Erwerbs-Obstbau https://doi.org/10.1007/s10341-017-0351-9

ORIGINAL ARTICLE

Seed Oil Quantity and Fatty Acid Composition of Different Sea Buckthorn (Hippophae Rhamnoides L.) Wild Populations in

Ali Kuhkheil1 · Ali Mehrafarin2 · Vahid Abdossi1 · Hassanali Naghdi Badi2

Received: 4 December 2016 / Accepted: 15 September 2017 © Springer-Verlag GmbH Deutschland 2017

Abstract Sea buckthorn (Hippophae rhamnoides L.) is ploited for selection of suitable genotypes to improvement a valuable multipurpose medicinal plant belonging to and commercial exploitation of this plant. Elaeagnaceae family. In this study, oil content and fatty acid composition of seed oil from 20 sea buckthorn wild Keywords Hippophae rhamnoides · Hierarchical cluster · populations of Iran were evaluated in 2014 and 2015. Oil Populations · Seed oil · Fatty acids extraction was done using n-hexane solvent in a Soxhlet apparatus and then GC analysis of fatty acid methyl esters was performed. The highest and lowest amount of seed oil Kernölmenge und Fettsäurezusammensetzung content in two years were obtained from Sarein and Kejel verschiedener, im Iran wild wachsender, populations, respectively (3.88 to 8.63% in 2014 and from Sanddorn-Herkünften (Hippophae rhamnoides L.) 4.07 to 9.66% in 2015). Importance of seed oil is related to it’s highly amount of unsaturated fatty acids. Maxi- Schlüsselwörter Hippophae rhamnoides · Hierarchisches mum content of oleic acid cis were existed in Kelavenga Cluster · Bestände · Kernöl · Fettsäuren (21.19%) on 2014. The highest value of oleic acid trans were related to Shahrestanak (6.34%) in 2015. Maximum content of linoleic acid in 2014 were existed in Baladeh Introduction (42.03%) on 2015. The highest linolenic acid content were obtained from Dehdar (30.73%) in 2014. Factor analysis Hippophae rhamnoides L., known as sea buckthorn is a de- based on PCA revealed, first three components (PC1–PC3) ciduous shrub or tree belonging to Elaeagnaceae family. It explained 63.86% of the total variation. The first compo- is a thorny and nitrogen fixing plant with high nutraceu- nent (PC1) was contributed by some traits such as seed oil tical and therapeutical properties. It is widely distributed percentage, Palmitoleic acid, oleic acid trance and linoleic in temperate zone of Asia, Europe and North America. It acid contents with about 29% of total variation. Hierarchi- also grows in a distinct area from the Elburz Mountains cal cluster analysis divided the populations into four main in Persia to Caucasia and eastern Turkey (Rousi 1971). Its groups with high diversity. Wide range of variation across flower, seed, leaf and fruit possesses an exclusive composi- the sea buckthorn populations in seed oil traits could be ex- tion of vitamins, nutrients and essential fatty acids. Growth and performance of plants are under influence of differ- ent factors such as genetic characteristics, regional climate and geographical location, etc. Furthermore, previous stud-  Hassanali Naghdi Badi ies have demonstrated that medicinal plants produce vari- [email protected] ous contents of secondary metabolites in different environ- 1 Department of Horticulture, Science and Research Branch, ments, resulting in differences in their medicinal qualities Islamic Azad University, , Iran (Dong et al. 2011). 2 Medicinal Plants Research Centre, Institute of Medicinal Oil of sea buckthorn seeds is the most valuable product Plants, ACECR, , Iran of this plant. Extracted oil from pulp and seed of sea buck-

K A. Kuhkheil et al. thorn presented different fatty acid composition according brane after oxidation due to attack of free radicals. Sea to the subspecies, origins, cultivation activities, harvesting buckthorn seed oil is very high in these two essential fatty time of the berries, and the extraction method (Yang and acids. Seed oil accumulates at a very rapid rate with the on- Kallio 2002b). There are two sources of oil in sea buckthorn set of maturation to a maximum and thereafter is constant fruit, the seed which contains 6.47–20.2% (w/w) oil, and the or decreases as the fruit becomes mature and ripens (Li and oil held in the pulpy fruit parts surrounding the seed which Beveridge 2003). The seed oil from different fruit origins is termed pulp oil (Li and Beveridge 2003). Of course, its vary in linoleic (37–44%), linolenic (27–31%), palmitic pulp oil contains more saturated fatty acids while seed oil (7–9%), stearic (2.5–3%), and vaccenic acids (2.2–2.8%) contains highly amount of unsaturated fatty acids. So the (Yang and Kallio 2001). seed oil has more important therapeutic effects such as pre- Despite important of sea buckthorn seed oil, the study venting heart disease and arthritis (Yang and Kallio 2002a). about sea buckthorn seed oil quantity and quality of Iran Solvent extraction using hexane, chloroform, acetone and aren’t well documented. The present study was carried to methanol in a soxhelt apparatus and also supercritical CO2 determine the variations of seed oil content and fatty acid extraction are the main methods used for sea buckthorn oil composition in wild populations of sea buckthorn growing extraction, both in industrial and laboratory scale (Gutier- in Iran. The results of this study are useful to identify suit- rez et al. 2008). Hexane has the advantage of low toxicity able sea buckthorn populations when organizing the berry and easy removal from the extracted oil with distillation breeding programs and also provides important information procedures (Li and Beveridge 2003). for food and pharmaceutical industry. The sea buckthorn grown widely in central and Northern and have used in Iranian folk medicine. In several studies, oil content and major fatty acid composition Material and Methods of the oils extracted from seeds of sea buckthorns popula- tions growing in different regions of the world were inves- Plant Material and Collection tigated. According to most studies, the predominant fatty acids in seed oil are polyunsaturated fatty acids (65–72%) 20 sea buckthorn populations were collected and evaluated such as linoleic and linolenic acids (Cenkowski et al. 2006; from their different natural habitat in Iran in middle October Fatima et al. 2012; Gutierrez et al. 2008; Li and Beveridge 2014 and 2015. Voucher specimens have been deposited in 2003). The human body absolutely requires these polyun- the herbarium of Medicinal Plants Institute (MPI), ACECR, saturated fatty acids. Both fatty acids repair the cell mem-

Table 1 Geographical origins of H. rhamnoides populations Population No Herbarium No Region originated Latitude (N) Longitude (E) Altitude (m) 1 MPIH-4529 Ardebil-Sarein 38° 050 4200 N 48° 07 3900 E 1474 2 MPIH-4528 Ardebil-Kejel 37° 250 2500 N 48° 100 4800 E 1703 3 MPIH-4515 -Bozaj 36° 120 4200 N 50° 480 3500 E 2298 4 MPIH-4511 Alborz-Parachan 36° 140 4500 N 50° 560 4900 E 2339 5 MPIH-4507 Alborz-Khodkavand 36° 080 3500 N 50° 490 5900 E 2231 6 MPIH-4510 Alborz-Dehdar 36° 110 2200 N 50° 030 0600 E 2328 7 MPIH-4516 Alborz-Shahrestanak 35° 580 2300 N 51° 210 2800 E 2220 8 MPIH-4512 Alborz-Shahrak 36° 100 3200 N 50° 460 4700 E 1830 9 MPIH-4517 Tehran-Jajrood 35° 450 5300 N 51° 410 3500 E 1481 10 MPIH-4519 Tehran-Dizin 36° 060 1800 N 51° 210 1800 E 2380 11 MPIH-4530 Zanjan-Zanjan 36° 380 1300 N 48° 300 3300 E 1675 12 MPIH-4518 Semnan-Shahmirzad 35° 470 0800 N 53° 180 5400 E 2191 13 MPIH-4525 Qazvin-Zarabad 36° 290 3600 N 50° 260 1400 E 1802 14 MPIH-4526 Qazvin-MoallemKelaye 36° 270 1300 N 50° 280 4400 E 1615 15 MPIH-4527 Qazvin-Razmian 36° 320 1600 N 50° 120 5700 E 942 16 MPIH-4524 Gilan-Astara 38° 260 5200 N 48° 480 2200 E10 17 MPIH-4520 Mazandaran-Baladeh 36° 110 2400 N 51° 470 3500 E 2070 18 MPIH-4521 Mazandaran-Kelavenga 36° 110 1900 N 51° 260 1100 E 2817 19 MPIH-4522 Mazandaran-Gachsar 36° 060 5400 N 51° 190 3200 E 2293 20 MPIH-4523 Mazandaran-Yoosh 36° 110 2700 N 51° 420 4800 E 2180

K Seed Oil Quantity and Fatty Acid Composition of Different Sea Buckthorn ( Hippophae Rhamnoides L.) Wild Populations in Iran

Karaj, Iran. geographical origins of the 20 sea buckthorn and solvents used in this study has prepared from Merck populations and their GPS coordinates are shown in Table 1. Company, Germany.

Oil Extraction Data Analysis

10 g of the dried seeds were milled and placed in an extrac- Analysis of variance was performed for all traits by SPSS tion thimble and extracted with organic solvent n-hexane statistics (ver. 22) software. ANOVA analysis and mean using a 250 ml capacity soxhlet apparatus for 8 h (60 °C) in comparison of the seed oil content and fatty acid composi- 3 replications (AOCS 1989). The oil was then separated by tion were done by using Duncan multiple range tests at p Ä rotary-evaporated under reduced pressure at 35 °C. 0.05 significant level. In order to determine the most vari- able characters among the populations, factor analysis based GC Analysis of Fatty Acid Methyl Esters on principal component analysis (PCA) was performed. Hi- erarchical cluster analysis of studied populations was based Determination of the fatty acids was done by gas chromato- on the Euclidean distances of traits using Wards method. graphic measurement of the prepared samples. We used The simple correlation coefficient was calculated to deter- a Unicam 4600 GC instrument was equipped with a flame mine the relationships between the studied traits using the ionization detector and a split/splitless injector. A fused-sil- Pearson correlation coefficient. ica capillary column BPX70 (SGE, Melbourne, Australia) with 30 m length, 0.22 mm internal diameter and 0.25 µm thickness was used for analysis. Injector and detector tem- Results and Discussion peratures were 230 and 250 °C, respectively. Oven condi- tions were 180 °C increased to 220 °C at a rate of 2 °C/min Seed oil content and all of fatty acid composition (including and maintained for 5 min. Helium was the carrier gas and myristic acid (P < 0.01), palmitic acid (P < 0.01), Palmi- nitrogen used as the make-up gas at a flow rate of 30 ml/min. toleic acid (P < 0.01), stearic acid (P < 0.01), oleic acid cis The quantification of fatty acid methyl esters composition (P < 0.01), oleic acid trance (P < 0.05) and linoleic acid (P < was realized by integration of the FID peak area and com- 0.01)) except linolenic acid changed significantly in the ex- paring their retention times with standards methyl esters perimental years. Also, variance analyses showed that the to be expressed by percentage (El-Adawy et al. 1999). All various populations had significantly differences in respect the GC analyses were run in triplicate and the average val- of seed oil content and fatty acid composition (P < 0.01) in ues were reported in the work. All of chemical materials two studied years (Table 2). The average seed oil content was 6.09% and 6.89% in 2014 and 2015 year, respectively.

Table 2 Analysis of variance for effect of sea buckthorn populations and experimental years on seed oil content fatty acid composition and their interaction Source of Year Degree Mean Squares Variation of Seed Myristic Palmitic Palmitoleic Stearic Oleic Oleic Linoleic Linolenic Freedom Oil acid acid acid acid acid cis acid acid acid (df) (%) Trance Population 2014 19 5.729** 0.005** 13.697** 38.884** 3.541** 23.566** 1.625** 52.304** 25.839** (Treat) 2015 19 7.459** 0.017** 23.342** 24.09** 6.376** 15.247** 2.75** 36.606** 23.111** Means 19 12.706** 0.012** 27.947** 36.661** 7.243** 27.654** 2.016** 57.231** 29.448** Year (Y) – 1 18.965** 0.014** 46.635** 47.511** 34.563** 989.674** 0.683* 112.162** 0.104ns Block 2014 2 0.067ns 0.00002ns 0.025ns 1.123ns 0.027ns 0.081ns 0.002ns 0.298ns 3.330ns (REP) 2015 2 0.23ns 0.002ns 0.484ns 0.753ns 0.032ns 0.055ns 0.097ns 0.051ns 1.948ns Means 2 0.095ns 0.0008ns 0.365ns 1.846ns 0.0001ns 0.134ns 0.063ns 0.062ns 5.129ns P ×Y – 19 0.481ns 0.011** 9.093** 26.312** 2.675** 11.159** 2.359** 31.679** 19.501** Error 2014 38 0.709 0.0004 0.980 2.316 0.295 1.620 0.135 3.304 2.514 2015 38 0.719 0.0013 1.64 1.815 0.511 1.429 0.229 3.989 1.396 Means 78 0.701 0.0009 1.28 2.013 0.394 1.486 0.179 3.56 1.909 CV (%) 2014 – 13.824 7.284 6.281 23.561 19.505 8.255 9.233 5.761 6.673 2015 – 12.314 12.088 7.530 17.457 18.529 12.355 11.586 5.964 4.960 Means – 12.903 10.197 6.906 20.016 18.899 9.715 10.434 5.802 5.808 ns non-significant, *significant at 0.05, **significant at 0.01 probability level

K A. Kuhkheil et al. b a a a b b a b b a b a b a a 6.09 3.32 6.89 6.49 9.68 31.55 6.46b 17.01 23.79 32.52 7.09 0.31 0.28 7.72 2.79 15.42 4.13 23.76 0.29 12.55 33.49 16.38 3.86 3.98 4.06 23.82 15.76 def c abcd defg def def fgh cde bcde cd bc e def de bcd de cd efg a bcde f b bc a efg ab a 5.89 3.15 7.03 6.46 10.19 33.50 7.19 19.05 23.59 33.20 5.15 0.50 0.31 3.11 2.98 14.59 5.13 21.39 0.40 12.39 32.90 17.15 3.33 4.79 4.96 25.80 15.26 abcd c ab defg efgh efg a a def defgh bcd ef fg fgh f fg efg fg gh bcde ab c efghi bc ef def def 4.35 2.60 4.67 4.51 11.17 33.58 10.16 17.41 22.79 31.43 0.32 0.34 11.79 1.69 14.61 3.85 22.93 0.33 12.89 29.29 15.22 10.97 3.51 3.68 3.76 22.66 13.02 cde gh defg ab i a a a ef cde cdef cde fg cd efg fgh f bcd hij ab bc ghijk cdef efg bc abc cde 4.44 3.65 5.01 4.72 10.66 24.39 1.73 17.34 26.95 26.95 0.35 0.25 9.45 2.00 21.19 3.39 29.88 0.30 15.93 29.51 16.67 5.59 5.29 4.56 3.97 24.02 16.01 cd j cd ab f efgh fgh a h ab bcde cde efg bc cd fgh abc fgh abcd ef bc bcd ef de efg efg bcd 6.47 2.48 7.31 6.89 8.01 32.86 9.62 11.11 24.62 37.44 0.24 0.32 3.94 2.17 12.96 4.77 22.14 0.28 10.49 42.03 13.77 6.78 2.80 3.51 4.14 27.11 16.43 cd ab h f fgh gh b a gh a de abcd fg ab bc gh bcd bcd efgh bc bc defgh cde bc fg def cdef 7.22 2.28 8.56 7.89 7.65 32.43 8.73 20.26 21.27 0.33 0.27 8.92 2.08 12.68 4.02 21.19 0.30 10.16 34.97 19.54 8.82 2.49 3.82 3.92 21.36 18.82 33.70 cd abc fgh cde defgh bcd bcd ab fgh a def abc defg ab b defg f ghij cd bc defgh h h fg efg a ab 7.65 Razmian Astara Baladeh Kelavenga Gachsar Yoosh Mean 2.86 8.28 7.97 9.42 32.21 2.50 20.14 22.64 0.25 6.74 2.65 15.91 4.04 22.96 0.22 0.18 12.66 34.10 19.22 4.62 3.07 5.21 4.63 22.31 18.31 33.16 fg a cdef gh cde gh fg b bc defg ab fgh efg b bc de hi cdefg def bc b bcde bcde bc a def ab 5.30 kelaye 4.64 7.30 6.30 5.93 26.90 12.11 17.86 21.92 0.29 4.89 2.30 15.99 5.39 20.53 0.31 0.32 10.96 35.33 17.97 8.50 6.99 3.79 4.59 23.31 18.09 31.11 ef defg bcd ab def bcde bcd f cde ab bcde bc bcde hi def def ef abc a b jk bcd def efg h g 9def 5.8 Zarabad Moallem 3.06 5.66 5.78 28.86 9.26 17.51 25.42 0.36 10.80 2.86 14.69 2.91 25.05 0.32 0.29 12.78 28.57 16.88 10.03 3.26 10.87 2.67 2.79 25.79 16.25 28.71 a cd defg cd ef ef cdefg bc def bc de de abc ab ab de cdef a b fg gh h efg ab i cde a 7.84 mirzad 75.67 8.64 8.24 32.54 6.21 16.84 24.68 0.30 5.14d 4.88 13.52 5.46 23.16 0.26 0.22 9.19 34.82 16.10 5.68 6.46 4.86 4.05 4.76 26.20 15.35 33.68 bility. h ab a cd ab cde bcd defg def a a cdef ab i efg g cdef ef abc bc ijk def fg g gh def ef 5.31 2.15 6.13 5.72 22.21 11.44 21.32 24.98 0.33 11.11 2.30 15.85 3.06 25.91 0.29 0.25 11.66 30.75 19.70 11.28 1.99 7.48 3.87 3.46 24.05 18.09 26.48 cde ab gh cdef ab ef efgh cdef bcde efg ab bcd cde efg efg defg efg abc bc hijk b bc efg def def ef 8efg 5.2 2.76 5.56 5.42 31.26 15.17 23.74 0.32 10.36 2.53 14.09 3.24 22.14 0.35 0.37 12.02 32.58 15.46 10.27 2.98 9.96 3.73 3.48 10.18 25.35 15.74 31.92 a fgh bc bc efgh a cdef h ab a f bcd abc defg ab bc k ef b bcde ef bc h efg fgh cde f 7.55 2.82 8.28 7.91 39.72 15.45 25.59 0.20 3.95 3.07 18.05 4.70 22.28 0.28 0.36 15.57 30.98 13.57 2.83 2.58 13.09 3.28 3.99 1.73 28.90 11.69 35.35 bc hi bcd def b cdef bc h cdef f cd bcde bc bcd cd fghij bc bc cdef bcde bc def bc efg ab ab ef Shahrak Jajrood Dizin Zanjan Shah- 6.75 4.12 7.01 6.88 13.53 25.25 0.26 8.87 2.59 14.93 4.42 26.55 0.31 0.36 11.76 34.63 12.96 6.35 5.64 8.59 4.87 4.64 3.83 23.95 12.39 34.58 34.61 a h cd fgh b ef ab abcd h abcd cde gh ab a a efgh def a ab a bc b gh abc efg a ef tanak 8.20 5.79 9.29 20.74 21.19 0.27 4.70 5.64 16.66 8.75 6.34 21.65 0.33 0.39 14.14 29.54 18.98 4.25 5.94 11.63 3.38 4.86 3.80 20.73 17.22 31.38 30.46 cdef a ef a defg bcdef cdef bc e def gh fg defg defg cdefg ab bc fg k cdef cdef def ef efg ab def f 4.69 Dehdar Shahres- 5.52 17.81 28.28 0.29 9.93 2.27 14.20 5.11 2.54 30.73 0.30 0.31 11.41 31.52 16.49 6.37 3.46 8.62 4.91 3.73 2.82 25.83 15.17 2.86 29.62 30.57 hi h ab a h a b h abcd efg f ab abc efg cdef ijk bc efg fghij fg def defg efg a abc f efg 5.27 kavand 5.78 13.76 21.52 0.23 10.40 2.39 21.07 5.52 3.77 20.02 0.26 0.29 16.21 34.89 6.06 12.72 2.52 11.35 5.15 4.46 1.73 23.02 11.68 2.45 37.74 36.31 efg ef bcd bc bcd cde a fgh abc c de cdef efg bc abc cde bc de defgh bc bcd efg efg bcd bcd ef efg 5.28 7.38 16.40 0.32 6.45 2.14 13.51 6.33 4.04 25.28 0.33 0.33 13.37 34.24 5.18 16.86 2.97 13.24 4.35 4.20 3.90 22.33 17.32 2.55 33.18 33.71 23.80 ij g bcdef a gh bcde cde bc a cde def cde defgh de bc de ghijk ef defg h cd efg efg ef ef bcd defg 5.94 6.59 12.23 0.28 6.28 2.54 10.03 6.26 3.40 23.96 0.28 0.28 9.30 40.87 7.61 14.10 3.23 8.58 3.64 3.52 8.94 25.13 15.96 2.89 34.65 37.76 24.54 ab ab fgh a bcd ab cd abc i abcde fg gh i bcde g g fghi c h cdefg gh h de efg def abc defg 3.88 4.07 0.26 10.98 1.73 19.67 3.97 4.29 21.39 0.22 0.18 16.10 34.23 10.22 17.59 3.90 12.53 3.44 3.86 9.47 15.77 17.04 2.81 26.99 30.61 18.58 18.14 ef bc cdef bcd cde fgh de cd def cdef bcde a a jk cd a a efghi gh efg efg cd defg gh f ef b Results of means comparisons for seed oil traits among studied Hippophae rhamnoides populations during 2014 and 2015 harvesting season 2014 8.63 2015 9.66 2014 14.18 2014 0.23 2015 6.54 2014 26.07 2014 4.90 Mean 9.14 Mean 11.94 2015 3.86 2015 34.00 Mean 16.73 Mean 0.24 2015 0.25 Mean 5.20 2015 22.79 2015 4.76 2014 15.36 2015 9.70 2014 2.89 Mean 3.37 2014 3.86 2014 32.51 Mean 33.25 Mean 24.43 Mean 4.83 2015 18.09 means in each column followed by the same letter (a–h) are not significantly different according to Duncan’s multiple range test at the 5% level of proba Table 3 Treatment (Population) Traits YearSeed Oil Sarein(%) Kejel Bozaj Parachan Khod- a–h Oleic acid cis Myristic acid Linolenic acid Stearic acid Palmitic acid Oleic acid Trance Palmi- toleic acid Linoleic acid K Seed Oil Quantity and Fatty Acid Composition of Different Sea Buckthorn ( Hippophae Rhamnoides L.) Wild Populations in Iran

Based on results of mean comparison, the highest amount of Same to our work, several studies on sea buckthorn seed oil content was obtained from Sarein population in two oil showed that both the seeds and soft parts are rich in years as 8.63% and 9.66% for 2014 and 2015 year, respec- oleic acid. In first study year, the content of oleic acid cis tively. The lowest amount of oil content was obtained from (15.42%) was more than the second year (9.68%). The max- the Kejel population as 3.88% and 4.07% for 2014 and 2015 imum and minimum content of this fatty acid in 2014 were year, respectively (Table 3). The rate of oil accumulation in existed in Kelavenga (21.19%) and Bozaj (10.03%) pop- the pulp and seed varies depending on environmental (cli- ulations, respectively. Also, the maximum and minimum matic and meteorological) factors (Li and Beveridge 2003). content of it in 2015 were in Parachan (13.24%) and Shah- In accordance to our study results on sea buckthorn seeds, mirzad (4.86%), respectively (Table 3). It was found that Sabir et al. (2005) were found the oil content ranging from oleic acid trans content in second year (4.13%) was more 7.69 to 13.7% between studied populations from different than the first year (3.98%). The highest (5.21%) and lowest areas of northern Pakistan. (2.67%) value of oleic acid trans in 2014 year were related It was found that the myristic acid content (0.31%) in to Razmian and Zarabad populations, respectively. But, the second year was more than first year (0.28%). Regard- highest (6.34%) and lowest (2.54%) value of this trait in ing with this parameter, the highest (0.36%) and lowest 2015 year were observed in Shahrestanak and Dehdar pop- (0.20%) value of myristic acid in 2014 year were related ulations, respectively (Table 3). to Zarabad and Jajrood populations, respectively. Also, the The higher content of linoleic acid was observed in sec- highest (0.50%) and lowest (0.18%) value of this trait in ond year (33.49%) in compare to first year (31.55%). The 2015 year were related to Yoosh and Razmian populations, maximum and minimum content of this fatty acid in 2014 respectively (Table 3). Previously, Cenkowski et al. (2006) were occurred in Jajrood (39.72%) and Zanjan (22.21%) and Gutierrez et al. (2008) reported that myristic acid wasn’t populations, respectively. Whereas, the maximum and min- present in seed oil of sea buckthorn, whereas Francis (2012) imum content of it in 2015 were in Baladeh (42.03%) and and Li and Beveridge (2003) found that myristic acid was Zarabad (28.57%), respectively (Table 3). an inseparable part of fatty acid composition of seed oil. The average of linolenic acid content was 23.76% and The average of palmitic acid was 15.76% and 17.01% 23.82% in 2014 and 2015 year, respectively. The highest in 2014 and 2015 year, respectively. Based on results of and lowest linolenic acid content in two years were related mean comparisons, the highest palmitic acid content were to Dehdar (28.28%) and Kejel (18.58%) populations, re- obtained from Astara (18.82%) and Zanjan (21.32%) pop- spectively. ulations in 2014 and 2015 years and lowest content of Previously, several studies indicated high content of un- this fatty acid were related to Khodkavand (11.68%) and saturated fatty acids (32–40% linoleic, 20–39% linolenic, Baladeh (11.11%) in 2014 and 2015 years (Table 3). and 13–17% oleic acids) and lower concentration of satu- Palmitoleic acid had a higher average content in 2015 rated fatty acids (8–13% palmitic and 3–8% steric acids) in year (7.72%) in compare with 2014 year (6.46%). Also, oil seed of H. rhamnoides subspecies sinensis and H. rham- maximum Palmitoleic acid content in 2014 and 2015 noides subspecies rhamnoides (Yang and Kallio 2002a; Li were reported from Moallemkelaye (12.11%) and Gach- and Beveridge 2003; Gutierrez et al. 2008). Linoleic acid sar (11.79%) populations, respectively. Whereas minimum (omega 6) and linolenic acid (omega 3) are the essential content of this fatty acid were in Kelavenga (1.73%) and fatty acids of human body and they carry all fat soluble vi- Yoosh (3.11%) populations in 2014 and 2015 years, re- tamins i. e., vitamin A, D, E and K and also their important spectively (Table 3). Palmitoleic acid is characteristic of function is to promote cognitive function and bone health the oils of the fruit coat and pulp, but is low in seed oils (Kumar er al. 2011). (Gao et al. 2000). Vereschagin et al. (1998) reporterd that Palmitoleic acid in the triacylglycerol of pulp/seed oil in Principal Components Analysis (PCA) wild sea buckthorn fruit of Central Asia and the Baltics (55 and 42%, respectively) higher than in the Caucasian Factor analysis was used based on principal components to mountain regions (16%). provide a reduced dimension model indicating differences Mean comparison results showed the stearic acid con- measured among groups. PCA allows to evaluate multi- tent (3.86%) in second year was higher than the first year collinear data and to determine the traits most suitable for (2.79%). The highest (5.64%) and lowest (1.69%) value classification. PCA indicated five components with explain- of stearic acid in 2014 year were related to Shahrestanak ing 88.31% of the total variance. The first three components and Gachsar populations, respectively. Also, the highest (PC1–PC3) explained 63.86% of the total variation (Ta- (6.99%) and lowest (1.99%) value of this trait in 2015 year ble 4). In the first PC (PC1), some characters such as seed were related to Moallemkelaye and Zanjan populations, re- oil percentage, and Palmitoleic acid, oleic acid trance and spectively (Table 3). linoleic acid content showed the highest variance. Also, in

K A. Kuhkheil et al.

Table 4 Eigenvectors of the first three principal component axes PC2, myristic acid and Linoleic acid contents showed the from PCA analysis of fatty acid quantity and quality variables in highest variance. While, the highest variance were observed studied H. rhamnoides populations for oleic acid cis and Linolenic acid contents in PC3. Character Component 123 Cluster Analysis Seed Oil 0.81** 0.22 0.25 Palmitic acid –0.15 –0.01 0.39 Cluster analysis based on Wards method at similarity co- Myristic acid –0.19 0.89** 0.12 efficient of 10, divided populations into four main groups Palmitoleic acid –0.83** 0.10 0.10 with high diversity (Fig. 1). The first main group was di- Stearic acid 0.61 0.48 0.22 vided into six populations. The first group (A) consisted of Oleic acid cis –0.16 0.03 –0.76** populations from Dizin, Gachsar, Zanjan, Zarabad, Dehdar Oleic acid Trance 0.69** 0.22 –0.28 and Kelavenga with similar characteristics such as seed oil Linoleic acid 0.59** –0.64** –0.14 content and linoleic acid. The second group (B) was com- Linolenic acid –0.03 –0.41 0.67** prised of Kejel population. Some of the prominent charac- Eigenvalue 2.63 1.70 1.42 teristics of this population are seed oil content, oleic acid % of variance 29.20 18.91 15.75 cis and linolenic acid which made it distinct among the Cumulative% 29.20 48.11 63.86 other populations. The third group (C) was comprised of ** Eigenvalues are significant ≥0.50 Parachan, Yoosh, Moallemkelaye, Astara, Sarein, Razmian and Shahrestanak populations. At last fourth group con- sisted of Khodkavand, Jajrood, Bozaj, Baladeh, Shahrak and Shahmirzad with similar characteristics such as myris- tic acid and palmitic acid.

Fig. 1 Wards cluster analysis of H. rhamnoides population based on seed oil traits

K Seed Oil Quantity and Fatty Acid Composition of Different Sea Buckthorn ( Hippophae Rhamnoides L.) Wild Populations in Iran

Table 5 Correlations between seed oil characteristics in Hippophae rhamnoides populations Variables Altitude Seed Oil Myristic Palmitic Palmitoleic Stearic Oleic acid Oleic acid Linoleic Linolenic acid acid acid acid cis Trance acid acid

Altitude 1 – –––––––– Seed Oil –0.453* 1–––––––– Myristicacid0.331–0.1631––––––– Palmitic acid –0.431 0.148 –0.007 1 – – –––– Palmitoleic acid –0.071 –0.603** 0.1050.1951––––– Stearic acid 0.153 0.530* –0.0300.113–0.3831–––– Oleicacidcis0.211–0.328–0.110–0.097–0.200–0.1441––– Oleic acid Trance 0.035 0.328 0.092 0.006 –0.557* 0.415 0.046 1 – – Linoleic acid –0.112 0.368 –0.152 –0.625** –0.395 –0.091 –0.259 0.282 1 – Linolenic acid 0.360 –0.012 0.180 –0.283 –0.227 –0.021 –0.229 –0.291 –0.119 1 *p<0.05, **p<0.01

Correlations Between the Characters Conclusion

Simple correlation coefficient analysis showed the existence In this study, the nutritional qualities of seed oil from sea of significant positive and negative correlations among seed buckthorn populations in several region of Iran were eval- oil characteristics (Table 5). We mentioned some of more uated. There was a wide variability in seed oil quality and important correlations between them. Seed oil content ex- quantity of different H. ramnoides populations in studied hibited negative correlation with altitude of populations po- regions of Iran. In other word, the results of this research sition (r = –0.453 p < 0.05) and Palmitoleic acid content revealed valuable information about the fatty acid quan- (r = –0.603 p < 0.01) and positive correlation with stearic tity and quality in the oils extracted from berry seeds of acid content (r = 0.530 p < 0.05). Palmitic acid had negative sea buckthorn wild populations in Iran. So that, the highest correlation with linoleic acid (r = –0.625 p < 0.01). Also value of seed oil content (%) were observed in Sarein pop- Palmitoleic acid showed negative correlation with oleic acid ulation in both experimental years (8.63% and 9.66% for trance (r = –0.557 p < 0.05). 2010 and 2011 years). Since the seed oil important is be- Same to our results, Yang (1989) explained that region cause of highly amount of unsaturated fatty acids (oleic altitude causes decrease in seed oil content above about acid, linoleic acid and linolenic acid), populations with 2500 m asl. Li and Beveridge (2003) mentioned negatively high content of these fatty acid are more valuable. Max- correlation between proportion of linolenic acid with those imum content of oleic acid cis in 2014 and 2015 were ex- of oleic and linoleic acids in seed oil. In present study, sim- isted in Kelavenga (21.19%) and Parachan (13.24%) popu- ilar results were obtained. In accordance to our study, fatty lations, respectively. the highest value of oleic acid trans in acid composition of fruit pulp and seeds oils was perviously 2014 and 2015 years were related to Razmian (5.21%) and studied in four different cultivars of sea buckthorn in dif- Shahrestanak (6.34%) populations, respectively. Maximum ferent regions of Himalaya with different altitude ranges. content of linoleic acid in 2014 were existed in Jajrood In the seed oils, the unsaturated fatty acids such as linoleic (39.72%) population, whereas maximum content of it in acid, oleic acid and α-linolenic acid constitute higher pro- 2015 were in Baladeh (42.03%). the highest linolenic acid portion (86.5–51.7%) with exception in H. salicifolia.This content were obtained from Dehdar (30.73%) and Khodka- investigation results showed that seed oils of H. rhamnoides vand (20.02%) populations in 2014 and 2015 years. Factor particularly from higher altitude area could be considered analysis indicated five components with explaining 88.31% excellent sources of linoleic and α-linolenic acids compare of the total variance. In the first PC some traits such as with lower altitude. (Singh and Gupta 2015). Also, Shah seed oil percentage, Palmitoleic acid, oleic acid trance and et al. (2007) reported the variation of seed oil content be- linoleic acid contents showed the highest variance. Hierar- tween different locations of Pakistan (7.03–12.86%). They chical cluster analysis divided populations into four main explained the huge difference in the range of oil content is groups with high diversity. Some positive or negative cor- due to altitude variations and genetics make of sea buck- relation between seed oil traits can be used in the future thorn ecotypes as well. breeding programs. Wide range of variation across the sea buckthorn populations can be exploited for selection of suit- able genotypes to improvement and commercial exploita- tion of this plant.

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