АннотацияSafe and healthy nutrition has a beneficial effect on human well-being. Various foods, such as berries, are known to inhibit cancer-promoting pre-proliferative signals. Among European fruit and berry crops, raspberries demonstrate one with the widest ranges of biologically active substances. Extraction remains a reliable method of obtaining biologically active substances from plant materials. The research objective was to obtain a semi-finished raspberry product by using microwave and ultrasonic processing and to study its antioxidant, anti-carcinogenic, sensory, physico-chemical, and microbiological properties. The raspberry extracts were obtained by maceration, ultrasound treatment, and microwave processing. After that, the samples underwent a comparative analysis of their antioxidant properties. The ultrasonic method gave the best results. A set of experiments made it possible to define the optimal technological modes for the extraction process: ethanol = 50%, ultrasonic radiation = 35 kHz, temperature = 40 ± 5°C, time = 120 min, water ratio = 1:10. A set of experiments on cell cultures demonstrated that the raspberry extract was able to reduce the expression of the anti-inflammatory COX-2, iNOS, and IL-8 genes. Hense, we recommend further studies of the effect of the raspberry extract on the induced expression of COX-2, iNOS, and IL-8. In addition, its anticarcinogenic properties have to be studied in vivo.
Ключевые словаExtraction of plant materials , phenolic substances , PRC-analysis , expression of anti-inflammatory genes , inhibition , ultrasound , microvaves
In many ways, human well-being is associated with safe and healthy food. Food safety is often understood as the absence of harmful microorganisms and chemicals, including synthetic additives, while its healthiness is often identified with naturalness and freshness . The increasing demand for safe and natural food has become a major problem for food science and industry over the past decades . Despite the use of various traditional and modern antibacterial agents, as well as chemical preservatives, about a third of the population of industrialised countries suffer from foodborne diseases . Meanwhile, more and more consumers demand fresh, ready-to-eat, minimally processed foods that contain neither chemical preservatives nor synthetic additives . For modern foods, it is not enough to be a source of energy: modern food has to be functional, e.g. to possess antioxidant or anti-carcinogenic properties. This trend has triggered multiple studies in the field of food processing, as well as an active search for alternative natural supplements with a wide spectrum of physiological properties .
In the process of evolution, plants developed natural mechanisms of defense against microbial infections and other harmful environmental factors. Plants are known to produce antimicrobial peptides, lectins, polyphenols, terpenoids, essential oils, and other biologically active compounds. According to some studies, phenolic substances obtained from berries can act as a new type of food components that can inhibit a wide range of pathogens, e.g. Salmonella, Escherichia, and Staphylococcus .
Cancer is a serious social problem in many parts of the world . According to the World Health Organization, cancer is responsible for about 7.6 mln (13%) of the 59 mln annual deaths. By 2030, cancer mortality worldwide is expected to reach 11.8 mln per year. Most cancer deaths are caused by five basic behavioral and eating habits, including a low intake of fruits and vegetables. Bad eating habits, obesity, and sedentary lifestyle have been proven to increase the risk of oncological diseases . Therefore, a healthy diet is one of the most important changes in one’s lifestyle that can reduce the risk of cancer .
Recent epidemiological and basic studies have demonstrated the anticarcinogenic properties of fruit components. They can inhibit pro-proliferative signals that cause or stimulate the growth of tumors or cancer cells .
Raspberry polyphenols consist mainly of anthocyanins and tannins . Raspberries are a rich source of cyanidin glycoside. A high content of ellagitannin releases free ellagic acid during hydrolytic processes . These components are responsible for about 60% of the antioxidant potential . According to Landele, raspberries owe their antioxidant, antimicrobial, and anti-inflammatory properties to ellagic acid and ellagotanin . Thus, raspberries and their semi-finished products can prevent many diseases, if included in foods.
Raspberries contain a wide range of phytonutrients with antioxidant, antitumor, anti-neurodegenerative, and anti-inflammatory properties [17, 18]. The following substances are responsible for the hemotherapeutic and prophylactic components: vitamins C and E, folic acid, calcium, selenium, β-carotene, α-carotene, lutein, such polyphenols as ellagic acid, ferulic acid, p-coumaric acid, anthocyanins, quercetin, and kaempferol, and such phytosterols as β-sitosterol and stigmasterol .
A raspberry ethanol extract suppresses cell proliferation in squamous cell carcinoma without altering cell viability and inhibits the expression of vascular endothelial growth factor. In addition, it also inhibits nitric oxide synthase activity and indicates apoptosis and terminal differentiation . These data suggest that raspberry extract can be used for chemoprophylaxis in people with oral cavity dysplasia.
Solvent extraction can be used to obtain polyphenols . Unfortunately, it has several drawbacks. For instance, it requires a larger amount of organic solvents and a longer extraction time. Moreover, the solvent can have a negative effect on human health and degrade the target compounds. However, there are several alternative methods that can either eliminate or reduce these disadvantages .
There are several factors that affect the efficiency of extraction of biologically active components from plant materials. They include technology, solvent type, time, temperature, material vs. solvent ratio, etc. However, it is technology that remains crucial. The traditional extraction technologies, such as Soxhlet extraction and maceration, require a lot of solvent, time, and energy, but are popular and effective. However, new extraction technologies are being actively introduced, e.g. ultrasonic, microwave, infrared, and fluid supercritical extractions. They are energy saving and environmentally friendly, according to one of the latest books on the extraction of biological active substances from plant and animal raw materials . Still, an optimal extraction technology should be simple, safe, reproducible, inexpensive, and suitable for industrial use .
Ultrasonic (US) extraction is a fairly cheap method that requires minimal hardware design . It destroys cell walls (lysis) and disintegrates individual cellular structures and the cell as a whole, which increases the number of components that enter the extract. US produces a mechanical effect: the solvent penetrates into the matrix of berries, thus increasing the area of the contact surface between the solid and the liquid phases . Moreover, US waves can cause some undesirable chemical processes that can change the chemical composition, degrade the target compounds, and cause free radicals in gas bubbles . Therefore, a set of experiments is required to define the optimal extraction conditions, i.e. time, temperature, power, and ultrasonic frequency.
Microwave (MW) radiation is another possible way to increase extraction efficiency . MW radiation is a popular means of extraction, as far as low-molecular compounds from plant raw material are concerned.
The research objective was to obtain a semi-finished raspberry product using MW and US processing, as well as to study its antioxidant, anti-carcinogenic, sensory, physico-chemical, and microbiological properties.
ОБЪЕКТЫ И МЕТОДЫ ИССЛЕДОВАНИЯ
The experiments were performed on the premises of the Department of Technology and Catering at the Samara State Technical University (Samara, Russia). The anti-inflammatory and cytostatic, or cytotoxic, properties were determined in the N.N. Blokhin National Medical Research Oncology Center (Moscow, Russia).
The research featured a variety of fresh raspberries (Rúbus idáeus L.) harvested in the Samara region (53°12′N - 50°06′E) in 2017. The raspberries were provided by the Research Institute of Horticulture and Medicinal Plants ‘Zhigulyovskie Sady’ (Samara, Russia).
Determination of the antioxidant properties indicators.
Chemicals and reagents. The experiment used ethanol and distilled water. The Folin-Ciocalteu reagent (FCR) and the gallic acid were provided by the Fluka company (Germany). The DPPH (2,2-diphenyl-1-picrylhydrazyl), sodium nitrite, aluminum chloride, sodium carbonate, and linoleic acid were ordered from Sigma-Aldrich, Inc. The 2,4,6-tri(2-pyridyl)-s-triazine (TPTZ) was purchased from Fluka Chemicals (Spain). Other chemicals included hydrochloric acid, potassium chloride, acetic acid, sodium acetate, sodium phosphate, ferric chloride (II), ferric chloride (III), and ammonium rodanide.
Phenolic compounds. The content of total phenols was estimated using a modified version of the FCR method . Gallic acid was used as a standard: an aqueous solution of gallic acid (200 mg in 1000 cm3) was diluted with distilled water to obtain the concentrations appropriate for the calibration curve. The experiment involved 0.50 cm3 of the analysed substance or standard gallic acid, 4.00 cm3 of distilled water, 0.25 cm3 of FCR reagent, and 0.25 cm3 of a saturated aqueous solution of sodium carbonate. The samples were shaken and kept in the dark at room temperature for 30 min. The absorption coefficient was determined at 725 nm with a spectrophotometer. Results were expressed in mg equivalent of gallic acid per 100 g of dry weight. The experiment was performed in triplicate.
Flavonoids. The content of flavanoids was determined using a modified method described Demidova et al . 0.50 cm3 of the analysed substance or standard catechin solution was put in a 10 cm3 measuring tube. After that, 2.50 cm3 of distilled water was added at the time zero followed by 0.15 cm3 of a 5% aqueous solution of sodium nitrate. After 5 minutes, 0.30 cm3 of a 10% aqueous solution of aluminum chloride was added and kept for another 5 min. The absorption coefficient was measured at 510 nm. The content of flavonoids was expressed in mg equivalent of catechin per 100 g of dry weight. The experiment was performed in triplicate.
Anthocyanins. To define the total content of anthocyanins, the absorption coefficient was measured at two different pH values (1.0 and 4.5) at 515 and 700 nm . The content of anthocyanins was expressed in mg equivalent of cyanidin-3-glycoside per 100 g of dry matter. The experiment was performed in triplicate.
Antioxidant activity in the linoleic acid system. The antioxidant activity in the linoleic acid system was determined according to the method described Karabegovic . 0.5 cm3 of ethanol, 0.5 cm3 of distilled water, 1 cm3 of linoleic acid, and 2 cm3 of phosphate buffer (pH 7.0) were added to 1.0 cm3 of the analysed substance. The mixture was kept at 40°C for 120 h. Then an aliquot part (0.1 cm3) was isolated from the mixture. After that, 9.7 cm3 of 75% ethanol and 0.1 cm3 of a 30% ammonium rhodanide solution were added to the aliquot and allowed to stand for 4 min. Subsequently, 0.1 cm3 of ferric chloride (II) solution was added to the mixture (0.2 M in 3.5% of HCl). A spectrophotometer was used to measure the optical density of the mixture at 500 nm. The control sample contained all the reagents but the extract. The antioxidant activity was expressed in percent of inhibition of linoleic acid oxidation. The experiment was performed in triplicate.
- Taylor TM. Handbook of Natural Antimicrobials for Food Safety and Quality. USA: Elsevier Science, 2014. 442 p.
- Bobinaite R, Viškelis P, Šarkinas A, Venskutonis PR. Phytochemical composition, antioxidant and antimicrobial properties of rasberry fruit, pulp, and marc extracts. CyTA – Journal of Food. 2013;11(4):334–342. DOI: https://doi.org/10.1080/19476337.2013.766265.
- Wallace CA, Sperber WS, Mortimore SE. Food Safety for the 21st Century: Managing HACCP and Food Safety Throughout the Global Supply Chain. Wiley; 2018. 477 p. DOI: https://doi.org/10.1002/9781444328653.
- Kapetanakou AE, Skandamis PN. Applications of active packaging for increasing microbial stability in foods: natural volatile antimicrobial compounds. Current Opinion in Food Science. 2016;12:1–12. DOI: https://doi.org/10.1016/j.cofs.2016.06.001.
- Boo H-O, Hwang S-J, Bae C-S, Park S-H, Heo B-G, Gorinstein S. Extraction and characterization of some natural plant pigments. Industrial Crops and Products. 2012;40(1):129–135. DOI: https://doi.org/10.1016/j.indcrop.2012.02.042.
- Tian Y, Liimatainen J, Puganen A, Alakomi H-L, Sinkkonen J, Yang B. Sephadex LH-20 fractionation and bioactivities of phenolic compounds from extracts of Finnish berry plants. Food Research International. 2018;113:115–130. DOI: https://doi.org/10.1016/j.foodres.2018.06.041.
- Siegel R, Naishadham D, Jemal A. Cancer statistics, 2012. CA Cancer Journal for Clinicians. 2012;62(1):10–29. DOI: https://doi.org/10.3322/caac.20138.
- Zanini S, Marzotto M, Giovinazzo F, Bassi C, Bellavite P. Effects of Dietary Components on Cancer of the Digestive System. Critical Reviews in Food Science and Nutrition. 2015;55(13):1870–1885. DOI: https://doi.org/10.1080/10408398.2012.732126.
- Kushi LH, Doyle C, McCullough M, Rock CL, Demark-Wahnefried W, Bandera EV, et al. American Cancer Society guidelines on nutrition and physical activity for cancer prevention: reducing the risk of cancer with healthy food choices and physical activity. CA Cancer Journal for Clinicians. 2012;62(1):30–67. DOI: https://doi.org/10.3322/caac.20140.
- Zhao T-T, Jin F, Li J-G, Xu Y-Y, Dong H-T, Liu Q, et al. Dietary isoflavones or isoflavone-rich food intake and breast cancer risk: A meta-analysis of prospective cohort studies. Clinical Nutrition. 2019;38(1):136–145. DOI: https://doi.org/10.1016/j.clnu.2017.12.006.
- Teng H, Fang T, Lin Q, Song H, Liu B, Chen L. Red raspberry and its anthocyanins: Bioactivity beyond antioxidant capacity. Trends in Food Science and Technology. 2017;66:153–165. DOI: https://doi.org/10.1016/j.tifs.2017.05.015
- Golubtsova YuV. Physical and chemical indicators and merchandasing assessment of wild strawberry, gooseberry, cherry, raspberry, banana, wild rose and kiwi. Foods and Raw Materials. 2017;5(1):154–164. DOI: https://doi.org/10.21179/2308-4057-2017-1-154-164.
- Zhidyokhina TV. Industrial assortment of raspberry and its productivity in the black-earth region. The Bulletin of KrasGAU. 2015;109(10):131–135. (In Russ.).
- Miret JA, Munné-Bosch S. Abscisic acid and pyrabactin improve vitamin C contents in raspberries. Food Chemistry.2016;203:216–223 DOI: https://doi.org/10.1016/j.foodchem.2016.02.046.
- Zhang L, Li J, Hogan S, Chung H, Welbaum GE, Zhou K. Inhibitory effect of raspberries on starch digestive enzyme and their antioxidant properties and phenolic composition. Food Chemistry. 2015;119:592–599. DOI: https://doi.org/10.1016/j.foodchem.2009.06.063.
- Landele JM. Ellagitannis, ellagicacid and their derived metabolites: A review about source, metabolism, functions and health. Food Research International. 2011;44(5):1150–1160. DOI: https://doi.org/10.1016/j.foodres.2011.04.027.
- Luchina NA. Technological Properties of Raspberries Grown in the Novosibirsk Region. Food Industry. 2015;(8):22–24. (In Russ.).
- Zhbanova EV. Biochemical characterization of fruits from raspberry vor gene pool under the circumstances of the Central Chernozem Zone (Michurinsk). Collection of scientific works SNBG. 2017;144–1:182–186. (In Russ.).
- Xiao T, Guo Z, Bi X, Zhao Y. Polyphenolic profile as well as anti-oxidant and anti-diabetes effects of extracts from freeze-dried black raspberries. Journal of Functional Foods. 2017;31:179–187. DOI: https://doi.org/10.1016/j.jff.2017.01.038.
- Terletskaya VA, Rubanka EV, Zinchenko IN. Influence of technological factors on the process of black chokebery extraction. Food Processing: Techniques and Technology. 2013;31(4):127–131. (In Russ.).
- Polyakov VA, Abramova IM, Zenina GP, Aristarhova TYu, Stekanova GV. Ripple Effect on the Extraction Process in the Preparation of Fruit Drinks Fortified for Distillery. Beer and beverages. 2016;(6):42–45. (In Russ.).
- Khramtsov AG, Evdokimov IA, Lodygin AD, Budkevich RO. Technology development for the food industry: a conceptual model. Foods and Raw Materials. 2014;2(1):22–26. DOI: https://doi.org/10.12737/4121.
- Meireles MAA. Extracting Bioactive Compounds for Food Products. Theory and Applications. Bova Raton: CRC Press; 2009. 464 p.
- Vongsak B, Sithisarn P, Mangmool S, Thongpraditchote S, Wongkrajang Y, Gritsanapan W. Maximizing total phenolics, total flavonoids contents and antioxidant activity of Moringa oleifera leaf extract by the appropriate extraction method. Industrial Crops and Products. 2013;44:566–571. DOI: https://doi.org/10.1016/j.indcrop.2012.09.021.
- Khoei M, Chekin F. The ultrasound-assisted aqueous extraction of rice bran oil. Food Chemistry. 2016;194:503–507. DOI: https://doi.org/10.1016/j.foodchem.2015.08.068.
- Nipornram S, Tochampa W, Rattanatraiwong P, Singanusong R. Optimization of low power ultrasound-assisted extraction of phenolic compounds from mandarin (Citrus reticulata Blanco cv. Sainampueng) peel. Food Chemistry. 2018;241:338–345. DOI: https://doi.org/10.1016/j.foodchem.2017.08.114.
- Espada-Bellido E, Ferreiro-González M, Carrera C, Palma M, Barroso CG, Barbero GF. Optimization of the ultrasound-assisted extraction of anthocyanins and total phenolic compounds in mulberry (Morus nigra) pulp. Food Chemistry. 2017;219:23–32. DOI: https://doi.org/10.1016/j.foodchem.2016.09.122.
- Markin VI, Cheprasova MIu, Bazarnova NG. Basic directions of use microwave radiation in the processing of plant raw material (review). Chemistry of plant raw material. 2014;(4):21–42. (In Russ.). DOI: https://doi.org/10.14258/jcprm.201404597.
- Cai M, Hou W, Lv Y, Sun P. Behavior and rejection mechanisms of fruit juice phenolic compounds in model solution during nanofiltration. Journal of Food Engineering. 2017;195:97–104. DOI: https://doi.org/10.1016/j.jfoodeng.2016.09.024.
- Demidova AV, Makarova NV. Influence of blanching on the physical and chemical properties and antioxidant activity of fruit raw materials cherries, plums, blank chokeberry, strawberry. Food industry. 2016;(2):40–43. (In Russ.).
- Strycova AD, Makarova NV. Frozen Berries – Effective Antioxidant for Whole Year. Food industry. 2013;(3):28–31. (In Russ.).
- Karabegovic IT, Stojičević SS, Veličković DT, Todorović ZB, Nikolić NT, Lazić ML. The effect of different extraction techniques on the composition and antioxidant activity of cherry laurel (Prunus laurocerasus) leaf and fruit extracts. Industrial Crops and Products. 2014;54:142–148. DOI: https://doi.org/10.1016/j.indcrop.2013.12.047.
- Cheigh C-I, Chung E-Y, Chung M-S. Enhanced extraction of flavanones hesperidin and narirutin from Citrus unshiu peel using subcritical water. Journal of Food Engineering. 2012;110(3):472–477. DOI: https://doi.org/10.1016/j.jfoodeng.2011.12.019.
- M’hiri N, Ioannou I, Mihoubi Boudhrioua N, Ghoul M. Effect of different operating conditions on the extraction of phenolic compounds in orange peel. Food and Bioproducts Processing. 2015;69:161–170. DOI: https://doi.org/10.1016/j.fbp.2015.07.010.
- Yao C-Y, Yang J-Y, Xu Z-L, Wang H, Lei,H-T, Sun Y-M, et al. Indirect Competitive Enzyme-Linked Immunosorbent Assay for Detection of Tylosin in Milk and Water Samples. Chinese Journal of Analytical Chemistry. 2018;46(8):1275–1281. DOI: https://doi.org/10.1016/S1872-2040(18)61106-5.
- Pal V, Saxena A, Singh S, Kumar S, Goel AK. Development of a TaqMan Real-Time Polymerase Chain Reaction Assay for Detection of Burkholderia mallei. Journal of Equine Veterinary Science. 2017;58:58–63. DOI: https://doi.org/10.1016/j.jevs.2017.08.008.
- PCR Primers for Gene Expression Detection and Quantification [Internet]. [cited 2018 Sep 01]. Available from: http://pga.mgh.harvard.edu/primerbank/.
- Luo , Zhang H. The role of proinflammatory pathways in the pathogenesis of colitis-associated colorectal cancer. Mediators of Inflammation. 2017;2017. DOI: https://doi.org/10.1155/2017/5126048.
- Nile SH, Ko EY, Kim DH, Keum Y-S. Screening of ferulic acid related compounds as inhibitors of xanthine oxidase and cyclooxygenase-2 with anti-inflammatory activity. Revista Brasileira de Farmacognosia. 2016;26(1):50–55. DOI: https://doi.org/10.1016/j.bjp.2015.08.013.
- Rennert K, Steinborn S, Gröger M, Ungerböck B, Jank A-M, Ehgartner J, et al. A microfluidically perfused three dimensional human liver model. Biomaterials. 2015;71:119–131. DOI: https://doi.org/10.1016/j.biomaterials.2015.08.043.
- Paccaud JP, Schifferli JA, Baggiolini M. NAP-1/IL-8 induces up-regulation of CR1 receptors in human neutrophil leukocytes. Biochemical and Biophysical Research Communications. 1990;166(1):187–192. DOI: https://doi.org/10.1016/0006-291X(90)91929-M.
- Souza GR, Cunha TM, Silva RL, Lotufo CM, Verri WA, Funez MI, et al. Involvement of nuclear factor kappa B in the maintenance of persistent inflammatory hypernociception. Pharmacology, Biochemistry and Behavior. 2015;134:49–56. DOI: https://doi.org/10.1016/j.pbb.2015.04.005.
- Alfaro C, Sanmamed MF, Rodríguez-Ruiz ME, Teijeira Á, Onate C, González Á, et al. Interleukin-8 in cancer pathogenesis, treatment and follow-up. Cancer Treatment Reviews. 2017;60:24–31. DOI: https://doi.org/10.1016/j.ctrv.2017.08.004.
- Vannini F, Kashfi K, Nath N. The dual role of iNOS in cancer. Redox Biology. 2015;6:334–343. DOI: https://doi.org/10.1016/j.redox.2015.08.009.