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ISSN : 1225-8504(Print)
ISSN : 2287-8165(Online)
Journal of the Korean Society of International Agricultue Vol.27 No.3 pp.381-392
DOI : https://doi.org/10.12719/KSIA.2015.27.3.381

Comparison in Antioxidant Activities and Total Polyphenol Contents of Safflower (Carthamus tinctorius L.) Germplasm Collection

Jung Sook Sung, Ho Cheol Ko, On Sook Hur, Sang Gyu Kim, Jung Ro Lee, Binod P. Luitel, Jae Gyun Gwag, Hyung Jin Baek, Kyoung Yul Ryu†
National Agrobiodiversity Center, National Academy of Agricultural Science, Rural Development Administration, Jeonju 54874, Korea
Corresponding author (Phone) +82-63-238-4930 (kyryu@korea.kr)
August 6, 2015 September 16, 2015 September 18, 2015

Abstract

This study was undertaken to select genetic resources of safflower with high antioxidant activities. A total of 100 accessions were obtained from the National Agrobiodiversity Center (NAC) of Korea. Total polyphenol contents (TPC) and antioxidant activities of 1,1-diphenyl-2-picryl-hydrazil (DPPH) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) in 75 % ethanol seed extract of safflower germplasm were determined. TPC ranged from 14.2 ± 0.41 to 81.6 ± 1.56 μg GAE mg-1dry weight (dw). Safflower seed extracts showed variation in DPPH antioxidant activities ranging from 1.6 ± 0.07 to 14.1 ± 0.23 μg ASC mg-1 dw. Antioxidant activities of ABTS ranged from 34.5 ± 0.70 to 156.5 ± 3.06 μg Trolox mg-1 dw. ABTS values showed significant positive correlation (r=0.954**) with DPPH activity. Group I (8 accessions) having high antioxidant activities were obtained by the matrix of calculated distances of TPC and antioxidant activities. K185841, K185879, and K185863 among group I germplasm had higher TPC and antioxidant activities than other accessions. These genotypes are potential sources of natural antioxidants. Therefore, this study provides valuable information for safflower breeders and growers to develop new varieties and produce functional foods.


수집홍화유전자원의 총폴리페놀 함량 및 항산화 활성 비교

성 정숙, 고 호철, 허 온숙, 김 상규, 이 정로, Binod P. Luitel, 곽 재균, 백 형진, 류 경열†
농촌진흥청 국립농업과학원 농업유전자원센터

초록


    Rural Development Administration
    PJ00937304

    Safflower (Carthamus tinctorius L.), originated from Egypt and Afghanistan, belongs to Asteraceae family. It has been used as medicinal materials in Korea, China, and Japan (Lee, 1980). Safflower is most commonly known as ‘Kusan’ (India, Pakistan), ‘Suff’ (Ethiopia), ‘Le carthame’ (France), and ‘Honghua’ (China and Korea). Safflower floret contains yellow and red quinochalcone natural dyes such as safflower yellow A, safflower yellow B, safflomin C, precarthamin, and carthamin (Cho and Hahn, 2000). Safflower seed is known to have plenty α-linoleic acid. It has been used as cooking oil in Europe. Safflower seed is rich in linoleic acid, protein, and dietary fiber. It has been clinically used for treating osteoporosis or rheumatism in Korea (Kim et al., 2007).

    Natural antioxidant and antioxidant enzymes are highly useful substances. They play critical roles in the defensive mechanism against environmental stress. The antioxidant activity of a specific substance can be measured in several ways. DPPH radical scavenging activity method is relatively simple. And it can measure large volume (Ryu et al., 2006). Methods using stable ABTS or DPPH radicals have been widely used to evaluate free radical scavenging ability of antioxidant substances (Nabavi et al., 2009). Both methods are characterized by excellent reproducibility under certain assay conditions. However, they might show significant differences in their responses to antioxidant (Arnao, 2000). Chemical compositions of different section of safflower and their effect have been studied previously (Kim et al., 2000; Kim et al., 2003; Kim et al., 2007; Kim et al., 2014). Antioxidant properties of twelve phenolic compounds in the seeds of safflower have been studied by Kim et al. (2007). Safflower seed extract exhibits remarkable radical scavenging activities, ferric reducing antioxidant power (FRAP), and reducing power in dose-dependent manners (Yu et al., 2013). Regarding the content of polyphenolic compound, Korean safflower flowers contains 13.85% water soluble extract and 9.70% MeOH extract, which is higher than those in a Chinese variety, 9.39% and 7.04%, respectively (Park and Park, 2003).

    The antioxidant activities of safflower leaves and seeds have been previously studied (Golkar et al., 2009; Yu et al., 2013). However, few research has been peformed on the antioxidant activity of safflower germplasm gathered from various areas. Bio-active compounds in safflower seed genotypes collected at different geographical locations may vary. Hence, the objective of this study was to investigate and compare the antioxidant activity of safflower genotypes collected from two different geographical locations, to select potential genetic resources of safflower for breeding sources or functional materials.

    MATERIALS AND METHODS

    Materials

    One hundred safflower accessions were obtained from the National Agrobiodiversity Center (NAC) of RDA. All accessions were collected from two collection areas, a northwest area (C I, n = 35) under the influence of mediterranean climate and a northeast area (C II, n = 65) that is affected by continental climate, adjacent to the northern part of the Saudi Arabian Peninsula (Table 1). These accessions were grown in the experimental field of Hwaseong, Gyeonggi-do in 2014. Planting distance was 30 × 20 cm. Intercultural operations followed the recommendation of RDA.

    Chemicals

    1,1-diphenyl-2-picryl-hydrazil (DPPH), L-ascorbic acid, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS), 6-hydroxy-2,5,7,8-tetramethylchroman- 2-carboxylic acid (Trolox), Folin-Ciocalteu reagent, and gallic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). All other reagents were of analytical grade.

    Sample preparation

    Crude extracts were obtained from 7 g dried of safflower seeds using ASE-200 (Dionex, Sunnyvale, California, USA) extractor (Piao et al., 2013). Extractions were performed in 40 ml 75% ethanol under nitrogen gas 1,500 psi and 70°C. Extracted samples were dried using a Genevac HT-4X vaccum concentrator (Ipswich, Suffolk IP1 5AP, UK) .

    Polyphenol assay

    Folin-Ciocalteu method was used for total polyphenol determination based on optimized conditions of Waterhouse (2002) with some modifications. Briefly, Folin-Ciocalteu reagent (100 μl) was added to 100 μl of sample solution and incubated at room temperature for 3 min. Absorbance was measured at 750 nm using an ELISA reader (Epoch, Bio-Tek, Winooski, VT, USA). Distilled water was used as blank. Results were expressed as milligrams of gallic acid equivalents (GAE) per gram dried weight sample (μg GAE mg-1 dw). Each extract sample was tested in triplicates.

    DPPH assay

    The free radical scavenging activity of extracts was assessed by the DPPH method proposed by Piao et al. (2013). Briefly, DPPH solution (150 μl at 1.5 × 10-4 M, in anhydrous ethanol) was added to 100 μl of sample solution. The mixture was shaken vigorously and left to stand at 25°C in the dark for 30 min. The absorbance of sample was measured at 517 nm using a spectrophotometer (Epoch, Bio-Tek, Winooski, VT, USA). DPPH free radical scavenging activity was calculated using the following equation :

    Radical scavenging activity (%) = 1− A sample A sample blank / A control A control blank × 100

    The radical scavenging effect was expressed as μg Lascorbic acid equivalent antioxidant capacity (ASC) per mg dried extract (μg ASC mg–1 dw).

    ABTS assay

    ABTS radical scavenging activity was estimated using the method of Re et al. (1999) with some modifications. Briefly, ABTS radical cation was generated by adding 7 mM ABTS to 2.45 mM potassium persulphate followed by overnight incubation in the dark at room temperature. The ABTS radical cation solution was diluted with methanol to obtain an absorbance of 0.7 ± 0.02 at 734 nm. Diluted ABTS radical cation solution (190 μl) was added to 10 μl of sample solution. After 6 min, the absorbance value was measured at 734 nm using a spectrophotometer (Epoch, Bio-Tek, Winooski, VT, USA). Scavenge capability of ABTS radical was calculated using the following equation :

    ABTS scavenging activity (%) = 1− A sample A sample blank / A control A control blank × 100

    The radical scavenging effect of each sample was reported as Trolox equivalent antioxidant activity by comparing the changes in absorbance at 734 nm of reaction mixtures containing safflower seed extract or Trolox equivalent (μg Trolox mg–1 dw).

    Statistical analysis

    Each sample was analyzed in triplicates. Data were reported as means ( ± S.D). T-Test for testing significant differences between safflower accessions collected from the two areas and Duncan’s multiple range test (DMRT) were carried out to test any significant differences among safflower accessions gathered from different collection areas using the R statistical software environment (http:// www.r-project.org). Correlation coefficients were calculated to describe the relationship between DPPH and ABTS activities.

    Results

    TPC, DPPH antioxidant activities, and ABTS activities of seed extracts of 100 safflowers are summarized in Table 2. TPC of seeds varied across the studied genotypes. TPC ranged from 14.2 ± 0.32 μg GAE mg–1 dw (K185868) to 81.6 ± 1.56 μg GAE mg–1 dw (K185841). Similarly, the antioxidant activities of DPPH varied from 1.6 ± 0.07 μg ASC mg–1 dw (K184608) to 14.1 ± 0.23 μg ASC mg–1 dw (K185841). Safflower seed extracts also showed wide variations in ABTS antioxidant activities, ranging from 34.5 ± 0.70 μg Trolox mg–1 dw (K184576) to 156.5 ± 3.06 μg Trolox mg–1 dw (K185841).

    T-test (p < 0.05) indicated that total polyphenol content, DPPH and ABTS antioxidant activities in accessions collected from the northwest area were 31.0 ± 12.61 μg GAE mg–1 dw, 5.0 ± 2.37 μg ASC mg–1 dw, and 73.4 ± 23.86 μg Trolox mg–1 dw, respectively. They were not significantly different from those (TPC, DPPH, and ABTS were 34.2 ± 12.74 μg GAE mg–1 dw, 5.8 ± 2.36 μg ASC mg–1 dw, and 77.9 ± 25.47 μg Trolox mg–1 dw, respectivley) collected from the northeast area of the Saudi Arabian Peninsula (Table 3). However, TPC, DPPH, and ABTS of top five accessions (K185841, K185879, K185876, K185863, and K185836) of the northeast collections were significantly higher than antioxidant activities of top five accessions collected from the northwest collections area of the Saudi Arabian Peninsula.

    The TPC in two accessions (K185841, K185879) from the northeast area of the Saudi Arabian Peninsula were more than 70.0 μg GAE mg–1 dw. Howerver, six accessions from the northwest and five accessions from the northeast had less than 20.0 μg GAE mg–1 dw of the TPC The TPC were less than 20.0 μg GAE mg–1 dw in 17.1% of the northwest accessions. In 57.1% of accessions from the northwest, the TPC were from 20.1 to 40.0 μg GAE mg–1 dw. In 83.0% of the northeast collections, the TPC were from 20.1 to 50.0 μg GAE mg–1 dw (Fig. 1).

    The DPPH activities and ABTS showed skew-normal distributions (Fig. 2 and Fig. 3, respectively). DPPH value was higher (14.1 μg ASC mg–1 dw) in K185841 collected from the northeast. However, it was lower than 2.0 μg ASC mg–1 dw in K184608 collected from the same area. About 88.5% of accessions collected from the northwest were clustered at 2.1 ~ 8.0 μg ASC mg–1 dw. A total of 87.8% of accessions collected from the northeast were distributed in the same range (Fig. 2). ABTS activities in accessions K185841, K185879, and K185863 collected from the northeast were more than 140 μg trolox mg–1 dw except two accessions (K185868, and K184608) collected from the northeast and K184576 collected from the northwest that had less than 40.0 μg trolox mg-1 dw. ABTS activities were from 40.1 to 100.0 μg trolox mg-1 dw in 82.9% of northwest accessions and 80.0% of northeast accessions. ABTS values showed positive significant (r = 0.954**) correlation with DPPH activity of the 100 accessions of safflower seeds used in this study (Fig. 4). In addition, agro-morphological traits of 100 safflower accessions collected from two areas are presented as appendix 1.

    Clustiering of 100 safflower accessions, using the R statistical software environment, was performed based on the matrix of calculated distances (Fig. 5). The tree showed three major clades. Group I were composed 8 accessions. In group I, average value of TPC, DPPH, and ABTS activities were 64.0 ± 9.7 μg GAE mg–1 dw, 11.1 ± 1.4 μg ASC mg–1 dw, and 134.3 ± 14.4 μg Trolox mg–1 dw, respectively. Group II composed 38 accessions were 38.3 ± 6.6 μg GAE mg–1 dw, 6.7 ± 1.1 μg ASC mg–1 dw, and 88.0 ± 11.8 μg Trolox mg–1 dw, respectively. And group? germplasm included 54 accessions were 24.9 ± 5.3 μg GAE mg–1 dw, 3.8 ± 0.9 μg ASC mg-1 dw, and 59.4 ± 11.7 μg Trolox mg–1 dw, respectively (Table 4). In this study, TPC and antioxidant activities in group1 germplasm were found to be significantly higher (P < 0.05) than those from group2 and group3 germplasm. Antioxidant activities of K185841, K185879, and K185863 were higher than other accessions in the group1. They collected from northeast area of the Saudi Arabian Peninsula.

    Discussion

    Natural antioxidant such as, L-ascorbic acid, α-tocopherol, β-carotene, and polyphenolics play important physiological roles in the inhibition of various oxidative stressmediated degenerative diseases in living systems (Fred, 1994; Larson, 1987). Phenolic compounds are important due to their functions in scavenging free radicals in human body (Islam et al., 2003). To find out useful genetic resource in collected Perilla frutescens germplasms, phenolic compounds and agronomic traits have been evaluated previously (Seong et al., 2015).

    The objective of this study was to select potential genetic resources of safflower germplasm for breeding purpose. We analyzed the TPC, DPPH and ABTS in the seed extracts of 100 accessions collected from the northeast and the northwest regions of Saudi Arabian Pennisula. TPC in the samples collected from the northwest varied from those collected from the northeast. In this study, seed extracts of northeast collections had higher level of average TPC than those collected from the northwest. However, the difference was not, significant (Table 3). Genotypes and temperatures might have contributed such difference in TPC. Genetic pedigree of safflower needs to be analyzed to elucidate such difference in phenolic compound contents. Difference in TPC in soybean genotypes collected from two locations has been reported by Mujic et al. (2011). Jang et al. (2008) have reported that the antioxidant capacities are increased with increasing levels of phenolic contents are increased in Agrimonia pilosa. Our study also showed that there was a positive correlation between TPC of seed extracts and their DPPH activities (r = 0.941**) or ABTS activities (r = 0.898**). We observed that genotypes with strong TPC also had stronger DPPH antioxidant activity, and vice-versa. This is in consistent with the findings of Malencic et al. (2007). DPPH antioxidant activity had meagre differences between the two geographical areas. However, it had greater variations among genotypes. The variations in DPPH antioxidant activities ranged from 1.6 μg ASC mg–1 dw to 14.1 μg ASC mg–1 dw in genotypes. Likewise, variations in ABTS were observed in genotypes collected from the two locations (Table 2). Our results revealed significant positive correlation between DPPH and ABTS antioxidant activities (r = 0.954**). Pasko et al. (2009) have reported a strong positive correlation between ABTS and DPPH antioxidant activity in amaranth and quinoa seeds. In the study of Piao et al. (2013), they have also reported the similar fineddings.

    In clustiering of 100 safflower accessions, the tree showed three major clades. TPC and antioxidant activities in group I germplasm were significantly higher than other groups. From the results, K185841, K185879, and K185863 were recommended as potential sources of safflower breeding or natrual antioxidants due to their highest antioxidant activities among accessions. Hence, this study provides valuable information for safflower breeders and growers to develop new varieties and produce functional foods.

    적 요

    사우디아라비아 북부지역에서 수집된 홍화 총 100자원에 대 한 총폴리페놀 함량 및 항산화 활성을 평가하여 항산화활성이 높은 홍화자원을 선발하고자 연구를 수행하였으며 그 결과는 다음과 같다.

    1. 총 100자원의 홍화유전자원에 대한 총폴리페놀 함량은 14.2 ± 0.41 μg GAE mg–1dw에서 81.6 ± 1.56μg GAEmg–1dw 까지 분포하였다. DPPH 라디컬 소거능은 1.6 ± 0.07 μg ASC mg–1 dw에서 14.1 ± 0.23 μg ASCmg–1 dw까지 활성을 나 타냈다. ABTS 활성은 34.5 ± 0.70 μg Trolox mg–1 dw에서 156.5 ± 3.06 μg Trolox mg–1 dw까지 평가되었다. 항산화활성 을 측정한 DPPH와 ABTS 활성간에는 상당한 유의성(r = 0.954**)을 보였다.

    2. 홍화자원 100자원의 총폴리페놀 함량 및 항산화활성 결 과값에 대한 상호관계를 분석하여 활성이 높은 그룹(Group1) 을 얻었다. Group1에 포함된 8개의 홍화자원은 나머지 그룹에 속한 자원들에 비해 항산화활성이 높았으며, 이중 K185841, K185879, K185863은 Group1내의 다른 자원들보다도 항산화 활성이 상당히 높아 홍화육종이나 기능성물질연구에 좋은 소 재가 될 것으로 사료된다.

    Figure

    KSIA-27-381_F1.gif

    Distribution of polyphenol contents in seed extracts of the 100 safflower accessions.

    KSIA-27-381_F2.gif

    Distribution of DPPH antioxidant activities in seed extracts of 100 safflower accessions.

    KSIA-27-381_F3.gif

    Distribution of ABTS antioxidant activities in seed extracts of 100 safflower accessions.

    KSIA-27-381_F4.gif

    Relationship between DPPH and ABTS antioxidant activities of seed extracts of the 100 safflower accessions.

    KSIA-27-381_F5.gif

    Hierarchical clustering analysis of total polyphenol contents and antioxidant activities in the 100 safflower accessions.

    Table

    Accession numbers and collection sites of 100 safflower seeds used in this study.

    *C.S. : Collection Site, C I: Northwest site of the Saudi Arabian Peninsula, C II: Northeast site of the Saudi Arabian Peninsula.

    Total polyphenol contents, DPPH, and ABTS antioxidant activities of seed extracts from 100 safflower accessions.

    *Mean ± SD (standard deviation),
    a)μg GAE mg–1;
    b)μg ASC mg–1;
    c)μg Trolox mg–1
    LSD p<0.05 :
    x)1.1;
    y)0.2;
    z)2.5

    Comparison of total polyphenol contents and antioxidant activities in seed extracts of 100 safflower accessions collected at two areas.

    *Values are expressed Means ± SD
    nsnot significant

    Average values of each groups based on antioxidant activities.

    Means within column with the same letters were not significantly different at p < 0.05.

    Agro-morphological traits of 100 safflower germplasms from two collection areas.

    *The spines of bract were recorded in 0 to 2 score, 0 = absence of spine or spineless, 1 = short spine (< 2 mm), and 2 = long spine (> 2 mm).
    ※The seeds were sown in April 1, 2014 and transplated in April 25, 2014.

    Reference

    1. Arnao MB (2000) Some methodological problems in the determination of antioxidant activity using chromogen radicals: A practical case , Trends in Food Science & Technology, Vol.11 ; pp.419-421
    2. Cho MH , Hahn TR (2000) Purification and characterization of precarthamin decarboxylase from the yellow petals of Carthamus tinctorius L , Archives of Biochemistry and Biophysics, Vol.382 ; pp.238-244
    3. Briviba K , Sies H , Fred B (1994) Nonenzymatic antioxidant defense systems Natural antioxidants in human health and disease , Academic Press, ; pp.107-120
    4. Golkar P , Arzani A , Rezaei AM , Yarali Z , Yousefi M (2009) Genetic variation of leaf antioxidant and chlorophyll content in safflower , African Journal of Agricultural Research, Vol.4 ; pp.1475-1482
    5. Islam MS , Yoshimoto M , Ishiguro K , Okuno S , Yamakawa O (2003) Effect of artificial shading and temperature on radical scavenging activity and polyphenolic composition in sweetpotato (Ipomoea batatas L) leaves , Journal of the AmericanSociety for Horticultural Science, Vol.128 ; pp.182-187
    6. Jang SH , Yu EA , Han KS , Shin SC , Kim HK , Lee SG (2008) Changes in total polyphenol contents and DPPH radical scavenging activity of Agrimonia pilosa accoring to harvest time and various part , Korean Journal of Medicinal Crop Science, Vol.16 ; pp.397-401
    7. Kim EO , Lee JH , Lee SK , Lee JY , Choi SW (2007) Antioxidant properties and quantification of phenolic compounds from Safflower (Carthamus tinctorius L) seeds , Food Science and Biotechnology, Vol.16 ; pp.71-77
    8. Kim HJ , Jun BS , Kim SK , Cha JY , Cho YS (2000) Polyphenolic compound content and antioxidative activities by extracts from seed, sprout and flower of safflower (Carthamus tinctorius L) , Journal of Food Science and Nutrition, Vol.29 ; pp.1127-1132
    9. Kim JH , Kim JK , Kang WW , Ha YS , Choi SW , Moon KD (2003) Chemical compositions and DPPH radical scavenger activity in different sections of safflower , Journal of Food Science and Nutrition, Vol.32 ; pp.733-738
    10. Kim YO , Lee SW , Yang SO , Na SW , Kim SK , Chung JH (2014) Neuroprotective effects of the extracts from the aerial parts of Carthamus tinctorius L. on transient cerebral global ischemia in rats , Korean Journal of Medicinal Crop Science, Vol.22 ; pp.46-52
    11. Larson RA (1987) The antioxidants of higher plants , Phytochemistry, Vol.27 ; pp.969-978
    12. Lee CB (1980) Picture book of korean plants , Baekyang Publishers, ; pp.779
    13. Malencic D , Popovic M , Miladinovic J (2007) Phenolic content and antioxidant properties of soyabean (Glycine max L. Merr.) seeds , Molecules, Vol.21 ; pp.576-81
    14. Mujic I , Sertovic E , Jokic S , Saric Z , Alibabic V , Vidovic S , Zivkovic J (2011) Isoflavone content and antioxidant properties of soybean seeds , Croatian Journal of Food Science and Technology, Vol.3 ; pp.16-20
    15. Nabavi SM , Ebrahimzadeh MA , Nabavi SF , Fazelian M , Eslami B (2009) In vitro antioxidant and free radical scavenging activity of Diospyros lotus and Pyrus boissieriana growing in Iran , Pharmacognosy Magazine, Vol.5 ; pp.122-126
    16. Park GS , Park EJ (2003) Comparison of the chemical compositions of Korean and Chinese safflower flower (Carthamus tinctorius L) , Korean Journal of Food and Cookery Science, Vol.19 ; pp.603-608
    17. Pasko P , Barton H , Zagrodzki Gorinstein S , Folta M Zachwieja (2009) Anthocyanins, total polyphenols and antioxidant activity in amaranth and quinoa seeds and sprouts during their growth , Food Chemistry, Vol.115 ; pp.994-998
    18. Piao XM , Jang EK , Chung JW , Lee GA , Lee HS , Sung JS , Jeon YA , Lee JR , Kim YG , Lee SY (2013) Variation in antioxidant activity and polyphenol content in tomato stems and leaves , Plant Breeding and Biotechnology, Vol.1 ; pp.366-373
    19. Re R , Pellegrini N , Proteggente A , Pannala A , Yang M , Rice-Evans C (1999) Antioxidant activity applying animproved ABTS radical cation decolorization assay , Free Radical Biology and Medicine, Vol.26 ; pp.1231-1237
    20. Ryu SW , Jin CW , Lee HS , Lee JY , Sapkota K , Lee BG , Yu CY , Lee MK , Kim MJ , Cho DH (2006) Changes in total polyphenol, total flavonoid contents and antioxidant activities of Hibiscus cannabinus L , Korean Journal of Medicinal Crop Science, Vol.14 ; pp.307-310
    21. Seong ES , Seo EW , Chung IM , Kim MJ , Kim HY , Yoo JH , Choi JH , Kim NJ , Yu CY (2015) Growth characteristics and phenol compounds analysis of collected Perilla frutescens resources from China and Japan , Korean Journal of Medicinal Crop Science, Vol.23 ; pp.132-137
    22. Wrolstad RE , Waterhouse AL (2002) Determination of total phenolics Current protocols in food analytical chemistry , John Wiley & Sons Inc, ; pp.1-4
    23. Yu SY , Lee YJ , Kim JD , Kang SN , Lee SK , Jang JY , Lee HK , Lim JH , Lee OH (2013) Phenolic composition, antioxidant activity and anti-adipogenic effect of hot water extract from safflower (Carthamus tinctorius L) seed , Nutrients, Vol.5 ; pp.4894-4907