran et al. 1995). The amino acids were determined and identified in Cistanche solesa (Jiao et al. 1989). Fatty acid was reported and tocochromanols composition in seeds of Orobanche family was recently investigated (Velasco et al. 2000). Information on the lipid composition of Cistanche phelypaea, however, is inadequate and data available are incomplete. In the present study, we analyzed the whole plant, to obtain informative profile of the lipids in Cistanche phelypaea which will serve as a basis for further detailed chemical investigation and nutritional evaluation of the herb.
Intact plants were dried and ground. The total lipids were extracted by petroleum ether at 60–80 °C using Soxhlet apparatus. The solvent was evaporated to dryness on a rotary evaporator at 40 °C under reduced pressure. 2.2.2 Gas liquid chromatography analysis of fatty acid methyl esters Fatty acids were transesterified into methyl esters by heating in borontrifluoride (10 % solution in methanol, Merck, Darmstadt, Germany) according to the procedure reported by Metcalfe et al. (1966). Fatty acids methyl esters (FAME) were identified on a Shimadzu GC-14A equipped with flame ionization detector and C-R4AX chromatopac integrator (Kyoto, Japan). The flow rate of the carrier gas (helium) was 0.6 mL/min and the split value with a ratio of 1:40 was used. A sample of 1 lL was injected on a 30 m 9 0.25 mm 9 0.2 g film thickness Supelco SP M-2380 (Bellefonte, PA, USA) capillary column. The injector and detector temperature was set at 250 °C. The initial column temperature was 100 °C programmed by 5 °C/min until 175 °C and kept for 10 min at 175 °C, then 8 °C/min until 220 °C and kept 10 min at 220 °C. A comparison between the retention times of the samples with those of authentic standard mixture (Sigma, St. Louis, MO, USA; 99 % purity specific for GLC), run on the same column
Cistanche phelypaea
under the same conditions, was made to facilitate identification. The quantification of each fatty acid was carried out by comparing the peak area of its methyl ester with that of methyl nonadecanoate without application of any correction factor. 2.2.3 Gas liquid chromatography analysis of sterols Separation of sterols (ST) was performed after saponification of the oil samples (Ramadan and Mo ¨rsel 2002a). After the addition of cholesterol acetate (1.5 mg; Sigma, MO, USA) as an internal standard lipid (250 mg) probes were refluxed with 5 mL ethanolic KOH solution (6 %, w/v) and a few antibumping granules for 60 min. The unsaponifiables were firstly extracted three times with 10 mL of petroleum ether. The extracts were combined and washed three times with 10 mL of neutral ethanol/ water (1:1, v/v) then dried overnight with anhydrous sodium sulphate. The extract was evaporated in a rotary evaporator at 25 °C under reduced pressure, and then the solvent was completely evaporated under nitrogen. Gas chromatographic analysis of unsaponifiables was carried out using a Mega Series (HRGC 5160, Carlo Erba Strumentazione; Milan, Italy) equipped with FID. The column was ID phase DB 5, packed with 5 % phenylmethylpolysiloxan (J&W scientific; Falsom, CA, USA), 30 m length, 0.25 mm i.d. 1.0 pin film thickness with carrier gas (helium) flow rate 38 mL/min and splitless injection. The detector and injector were set at 280 °C. The oven temperature was kept constant at 310 °C and the injected volume was 2 lL. All ST homologues eluted within 45 min and total analysis was set at 60 min to assure the elution of all ST. The quantification of sterol compounds was carried out with a cholesterol acetate internal standard and calculated by applying the detector response of sitosterol. The repeatability of the analytical procedure was tested and the relative standard deviation of three repeated analyses of a single sample was \5 %. Quantitative analyses were performed with a Shimadzu (C-R6A Chromatopac; Kyoto, Japan) integrator. 2.2.4 Gas liquid chromatography analysis of Hydrocarbons 2.2.4.1 Preparation of unsaponifiable matter Unsaponifiable matter was determined according to the method described in AOAC (2000). A known weight of the oil (5 g) was dissolved in ethanol (30 mL), then 1.5 mL alcoholic KOH (50 %) was added. The oil was saponified on a water bath for 30 min under reflux
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air condenser. The alcoholic solution was concentrated and quantitatively transferred into separator funnel using a total of 50 mL distilled water and 50 mL petroleum ether. The unsaponifiable matter was extracted three times with petroleum ether, washed several times with distilled water dried over anhydrous sodium sulphate and then filtered into a weighed flask. The solvent was evaporated using a boiling water bath and the flask was dried at 105 °C until constant weight was reached. 2.2.4.2 Analysis of the hydrocarbons by GLC The hydrocarbon compounds were identified using a PYE UNICAM PRO-GC gas chromatograph equipped with flame ionization detector (FID). The column used for separating the unsaponifiable matter was a (OV-17) 1.5 m 9 4 mm i.d. fused silica capillary column coated with methyl phenyl silicone fluid. The following chromatographic conditions were performed: Split ratio 1: 200, sample size 1 mL, carrier gas nitrogen at a flow of 1 mL/min, injection temperature 250 °C, oven programmed from 70 °C to 270 °C at 5 °C/min intervals followed by 20 min at 270 °C, detector temperature was 300 °C; auxiliary (detector make up) gas nitrogen flow rate at 30 mL/min, hydrogen and air flow rates were 33 and 330 mL/min, respectively. The peak areas were measured using Hewlett-Packard 3392 integrator. 2.2.5 Normal phase high performance liquid chromatography (NP-HPLC) analysis of tocopherols 2.2.5.1 Procedure NP-HPLC was selected to avoid extra sample treatment (e.g., saponification). Analysis was performed with a solvent delivery LC-9A HPLC (Shimadzu, Kyoto, Japan). The chromatographic system included a model 87.00 variable wavelength detector and a 250 9 4 mm i.d. LiChrospher-Si 60, 5 lm, column (Knauer, Berlin, Germany). Separation of all components was based on isocratic elution where the solvent flow rate was maintained at 1 ml/ min at a column back-pressure of about 65–70 bar. The solvent system selected for tocopherols elution was isooctane/ethylacetate (96:4, v/v) with detection at 295 nm. 20 lL of the diluted solution of TL in the selected mobile phase were directly injected into the HPLC column. Tocopherols were identified by comparing their retention times with those of authentic standards. 2.2.5.2 Preparation of standard curves Standard solutions of tocopherols were prepared by serial
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dilution to concentration of approximately 5 mg/mL of vitamin E. Standard solutions were prepared daily from a stock solution which was stored in the dark at -20 °C. 20 lL was injected and peaks areas were determined to generate standard curve data.
Table 1 Relative percentages of fatty acids in Cistanche phelypaea oil
2.2.5.3 Quantification All quantitation was carried out by peak area measurement using Shimadzu C-R6A chromatopac integrator (Kyoto, Japan). Standard curves (concentration versus peak area) were calculated from six concentrations levels by linear regression. Based on the established chromatographic conditions, repeated injections of different concentrations of the standard tocopherols were made three times onto the HPLC system. All work was carried out under subdued light conditions. All results presented are mean values of at least three experiments, wherein no statistically significant difference (P [ 0.05) was found among the experiments.
C 15:0 Pentadecanoic acid
0.60 ± 0.02
Fatty acids
Percent
C 10:0 Capric acid
3.82 ± 0.08
C 12:0 Lauric acid
0.63 ± 0.02
C 14:0 Myristic acid
3.94 ± 0.06
C 16:0 Palmitic acid
25.0 ± 0.35
C 16:1n-7 Palmetoleic acid
2.24 ± 0.02
C 17:0 Margaric acid C 18:0 Stearic acid
0.58 ± 0.02 3.55 ± 0.09
C 18:1n-9 Oleic acid
28.1 ± 0.22
C 18:2n-6,9 Linoleic acid
16.6 ± 0.16
C 18:3n-3,6,9 Linolenic acid
1.53 ± 0.03
C 22:0 Arachidic acid
0.82 ± 0.02
C 22:2 Docosadienoic acid
1.34 ± 0.02
C 23:0 Tricosanoic acid
2.03 ± 0.04
C 24:1 Teracosenoic acid
0.47 ± 0.01
C 26:0 Cerotic acid
1.66 ± 0.03
C 30:0 Melissic acid
0.49 ± 0.01
Total unknown compounds
6.35 ± 0.02
3 Results and discussion Data concerning the lipid composition of the genus Orobanche are scarce. Total lipids extracted from the whole plant of Orobanche were found to be 10 g/kg on dry weight basis. This is the first time to study the lipid profile of the genus Orobanche. 3.1 Fatty acid composition Data about the qualitative and quantitative composition of fatty acids are summarized in Table 1. Among total lipids present in the Cistanche phelypaea, fatty acid profile evinces the lipids as a good source of the nutritionally essential linoleic acid, wherein the ratio of oleic acid to linoleic acid was about 3:2. Oleic acid (28.1 %) was the dominating fatty acid followed by palmitic acid (25.0 %) and linoleic acid (16.6 %). Aurand et al. (1987) mentioned that the nutritional value of linoleic acid is due to its metabolism at tissue levels which produces the hormone-like prostaglandins. The activity of these prostaglandins includes lowering of blood pressure and constriction of smooth muscle. The majority of fatty acids were unsaturated fatty acids (50.4 % of total fatty acids), while saturated fatty acids (mainly palmitic acid) were about 43.2 % of the total fatty acids. The fatty acid composition and high amounts of unsaturated fatty acids makes Cistanche phelypaea to a special herb suitable for nutritional applications.
3.2 Sterols and hydrocarbons composition The analysis of the free sterols provides rich information about the quality and the identity of the oil investigated. In fixed oils, neither cultivation of new breeding lines nor environmental factors have been found to alter content and composition of free sterols significantly in contrast to the fatty acid composition, which has been changed dramatically by breeding programmes (Lechner et al. 1999; Ramadan et al. 2006). Moreover, sterols comprise the bulk of the unsaponifiables in many oils. They are of interest due to their impact on health. Recently, sterols have been added to vegetable oils as an example of a successful functional food (Ntanios 2001; Ramadan et al. 2009). This type of products is now available and has been scientifically proven to lower blood LDL-cholesterol by around 10–15 % as part of a healthy diet (Jones et al. 2000). The content and composition of most of sterols in Cistanche phelypaea oil are presented in Table 2. High levels of sterols were estimated in the oil, which made up 29.4 g/kg oil. b-sitosterol, D7-avenasterol, stigmasterol and D5-avenasterol were the major components. The main component was b-sitosterol which represented ca. 77.4 % of the total sterol content. Other components, e.g. stigmasterol, D7-avenasterol and D5-avenasterol, were present at approximately equal amounts (6-9 % of total sterols).
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Table 2 Sterol composition (g/kg) of Cistanche phelypaea oil Compound
(g/kg)
Stigmasterol
2.10 ± 0.04
Table 4 Tocopherol composition (g/kg) of Cistanche phelypaea oil Compound
(g/kg)
b-Sitosterol
22.8 ± 0.19
a-Tocopherol
0.75 ± 0.02
D5-Avenasterol
1.89 ± 0.03
b-Tocopherol
2.19 ± 0.07
2.65 ± 0.05
c-Tocopherol
0.14 ± 0.01
d-Tocopherol
0.27 ± 0.01
7
D -Avenasterol
Table 3 Relative percentage of hydrocarbons in Cistanche phelypaea oil Hydrocarbons
Percent
Dodecane (C12)
0.43 ± 0.06
Pentadecane (C15)
5.37 ± 0.09
Octadecane (C18)
0.62 ± 0.03
(3.35 g/kg). b-Tocopherol was the major component followed by a-tocopherol. Both tocopherols isomers comprised more than 87 % of total vitamin E content in the oil. c- and d-tocopherols were detected in small amounts in the oil accounting for 14–16 % of the total vitamin E content. High levels of vitamin E detected in the oils may contribute to the great stability towards oxidation.
Eicosane (C20)
4.11 ± 0.07
Uneicosane (C21)
19.3 ± 0.20
Doeicosane (C22)
0.69 ± 0.03
Hexaeicosane (C26) Octaeicosane (C28)
23.8 ± 0.33 2.61 ± 0.12
4 Conclusions The trend towards natural ingredients and products promoting health is likely to increase. The data obtained will be important as an indication of the potentially nutraceutical and economical utility of Cistanche phelypaea. Cistanche phelypaea provides low yields of oil, but is a rich source of essential fatty acids, sterols, and fat-soluble vitamins.
Triacontane (C30)
3.04 ± 0.03
Dotriacontane (C32)
18.0 ± 0.25
Tetratriacontane (C34)
1.20 ± 0.03
Hexatriacontane (C36)
2.44 ± 0.07
Total unknown compounds
18.2 ± 0.19
Hydrocarbons profile of Cistanche phelypaea was estimated using gas liquid chromatography in the unsaponifiable matter and presented in Table 3. The main identified compounds were C21, C26 and C32 which account together for about 61.2 % of total identified hydrocarbons. Small amounts of C12, C18 and C22 were also detected in the unsaponifiable matter of Cistanche phelypaea. The picture of the hydrocarbons may be of value in the chemotaxonomy of plants. 3.3 Tocopherols composition Nutritionally important components such as tocopherols (vitamin E) improve the stability of oils. Data about the qualitative and quantitative composition of vitamin E are summarized in Table 4. NP-HPLC technique was used to eliminate column contamination problems and allow the use of a general lipid extraction for tocopherols separation (Ramadan and Mo ¨rsel 2002b, 2003). In our study, saponification of oil samples was not required, which allowed shorter analysis time and greater vitamin stability during analysis. All tocopherol derivatives were identified in both samples. Vitamin E levels were high in the oil
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