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� Springer Basel AG 2010

Abstract Oil extracted from the wild plant of Cis-tanche phelypaea was analyzed for its fatty acid,sterol, hydrocarbon and tocopherol contents. Totallipids (TL) content was 10 g/kg (on dry weight basis).The majority of fatty acids were of the unsaturatedtype (50.4 % of total fatty acids), while the saturated(mainly palmitic acid) were about 43.2 % of the totalfatty acids. Oleic acid was the dominating fatty acidfollowed by palmitic and linoleic acids. Highamounts of sterols were found in the oil with themain 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 hydrocarboncompounds were C21, C26 and C32 constituting about61.2 % of total hydrocarbons. Small amounts of C12,C18 and C22, however, were also detected. Tocopherollevels were high in the oil (3.36 g/kg oil), whereinb-tocopherol was the main component followed bya-isomer. Both tocopherol components comprisedmore than 87 % of total vitamin E content in the oil.Furthermore, c- and d-tocopherols were detected insmall amounts in the oil accounting for 14–16 % ofthe total vitamin E content. Information provided bythe present work will be of importance for foodapplications and chemotaxonomy of Cistanchephelypaea.

Keywords Cistanche phelypaea � Oil � Fatty acids �Sterols � Hydrocarbons � Tocopherols

1 Introduction

The genus Cistanche that belongs to the familyOrobanchaceae includes 16 species. They form anattractive group of phanerogamic root parasites.The occurrence of the genus is restricted to certainarid and semi arid regions of Africa, Asia and theMediterranean area including parts of SouthernEurope. The family Orobanchaceae represents twogenera Cistanche and Orobanche. The genus Cis-tanche is represented in Egypt by three speciesnamely C. phelypaea (Fig. 1), C. tubulosa and C. vi-olacea according to Tackholm (1974). Many edibleand medicinal uses are attributed to this genus. Thewhole plant of Cistanche tubulosa is used medicallyin Pakistan as a remedy for diarrhea and sores(Kobayashi et al. 1987). Shoots and stems of Cistan-che species can be used for food applications and asa tonic in the traditional Chinese medicine for thedeficiency of the kidney. Moreover, it is used forcold sensation in the lions and knees, female ste-rility, and constipation due to dryness of the bowel inthe senile (Mabberley 1997; Namba 1994). The genusOrobanche (Orobanchaceae) comprises about 100species of haloparasitic plants. Beack-Mannagetta(1930) proposed a subgeneric classification of thegenus in four sections: Gymnocaulis Nutt., Myzorrhiza(Phil.) Beck., Trionychen Wallr. and Osproleon Wallr.The latter is known nowadays as sect. Orobanche

Dr. M. F. Ramadan (&) � Dr. H. T. M. Hefnawy �A. M. GomaaBiochemistry Department, Faculty of Agriculture,Zagazig University, 44511 Zagazig, Egypte-mail: hassanienmohamed@yahoo.com

J. Verbr. Lebensm. (2011) 6:333–338DOI 10.1007/s00003-010-0648-1

Journal fur Verbraucherschutz und LebensmittelsicherheitJournal of Consumer Protection and Food Safety

according to the rules of the International Code ofBotanical Nomenclature (Greuter 1988). Many bio-logical activities were reported for some species ofthis family including antimicrobial, anti-inflamma-tory activities (Endo et al. 1982), antispasmodic,smooth muscle relaxant (El-Shabrawy et al. 1989) andcardioactivity (Pennacchio et al. 1969). Furthermore,Orobanche is eaten, like asparagus, as mentionedrecently by Rubiales et al. (1999).

The plant contains a number of constituentswhich include phenylethanoid glycosides (some-times called phenylpropanoids), iridoids (Xiong et al.1996) and polysaccharides (Naran et al. 1995). Theamino acids were determined and identified inCistanche solesa (Jiao et al. 1989). Fatty acid wasreported and tocochromanols composition in seedsof Orobanche family was recently investigated(Velasco et al. 2000). Information on the lipid com-position of Cistanche phelypaea, however, isinadequate and data available are incomplete. In thepresent study, we analyzed the whole plant, toobtain informative profile of the lipids in Cistanchephelypaea which will serve as a basis for furtherdetailed chemical investigation and nutritionalevaluation of the herb.

2 Materials and methods

2.1 Materials

The whole wild plants of Cistanche phelypaea werecollected from South Sinai (Egypt) during April 2008.The plants were kindly authenticated by Prof. Dr. El-Hadidy, Faculty of Science, Cairo University, Egypt.Herbarium specimens were kept at the Departmentof Agricultural Biochemistry, Faculty of Agriculture,Zagazig University. Standards used for sterols cha-racterization, cholesterol, b-sitosterol, stigmasterol,lanosterol, ergosterol, campesterol, D5-avenasteroland D7-avenasterol were purchased from Supelco(Bellefonte, PA, USA). Standards used for the charac-terization of vitamin E (a-, b-, c- and d-tocopherol)were purchased from Merck (Darmstadt, Germany).All reagents and chemicals used were of the highestpurity available.

2.2 Methods

2.2.1 Extraction of total lipids (TL)

Intact plants were dried and ground. The total lipidswere extracted by petroleum ether at 60–80 �C usingSoxhlet apparatus. The solvent was evaporated todryness on a rotary evaporator at 40 �C underreduced pressure.

2.2.2 Gas liquid chromatography analysis of fatty acidmethyl esters

Fatty acids were transesterified into methyl esters byheating in borontrifluoride (10 % solution in metha-nol, Merck, Darmstadt, Germany) according to theprocedure reported by Metcalfe et al. (1966). Fattyacids methyl esters (FAME) were identified on aShimadzu GC-14A equipped with flame ionizationdetector and C-R4AX chromatopac integrator (Kyoto,Japan). The flow rate of the carrier gas (helium) was0.6 mL/min and the split value with a ratio of 1:40was used. A sample of 1 lL was injected on a30 m 9 0.25 mm 9 0.2 g film thickness Supelco SPM-2380 (Bellefonte, PA, USA) capillary column. Theinjector and detector temperature was set at 250 �C.The initial column temperature was 100 �C pro-grammed by 5 �C/min until 175 �C and kept for10 min at 175 �C, then 8 �C/min until 220 �C and kept10 min at 220 �C. A comparison between the reten-tion times of the samples with those of authenticstandard mixture (Sigma, St. Louis, MO, USA; 99 %purity specific for GLC), run on the same column

Fig. 1 The spectacular plant is a member of the broomrape(Orobancheaceae) family but has a lupin like shape, five lobedtwo lipped flowers and can grow half a meter tall

334 M. F. Ramadan et al.

under the same conditions, was made to facilitateidentification. The quantification of each fatty acidwas carried out by comparing the peak area of itsmethyl ester with that of methyl nonadecanoatewithout application of any correction factor.

2.2.3 Gas liquid chromatography analysis of sterols

Separation of sterols (ST) was performed aftersaponification of the oil samples (Ramadan andMorsel 2002a). After the addition of cholesterol ace-tate (1.5 mg; Sigma, MO, USA) as an internal standardlipid (250 mg) probes were refluxed with 5 mL etha-nolic KOH solution (6 %, w/v) and a few anti-bumping granules for 60 min. The unsaponifiableswere firstly extracted three times with 10 mL ofpetroleum ether. The extracts were combined andwashed three times with 10 mL of neutral ethanol/water (1:1, v/v) then dried overnight with anhydroussodium sulphate. The extract was evaporated in arotary evaporator at 25 �C under reduced pressure,and then the solvent was completely evaporatedunder nitrogen. Gas chromatographic analysis ofunsaponifiables 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 sci-entific; Falsom, CA, USA), 30 m length, 0.25 mm i.d.1.0 pin film thickness with carrier gas (helium) flowrate 38 mL/min and splitless injection. The detectorand injector were set at 280 �C. The oven tempera-ture was kept constant at 310 �C and the injectedvolume was 2 lL. All ST homologues eluted within45 min and total analysis was set at 60 min to assurethe elution of all ST. The quantification of sterolcompounds was carried out with a cholesterol ace-tate internal standard and calculated by applying thedetector response of sitosterol. The repeatability ofthe analytical procedure was tested and the relativestandard deviation of three repeated analyses of asingle sample was \5 %. Quantitative analyses wereperformed with a Shimadzu (C-R6A Chromatopac;Kyoto, Japan) integrator.

2.2.4 Gas liquid chromatography analysisof Hydrocarbons

2.2.4.1 Preparation of unsaponifiable matter Unsa-ponifiable matter was determined according to themethod described in AOAC (2000). A known weightof the oil (5 g) was dissolved in ethanol (30 mL), then1.5 mL alcoholic KOH (50 %) was added. The oil wassaponified on a water bath for 30 min under reflux

air condenser. The alcoholic solution was concen-trated and quantitatively transferred into separatorfunnel using a total of 50 mL distilled water and50 mL petroleum ether. The unsaponifiable matterwas extracted three times with petroleum ether,washed several times with distilled water dried overanhydrous sodium sulphate and then filtered into aweighed flask. The solvent was evaporated using aboiling water bath and the flask was dried at 105 �Cuntil constant weight was reached.

2.2.4.2 Analysis of the hydrocarbons by GLC Thehydrocarbon compounds were identified using a PYEUNICAM PRO-GC gas chromatograph equipped withflame ionization detector (FID). The column used forseparating the unsaponifiable matter was a (OV-17)1.5 m 9 4 mm i.d. fused silica capillary columncoated with methyl phenyl silicone fluid. The fol-lowing chromatographic conditions were performed:Split ratio 1: 200, sample size 1 mL, carrier gas nitro-gen at a flow of 1 mL/min, injection temperature250 �C, oven programmed from 70 �C to 270 �C at5 �C/min intervals followed by 20 min at 270 �C,detector temperature was 300 �C; auxiliary (detectormake 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 usingHewlett-Packard 3392 integrator.

2.2.5 Normal phase high performance liquidchromatography (NP-HPLC) analysisof tocopherols

2.2.5.1 Procedure NP-HPLC was selected to avoidextra sample treatment (e.g., saponification). Analysiswas performed with a solvent delivery LC-9A HPLC(Shimadzu, Kyoto, Japan). The chromatographic sys-tem included a model 87.00 variable wavelengthdetector and a 250 9 4 mm i.d. LiChrospher-Si 60,5 lm, column (Knauer, Berlin, Germany). Separationof all components was based on isocratic elutionwhere 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 elutionwas isooctane/ethylacetate (96:4, v/v) with detectionat 295 nm. 20 lL of the diluted solution of TL in theselected mobile phase were directly injected into theHPLC column. Tocopherols were identified by com-paring their retention times with those of authenticstandards.

2.2.5.2 Preparation of standard curves Standardsolutions of tocopherols were prepared by serial

Cistanche phelypaea 335

dilution to concentration of approximately 5 mg/mLof vitamin E. Standard solutions were prepared dailyfrom a stock solution which was stored in the dark at-20 �C. 20 lL was injected and peaks areas weredetermined to generate standard curve data.

2.2.5.3 Quantification All quantitation was carriedout by peak area measurement using ShimadzuC-R6A chromatopac integrator (Kyoto, Japan). Stan-dard curves (concentration versus peak area) werecalculated from six concentrations levels by linearregression. Based on the established chromato-graphic conditions, repeated injections of differentconcentrations of the standard tocopherols weremade three times onto the HPLC system. All work wascarried out under subdued light conditions.

All results presented are mean values of at leastthree experiments, wherein no statistically signifi-cant difference (P[0.05) was found among theexperiments.

3 Results and discussion

Data concerning the lipid composition of the genusOrobanche are scarce. Total lipids extracted from thewhole plant of Orobanche were found to be 10 g/kgon dry weight basis. This is the first time to study thelipid profile of the genus Orobanche.

3.1 Fatty acid composition

Data about the qualitative and quantitative com-position of fatty acids are summarized in Table 1.Among total lipids present in the Cistanche phely-paea, fatty acid profile evinces the lipids as a goodsource of the nutritionally essential linoleic acid,wherein the ratio of oleic acid to linoleic acid wasabout 3:2. Oleic acid (28.1 %) was the dominatingfatty acid followed by palmitic acid (25.0 %) andlinoleic acid (16.6 %). Aurand et al. (1987) mentionedthat the nutritional value of linoleic acid is due toits metabolism at tissue levels which produces thehormone-like prostaglandins. The activity of theseprostaglandins includes lowering of blood pressureand constriction of smooth muscle. The majority offatty acids were unsaturated fatty acids (50.4 % oftotal fatty acids), while saturated fatty acids (mainlypalmitic acid) were about 43.2 % of the total fattyacids. The fatty acid composition and high amountsof unsaturated fatty acids makes Cistanche phely-paea to a special herb suitable for nutritionalapplications.

3.2 Sterols and hydrocarbons composition

The analysis of the free sterols provides rich infor-mation about the quality and the identity of the oilinvestigated. In fixed oils, neither cultivation of newbreeding lines nor environmental factors have beenfound to alter content and composition of free sterolssignificantly in contrast to the fatty acid composi-tion, which has been changed dramatically bybreeding programmes (Lechner et al. 1999; Ramadanet al. 2006). Moreover, sterols comprise the bulk ofthe unsaponifiables in many oils. They are of interestdue to their impact on health. Recently, sterols havebeen added to vegetable oils as an example of asuccessful functional food (Ntanios 2001; Ramadanet al. 2009). This type of products is now availableand has been scientifically proven to lower bloodLDL-cholesterol by around 10–15 % as part of a healthydiet (Jones et al. 2000). The content and compositionof most of sterols in Cistanche phelypaea oil arepresented in Table 2. High levels of sterols wereestimated in the oil, which made up 29.4 g/kg oil.b-sitosterol, D7-avenasterol, stigmasterol and D5-ave-nasterol were the major components. The maincomponent was b-sitosterol which representedca. 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 % oftotal sterols).

Table 1 Relative percentages of fatty acids in Cistanchephelypaea oil

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 15:0 Pentadecanoic acid 0.60 ± 0.02

C 16:0 Palmitic acid 25.0 ± 0.35

C 16:1n-7 Palmetoleic acid 2.24 ± 0.02

C 17:0 Margaric acid 0.58 ± 0.02

C 18:0 Stearic acid 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

336 M. F. Ramadan et al.

Hydrocarbons profile of Cistanche phelypaea wasestimated using gas liquid chromatography in theunsaponifiable matter and presented in Table 3. Themain identified compounds were C21, C26 and C32

which account together for about 61.2 % of totalidentified hydrocarbons. Small amounts of C12, C18

and C22 were also detected in the unsaponifiablematter of Cistanche phelypaea. The picture of thehydrocarbons may be of value in the chemotaxon-omy of plants.

3.3 Tocopherols composition

Nutritionally important components such as to-copherols (vitamin E) improve the stability of oils. Dataabout the qualitative and quantitative composition ofvitamin E are summarized in Table 4. NP-HPLCtechnique was used to eliminate column contami-nation problems and allow the use of a general lipidextraction for tocopherols separation (Ramadan andMorsel 2002b, 2003). In our study, saponification ofoil samples was not required, which allowed shorteranalysis time and greater vitamin stability duringanalysis. All tocopherol derivatives were identified inboth samples. Vitamin E levels were high in the oil

(3.35 g/kg). b-Tocopherol was the major componentfollowed by a-tocopherol. Both tocopherols isomerscomprised more than 87 % of total vitamin E contentin the oil. c- and d-tocopherols were detected in smallamounts in the oil accounting for 14–16 % of the totalvitamin E content. High levels of vitamin E detectedin the oils may contribute to the great stabilitytowards oxidation.

4 Conclusions

The trend towards natural ingredients and productspromoting health is likely to increase. The dataobtained will be important as an indication of thepotentially nutraceutical and economical utility ofCistanche phelypaea. Cistanche phelypaea provideslow yields of oil, but is a rich source of essential fattyacids, sterols, and fat-soluble vitamins.

References

AOAC (2000) Official Methods of Analysis of Association ofOfficial Agriculture Chemists, Washington, DC

Aurand LW, Woods AE, Wells MR (1987) Food composition andanalysis. AVI Book, Van Nostrand Reinhold Co., New York

Beack-Mannagetta G (1930) Orobanchaceae. In: Engler A (ed)Das Pflanzenreich, vol IV. Verlag Wilhelm Engelmann,Leipzig, pp 1–348

El-Shabrawy OA, Melek FR, Ibrahim M, Radwan AS (1989)Pharmacological evaluation of the glycosidated phenyl-propanoids containing fraction from Orobanche crenata.Arch Pharmacol Res 12:22–25

Endo K, Takahashi K, Hikino H (1982) Structure for forsythosideB, an antibacterial principal of Forsythia koreana stems.Heteroycles 19:261–265

Greuter W (1988) International code of botanical nomencla-ture. Koelt/Scientific Books, Konigstein

Jiao Y, Sun J, Pan X (1989) Identification and determination ofamino acids in Xinjiang Cistanche salsa. Zhongcaoyao20:322–325

Jones P, Raeini-Sarjaz M, Ntanios F, Vanstone C, Feng J, ParsonsW (2000) Modulation of plasma lipid levels and choles-terol kinetics by phytoesterol versus phytostanol esters.J Lipid Res 41:697–705

Kobayashi H, Oguchi H, Takizawa N, Miyaze T, Veno A,Usmanghani K, Ahmed M (1987) New phenylethanoid

Table 2 Sterol composition (g/kg) of Cistanche phelypaea oil

Compound (g/kg)

Stigmasterol 2.10 ± 0.04

b-Sitosterol 22.8 ± 0.19

D5-Avenasterol 1.89 ± 0.03

D7-Avenasterol 2.65 ± 0.05

Table 3 Relative percentage of hydrocarbons in Cistanchephelypaea oil

Hydrocarbons Percent

Dodecane (C12) 0.43 ± 0.06

Pentadecane (C15) 5.37 ± 0.09

Octadecane (C18) 0.62 ± 0.03

Eicosane (C20) 4.11 ± 0.07

Uneicosane (C21) 19.3 ± 0.20

Doeicosane (C22) 0.69 ± 0.03

Hexaeicosane (C26) 23.8 ± 0.33

Octaeicosane (C28) 2.61 ± 0.12

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

Table 4 Tocopherol composition (g/kg) of Cistanche phelypaeaoil

Compound (g/kg)

a-Tocopherol 0.75 ± 0.02

b-Tocopherol 2.19 ± 0.07

c-Tocopherol 0.14 ± 0.01

d-Tocopherol 0.27 ± 0.01

Cistanche phelypaea 337

glucosides from Cistanche tubulosa (Schrenk) Hook. F. PartI. Chem Pharm Bull 35:3309–3314

Lechner M, Reiter B, Lorbeer E (1999) Determination of free andesterified sterols in potential new oil seed crops bycoupled on-line liquid chromatography–gas-chromato-graphy. Fett/Lipid 101:171–177

Mabberley DJ (1997) The plant book. Cambridge UniversityPress, Cambridge

Metcalfe LC, Schmitz AA, Pleca R (1966) Rapid preparation offatty acid esters from lipids for gas chromatographicanalysis. Anal Chem 38:514–515

Namba T (1994) The Encyclopedia of Wakan-Yaku (TraditionolSino-Japanese Medicines) with color picture. vol II. Hoiku-sha, Tokyo, pp 16–17

Naran R, Ebringerova A, Hromadkova Z, Patoprsty V (1995)Carbohydrate polymers from underground parts of Cis-tanche deserticola. Phytochemistry 40:709–715

Ntanios F (2001) Plant-sterol-ester-enriched spreads as anexample of a new functional food. Eur J Lipid Sci Technol103:102–106

Pennacchio M, Alexander E, Syan YM, Chisalberti EL (1969) Theeffect of verbascoside on cyclic 30,50-adenosine monophos-phate levels in isolated rat heart. Eur J Pharmacol305:169–171

Ramadan MF, Morsel J-T (2002a) Neutral lipid classes of blackcumin (Nigella sativa L.) seed oils. Eur Food Res Technol214:202–206

Ramadan MF, Morsel J-T (2002b) Direct isocratic normal phaseassay of fat-soluble vitamins and b-carotene in oilseeds.Eur Food Res Technol 214:521–527

Ramadan MF, Morsel J-T (2003) Recovered lipids from pricklypear [Opuntia ficus-indica (L.) Mill] peel: a good source ofpolyunsaturated fatty acids, natural antioxidant vitaminsand sterols. Food Chem 83:447–456

Ramadan MF, Sharanabasappa G, Seetharam YN, Seshagiri M,Morsel J-T (2006) Characterisation of fatty acids andbioactive compounds of kachnar (Bauhinia purpurea L.)seed oil. Food Chem 98:359–365

Ramadan MF, Kinni SG, Rajanna LN, Seetharam YN, SeshagiriM, Morsel J-T (2009) Fatty acids, bioactive lipids andradical scavenging activity of Celastrus paniculatus Willd.seed oil. Sci Hort 123:104–109

Rubiales D, Cubero JI, Moreno MT, Sillero J (1999) Resistance toOrabanche: the state of the art. Sunta de Andalucia.Consejeria de Agriculturay pesca, Sevilla

Tackholm V (1974) Students’ flora of Egypt, 2nd edn. Cairo Univ.Press, Beirut

Velasco L, Goffman FD, Pusadas-Salva AJ (2000) Fatty acid andtocochromonols in seeds of Orobanche. Phytochemistry54:295–300

Xiong Q, Kadota S, Tani T, Namba T (1996) Antioxidative effectsof phenylethanoids from Cistanche deserticola. Biol PharmBull 19:1580–1585

338 M. F. Ramadan et al.