Bulletin UASVM Agricultue, 66 (2)/2009 Print ISSN 1843-5246; Electronic ISSN 1843-5386
HPLC- PDA and UV-VIS Spectrometry Analysis used to Fingerprint Sea Buckthorn (Hippophae rhamnoides L.) Berries Comparatively with Leaves and Seeds Extracts Raluca Maria PARLOG 1), Dan Cristian VODNAR1), Francisc Vasile DULF1), Loredana LEOPOLD1), Carmen SOCACIU1) 1)
University of Agricultural Sciences and Veterinary Medicine, Faculty of Agriculture, Department of Chemistry and Biochemistry, 3-5 Mănăştur Street, 400372,Cluj-Napoca, România,
[email protected]
Abstract: The carotenoids and chlorophylls from sea buckthorn (Hippophae rhamnoides L., ssp. Carpatica) berries, leaves and seeds were identified by HPLC –PDA and quantified by UV-VIS spectrometry. The HPLC chromatograms showed that among carotenoids, lutein (tR= 11.093 min) was the major compound followed by β carotene (tR= 25.04 min), zeaxanthin (tR= 11.51 min), small quantities of neoxanthin (tR= 6.93 min), violaxanthin (tR= 4.66 min) and traces of β cryptoxanthin (tR= 18.14 min).The identified chlorophylls were: chlorophyll a (tR= 17.55 min), chlorophyll b (tR =14.97 min) and pheophytin a (tR= 23.82 min). In accordance with the HPLC fingerprint, the chlorophyll content was: 0% in berries, 41.16 % in leaves and 16.36% in seeds. The total carotenoid concentrations were 293.59, 264.80 and 3.63 mg /kg fresh berries, leaves and seeds, respectively. Keywords: sea buckthorn, carotenoids, chlorophylls, HPLC-PDA, UV-VIS spectrometry
INTRODUCTION Sea Buckthorn is a deciduous shrub with yellow or orange berries and has been used for centuries, for food in both Europe and Asia. The leaves, berries and seeds of sea buckthorn have high nutritional and medicinal values (Sighn, 2006, Sighn, 2003). Nowadays, seabuckthorn is known to contain more than 190 kinds of bioactive substances which are much more abundant than in any other fruit or vegetable: vitamins (A, K, E, C, B1, B2, Folic acid), carotenoids, pigments, many lipids and fatty acids (including Omega 3, 6, 7, and 9), organic acids, amino acids, carbohydrates, tocopherols, flavonoids, phenols, terpenes, tannins and minerals (Sighn, 2006, Sighn, 2003, Zeb, 2004, Tiitinen et al., 2005, Shah et al., 2007, Socaciu, 2003). Among these bioactive compounds, carotenoids and chlorophylls are the essential pigments with acessory functions in the photosynthetic process and with considerable medicinal value (Beveridge et al., 1999). Carotenoids are important antioxidants and the major source of vitamin A. Carotenoids improve the physical and physiological capacity of the humans, acting as potential anti-cancerogenic, anti-mutagenic and anti-inflamatory agents (Sighn, 2006, Zeb, 2004, Xinga et al., 2002). These components, however, vary considerably among individual plants and regional origin of the species. Previous investigations have shown individual differences in chemical composition and content, which may be due to their genetic variability, climate and growing conditions, degree of ripening when harvested, storage conditions, parts of the plant and of the berry which is analyzed as well as the analytical methods used (Andresson et al., 2009, Dalija et al., 2008, Deepu et al., 2007). 409
The aim of our study was to determine the presence and total quantity of carotenoids and chlorophylls in the pulp of sea buckthorn berries, seeds and leaves at the end of ripening period. A further aim was to analyze their HPLC fingerprint in order to get informations regarding their specific metabolomic profile. MATERIALS AND METHOD Sample Collection All plant materials (seeds, fruits and leaves from Hippophae rhamnoides L., ssp. Carpatica) were collected from an experimental field at the Fruit Research Station from Bacau, Romania in the 1st week of October 2008, when they were handpicked fully matured. All the berries and leaves were kept in plastic pots and transported under refrigeration conditions to University of Agricultural Science and Veterinary Medicine, Cluj-Napoca, Romania. After 8 hours of refrigeration the samples were frozen and stored at -20 ºC until they were analyzed. Sample Preparation Freshly thawed berries were manually separated into two fractions, the seeds (SBS) and the fruit pulp (SBB), which were freezed immediately after separation. The leaves (SBL) were also freezed. Right before the extraction, the frozen samples were homogenized using an Ultraturax. Carotenoids and chlorophylls extraction Total carotenoids and chlorophylls were extracted from frozen SBS, SBB and SBL (10 g) using methanol:ethyl acetate:petroleum ether (1:1:1, v/v/v). The extraction was made under continuous stirring for 4 h in darkness conditions. The extract was washed successively with water, diethyl ether and NaCl saturated solution. The ether phase was evaporated using a rotavapor at 35ºC. The obtained oleoresin was dissolved in a known volume of ethyl acetate. Instrumentation and measurement protocols The qualitative analysis of extracts was done by HPLC coupled with PDA, while quantitative analysis was done using UV- VIS spectrometry. For HPLC analysis we used a HPLC-PDA Shimadzu chromatograph on a LiChrosorb RP 18 column (5 m). The mobile phase consisted in: (solvent A) acetonitrile: water 9: 1, v/v with 0.25% triethylamine and (solvent B) ethyl acetate with 0.25% triethylamine according to Pintea et al. (2005) with modifications. The binary gradient was as presented in Tab. 1. The flow rate was 1 mL/min, and the effluent was monitored at 450 nm for carotenoids and 660 nm for chlorophylls. All solvents were HPLC-grade and chemicals were analytical grade. Tab. 1 The HPLC gradient program used for both (A and B) solvents Time / min 0-16 17-54
%A 85 38
410
%B 15 62
55-56 57-60 60-60.01 60.01
38 85 85
62 15 15 STOP
The peak identification was made on the basis of their HPLC retention times Rt (min), compared with those of authentic standards and by their specific spectra recorded by the PDA as shown in Tab. 2. Tab. 2 Specific spectral maximum absorption and retention time for carotenoid and chlorophylls standards Signal
Standards
6.93 7.27 11.093 11.51
415 ,439, 468 415, 436, 436 444, 472 450, 477
18.14
451, 477
Neoxanthin Violaxanthin Lutein Zeaxanthin β criptoxanthin β carotene Chlorophyll b Chlorophyll a Pheophytin a
25.034
452, 478
14.97
458, 646
17.55
431, 665
23.82
409, 665
CHLORO PHYLL
Absorbtion Maxima (nm)
CAROTENOID
Rt (min)
Total carotenoid contents were estimated spectrophotometrically using a Jasco V 530 spectrophotometer. The results were calculated using the absorption values at 450 nm according to the formula: X= (A x Y x 1000) x dilution / (2500 x 100), X= weight of carotenoids in the sample (mg), A= absorbance (λ max= 450 nm) Y= volume of the sample (ml) 2500= A1%1cm (specific absorbance of coloured carotenoids) All data were processed with specific softwares (Shimadzu LC Solution and Spectra Manager for Windows 95/NT). RESULTS AND DISCUSSION 1. HPLC fingerprint and qualitative analysis The free carotenoids and chlorophylls identified in SBB, SBL and SBS extracts were expressed as area percent resulted from the HPLC – PDA chromatograms (Fig.1. A, B and C) and presented in Tab. 3.
411
300000
1600000
A 1400000
6
B
SBL carotenoids at 450 nm SBL chlorophyls at 660 nm
SBB carotenoids at 450 nm SBB chlorophyls at 660 nm
250000
8 1200000
200000
Carotenoid esters ZE
6 A.U.
3 800000
150000
9 600000
LE
100000
4
2
400000
50000
5
1 200000
4
CE
7 0
0 0
10
20
10
30
20
30
40
50
Retention time (min)
Retention time (min)
20000
6
3
C
SBS carotenoids at 450 nm SBS chlorophyls at 660 nm
8
15000
A.U.
A.U.
1000000
9 10000
4 5000
2 1
5
7
0 0
10
20
30
40
50
Retention time (min)
Fig. 1. HPLC - PDA carotenoids and chlorophylls fingerptint, recorded at 450 and 660 nm, respectivetly for: (A) SBL, (B) SBB and (C) SBS Tab. 3 The percentage of carotenoids and chlorophylls identified in SBL, SBB and SBS as calculated using the software
Signal 1 2 3 4 5 6 7 8 9
Area (%)
Identification
Neoxanthin Violaxanthin Lutein Zeaxanthin β criptoxanthin β carotene Chlorophyll b Chlorophyll a Pheophytin a
412
SBL 4.66 8.30 27.13 -
SBB 2.28
SBS 1.62 1.99 16.80 3.28
-
-
traces
14.14
13.71
15.49
19.15
-
8.43
4.42
-
2.13
1.27
-
1.007
As it is shown in Fig. 1 (A) and table 3 the carotenoids in SBL are mainly represented by free lutein (3) followed by β carotene (6), violaxanthin (2) and neoxanthin (1). Regarding the chlorophylls, the highest percent was found for chlorophyll b (7) followed by chlorophyll a (8) and pheophytin a (9). According to Andersson et al., (2009) pheophytin a is an inversely related marker of the degree of SB berries ripening confirming that SBB were piked when they were fully maturated. In Fig.1 (B) the carotenoids found in SBB were: β carotene (6) followed by zeaxanthin (4). According to literature studies the small quantities of lutein and zeaxanthin might be due to their esterification during the ripening process (Pintea et al., 2005, Sighn, 2006, Olteanu et al., 2008). The criptoxanthin (CE), lutein (LE) and zeaxanthin (ZE) esters were detected in SBB extracts as it is shown in figure 1 (B). (Pintea et al., 2005, Socaciu, 2003). The chlorophylls were not present, the berries being fully maturated. Fig. 1 (C) represents the HPLC of carotenoids and chlorophylls fingerprint for the SBS extract. β carotene (6) was found in the highest proportion followed by lutein (3), zeaxanthin (4), violaxanthin (2), neoxanthin (1) and traces of β criptoxanthin (5). Small percentages of esters (CE, LE and ZE) were also identified. The chlorophylls were: chlorophyll b (7), chlorophyll a (8) and pheophytin a (9). 2. Quantitative analysis The total carotenoid content of unsaponified extract of SBB, SBS and SBL were estimated spectrophotometrically. The results were in agreement with other studies. (Zeb, 2004, Cenkowski et al., 2006, Andersson et al., 2009, Sighn, 2006). The total carotenoids concentration was 4.34, 293.59 and 450.04 mg /kg fresh sample for SBS, SBB and SBL respectively. In our study, highest concentrations of carotenoids were estimated in SBL. 3. Comparative analysis The correlation between the quantitative content of the samples with the qualitative analysis obtained from the HPLC chromatograms showed that the quantitative results were overestimated. This follows from the overlapping of carotenoids and chlorophylls (in SBS, SBB and SBL) at 450nm and 660 nm which indicated the presence of chlorophylls in the carotenoid extract. So in accordance with the HPLC fingerprint, the total chlorophylls percentage found in carotenoid extract was 16.36% in SBS, 0% in SBB and 41.16 % in SBL. Furthermore, we corrected these values by subtracting the chlorophylls concentration obtained from the HPLC chromatograms recorded at 450 nm (characteristic for carotenoids). The final corrected total carotenoid values became 3.63 and 264.80 mg /kg fresh SBS and SBL, respectively. During the early stages of maturation the carotenoids may be masked by the chlorophylls which are decreasing during ripening. CONCLUSIONS Using HPLC to fingerprint carotenoids and chlorophylls from SB leaves, SB berries and SB seeds we noticed that the highest quantity of free carotenoids and chlorophylls was found in SB leaves extract. The mature SB berries extract had the highest quantity of carotenoid esters (criptoxanthin, lutein and zeaxanthin esters) and undetectable chlorophylls.
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The SB seeds extract contained all free forms of investigated carotenoids. Among them β carotene was detected in the highest percentages. The total carotenoids content was determined and corrected by substracting the chlorophylls concentraction. Further investigations will be focused on other sea buckthorn Romanian species, in order to obtain their metabolomic profile. REFERENCES 1. Andersson, S. C., M. E. Olsson, E. Johansson, K. Rumpunen, (2009). Carotenoids in Sea Buckthorn (Hippophae rhamnoides L.) Berries during Ripening and Use of Pheophytin a as a Maturity Marker. J. Agric. Food Chem. 57: 250–258. 2. Beveridge, T., T. S. C. Li, B. D. Oomah, A. Smith (1999). Sea Buckthorn Products: Manufact,ure and Composition. J. Agric. Food Chem. 47: 3480-3488. 3. Cenkowski, S., R. Yakimishen, R. Przybylski, W.E. Muir (2006). Quality of extracted sea buckthorn seed and pulp oil. Canadian Biosystems Engineering. 48. 4. Dalija, S., D. Karklina, I. Gailite, S. Ruisa, I. Krasnova, G. Heidemane (2008). Changes of biochemical compounds in seabuckthorn marc during storage. Foodbalt. 109-113. 5. Deepu, M., T. Parimelazhagan, S. Gomez, Z. Ahmed (2007). Characterization of Seabuckthorn (Hippophae spp.) genetic resources in India using morphological descriptors. PGR Newsletter FAOBioversity.149: 22 – 26. 6. Olteanu, Z., M. M. Zamfirache, L. Oprică, E. Truţă (2008). Comparative study of behaviour of some biochemical parameters in different phenophases of seabuckthorn cultivars. Analele Ştiinţifice ale Universităţii „Alexandru Ioan Cuza”. TOM IX : 47-54. 7. Pintea, A., A. Varga, P. Stepnowski, C. Socaciu, M. Culea, H. A. Diehl (2005). Chromatographic analysis (HPLC, GC) of Carotenol Fatty Acids Esters in Physalis alkekengi and Hippophae rhamnoides. Phytochemical Analysis.16:188-195. 8. Shah, A. H., D. Ahmed, M. Sabir, S. Arif, I. Khaliq, F.Batool (2007). Biochemical and Nutritional Evaluations of sea buckthorn (Hyppophae Rhamnoides L. spp. Turkestanica) from different locations of Pakistan. Pak. J. Bot. 39(6): 2059-2065. 9. Singh, V., (2003). Seabuckthorn (Hippophae L.): A Multipurpose Wonder Plant: Vol. 1: Botany, Harvesting and Processing Technologies. Indus New Delhi. 10. Singh, V., (2006). Seabuckthorn (Hippophae L.): A Multipurpose Wonder Plant: Vol. II: Biochemistry and Pharmacology. Daya Pub. New Delhi. 11. Socaciu, C., (2003). Bioactive compounds in seabuckthorn: cellular and molecular investigations and their applications. Proc. Int. Symposium Bull. USAMV. 59: 12-20. 12. Tiitinen, K. M., M. Hakala, H. P. Kallio (2005). Quality Components of Sea Buckthorn (Hippophae rhamnoides) Varieties. J. Agric. Food Chem. 53:1692-1699. 13. Xinga, J., B. Yang, Y. Donga, B. Wanga, J. Wanga, H. P. Kallio (2002). Effects of sea buckthorn (Hippophae¨ rhamnoidesL.) seed and pulp oils on experimental models of gastric ulcer in rats. Fitoterapia. 73: 644–650. 14. Zeb, A., (2004). Chemical and Nutritional Constituents of Sea Buckthorn Juice. Pakistan Journal of Nutrition 3(2): 99-106. 15. Zeb, A., (2004). Important Therapeutic Uses of Sea Buckthorn (Hippophae): A Review. Journal of Biological Sciences 4(5): 687-693.
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