Preservation of polyphenolic antioxidants from Goji berries (Lycium barbarum L.) affected by different drying techniques

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1 Proceedings of the 214 International Conference on Food Properties (ICFP214) Kuala Lumpur, Malaysia, January 24 26, 214 Preservation of polyphenolic antioxidants from Goji berries (Lycium barbarum L.) affected by different drying techniques Anaïs Chassaing, Draženka Komes, Arijana Bušić, Ana Belščak-Cvitanović, Aleksandra Vojvodić Department of Food Engineering, Faculty of Food Technology and Biotechnology, Pierottijeva 6, 1 Zagreb, Croatia Abstract Health benefits of Goji berries (Lycium barbarum L.) consumption, recognized for centuries in traditional Chinese medicine, are increasingly becoming popular among the modern consumers, with bioactive polysaccharides and pigments being the most studied compounds so far. Due to the lack of data on other bioactive constituents, polyphenolic antioxidants in particular, dried nature of Goji fruit as the most popular marketing form as well as thermolability of polyphenolics, the effect of different drying techniques on the latter was studied. Upon selecting the most suitable extraction procedure (8% acetone assisted conventional extraction), HPLC analysis have shown vanillic acid derivatives as the most abundant components of both fresh and dried Goji berry extracts. Freeze drying enabled the best preservation of these compounds, while oven drying at 6 C was shown to be the most detrimental. Keywords Antioxidant capacity, Drying technique, Extraction, Goji berries, Polyphenols 1. Introduction Over the last years, Goji berries (Lycium barbarum L.) have become extremely popular due to their nutritionally rich composition including the presence of various vitamins, minerals, antioxidants and amino acids [1], but also due to increasing consumers demand for natural products with potential health benefits. Owing to the diverse bioactive profile, especially the presence of carotenoids, polysaccharides and polyphenolic compounds, Goji berries exert numerous biological and health-related activities [2-5], which have been appreciated for centuries in traditional Chinese herbal medicine. Newer scientific studies linked the consumption of Goji berries to beneficial medicinal properties in age-related diseases [6], cancer prevention, immunity enhancement [7] and neuroprotective properties [8]. Polysaccharides from L. barbarum also induce maturation of dendritic cells with strong immunogenicity, enhancing Th1 and Th2 response [9], and their anticancer and immunomodulatory effects have recently been reviewed [1]. Although they are most often consumed as fresh or dried, Goji berries are also added to processed products such as yogurts, jams, beverages, canned fruits, and jellies [11], while concentrated extracts and infusions prepared from the berries have a history of use as ingredients in various soft or alcoholic drinks that were marketed for their medicinal benefits. Among the chemical constituents of Goji berries, water-soluble glycoconjugates, (L. barbarum polysaccharides or LBP) are the most extensively studied components, estimated to comprise 5-8% of dried fruits [12]. The reddish-orange color of L. barbarum fruits is derived from a group of carotenoids, which make up only.3.5% of the dried fruit [13]. A total of 11 free carotenoids and 7 carotenoid esters were detected from unsaponified and saponified L. barbarum extracts, with zeaxanthin being the predominant carotenoid and comprising about one-third to one-half of the total carotenoids. According to the available data, so far the bioactive properties of Goji berries have been mainly evaluated on the basis of polysaccharides and carotenoids, while the polyphenolic content and composition are known in a much poorer extent. Moreover, since Goji berries are still most often marketed and consumed as dried fruits, the effect of drying techniques on the content of beneficial compounds must be clarified. Therefore, the objective of this study was to evaluate the influence of different drying techniques (oven drying, air-drying at room temperature, freeze drying and infrared drying) on bioactive profile of Goji berries in order to define which drying technique has the highest preservation output on the polyphenolic compounds of this fruit.

2 2. Materials and methods 2.1. Chemicals Folin-Ciocalteau, formic acid and acetone were supplied from Kemika (Croatia). DPPH (2,2-diphenyl-1- picrylhydrazyl) and Trolox (6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid) were supplied from Fluka (Switzerland). ABTS (2,2-azino-bis(3-ethyl-benzthiazoline-6-sulphonic acid)diammonium salt), chlorogenic acid, caffeic acid, p-coumaric acid and gallic acid were obtained from Sigma (Sigma Aldrich Chemie, Germany). Methanol and acetonitrile were supplied from Panreac (Spain). Hydrochloric acid (37%) and ethanol (96%) were supplied from Carlo Erba Reagenti (Italy). Quercetin and vanillic acid were obrained from Acros Organics (USA). Formaldehyde sodium hydroxide (NaOH) and sodium carbohydrate (Na 2 CO 3 ) were supplied from T.T.T (Croatia) Sample preparation Goji berries (Lycium barbarum L.) analysed in this study were grown in the northwestern Croatia, and were harvested in 212. Several extraction techniques and solvents were employed in order to find the extraction procedure that provides the best extraction efficiency of polyphenolic compounds from fresh Goji berries. Therefore, ultrasound extraction using 5% and 7% ethanol according to a modified method of Belščak et al. [15], conventional extraction by stirring using 8% acetone according to a method of Makris and Kefalas [16], as well as acid and alkali hydrolysis using modified methods of Perva-Uzunalić et al. [17] and Pellegrini et al. [18] were performed. Dried Goji berries were prepared using several drying techniques (freeze drying (FD), air drying (AirD), oven drying (OD4 C, OD6 C), infrared drying- without (IR) and with ultrasound pretreatment of 1 minutes (IR+ut)), and the obtained samples were submitted to the optimized extraction technique Determination of dry matter Dry matter (DM) content was determined gravimetrically as the residue remaining after drying until constant mass, according to the official AOAC method [14]. Dry matter content was expressed as a percentage of the initial sample weight and used in calculations of other analyzed parameters Determination of total polyphenol (TPC) and total flavonoid (TFC) content Total polyphenols content (TPC) was measured spectrofotometrically using Folin-Ciocalteu s reagent according to a modified method of Lachman et al. [19]. Total flavonoids content was determined using the formaldehyde precipitation. Precipitated flavonoids were separated from the solution by filtration and total nonflavonoids (TNC) remained in the filtrate, which was subsequently analyzed according to the above mentioned procedure. The results were calculated as the difference between TPC and TNC. All measurements were performed in triplicate and expressed on dry matter (DM) basis as mg/g DM of gallic acid equivalents (GAE) HPLC analysis of polyphenolic compounds HPLC analysis of Goji berries extracts were carried out on Agilent 12 Series HPLC system (Agilent Technologies, Santa Clara, USA) accompanied with photodiode array detector (Agilent Technologies, Santa Clara, USA) using a reversed-phase column Zorbax Extended C-18 (25 x 4.6 mm, 5 µm i.d.) (Agilent Technologies, Santa Clara, USA). The mobile phase consisted of 2% formic acid in acetonitrile (solvent A) and 2% formic acid in deionized water (solvent B) at a flow rate of 1 ml/min. Gradient elution of injected 2µL of the sample was performed starting at 9% A and 1% B, 6% A and 4% B at 25 min, 3% A and 7% B at 4 min and becoming isocratic for 5 min. Chromatograms were recorded at 278 nm with scanning option between 2-4nm, in order to obtain peak spectra. Polyphenolic compounds were identified by comparing the retention times and spectral data with those of standards. Data acquisition was conducted using the ChemStation software (Agilent Technologies, Santa Clara, USA). All analyses were repeated three times and the results were expressed as mg /g DM. Prior to injection, all extracts were filtered through.45µm cellulose acetate (CA) filter (Macherey-Nagel, Germany) Determination of antioxidant capacity Antioxidant capacity of Goji berry extracts was determined using the DPPH radical scavenging assay described by Brand-Williams et al. [2]. The free radical scavenging capacity using the DPPH radical reaction was evaluated by measuring the absorbance at 515 nm after 3 min of reaction at room temperature. The results were expressed as µmol Trolox equivalents/g DM. All measurements were performed in triplicate. Trolox equivalent antioxidant capacity (TEAC) of samples was also estimated by the ABTS radical cation decolorization assay monitored at 734 nm after 6 min of reaction time, described by Re et al. [21]. The results, obtained from triplicate analyses, were expressed as Trolox equivalents (μmol Trolox/g DM).

3 3. Results and discussion Although it was previously stated that polysaccharides and carotenoids are the most extensively studied bioactive compounds of Goji berries, polyphenols attribute to the functional properties of this fruit by their excellent antioxidant properties. As secondary plant metabolites also known to provide colour to the plant, and known as highly reactive and labile compounds sensitive to numerous factors among which temperature is the most crucial one, different drying techniques employing high temperatures may significantly affect the content of polyphenolic compounds. For that matter finding the best drying technique that would enable the highest preservation of these compounds needs to be determined. Also, in order to provide reliable information about the exact polyphenolic content of Goji berries, several extraction procedures were employed using different extraction solvents and techniques, since it is well established that the extraction efficiency of polyphenols depends on the extraction parameters µmol Trolox/g DM ut5% EtOH ut7% EtOH c8%acon hhcl hnaoh ABTS DPPH TPC mg GAE/g DM 1 1 µmol Trolox/g DM mg GAE/g DM 2 2 FD AirD OD4 C OD6 C IR IR+ut ABTS DPPH TPC ut5%, ut7% - Goji berries extracted in ultrasound bath using 5% and 7% EtOH, c8% AcON conventionally extracted berries using 8% acetone, hhcl-acid hydrolysis using 6M HCl, hnaoh alkali hydrolysis using.25m NaOH Figure 1: Antioxidant capacity (µmol Trolox/g DM)) and total polyphenols content (TPC mg GAE/g DM) of fresh Goji berries extracted using different extraction procedures and solvents (a) and dried using different drying techniques (b) As can be seen in Figure 1a, the highest TPC was obtained in the acid hydrolizate (hhcl). Both acid and alkali hydrolyzates exhibited significantly (p<.5) higher TPC when compared to non-hydrolyzed extracts (ut5% and ut7%, c8% AcON), which might lead to conclusion that the extraction of polyphenolic compounds was enhanced by employing acid or alkali. However, this result might be attributed to the non-selectivity of Folin-Ciocalteu reagent [22]. Namely, since this reagent reacts not only with polyphenols but also with other reducing compounds such as carotenoids, amino acids, sugars, vitamin C and Cu(I) [23], interferences from carotenoids and polysaccharides (which are hydrolyzed and degraded to smaller units or reducing sugars) present in Goji berries may have contributed the overestimation of the TPC. Antioxidant capacity of differently extracted Goji berries did not exhibit the same trend as the TPC, as it might be expected, which was confirmed by a low linear correlation obtained between the results (r TPC/ABTS =.128). This indicates that compounds other than polyphenols, may have contributed to the observed antioxidant capacity, which is not surprising considering the rich bioactive profile of Goji berries and a high content of polysaccharides and carotenoids which also display antioxidant properties. The discrepancy in the

4 sample ranking based on the two applied antioxidant assays (also observable by negative correlation coefficient (r DPPH/ABTS = -.323) may be attributed to the differences in the method principle and the affinity of a compound to quenche a certain radical (ABTS or DPPH). Taking into account possible overestimation of TPC results in the case of acid and alkali hydrolyzates, the conventional extraction using 8% acetone was selected as the extraction method for the analysis of polyphenolic content in differently dried Goji berries, complemented by the simplicity of the procedure and the results similar to the ultrasonically extracted samples using hydroalcoholic solvents. According to the results presented on Figure 1b, oven dried Goji berries at 4 C exhibited the highest TPC, while air dried Goji berries were the best radical scavengers. The discrepancy between the results of these assays may again be attributed to the assay principle and the interactions of the reagents and radicals with the bioactive Goji berries constituents. Although it would be expected that freeze dried sample might produce the highest TPC and antioxidant capacity, since this technique is regarded as the best in terms of preservation of bioactive compounds, it was ranked third with 7.45 mg GAE/g DM of total polyphenols, behind the oven dried (4 C) and infrared dried (with 1 min of ultrasound pre-treatment) samples. Table 1: The content of total flavonoids (TFC) as well as individual polyphenolic in fresh Goji berries extracts prepared using different solvents and techniques TFC Individual polyphenolic compounds (mg/g DM) mg GAE/g DM ΣVAD ΣChlAD CaffA p-couma ΣQueD ut5%etoh 1.4± ± ± ±.12.16±.2 2.5±.9 ut7%etoh 9.52± ±.85.83±.1 n.d. n.d. 1.17±.3 c8%acon 9.82± ± ±.9.3±.2.8±.4 1.6±.11 hhcl 2.2±.37 n.d. 1.56±.6.3±. n.d. n.d. hnaoh 14.1± ± ±.8 n.d. n.d. n.d. VAD - vanillic acid derivatives, ChlAD - chlorogenic acid derivatives, CaffA - caffeic acid, p-couma - p-coumaric acid, QueD - quercetin derivatives n.d. - not detected Using the HPLC analysis, as presented in Table1, five polyphenolic compounds were detected, two of which were detected as individual compounds, while the remaining three represented groups of similar compounds compiled as derivatives. Among the detected compounds, phenolic acids were predominant with representatives from both hydroxycinnamic (caffeic acid and chlorogenic acid) as well as hydroxybenzoic acids (vanillic acid and corresponding derivatives and p-coumaric acid). Among other polyphenols, a number of quercetin-like compounds was detected and subsequently quantified as quercetin derivatives. Polyphenolic profiling of Goji berries extracts revealed vanillic acid derivatives (VAD) as the most represented, exhibiting the highest content of mg/g DM in extracts obtained using the ultrasound extraction technique and 7% EtOH as a solvent. Within the same extraction technique, more diluted hydroalcoholic solution however resulted in notable reduction of VAD extraction efficiency, but at the same time the content of other polyphenols increased. Other extraction methods, namely conventional techniques employing stirring at room temperature with acid or alkali hydrolysis, provided poorer phenolic profiles, detecting only two out of five compounds, while the conventional 8% acetone extraction exhibited results most similar to the ultrasound extraction employing 5% ethanol. Anyhow, the extraction with alkali hydrolysis (hnaoh), although exerting a poor phenolic profile, exhibited rather high content of chlorogenic acid (2.28 mg/g DM), in line with c8%acon sample. Concerning the total flavonoid content (TFC), it was well correlated with TPC, showing the highest content for aqueous conventional extraction accompanied by acid hydrolysis (hhcl) and followed by alkali hydrolysis (hnaoh). Table 2: The content of total flavonoids (TFC) and individual polyphenolic compounds in 8% acetone extracts of differently dried Goji berries TFC Individual polyphenolic compounds (mg/g DM) mg GAE/g DM ΣVAD ΣChlAD CaffA p-couma ΣQueD FD 6.87± ± ±.6.11±..74± ±.11 AirD 6.24± ±.73.83±.2.7±..1±. 1.65±.13 OD4 C 9.77±.36.83±.3.31±.2 n.d..5±..31±.1

5 OD6 C 6.24±.2.51±.2.17±.2 n.d..3±..8±.1 IR 6.27± ±.4.4±.1 n.d..7±..13±.1 IR+ut 7.95± ± ±.11.26±.1.6± ±.9 VAD - vanillic acid derivatives, ChlAD - chlorogenic acid derivatives, CaffA - caffeic acid, p-couma - p-coumaric acid, QueD - quercetin derivatives n.d. - not detected Using the 8% acetone assisted conventional extraction, the obtained results from HPLC analysis of individual polyphenolic compounds revealed the presence of the same polyphenolic profile (Table 2) as in the case of fresh Goji berries extracts (Table 1). Concerning the impact of drying techniques, it can be observed that drying in general resulted in decrease of both TFC and individual polyphenolic compounds, with oven drying at 6 C as the technique exhibiting the highest rate of individual polyphenolic compounds losses, over 9% (or not detected). On the contrary, freeze drying in general was shown to be the most suitable technique for maintaining high contents of polyphenolics, in line with the fresh fruit extract. The VADs were found to be the most representative compounds, exhibiting the highest contents in freeze dried, air dried (AirD) and infra-red dried (IR+ut) samples, respectively. The obtained results are in line, or slightly higher than the fresh reference sample (c8%acon) where VADs were also the predominant polyphenolics. Among other detected compounds, except for caffeic acid, the highest contents were also quantified in freeze-dried sample, not surprisingly since this non-thermal drying technique is well known for good preservation of polyphenolic antioxidants. Furthermore, AirD and IR+ut samples also provided rather high content of preserved polyphenolic compounds, with the latter drying technique being in general more benefitial for that matter. Comparing these results to the ones obtained for TPC (Figure 1b), it can be observed that FD, AirD and IR+ut samples are in rather good correspondence, with smaller discrepancies due to the robustness of Folin- Ciocalteau method. On the other hand, the high TPC results for OD4 C sample did not correspond neither to the contents of individual polyphenolics, nor the polyphenolic profile was as complete as in other samples, which can only be partially attributed to the method accuracy, but further insight is certainly needed. However, when employing higher temperatures in oven drying, the HPLC analysis clearly revealed a decrease of all detected polyphenolics (Table 2). The obtained results for TFC follow the trend of TPC values for dried samples, showing the OD4 C as the most abundant in total flavonoids, followed by IR+ut sample and subsequently the rest of dried samples, all exhibithing similar values. As in the case of TPC, further insight in the composition of the obtained extracts is needed due to the possible interferences in spectophotometrical methods. 4. Conclusion Among the investigated procedures for the extraction of polyphenolic compounds from Goji berries, 8%AcON assisted conventional extraction was selected as the most suitable. Discrepancies in the results of TPC and antioxidant capacity analysis of different extracts of fresh Goji berries implied a complex composition containing other constituents contributing to the obtained values apart from polyphenolics. HPLC analysis revealed the predominance of phenolic acids with vanillic acid derivatives as the most represented. Oven drying at 6 C was shown to have the most impact on polyphenolic compounds losses, while upon freeze drying the analyzed parameters were the best preserved. Taking into account the expensiveness of freeze drying technique, infra-red drying and oven air drying impose good alternatives according to the obtained results. Furthermore, since air drying is not easily controlled and it is rather time consuming, the infra-red drying, accompanied with ultrasonic pretreatment for 1 min can be regarded as the best alternative as it shortened the time of drying and in the same time exhibited the most similar results to the freeze drying. 5. Acknowledgements Fresh Goji berries were kindly donated by Mr. Željko Avar, commercial grower and producer of Lycium barbarum L. plants and fruits. References 1. Yao, X., Peng, Y., Xu, L. J., Li, L., Wu, Q. L., and Xiao, P. G., 211, Phytochemical and Biological Studies of Lycium Medicinal Plants, Chemistry & Biodiversity, 8(6),

6 2. Ren, B., Ma, Y., Sheng, Y., and Gai, B., 1995, Protective Action of Lycium barbarum L. (LbL) and Betaine on Lipid Peroxidation of Erythrocyte Membrane Induced by H2O2, China Journal of Chinese Materia Medica, 2(5), Kim, H. P., Lee, E. J., Kim, Y. C., Kim, J., Kim, H. K., Park, J. H., Kim, S. Y., and Kim, Y. C., 22, Zeaxanthin Dipalmitate from Lycium chinense Fruit Reduces Experimentally Induced Hepatic Fibrosis in Rats, Biological & Pharmaceutical Bulletin,, 25(3), Wang J. H., Wang H. Z., Zhang M., and Zhang S. H., 22, Anti-Aging Function of Polysaccharides from Fructus Lycii, Acta Nutrimenta Sinica, 24(2), Wang J. H., Wang H. Z., Zhang M., and Zhang S. H., 22, Effect of Lycium barbarum Polysaccharides (LBP3) on Lipid Peroxidation in Mice, Chinese Journal of Veterinary Science, 22, Chang, R. C. C., and So, K. F., 28, Use of Anti-Aging Herbal Medicine, Lycium barbarum, Against Aging-Associated Diseases. What Do We Know So Far?, Cellular and Molecular Neurobiology, 28(5), Gan, L., Zhang, S. H, Yang, X. L., and Xu, H. B., 24, Immunomodulation and Antitumor Activity by a Polysaccharide Protein Complex from Lycium barbarum, International Immunopharmacology, 4(4), Chan, H. C., Chang, R. C., Koon-Ching, Ip. A., Chiu, K., Yuen, W. H., Zee, S. Y., So, K. F., 27, Neuroprotective Effects of Lycium barbarum Lynn on Protecting Retinal Ganglion Cells in an Ocular Hypertension Model of Glaucoma, Experiemental Neurology, 23(1), Chen, Z., Tan, B. K. H., and Chan, S. H., 28, Activation of T Lymphocytes by Polysaccharide Protein Complex from Lycium barbarum L., International Immunopharmacology, 8(12), Tang, W. M., Chan, E., Kwok, C. Y., Lee, Y. K., Wu, J. H., Wan, C. W., Chan, R. Y., Yu, P. H., and Chan, S. W., 212, A Review of the Anticancer and Immunomodulatory Effects of Lycium barbarum Fruit, Inflammopharmacology, 2(6), He, N., Yang, X., Jiao, Y., Tian, L., and Zhao, Y., 212, Characterisation of Antioxidant and Antiproliferative Acidic Polysaccharides from Chinese Wolfberry Fruits, Food Chemistry, 133(3), Wang, Q., Chen, S., and Zhang, Z., 1991, Determination of Polysaccharide Contents in Fructus Lycii, Chinese Traditional and Herbal Drugs, 22(2), Peng, Y., Ma, C., Li, Y., Leung, K. S., Jiang, Z. H., and Zhao, Z., 26, Quantification of Zeaxanthin Dipalmitate and Total Carotenoids in Lycium Fruits, Plant Foods for Human Nutrition, 6(4), AOAC, Official methods of analysis, Association of Official Analytical Chemist, Arlington, VA, USA, Belščak, A., Komes, D., Horžić, D., Kovačević Ganić, K., and Karlović D., 29, Comparative Study of Commercially Available Cocoa Products in Terms of Their Bioactive Composition, Food Research International. 42(5-6), Makris, D. P., and Kefalas, P., 24, Carob Pods (Ceratonia siliqua L.) as a Source of Polyphenolic Antioxidants, Food Technology Biotechnology, 42(2), Perva-Uzuvalić, A., Škerget, M., Knez, Ž., Weinreich, B., Otto, F., and Grüner, S., 26, Extraction of Active Ingredients From Green Tea (Camellia sinensis): Extraction Efficiency of Major Catechins and Caffeine, Food Chemistry, 96(4), Pellegrini, N., Serafini, M., Salvatore, S., Del Rio, D., Bianchi, M., and Brighenti F., 26, Total Antioxidant Capacity of Spices, Dried Fruits, Nuts, Pulses, Cereals and Sweets Consumed in Italy Assessed by Three Different in vitro Assays, Molecular Nutrition and Food Research, 5(11), Lachman, J., Hosnedl, V., Pivec V., and Orsak, M., 1998, Polyphenols in Cereals and Their Positive and Negative Role in Human and Animal Nutrition, Proc. of the Cereals for Human Health and Preventive Nutrition Conference, July 7 11, Brno, Czech Republic, Brand-Williams W., Cuvelier M.E., and Berset C., 1995, Use of a Free Radical Method to Evaluate Antioxidant Activity, LWT- Food Science and Technology, 28(1), Re, R., Pellegrini, N., Proteggente, A., Pannala, A.,Yang, M., and Rice-Evans, C., 1999, Antioxidant Activity Applying an Improved ABTS Radical Cation Decolorization Assay, Free Radical Biology and Medicine, 26(9-1), Escarpa, A., and Gonzalez, M. C., 21, An Overview of Analytical Chemistry of Phenolic Compounds in Foods, Critical Reviews in Analytical Chemistry, 31(2), Vinson, J. A., Su, X., Zubik, L., and Bose, P., 21, Phenol Antioxidant Quantity and Quality in Foods: Fruits, Journal of Agricultural and Food Chemistry, 49(11),