Study On Horseradish (Armoracia Rusticana) Essential Oil And Comparison With The Related Species – Debreceni Horseradish (Armoracia Macrocarpa).

Study On Horseradish (Armoracia Rusticana) Essential Oil And Comparison With The Related Species – Debreceni Horseradish (Armoracia Macrocarpa). Doktori (PhD) értekezés Nguyen Minh Nhat Témavezető: Dr. Vasas Gábor DEBRECENI EGYETEM Természettudományi Doktori Tanács Juhász-Nagy Pál Doktori Iskola Debrecen, 2016. 1 2 Nyilatkozatok 3 Ezen értekezést a Debreceni Egyetem Természettudományi Doktori Tanács Juhász-Nagy Pál Doktori Iskola Biológia Programja keretében készítettem a Debreceni Egyetem természettudományi doktori (Ph.D) fokozatának elnyerése céljából. Debrecen, 2016.10.17 ………………………... Nguyen Minh Nhat Tanusítom, hogy Nguyen Minh Nhat doktorjelölt 20011-2016 között a fent megnevezett Doktori Iskola biológia programjának keretében irányításommal végezte munkáját. Az értekezésben foglalt eredményekhez a jelölt önálló alkotó tevékenységével meghatározóan hozzájárult. Az értekezés elfogadását javasolom. Debrecen, 2016.10.17 …….……………………... Dr. Vasas Gábor 4 Study On Horseradish (Armoracia Rusticana) Essential Oil And Comparison With The Related Species – Debreceni Horseradish (Armoracia Macrocarpa). Értekezés a doktori (Ph.D) fokozat megszerzése érdekében a biológia tudományágban Írta: Nguyen Minh Nhat, pharmacist Készült a Debreceni Egyetem Juhász-Nagy Pál Doktori Iskola (biológia doktori programja) keretében Témavezető: Dr. Vasas Gábor A doktori szigorlati bizottság: elnök: Dr. ………………………… ………………... tagok: Dr. ………………………… ……..…………. Dr. ………………………… ………………… A doktori szigorlat időpontja: 2016…………………. Az értekezés bírálói: Dr. ………………………… ………………… Dr. ……………………….... ………………… Dr. ………………………… ………………… 5 6 A bíráló bizottság: elnök: Dr. …………………………. ………………… tagok: Dr. …………………………. ………………… Dr. …………………………. ………………… Dr. …………………………. ………………… Dr. …………………………. ………………… Az értekezés védésének időpontja: 7 Table of contents Page Introduction 14 Chapter I: Literature Review 1. The plant 2. The origin 3. The relative species 4. The crop 5. Possible uses 6. Possible pharmacological studies 6.1. ITCs inhibit carcinogen-activating enzymes and induce carcinogen-detoxifying enzymes 6.2. ITCs trigger apoptosis pathways 6.3. ITCs inhibit cell cycle progression 6.4. ITCs inhibit pro-inflammatory and pro-carcinogen signaling factor 6.5. ITCs possess antimicrobial and antioxidant activities 6.6. ITCs are considered to be oxidants themselves 16 16 18 18 21 21 22 22 23 24 25 25 26 Chapter II: Essential Oil Distillation Technology 1. Small-scaled distillation 1.1. Materials and sample preparation 1.2. Methods 1.3. Results and discussion 2. Medium-scaled distillation 2.1 Materials and technology 2.2. Results and discussions 27 27 27 28 31 33 34 36 8 Chapter III: Analytical Studies 1. Background information 1.1. Glucosinolates (GLSs) 1.2. GLSs hydrolysis reaction 1.3. Isothiocyanates (ITCs) 2. Gas chromatography and mass spectrometry study on horseradish essential oil 2.1. Sample preparation and method 2.2. Result and discussions 3. Capillaries electrophoresis (CE) study 3.1. Material preparation and instrument 3.2. Method 3.3. Result and discussion 38 38 38 39 40 42 43 44 46 47 48 55 Chapter IV: Comparative Analysis of A. rusticana and A. macrocarpa 1. Background information 1.1. Myrosinase 1.2. Peroxidase 2. Liquid chromatography and mass spectrometry studies on glucosinolates 2.1. Materials and method 2.2. Results and discussion 3. Anatomical studies 3.1. Material and methods 3.2. Results and discussion 4. Gel electrophoresis studies 4.1. Materials and method 4.2. Results and discussions 71 71 71 72 74 74 75 78 79 79 83 84 84 9 Chapter I Literature Review 1. THE PLANT Armoracia rusticana, Cochlearia armoracia, and Armoracia lapathifolia are scientific names that refer to a perennial plant of Mustard family (genera-group Cardamineae of Brassicaceae) commonly known as horseradish (Mohlenbrock 1980).The plant can reach the height of 120cm. It has a hardy glabrous stem, from which wavy margin leaves arise directly (cauline leaf) following a circular arrangement pattern (basal rosette). Horseradish leaf is described to have a length of 30–100cm, a cordate base, long petiole, and the shape slightly varying from the lower to the uppermost leaf. Whereas a shorter petiole and a lobe shape with entire or serrate margin are characteristics of lower leaves, upper leaves have a narrow base, obtuse apex, oblong or lanceolate shape with crenate or serrate margin. The margin is linear or almost entire in the case of uppermost leaves (Mohlenbrock 1980).Horseradish has white, tetramerous flowers arranged in racemes and a smooth, brown angustiseptate fruit—a fruit flattened at a right angle to the septum, which usually contains very few (≤ 6) or no seeds. In addition, the lack of evidence that horseradish grows from seeds suggests sterility (Sampliner and Miller 2009). 16 Figure 1. Armoracia rusticana. Adapted from http://plantillustrations.org. 17 2. THE ORIGIN Horseradish is believed to be native to Eastern European countries (such as Romania or Ukraine) even though it can now be found throughout Europe. Horseradish occurrence was found only associated in areas where there are people. No information about the wild population of the plant has been found. It is suggested that the wild population of horseradish may have become extinct or it may have been derived from related species such as Armoracia macrocarpa and Armoracia sisymbroides (DC.) N.Busch ex Ganesh. Its ability to spread by rhizome could mean that horseradish is a problematic weed, though it is also possible that its wild population is just yet to be found (Sampliner and Miller 2009). 3. THE RELATIVE SPECIES Armoracia macrocarpa is native to Eastern Europe, specifically to the marshes of the Central Danube Basin (e.g., in Hungary, Czech Republic, Romania, and Bulgaria), whereas Siberia is the native land of Armoracia sisymbroides. Armoracia macrocarpa grows in reeds (Scirpo-Phragmitetum austro-orientale), wet meadows (Phalaridetum arundinaceae) and salt meadows (Agrosti (o)- Alopecuretum pratensis). It favors alkaline soils, salt tolerant, wet or water covered adherent loam, clay, salt soils, rich in nutrients and alkaline minerals. In Hungary, Armoracia macrocarpa can be found in the North Hungarian Mountains (Mátra margins), Great Hungarian Plain (Danube region, DanubeTisza köze region and Tiszántúl region) and South Transdanubia. It has a fragmented distribution, especially in northeastern parts of the country. Due to 18 its habitat specialization and a continuing decline in extent and quality of its habitat, Armoracia macrocarpa is classified as rare (Hungary, Romania) or very rare (Serbia). A. macrocarpa and A. rusticana are nearly identical. The flowers and fruits of A. macrocarpa are larger than those of A. rusticana; further, A. macrocarpa fruits contain numerous seeds, while the fruits of A. rusticana rarely contain seeds. Armoracia macrocarpa seems to have much larger inflorescences than A. rusticana .(Sampliner and Miller 2009). 19 Figure 2. Armoracia macrocarpa. Adapted from http://plantillustrations.org. 20 Armorcia sisymbrioides is distinguishable from other relative species by its whitish, glaucous leaves. Moreover, its cauline leaves are auriculate. Neither of Armoracia rusticana and A. macrocarpa have these features (Sampliner and Miller 2009). There is little or no information on these two species of Armoracia genus, nor on the relation between these species and horseradish. Research in this field could help improve current horseradish crops, via traditional breeding or biotechnological techniques. 4. THE CROP For propagation, the roots are used exclusively. Horseradish root is white, cylindrical or tapering, which can reach a length of 60 cm in loose soil and consist of several lateral roots. Deep, silty loam soils with good drainage and temperate climates are favorable conditions for both annual and perennial horseradish crops. For commercial production, horseradish is usually cultivated as an annual crop, which usually starts with plantation in early spring because the roots need the higher temperatures of the summer (15–27oC) and the lower temperatures of the end of summer and fall (11–22oC) to develop. Usually the whole root is harvested. In the case of perennial production, the underground shoot grown from original root is collected, and the original root is left in the field for regeneration. Major horseradish-growing countries are the United States and Hungary (Shehata et al. 2009). 5. POSSIBLE USES Due to the characteristic pungent, intensive lachrymatory odor and taste of the 21 root, horseradish is cultivated mostly for condiment production. It is also used as a type of food and for traditional medical purposes. For example, horseradish has been used to ease pain such as low back pain and pain associated with sciatica and rheumatism. It is also traditionally used as a urinary, gastrointestinal, and respiratory aid, for toothache, and as aphrodisiac. Combination of horseradish root and honey in warm water is made for treating influenza. However, the underlying mechanism of possible medicinal benefits of horseradish remains unclear (Sampliner and Miller 2009). 6. POSSIBLE PHARMACOLOGICAL STUDIES Isothiocyanates (ITCs) are the products of hydrolysis reaction of glucosinolates (GLSs), which occurs when the plant tissues are damages. ITCs are potential anti-carcinogenic agents that not only inhibit the development of cancer cells but also eliminate established cancer cells (Zhang 2004). The mechanism underlying anti-carcinogenic effect of ITC is unclear even though there have been many suggested hypotheses based on observations in animal and human cell studies. There are yet no clinical trials on ITCs or their precursors, GLSs (Valgimigli and Iori 2009). 6.1. ITCs inhibit carcinogen-activating enzymes and induce carcinogendetoxifying enzymes. The underlying pharmacology mechanisms of ITCs on cancer cells could be due to the possibilities of inhibiting carcinogen-activating enzymes and inducing carcinogen-detoxifying enzymes. Cellular enzymes such as those belonging to the cytochrome P450 (CYP) family are known as carcinogen22 activating enzymes due to their ability to transform pro-carcinogens into carcinogens, the active form that can be harmful to cells. ITCs showed the ability to inhibit or down-regulate these enzymes. In a study where animals were treated with carcinogenic agent (nitrosamine), ITCs, especially arylalkyl ITCs such as PEITC, inhibited CYP enzymes that are necessary for nitrosamine activation (Hecht 2000). Phase II enzymes such as quinone reductase 1 (QR1), glutathione S-transferase (GST), and heme oxygensase 1 (HO-1) play important roles in cellular defense mechanism against oxidants and carcinogens and are known as carcinogendetoxifying enzymes. Inducing gene transcriptions of those enzymes is thought to be one of the anti-carcinogenic strategies used by ITC. Modulation of phase II enzymes is the consequence of the interaction between ITCs and the complex consisting of a nuclear transcription factor, Nrf2 (NF-E2–related factor 2), and a protein anchored to the actin cytoskeleton known as Keap1 (Kelch-like ECHassociated [erythroid cell-derived protein with CNC homology] protein 1). Binding of ITC to the complex involves the reaction of ITC and protein sulfhydryl residues of Keap1, leading to the dissociation of the complex into Nrf2 and Keap1, as it is observed that Nrf2 concentration is significantly increased when treating human hepatoma HepG2 cells with 25 μM AITC (Jeong et al. 2005). Free Nrf2 then translocates into the nucleus where together with other transcription factors, it interacts with antioxidant response element (ARE), resulting in the activation of gene transcription for carcinogendetoxifying enzymes (Dinkova-Kostova et al. 2002). 6.2. ITCs trigger apoptosis pathways. 23 Apoptosis or programmed cell death results from cleavage of specific cellular substrates caused by the activity of aspartate-specific cysteine protease known as caspase. Main signaling pathways leading to caspase activation are via death receptors such as tumor necrosis factor (TNF) receptors and via mitochondria. AITC (10 μM) activated caspase 9 (mitochondria pathway), caspase 8 (death receptor pathway), and caspase 12 (estrogen receptor [ER] pathway) in conjugation with caspase 3 activation in human leukemia HL60 cells (R. Yu et al. 1998). Mitochondrial pathway is regulated largely by members of Bcl2 family— antiapoptotic members (Bcl-2, Bcl-x L), proapoptotic members (Bax, Bak, Bok), and BH3-only protein (Bid, Bad, Bin). Changes in the regulators of mitochondria pathway were accompanied with ITCs treatment in several studies. Apart from the effect on regulators, ITCs are suggested to exert direct effect on mitochondria itself, causing the release of cytochrome c. By binding to a heme group, cytochrome c becomes holocytochrome c, which has the ability to activate caspases (Srivastava et al. 2003; Singh et al. 2004; Xiao et al. 2003; Fimognari et al. 2002; Chen et al. 1998; Xu and Thornalley 2001). 6.3. ITCs inhibit cell cycle progression. The key regulator molecules of cell proliferation through the cell cycle are cyclins – the regulatory proteins that activate a specific class of enzymes known as cyclin-dependent kinase (cdk). Together, they act as an activated complex that pushes the cell through certain stages of the cycle. Each stage of the cell cycle is marked with the activity of certain type of cyclin and kinases. AITC caused cell cycle arrest in the G1 phase and in the G2/M phase (Zhang, Tang, 24 and Gonzalez 2003; Tang and Zhang 2004; Smith et al. 2004). Cell cycle arrest after AITC treatment was accompanied with down-regulation of cyclin B, cdk1, cdc25B, cdc25C, and tubulin disruption, suggesting that the inhibitory effect of ITCs is a complex process. Cell types, dose and time of exposure may influence the inhibitory effect of ITCs (Clarke, Dashwood, and Ho 2008; Hwang and Lee 2006; Chiao et al. 2002). 6.4. ITCs inhibit pro-inflammatory and pro-carcinogen signaling factor. Cancer can be the consequence of chronic inflammation process in which proinflammatory and pro-carcinogen signaling factors secreted by cells play important roles. These factors are cell-derived mediators (e.g., nitric oxide (NO), prostaglandin E2 (PGE2), and tumor necrosis factor (TNF)). Inhibiting the production or secretion of those molecules is possibly one of the anticarcinogenic mechanisms by which ITCs act (Gerhäuser et al. 2003; Ippoushi et al. 2002). 6.5. ITCs possess antimicrobial and antioxidant activities. ITCs possess antibacterial effect against several bacteria, which is related to anticarcinogenic effects, e.g., against Helicobater pylori, one possible cause of stomach cancer (Zsolnai 1971). Bactericidal effect against Helicobacter pylori, Escherichia coli, Salmonella typhimurium, Staphylococcus aureus, Streptococcus mutans, Penicillium notatum, Bacillus cereus, and Vibrio parahaemolyticus was reported at AITC concentration of 3.8–16.7 μM, with an activity that was 7.8–20.5 times less than that of PEITC (Shin, Masuda, and Naohide 2004; Luciano and Holley 2009; Tunc et al. 2007). There are several 25 using direct heating mode. Regardless of different amount of added water, the plant materials near the bottom of the still charred, affecting the yield, quality and odor of essential oil. Attempt to decrease heating power together with extend the distillation duration led to the decomposition of essential oil's components due to the prolonged interaction with hot water. Moreover, the insufficient rate of steam production due to the low heating power (<90oC) causes the reflux of oil back to the sample containing flask, leading to decomposition reaction and poor oil quality. The same phenomenon was observed in apparatus using steam heating, plus the contact surface area of the plant materials and steam is limited, i.e. plant material tended to aggregate, preventing steam from heating the upper, inner layers. In fractional distillation, longer distance and flow back of essential oil droplets are probably the explaination for discrepancy in essential oil amount. In the improvised hydro-distillation using water bath heating, problems with burning and char of plant materials were solved. The flask was heated at constant temperature of boiling water (approximately 100oC). No material char was observed. The heating surface area was increased as the flask was almost entirely immersed into the boiling water. Produced steam was sufficient for the process. The yield of 0.071% (166.66 ± 27.54 μl) essential oil was extracted from fresh horseradish main roots (233 ± 38.74 g). For fresh lateral roots (151.67 ± 1.52 g), the yield of essential oil is 0.12% (187 ± 9.6 μl). The amount of water added following the ratio of 1:3 volume / material weight, results in the highest yield compared to other ratio in case of lateral root distillation. For the main roots distillation, less water was added (1:6 volume / material mass) because of their higher water content. The duration of 1.5 h was sufficient for complete extraction of essential oil. In addition to provide the stable yield of 32 essential oil, this extracting method comes with simplicity and the ease of use, i.e. required minimum attention during the operation. Figure 7. Comparing chart of horseradish essential oil yields from different distillation methods. Chart was made by LibreOffice Calc 5.2.1. 2. MEDIUM SCALED DISTILLATION The medium-scaled distillation technology was built based on the experiences from the laboratory-scaled extracting methods. As the amount of plant material is much higher in this case (approximately 15 kg), different adjustments on influencing factors (e.g. duration of the process, amount of added water, heating temperature…) were crucial in order to reach the similar yield (0.07%) of laboratory-scaled experiments. 33 0.00% 0.02% 0.04% 0.06% 0.08% 0.10% 0.12% 0.14% Yield (%) 2.1 Materials and technology The fresh fleshy horseradish roots were cultivated in Újléta and supplied by KELET PRODUCTION Zrt., Hungary. The plant materials (≈15.5 kg) were chopped, grind by commercial industrial grinder and transferred immediately into the still with great care due to the pungency of high material amount. The still and its components are made of stainless steel to avoid corrosiveness from essential oil. The plant material was mixed during distillation by a rotating frame with diagonal bars, which is introduced inside the still. Heating plates at the bottom (n=3) and heating rings on the wall (n=3) of the still ensure the sufficiency of heating surface area as well as heating power. The rotation (rpm) of mixing frame and power of heating plates as well as their activating order are accessible through a controlling panel located separately from the still. The spiral condenser was used to sufficiently process large amount of steam. 34 Figure 8. Diagram of mid-scaled distillation instrument. Diagram was made by INKSCAPE, version 0.91. Legends – 1: the tank, 2: heating rings, 3: heating plates, 4: rotating motor, 5: lid of the tank, 6,7: mixing frame with diagonal bars, 8: thermostat, 9: valve, 10: neck connector, 11: connecting tube, 12: condenser, 13: control panel. The important tested factors of extracting technology includes: amount of water added to the system, optimum temperature and heating mode. Different amounts of added water (0, 250, 500, 750 mL) were tested and were combined with various heating program (90, 92.5, 95, 97.5oC). Heating programs were designed based on the activating order of wall and bottom plates and the desired end-temperature of the system (90÷97.5oC). The extracted essential oil was separated from the watery distillate by using massive centrifugation (Beckman Avanti J-25) at 13000 rpm for 10min. 35 2.2. Results and discussions At the same heating program (95oC), experiments with different added water amounts showed that slightly better yield (0.03% yield) of essential oil was achieved when no water was added compared to 0.02% essential oil yield in the other cases (i.e. 0.25, 0.5, 0.75 L). This can be explained based on the large amount of water comes from the fresh roots. It is important to note that adding more water into the system would result in high amount of watery extract. The heating program was designed as following: heating rings on the wall of the tank is activated in order for the temperature in the still to reach 70oC (to decrease the heating duration of substance agglomerated at the bottom of the tank) and heating plates on the bottom is activated to reach desired end temperature. Low heating temperature (<95oC) results in prolonged duration of the distillation while high temperature (>97oC) more likely results in sample char. Good result was achieved when the setting temperature falls around 95oC, i.e. better yield (0.05%) compared to other temperature settings (i.e. 90oC – 0.02%, 92.5oC – 0.03% and 97.5oC – 0.03%). No sample burning was recorded but agglomerations of plant material were found at the bottom of the still by the end of the process, suggesting that the distillation may not reach it highest yield. However, this is an inevitable technical problem. The extracting process is considered to be completed in 2 h. Distillation of average 15 kg of fresh horseradish main roots using the optimal setting resulted in 12 mL essential oil (average yield 0.08%). On the other hands, distillation of fresh lateral roots (average yield of 0.01%) failed to achieve the expected yield regardless to different amount of added water. Various amounts oil were extracted from horseradish roots incubated in different period. In some cases, e.g., distillation 36 of roots stored for 21 days in cold room (4oC), better yield was achieved (i.e. 0.14%). However, due to the discrepancy of the results, different incubating periods and storing conditions are not a reliable method compared to the distillation using fresh roots. The newly developed extracting technology satisfied the requirements. It provides a stable yield of essential oil and similar to that from the laboratory-scaled experiments. It is easy to handling, operating and requires reasonable time of attention. The proposed technology is suitable for extracting high amount of essential oil from fresh horseradish roots. The investigation of the efficiency of the new method is discussed in the following chapter. 37 Chapter III Analytical Studies 1. BACKGROUND INFORMATION 1.1. Glucosinolates (GLSs) A. rusticana is rich in glucosinolates, the secondary products that play an important role in the plants defensive system. GLS is an organic anion that is stable and soluble in water (Oerlemans et al. 2006). GLS’ s structure consists of β-thioglucoside N-hydroxysulfates with a side chain (R) and a sulfur-linked βD-glucopyranose moiety (Figure 9). Based on the structure variety of the side chain (R), GLS can be classified into different groups, the most common are aliphatic, ω-methylthioalkyl, aromatic, and heterocyclic (indole) GLS (Fahey, Zalcmann, and Talalay 2001). Figure 9. General structure of glucosinolates. Adapted from www.wikipedia.org. 38 According to previous studies, eight different GLS have been found in horseradish (Figure 12b), in which sinigrin (2-propenyl or allyl glucosinolate) and gluconasturtiin (phenethylglucosinolate) were found in higher quantity compared with the others. Sinigrin is the main GLS found in horseradish and accounts for 74% of the total GLS in the plant material (Li and Kushad 2005). The quantity and quality of plant GLS content are significantly affected by the plant’s age and environmental factors (e.g. soil fertility, wound, pathogen challenge, etc…) (Björkman et al. 2011). 1.2. GLSs hydrolysis reaction GLSs hydrolysis is triggered when damage is done to the plant. This reaction requires an important enzyme called myrosinase, which is separated under normal conditions to avoid contact with GLS. Once hydrolysis is triggered, myrosinase is released and reacts with GLS, resulting in an unstable aglucone (thiohydroximate O sulfonate), which then gives rise to different products depending on reaction conditions and participation of other factors. For example at pH 7, 37–45 ◦C, and under the effect of myrosinase, the products of GLS hydrolysis reaction are mainly isothiocyanates (ITCs). Nitrile and epithionitrile are final products if the reaction takes place at pH 3 and 6, respectively, in the presence of Fe2+ ions and an epithiospecifier protein. The participation of thiocyanate-forming factor in the reaction results in the formation of thiocyanate. ITC, thiocyanate, nitrile, epithionitrile, and oxazolidinethione are possible products of GLS hydrolysis, of which ITC has recently attracted research, as a potential anticarcinogenic agent (Zhang 2010; Li and Kushad 2005). 39 Figure 10. Glucosinolates break-down pathways. Figure was made by Marvin JS version 16.9.12. 1.3. Isothiocyanates (ITCs) Structure and properties – The most common products yielded from hydrolysis of GLSs are ITCs that share a common structure consisting of –NCS group and side chain –R (figure 11). Because of the –NCS group, specifically the C atom of the group, ITCs possess electrophilic characteristics, i.e. they favor reactions with nucleophilic molecules (e.g. cellular peptides and amino acids), thus leading to possible pharmacological effects. Electrophilicity is influenced by the side chain –R because of the steric hindrance effect on the electrophilic C atom. In addition to electrophilicity, the side chain also influences the lipophilicity of the molecule (Zhang 2004). ITCs are more stable in acidic conditions than in neutral or alkaline conditions due to their reactions with water molecule’s OH− ions (Ohta, Takatani, and Kawakishi 1995). Organic solvents (e.g., hexane, acetone, and ethyl acetate) are more favorable for storage than aqueous solutions, in which decomposition of ITCs is temperature 40 dependent. The decomposition rate is fast at temperature 37oC, decreasing and stopping as the temperature decreases to −5oC. The ability to react readily with ethanol makes ITCs unstable in alcoholic solution. Addition of citric acid, sugar esters, or vegetable oil may stabilize a solution of ITCs (Ina et al. 1981). Figure 11. General structure of Isothiocyanate. Adapted from www.wikipedia.org. Horseradish ITCs – Allyl ITC (AITC) and 2-phenylethyl ITC (PEITC) are the most common ITC components found in horseradish root. Whereas AITC is also found to be present in both horseradish and wasabi (Wasabi japonica), PEITC is only found in horseradish, which may at least partly contribute to the difference in taste between the two species (A. Depree, M. Howard, and P. Savage 1998). AITC constitutes 78% of total horseradish ITCs. AITC is most likely responsible for the pungent, lachrymatory odor and taste of horseradish root. It is the final product from hydrolysis of sinigrin. Also known by the common name “mustard oil,” at room temperature AITC appears as a colorless liquid with a boiling point of 150oC and melting point of −80oC (Zhang 2010; E. Y. Yu et al. 2001). PEITC, also referred to as “phenethyl mustard oil”, is the colorless or light yellow final product of the hydrolysis of gluconasturtiin. 41 immersed in boiling water for 10 minutes (watercress) and 30 minutes (other vegetables) to completely inactivate the myrosinase. 10 mL of MeOH was added to the cooked plant material, followed by thorough homogenization, and centrifugation at 13000 rpm for 3 minutes. The supernatant was evaporated to dryness. Prior to analysis, the dried samples were resuspended in water, centrifuged and subjected to analysis by both CE and LC/MS - after dilution with water, if necessary. These extracts are referred to as “methanolic extracts”. The instrument – Method development was carried out on a PrinCE-C 700 capillary electrophoresis instrument. A 60 cm fused silica capillary with 50 µm (i.d.) was used. For myrosinase activity study, effective length was 7.2 cm (short-end injection). For determination of different glucosinolates or allyl isothiocyanate quantification from concentrated real matrices, effective length was 52.8 cm (long-end injection). Capillary preconditioning and postconditioning were previously described (Gonda et al. 2013), Sample injection was hydrodynamic (100 mbar × 0.25 min.). Sinigrin was quantified at 230, gluconasturtiin at 210, ITC derivates at 275 nm. The softwares – ChemAxon MarvinSketch was used for drawing chemical structures and reactions, while Calculator Plugins were used for structure property prediction and calculation. Version 6.2.3_b915, 2014 was used. Figures were generated using scripts in R 3.1.1. (R Development Core Team, 2009) using ggplot 0.9.3.1. 3.2. Method 3.2.1. Optimization of separation 48 The basic of the new method is to be able to separate GLSs and ITCs from the plant matrix. Solutions of pure sinigrin (SIN), gluconasturtiin (GNT) and methanol extract of horseradish roots were used. The starting background electrolyte (BGE) solution contained N-Tris(hydroxymethyl)methyl-3aminopropanesulfonic acid (TAPS, 20mM), sodium deoxycholate (250 mM), sodium tetraborate (15 mM), pH was 8.50. Influencing parameters (i.e., pH, concentration of electrolytes and surfactants, addition of organic solvents and polarity (short-end injection mode)) were taken into consideration in order to improve the speed of the operation with similar sensitivity and stability. 3.2.2. Derivatization study Figure 14. Derivatization scheme of isothiocyanates generated in-vial by myrosinase mediated decomposition of glucosinolates. Figure was made by Marvin JS version 16.9.12. Dithiocarbamate is the product of in-vial derivatization reaction of ITCs with mercaptoacetic acid (MAA) (figure 14). MAA is the suitable derivatizing agent for ITCs because it is inexpensive, miscible with water at any pH, and the product (dithiocarbamate) carries charges at the pH of BGE (pH = 9.0). The 49 study of optimal concentration of ascorbic acid (enzyme activator) and MAA was designed as following: 350 µg/mL AITC was derivatized in phosphate buffer (10 mM, pH 7.5) with the different concentrations of MAA (1, 5, 10 mM), and ascorbic acid (0, 1, 5, 10 mM). The working pH range of the reaction was investigated based on the quantification (using proposed CE-MEKC method) of the generated derivatized products in different pH – 350 µg/mL AITC was derivatized with MAA (5 mM), ascorbic acid (1 mM) in a pH series of 5.5-9.5 (acetate, phosphate or borate buffers in 10 mM end-concentration). The characterization of derivatization products by LC-MS was run on a Thermo Accela HPLC attached to a Thermo LTQ XL Linear Ion Trap MS (column: Hypersil Gold 50 mm × 2.1 mm × 1.9µm). ESI ionization parameters were as follows: heater temperature, 300 °C; sheath gas, N2; flow rate, 20 arbitrary units (arb); aux gas flow rate, 8 arb; spray voltage, 4 kV; capillary temperature, 275 °C; capillary voltage, -28.00 V, negative ion mode. Gradient components were A, water with 0.1% (v/v) formic acid; B, MeCN with 0.1% (v/v) formic acid. The time program was 10% B: 0 – 2 min, 10 – 90% B: 2 – 7 min, 90%B: 7-13 min, 90 – 10% B: 13 – 13.1 min, 10% B: 13.1 – 15 min. Flow rate was 300 µL/min. 1 µL of a derivatized sample (acetate buffer, pH 5.0, mercaptoacetic acid (5 mM), ascorbic acid (1 mM)) containing 1 µg/mL of allyl isothiocyanate and phenethyl isothiocyanate was injected. 50 Figure 15. Charge of isothiocyanates and their mercaptoacetic acid dithiocarbamate products in the pH range usually used for capillary electrophoresis. Calculations were done by ChemAxon MarvinSketch v6.2.3., using default method parameters. Abbreviations – AITC: allyl isothiocyanate; PEITC: phenethyl isothiocyanate; AITCp allyl isothiocyanate dithiocarbamate product; PEITCp: phenethyl isothiocyanate dithiocarbamate product. 3.2.3. Validation The validation of the new CE method was performed using short-end injection mode. For AITC, the 5-points calibration curve (4.5, 9, 45, 90, 450 µg/ml) was prepared and measured as following: 25 mg/ml AITC stock solution was prepared with MeCN, which then diluted with water to reach the desired concentration. These solutions were mixed with derivatization solution (NaH2PO4 (100 mM), mercaptoacetic acid (50 mM), ascorbic acid (10 mM), pH 7.50, following 9:1 ratio). In case of sinigrin and gluconasturtiin , 7-point 51 calibration curve (5, 10, 50, 100, 500, 1000, 5000 µg/ml) was prepared by dilution of these glucohydrolates with water. From the calibration curves, limit of detection (LOD), limit of quantitation (LOQ), coefficient of determination (R2) and regression equations were calculated. Reproducibility studies were designed as following: five injections of isothiocyanate derivatized solution (100 µg/ml), and 100µg/ml glucosinolate solution were introduced and measured per day in 3 days. The relative standard derivation (RSD) between the area under curve (AUC) and retention time was calculated by DAx 8.1. software. For accuracy study, sample of 10 µl inactivated cold buffer horseradish extract/100 µl volume was separately spiked with 1000 µg/ml standards and the recoveries were calculated. The absence of residual glucosinolates in these plant extracts was confirmed by injecting samples without adding glucosinolate standards. 3.2.4. Quantification of GLSs and AITC from real plant matrix Brussels sprouts, horseradish, radish and watercress methanol extract were measured by capillary electrophoresis (long end injection mode) for glucosinolate profile and LC-ESI-MS for sinigrin and gluconasturtiin comparative study. Glucosinolate determination by LC-ESI-MS was done on a Thermo Accela HPLC attached to a Thermo LTQ XL Linear Ion Trap MS, column: Kinetex XB-C18 (100 × 2.10 mm, 2.6 µm, Phenomenex). Five-point calibration curves of sinigrin and gluconasturtiin in water ranging from 0.5 to 40 µg/mL were used as calibration curves. Gradient components were A, water with 0.1% (v/v) formic acid; B, MeCN with 0.1% (v/v) formic acid. The time program was 5% B: 0 – 1 min, 5 – 25% B: 1 – 4 min, 25 – 60% B: 4-5 min, 60 – 5% B: 5 – 6 min, 5% B: 6-8 min. Flow rate was 250µL/min. 1µL of the 52 diluted methanol extract was injected, typically 5-100-fold dilutions with water were appropriate. The instrument was tuned automatically for sinigrin to obtain the optimal ESI parameters. ESI ionization parameters were as follows: capillary temperature, 275 °C; source heater temperature, 300 °C; sheath gas, N2; sheath gas flow, 30 arbitrary units (arb); aux gas flow, 5 arb; source voltage, 3 kV; capillary voltage, -1.00 V, negative ion mode. The new CE method is applied on the study of AITC content of food products (i.e., mustard sauce, two types of horseradish sauces and wasabi cream). The samples were composed of 100 mg of food product diluted with 100µl buffered deodorization solution ( NaH2PO4 (100 mM), ascorbic acid (10 mM), mercaptoacetic acid (50 mM), pH 7.5) and 800 µl water. After mixing and centrifugation, the supernatant was introduced directly to CE (long-end injection mode) for measurement. pH readjustment (to 7.5) is necessary if the products contain significant amount of vinegar. 3.2.5. Myrosinase activity determination and AITC release study The myrosinase activity study was designed as following: The myrosinaseinactivated plant extract obtained by buffer extraction of boiled plant, served as negative controls. The fresh plant extracts by cold buffer were diluted with buffer solution ( 100 mM NaH2PO4, 10 mM ascorbic acid, pH 6.50) in 9:1 and water. 5 µL GLS stock (10 mg/ml) was added to 190 µL of previous mixture. Addition of the substrate was the reaction start point, negative controls were obtained by using plant extracts that were previously boiled to inactivate the myrosinase. The reaction was run at 25 °C for 5 minutes, and then terminated by heating the test tubes to 100 °C for 5 minutes, followed by the sinigrin 53 concentration determination by CE after centrifugation at 13000 rpm for 1 min. Substrate concentration decrease was kept below 10%. The plant extracts were also checked for the presence of residual substrates (no sinigrin added). Protein determination from these extracts was done using Bradford's reagent with bovine serum albumine as standard. Under the same conditions, a series of different initial sinigrin concentrations were tested to obtain the Km constant for myrosinase. Tested initial concentrations were 20, 35, 50, 65, 100, 150, 225, 300µg/ml, the determination was run in three replicates. The added myrosinase containing 50-fold diluted horseradish extract was allowed to decompose sinigrin for 5, 8.75, 12.5, 16.25, 25, 37.5, 56.25, 75 minutes, respectively, keeping decomposed substrate below 10%, allowing the estimation of the initial reaction rate (v0). The Km value was calculated by fitting the Michaelis Menten equation (v0 = vmax[S] / Km+[S]) to the obtained data. For non-linear curvefitting, the nls package in R was used (n=3). The same vegetable extracts were also assayed for myrosinase activity by the widely used pH stat assay (Piekarska et al., 2013). The reaction mixture was the same as that for CE, except that it was not buffered: to 7.66 mL of water 80 µL of ascorbic acid solution (100 mM, pH adjusted to 6.50 with NaOH), 80 µL of plant extract (diluted if necessary) was added. After the pH drift stopped after a few minutes, the reaction was initialized by addition of the substrate (final concentration: 250 µg/mL). Thereafter, freshly prepared 1 µM NaOH was added under slow constant stirring to keep the pH at 6.50. The amount of NaOH consumed by the released H+ during glucosinolate decomposition was registered for 5 minutes. Extracts of the four vegetables (Brussels sprouts, horseradish, radish, watercress) were compared for the sinigrin aglycon – allyl isothiocyanate conversion rate. The reaction mixture was: 10 µL of buffered 54 derivatization solution (100mM NaH2PO4, 10 mM ascorbic acid, 50 mM mercaptoacetic acid, pH 7.5), 60 µL bidistilled water, 10 µL enzyme containing extract (not diluted) and 20 µL of sinigrin stock solution (10 mM). The experiment was designed to result in 2 mM allyl isothiocyanate if the conversion ratio is 100%. 3.3. Result and discussion 3.3.1.Optimization of separation A good resolution for GLSs (sinigrin / gluconasturtiin) was observed in horseradish methanol extract using the starting BGE and long end injection mode. However, the method could be further developed in the aspects of decreasing measuring time and increasing sensitivity. Completely removing or decreasing the concentration of BGE 's components can shorten the measuring time due to the possibility to increase the voltage. Completely removing of borate and decreasing sodium deoxycholate concentration (from 250 to 175 mM) showed no effect on the resolution between GLSs. Further decrease of analysis time is also achieved by increasing pH to 9.0, i.e., giving the ability to increase electroosmotic flow (EOF). At pH 9.0, CHES (N-Cyclohexyl-2aminoethanesulfonic acid) was used as a buffering agent (significant buffering capacity, low UV absorption). Other strategies such as addition of organic solvents, employing different surfactants (also as mixtures) resulted in loss of resolution and/or sensitivity for some analytes of interest. 55 Figure 16. Electropherograms of the different methods used during optimization on PrinCE-C 700 capillary electrophoresis. a) Background electrolyte was 20 mM TAPS, 15 mM Borate, 250 mM SDC, pH 8.5. b) Background electrolyte was 20 mM TAPS, 250 mM SDC, pH 8.5. c) Background electrolyte was 20 mM TAPS, 175 mM SDC, pH 8.5. 56 The most effective BGE contains CHES (20 mM), sodium deoxycholate (175 mM), pH 9.0, applied voltage 20 kV. It showed good resolution for sinigrin / gluconasturtiin, and no major interfering peak in the methanol extract of horseradish. Although AITC was separated from GLSs, because of its low specific absorbance, the limit of detection is so high which will be the problem for further study. Increasing the sensitivity of the method for AITC was the next step for method improvement. Figure 17. Electropherogram of a myrosinase inactivated (cooked) horseradish root extract, spiked with 500 ppm allyl isothiocyanate without derivatization, using the proposed CE-MEKC procedure in long-end injection, with 52 cm effective length. Background electrolyte was 20 mM CHES, 175 mM SDC, at pH 9.0. As detection wavelength, 230 nm was used. Note good resolution between GLSs, and low sensitivity and resolution for the underivatized AITC. Abbreviations: AITC, allyl isothiocyanate; G, gluconasturtiin (phenethyl glucosinolate); S, sinigrin; uG, unidentified glucosinolate. 3.3.2. Derivatization study 57 Table 4. Glucosinolate Content of Four Tested Vegetables, as Measured by the Proposed CE Method, or by LC-ESI-MS. Vegetable CE LC-ESI-MS CE LC-ESI-MS Sinigrin (µg/g (FW)) Gluconasturtiin (µg g-1 (FW)) Radish n.d n.d n.d n.d Brussels sprout 161 148.2 n.d n.d Watercress n.d n.d 162.9 153.7 Horseradish 2291.8 2784.4 248.5 244.3 Abbreviations: CE, capillary electrophoresis; FW, fresh weight. The application of proposed method showed the tested commercial condiments contain 369 – 418 µg/g AITC. The method provided a good resolution with simple sample preparation procedure, as plotted in figure 22. The presented long end injection method separates the analytes of interest within 15 minutes from real matrices. This falls in the range of the fastest HPLC methods available for determination of sinigrin and allyl isothiocyanate (Tsao et al. 2002). Typical methods last 20-25 minutes or more, consume 1mL/min solvent and require the removal of protein and fat before analysis (Budnowski et al. 2013; Herzallah and Holley 2012). Direct, simultaneous quantifications from complex matrices can be even longer (Song et al. 2005). Isothiocyanate determination – even when derivatized – can also be time demanding. The presented method is similar in speed to the fastest available CE methods for glucosinolates, an analysis time of 15-25 minutes is typical (Karcher and El Rassi 1999). However, the separation of isothiocyanate adducts usually requires more time (Bjergegaard et al. 1999). With the new method, separation of allyl isothiocyanate dithiocarbamate was also done within 15 minutes. The CE 64 screening is also faster as compared to GC-MS methods. The usual time of a GC-MS for different isothiocyanates measurement is 30-35 minute (Zhao, Tang, and Ding 2007). Figure 22. Detection of allyl isothiocyanate from food products in long-end injection mode. a., mustard (condiment); b., horseradish sauce with wasabi. Electropherograms obtained using the proposed capillary electrophoresis – micellar electrokinetic chromatography (CE-MEKC) method showing possible applications. Background electrolyte: CHES (20 mM), sodium deoxycholate (175 mM), pH 9.0. Isothiocyanates are present as dithiocarbamates during separation. Sample matrices: phosphate (10 mM, pH 7.5), ascorbic acid (1 mM), mercaptoacetic acid (5 mM). 3.3.5. Study of myrosinase activity and allyl isothiocyanate release of vegetable extracts The average Km values obtained by non-linear regression analysis of reaction velocity versus sinigrin concentration (at pH 6.50, 25 °C, 0.1 mM ascorbic acid) fell in the range of 0.129 ± 0.025µM. This is the same order of magnitude found for sinigrin in several previous studies for different myrosinases, thus it can be stated that the presented activity is truly that of myrosinase (Li and Kushad 65 2005; Nehmé et al. 2014). The activities (expressed in µmol sinigrin decomposed per minute (U)) of tested vegetables are shown in the table below. Activity of sinigrin decomposition ranged from 4.42 U/g fresh weight (watercress) to 208.26 U/g fresh weight (horseradish) in 10 mM phosphate, 1 mM ascorbic acid, pH 6.50, 25 °C, initial substrate concentration: 250µg/ml. The method was shown to be suitable to measure myrosinase activity from low activity mixtures without major interferences. The obtained myrosinase activities with sinigrin as the substrate were compared to those found in the widely used pH-stat assay. Under the same conditions (1 mM ascorbic acid, pH 6.50, at 25 °C, initial substrate concentration: 250 µg/mL), the myrosinase containing extracts of the vegetables had very similar activity (93.7% – 116.9%, average: 107.1%, table 5). Table 5. Myrosinase Activities of Four Tested Vegetables Using Sinigrin or Gluconasturtiin as Substrate, Measured By the Proposed CE Method and pH Stat Assay Vegetable Sinigrin, CE (U/g FW) Sinigrin, CE (U/mg protein) Sinigrin, pH stat (U/g FW) Sinigrin, pH stat (U/mg protein) Radish 10.31±1.31 3.41±0.43 11.00±0.94 3.64±0.31 Brussels sprout 7.72±0.6 0.96±0.07 6.83±0.24 0.85±0.03 66 Watercress 4.42±0.5 0.76±0.09 4.22±0.94 0.73±0.16 Horseradish 208.26±42.94 27.69±5.71 178.13±4.42 23.69±0.59 Vegetable Gluconasturtii n, CE (U/g FW) gluconasturtii n, CE (U/mg protein) S/G AR* ITC release (%) Radish 5.35±1.02 1.77±0.34 1.93 92±4.39% Brussels sprout 3.71±0.09 0.46±0.01 2.08 73.13±0.27% Watercress 6.01±0.12 1.04±0.02 0.73 102.13±0.94% Horseradish 197.94±33.98 26.32±4.52 1.05 98.25±3.02% One unit (U) of activity is defined as 1µmol per minute. Abbreviations: CE, capillary electrophoresis; FW, fresh weight; S/G AR: Ratio of myrosinase activity with sinigrin as the substrate / gluconasturtiin as the substrate. The presented values are mean ± SD of three measurements. Comparing the activity ratio of the same extracts with sinigrin and gluconasturtiin as the substrate, some level of specificity can be found: Brussels sprouts that contains sinigrin but no gluconasturtiin (Table 5) had 2.08-fold activity against sinigrin as compared to gluconasturtiin. Watercress containing gluconasturtiin but no sinigrin (Table 5) also showed some specificity towards its own glucosinolate. Horseradish, which contains both glucosinolates (Table 5), decomposed both with similar efficacy. Though the measured specificity is not that striking as it was previous described for Crambe abyssinica, it suggests that using sinigrin as the sole substrate for myrosinase activity determination may sometimes result in serious underor overestimation of the biologically 67 relevant myrosinase activity of plant extracts. Therefore, if possible, methods capable of using substrates other than sinigrin should be integrated into myrosinase activity tests. The phenomenon clearly requires more in-depth study, for which the current method can be used. 3.3.6. Discussion There are popular methods for myrosinase measurement, most of them are based on spectrophotometry instead of chromatographic separation. These include the indirect measurement of the glucose released (Wilkinson, Rhodes, and Fenwick 1984) or measuring the breakdown kinetics of the substrate (decrease of absorbance maximum). It is also possible to use the released H+ for quantification in a pH-stat assay (Piekarska et al. 2013). As glucosinolate absorbance maxima are usually around 210-230 nm, many compounds can interfere with quantification in UV-Vis, especially when using more concentrated raw extracts. Higher specificity can only be achieved by subjecting the reaction mixture to chromatographic separation. For this purpose, the CE methods can be used. They operate with a minimal amount of sample and are able to study decomposition of different glucosinolates, but neither can give information on the ITC release rate that is of primal biological significance. HPLC methods have excellent reproducibility and sensitivity, but are frequently time consuming and require much more reagents than CE measurements. The reaction mixtures used in this study are simple enough to use short-end injection, which results in less, but sufficient resolution, and less analysis time. In this case, the presented method is capable of separating a glucosinolate – isothiocyanate pair from the reagents in 2.5 minutes. With a capillary reconditioning applied after every sixth injection, a number of about 68 twelve injections per hour can be reached. This is comparable to the widely used assays' time demand, yet, chromatographic separation takes place, which gives the least interferences from for example ascorbic acid. For many vegetables, conversion of sinigrin to allyl isothiocyanate was not found to be 100% in the literature. The isothiocyanate yield from the glucosinolate aglycon can range from a few percent to near 100% (Piekarska et al. 2013). This is usually attributed to the presence of specifier proteins that cause the glucosinolate aglycon to rearrange into different volatile products such as nitriles, epithionitriles, thiocyanates among others. In the current study, four vegetable extracts (Brussels sprouts, horseradish, radish, watercress) were successfully compared for ITC conversion rate, with minimal amount of reagents. The procedure also does not require laborious sample preparation (e.g. liquid-liquid extraction) needed to study the ITC content by GC-MS as in previous studies. The amount of allyl isothiocyanate generated from the same amount of added sinigrin (1mM) significantly differed among the vegetable extracts (p<0.05, n=3, ANOVA). Conversion rate was found to range between 73.13±0.27% and 102.13±0.94%. The HPLC method successfully separates allyl isothiocyanate and sinigrin from model matrices with active myrosinase within 6 minutes, and was successfully used to measure ITC release (Vastenhout et al. 2014). However, it was not tested for the ability to measure myrosinase activity or ITC release from real matrices, and operates with a 1 mL/min solvent flow. Testing of real matrices would also require the removal of protein and fat as sample preparation steps. The advantages of the proposed CE method include saving time and solvents as compared to many HPLC methods. Hence, it is suitable as a screening method for glucosinolates and allyl isothiocyanate. The method was used as a higher specificity myrosinase assay 69 that also allows quantification of on-line generated isothiocyanates, the main bioactive products. Only 25-50 µg glucosinolate per sample is sufficient for a myrosinase study, which is especially important in the case of glucosinolates other than sinigrin. Analysis of factors affecting the glucosinolate – isothiocyanate conversion rate is also possible. 70 Chapter IV Comparative Analysis Of Armoracia rusticana And Armoracia macrocarpa 1. BACKGROUND INFORMATION 1.1. Myrosinase Myrosinase' s structure and properties – As mentioned earlier, myrosinase plays a crucial role in the hydrolysis reaction of GLS. Horseradish myrosinase (β-thioglucoside glucohydrolase) is an S-glucosidase enzyme whose (β/α) 8barrel structure consists of two similar subunits with a molecular weight of 65 kDa linked by a zinc atom (Zhang 2010). To make contact with GLS, horseradish myrosinase requires a hydroxyl group on C2 of the glucose moiety of GLS and a nucleophilic glutamate for catalytic activity (Fahey, Zalcmann, and Talalay 2001). In Brassicaceae, myrosinases are generally classified based on the place where they can be found in the plant. There are three subgroups in this enzyme family, myrosinase A, myrosinase B, and myrosinase C (MA, MB, and MC, respectively). MA and MC can be found only in the seed tissue, whereas MB can be found in most tissues of the plant. Considering this point, horseradish myrosinase should belong in the MB subgroup but classification gets more complicated because horseradish myrosinase possesses a water-soluble property, whereas MB myrosinase does not. This leads to the hypothesis that horseradish myrosinase may belong to an unidentified subgroup of the myrosinase family. Myrosinase is not substrate specific. Horseradish myrosinase not only catalyzes the hydrolysis of GLSs found in horseradish such 71 as sinigrin, but can also catalyze hydrolysis of GLSs found in different other species (e.g. in broccoli), with rates depending on the substrate (Li and Kushad 2005). Myrosinase' s activities – The optimum conditions for activity of myrosinase are when the temperature of the reaction falls in the range of 37–45oC and pH in the range of 5–8. Horseradish myrosinase activity was observed to increase at 23oC, remaining high (i.e. >80% of maximum) from 37oC, reaching its maximum at 45oC, decreasing at temperatures over 50oC, and the enzyme becomes inactive at temperatures above 70oC. Within the pH range, increasing myrosinase activity was noticed from a pH of 3–4, reaching maximum at pH 5.7 and the activity remains high (i.e. >80% of maximum) at pH range 5–8. Addition of 0.5mM ascorbic acid can significantly stimulate horseradish myrosinase, possibly due to a conformation change in the enzyme leading to increased substrate affinity of horseradish myrosinase and reaction velocity (Bones and Rossiter 2006). Loss in endogenous ascorbic acid was also suggested to cause the decrease of myrosinase activity (A. Depree, M. Howard, and P. Savage 1998). In another study on myrosinase isolated from horseradish grown in China, the best condition for myrosinase activity is at temperature of 65oC and pH 4.0, with ascorbic acid (2 mg/g powdered root) added and an incubation period of 120 min (Zi-Tao Jiang 2006). The inconsistency on the details required for optimal activity between these two studies may be related to differences in geographic regions where the horseradishes were grown. However, it can be concluded that temperature, pH, and ascorbic acid content are the main factors affecting the activity of the enzyme. 1.2. Peroxidase 72 Structure and properties – Apart from myrosinase, horseradish contains another enzyme that has gained interest: horseradish peroxidase (HP) – a hemecontaining enzyme that utilities hydrogen peroxide to oxidize a wide variety of organic and inorganic compounds, due to its larges-scaled commercial uses, for example as a reagent for organic synthesis and bio-transformation, as in coupled enzyme assays , chemiluminescent assays, immunoassay and the treatment of waste water. Fifteen HP isoenzymes have been identified from horseradish root. Based on their isoelectric point values, these HPs are referred to by codes as A1-3 (acidic), B1-3 and C1-C2 (neutral basic) and E1-E6 (basic). Among those, the C isoenzyme is the most abundant. Horseradish peroxidase isoenzyme C (HRPC) comprises a single polypeptide of 308 amino acid residues. The structure of the enzyme is largely α-helical and small region of β-sheet. HRPC contain the heme group (iron (III) protoporphyrin IX), located between the distal and proximal calcium binding domains. These metal centers are crucial for the structural and functional integrity of the enzyme. Although so little is known about the function of HP in plant, it is believed to be involved in the conversion of hydrogen peroxide to water and used by plants to regulate level of intracellular hydrogen peroxide. The radical products from HRP-calalysed reactions possibly involve cross-linking reactions (e.g. the formation of diferulate linkages from polymer-attached ferulate groups of polysaccharides or pectins, the formation of dityrosine linkages, etc.), which may be expressed in response to external factors such as the sounding of plant tissue. Water loss and invasion by pathogens can therefore be limited by the formation of a protective polymeric barrier such as suberin (Veitch 2004). 73 i.e. the narrow core of primary xylem is surrounded by an extensive but mainly parenchymatous secondary xylem in which a few tracheary elements occur (figure 23). The well-defined vascular cambium also produces centrifugally a largely parenchymatous secondary phloem. IKI stained cross sections revealed starch present in cross sections based on the reaction of iodine and the center of the helical starch molecules, giving the dark blue color (longer molecules) or more red color (shorter molecules; figure 24). The oil content in the cross sections of A. rusticana and A. macrocarpa were investigated by exploiting the fat-soluble property of Sudan III stain, which is physically attracted to hydrophobic structures (figure 25, 26). These cross-sections showed the similarity in structure of fresh roots from both species – A. rusticana and its relative, A. macrocarpa. 80 Figure 23. Cross sections of the fleshy roots of Armoracia rusticana and Armoracia macrocarpa stained with toluidine blue solution. The images were viewed with Olympus Provis AX70/A microscope. Figure 24. Cross sections of the fleshy roots of Armoracia rusticana and Armoracia macrocarpa stained with IKI solution. The images were viewed with Olympus Provis AX70/A microscope. 81 Figure 25. Cross section of the fleshy roots of Armoracia rusticana stained with Sudan III solution. The images were viewed with Olympus Provis AX70/A microscope. 82 Figure 26. Cross section of the fleshy roots of Armoracia macrocarpa stained with Sudan III solution. The images were viewed with Olympus Provis AX70/A microscope. 4. THE GEL ELECTROPHORESIS STUDIES Gel electrophoresis is a flexible method for separation and analysis of proteins, nucleic acids and other charged molecules. In electrophoresis, charged molecules are traveled through a porous gel by an applied electric field generated in a buffer which permeates the gel, and are separated based on their different electrophoretic mobilities. Variations in the gel and buffer make it possible to separate molecules not only based on their charges, but also on their molecular weight, isoelectric point and bio-specific affinity. The technique is fast, convenient, and inexpensive, and is used both as an analytical method and as a preparative procedure in the final stages of purification. 83 4.1. Materials and method Roots and leaves of A. rusticana and A. macrocarpa were grind by commercial electric mixer. The material (approx. 1 g) was then transferred to 2 ml Eppendorf tubes, followed by the addition of 1mL buffer (NaH2PO4/Na2HPO4 20 mM, 4oC, pH 6.55, VWR International Ltd.). The mixtures were aggressively mixed and centrifuged at 13000 rpm for 30 min using Heraeus Biofuge in order to obtain the supernatant. The protein content of the supernatant was assayed by the method of Bradford. 40 μg protein was loaded into each well of native 75% polyacrylamide gels. Electrophoresis was performed at 4oC. For myrosinase activity study, the gel was washed with distilled water and stained with solution (pH 8) containing 0.25 mL 20 mM KH2PO4/K2HPO4, 0.05 ml (169 mg/10 ml) Ascorbic acid, 0.5 mL 0.1% dye solution (methyl red), 1.25 (10 mg/mL) sinigrin and 2.95 mL water for 1-2 min. The myrosinase isoenzymes' band intensity were evaluated by ImageJ® and CP Atlas version 1.01 software. The data was graphically presented by Sigma plot 11.0 and Libreoffice Calc software. The activity of peroxidase was investigated using spectrophotometry (SHIMADZU, UV-1601). Each sample for spectrometry contains 970 μL 50mM KH2PO4/K2HPO4, 5 μL 3%H2O2, 20 μL 1 M pirogallol and 5 μL peroxidase enzyme from the supernatant. 4.2. Results and discussions Peroxidase activity – Data obtained from spectrometry measurement (figure 27) compared the peroxidase activity in leaves, young root and old root from A. rusticana and A. macrocarpa collected in july. In A. macrocarpa, the highest activity was observed in the old root (801.05 ± 31.04 ΔOD/min/mg protein), 84 followed by the young root (412.6 ± 42.4 ΔOD/min/mg protein) and the least activity was in the leave (55.75 ± 1.43 ΔOD/min/mg protein). In A. rusticana, the same order of peroxidase activities were measured, i.e. the highest activity was recored in the old root (671.25 ± 31.5 ΔOD/min/mg protein), followed by the young root (206.95 ± 1.2 ΔOD/min/mg protein) and the least activities was measured in the leave (90.6 ± 2.4 ΔOD/min/mg protein). According to spectrometry results, the peroxidase activity in A. macrocarpa was higher compared to that in A. rusticana. Different results were recorded in other measurements with roots and leaves of both species collected in octorber, in which peroxidase activity was higher in A. rusticana (352.8 ± 1.4 ΔOD/min/mg protein, 16.4 ± 0.01ΔOD/min/mg protein in root and leaf, respectively) compared to that of A. macrocarpa (232.9 ± 0.57 ΔOD/min/mg protein, 27.72 ± 0.18 ΔOD/min/mg protein in root and leaf, respectively). The different results in these studies could be related to age and time of harvesting of roots. Figure 27. a) Peroxidase activities measured by spectrophotometry. (A.m: Armoracia macrocarpa, A.r: Armoracia rusticana, L: Leaf, j.R: young root, o.R: old root). b) Peroxidase (E.C. 1.11.1.7) activity was visible due to dark red-colored purpurogallin bands showed on the gel. Picture was taken by 85 a) b) a) b) a) Olympus 4040 camera. Myrosinase activity – The data on myrosinase activity was calculated based on the band intensities on the PEG gel (pixel per area – ppa). In A. rusticana, myrosinase activities in the young root and leave were similar (1378.67 ± 113.2 ppa and 1342 ± 49.66ppa). The highest activity was measured in the old root (1881 ± 110ppa). In case of A. macrocarpa, the enzymatic activity was higher in the roots (1628 ± 31.32ppa), in which the old root has the higher activity, similarly to the case of A. rusticana. Comparing 2 species, myrosinase activity was overall higher in A. rusticana compared to its relative. The highest myrosinase activity measured in A. macrocarpa old roots was similar to the activity measured in leave and young roots of A. rusticana (figure 28). Figure 28. a) Gel electrophoresis study on the myrosinase activity in A. rusticana and its relative, A. macrocarpa. (1: A. rusticana young root, 2: A. rusticana old root, 3: A. rusticana leaf, 4: A. macrocarpa young root, 5: A. 86 b) a) macrocarpa old root, 6: A. macrocarpa leaf). b) Myrosinase activity measured by band intensities on gel. (A.r: Armoracia rusticana, A.m: Armoracia macrocarpa, L: leaf, y.R: young root, o.R: old root). Picture was taken by Olympus 4040 camera. 87 Chapter V Summary The first part of this study came up with a new technology to extracting high yield essential oil from fresh horseradish roots. With this method, for 15 kg of fresh horseradish root, 12ml of essential oil can be extracted (the yield of 0.08%). Together with the stable and high yield, the ease of use and its simplicity make sure that the new method is suitable for the horseradish essential oil production. Investigation on the quality of horseradish essential oil by gas chromatography and mass spectrometry showed the presences in high amount of 2 main substances: allyl isothiocyanate and 2-phenethyl isothiocyanate. The other three identified isothiocyanates are sec-butyl-, 3butenyland 4-pentenyl isothiocyanate. Investigation on the completion of the extraction (no glucosinolate / isothiocyanate found in the post-distilled mush) and the “trapped” content of isothiocyanate (5 ppm) in watery extract by gas chromatography and mass spectrometry confirmed that the new method is recommended for large scaled horseradish oil production. The second part of the study focuses on the development of new capillary electrophoresis (CE-MECK) method for simultaneous quantification of glucosinolates and isothiocyanates. The assay in short-end injection mode enables myrosinase quantification as well as glucosinolate' aglycon to isothiocyanate conversion rate estimation. The method uses sinigrin or gluconasturtiin as substrate, the main products of interest are derivatized to a more sensitively detectable dithiocarbamate product. It can also be a good alternative to established methods to quantify myrosinase activity from raw plant materials and similar matrices, as well as characterization of soluble 88 myrosinase enzymes, with respect to, for example, substrate specificity and pH optima. The method combines many advantages of frequently used methods: the specificity of chromatographic separations and the simplicity, low cost and time demand that is the property of the spectrophotometric assays. The final part of this study compares the anatomical structure, glucosinolate profile and the enzymatic activities in both A. rusticana and A. macrocarpa. Study on cross sections stained with toluidine blue solution, IKI solution and Sudan III from A. rusticana and A. macrocarpa showed the similarity in anatomical structures of the root of both species. Study on glucosinolate profile in both species by liquid chromatography and mass spectrometry showed that there are 6 identified glucosinolates in A. rusticana and 16 glucosinolates were tentatively identified in A. macrocarpa. Gluconasturtiin, glucobrassicin, glucocochlearin, glucoconringianin and glucoibarin are the five glucosinolates found in both species. The absence of sinigrin (parent glucosinolate of allyl isothiocyanate) can be the possible explanation for the difference in the taste and smell of these plants. The activities of peroxidase enzyme were recorded higher in A. rusticana compared to A. macrocarpa studied by spectrophotometry. However, the opposite results were also recorded, suggesting that peroxidase activity of both species heavily depends on the age and the time of harvesting of the roots. The old root had the highest activity compared to the young root and the leaf, where the lowest peroxidase activity was measured. The gel electrophoresis study on myrosinase enzyme activity showed higher activity in A. rusticana compared to A. macrocarpa. In A. rusticana, the old root had higher myrosinase activity than the young root and leaf, which had similar myrosinase activity. In A. 89 Oxide Production in Lipopolysaccharide-Activated Macrophages.” Life Sciences 71 (4): 411–19. doi:10.1016/S0024-3205(02)01685-5. Jeong, Woo-Sik, Young-Sam Keum, Chi Chen, Mohit R. Jain, Guoxiang Shen, Jung-Hwan Kim, Wenge Li, and Ah-Ng Tony Kong. 2005. “Differential Expression and Stability of Endogenous Nuclear Factor E2-Related Factor 2 (Nrf2) by Natural Chemopreventive Compounds in HepG2 Human Hepatoma Cells.” Journal of Biochemistry and Molecular Biology 38 (2): 167–76. Karcher, A., and Z. El Rassi. 1999. “Capillary Electrophoresis of Glucosinolates and Their Degradation Products.” Electrophoresis 20 (15–16): 3181–89. doi:10.1002/(SICI)1522-2683(19991001)20:15/16<3181::AIDELPS3181>3.0.CO;2-G. KJZER, ANDERS. 1963. “Mass Spectra of Isothiocyanates.” Acta Chem. Scand 17 (8). Kleinwächter, Maik, and Dirk Selmar. 2004. “A Novel Approach for Reliable Activity Determination of Ascorbic Acid Depending Myrosinases.” Journal of Biochemical and Biophysical Methods 59 (3): 253–65. doi:10.1016/j.jbbm.2004.03.005. Li, Xian, and Mosbah M. Kushad. 2005. “Purification and Characterization of Myrosinase from Horseradish (Armoracia Rusticana) Roots.” Plant Physiology and Biochemistry 43 (6): 503–11. doi:10.1016/j.plaphy.2005.03.015. Luciano, Fernando B, and Richard A Holley. 2009. “Enzymatic Inhibition by Allyl Isothiocyanate and Factors Affecting Its Antimicrobial Action against Escherichia Coli O157:H7.” International Journal of Food Microbiology 131 (2–3): 240–45. doi:10.1016/j.ijfoodmicro.2009.03.005. 96 Mohlenbrock, Robert H. 1980. Flowering Plants: Willows to Mustards. SIU Press. Murata, M., N. Yamashita, S. Inoue, and S. Kawanishi. 2000. “Mechanism of Oxidative DNA Damage Induced by Carcinogenic Allyl Isothiocyanate.” Free Radical Biology & Medicine 28 (5): 797–805. Nehmé, Reine, Hala Nehmé, Grégory Roux, Deimante Cerniauskaite, Philippe Morin, Patrick Rollin, and Arnaud Tatibouët. 2014. “Contactless Conductivity Detection for Screening Myrosinase Substrates by Capillary Electrophoresis.” Analytica Chimica Acta 807 (January): 153– 58. doi:10.1016/j.aca.2013.11.012. Nielsen, P. V., and R. Rios. 2000. “Inhibition of Fungal Growth on Bread by Volatile Components from Spices and Herbs, and the Possible Application in Active Packaging, with Special Emphasis on Mustard Essential Oil.” International Journal of Food Microbiology 60 (2–3): 219–29. Oerlemans, Kirsten, Diane M. Barrett, Carme Bosch Suades, Ruud Verkerk, and Matthijs Dekker. 2006. “Thermal Degradation of Glucosinolates in Red Cabbage.” Food Chemistry 95 (1): 19–29. doi:10.1016/j.foodchem.2004.12.013. Ohta, Yoshio, Kenichi Takatani, and Shunro Kawakishi. 1995. “Decomposition Rate of Allyl Isothiocyanate in Aqueous Solution.” Bioscience, Biotechnology, and Biochemistry 59 (1): 102–3. doi:10.1271/bbb.59.102. Piekarska, Anna, Barbara Kusznierewicz, Magdalena Meller, Karol Dziedziul, Jacek Namieśnik, and Agnieszka Bartoszek. 2013. “Myrosinase Activity in Different Plant Samples; Optimisation of Measurement Conditions for Spectrophotometric and pH-Stat Methods.” Industrial Crops and 97 Products 50 (October): 58–67. doi:10.1016/j.indcrop.2013.06.048. Sampliner, Danielle, and Allison Miller. 2009. “Ethnobotany of Horseradish (Armoracia Rusticana, Brassicaceae) and Its Wild Relatives (Armoracia Spp.): Reproductive Biology and Local Uses in Their Native Ranges.” Economic Botany 63 (3): 303–13. doi:10.1007/s12231-009-9088-1. Shehata, Ashraf, Richard M. S. Mulwa, Mohammad Babadoost, Mark Uchanski, Margaret A. Norton, Robert Skirvin, and S. Alan Walters. 2009. “Horseradish: Botany, Horticulture, Breeding.” In Horticultural Reviews, edited by Jules Janick, 221–261. John Wiley & Sons, Inc. http://onlinelibrary.wiley.com/doi/10.1002/9780470593776.ch4/summar y. Shin, Il Shik, Hideki Masuda, and Kinae Naohide. 2004. “Bactericidal Activity of Wasabi (Wasabia Japonica) against Helicobacter Pylori.” International Journal of Food Microbiology 94 (3): 255–61. doi:10.1016/S0168-1605(03)00297-6. Singh, Ajita V., Dong Xiao, Karen L. Lew, Rajiv Dhir, and Shivendra V. Singh. 2004. “Sulforaphane Induces Caspase-Mediated Apoptosis in Cultured PC-3 Human Prostate Cancer Cells and Retards Growth of PC-3 Xenografts in Vivo.” Carcinogenesis 25 (1): 83–90. doi:10.1093/carcin/bgg178. Smith, Tracy K., Elizabeth K. Lund, Mary L. Parker, Rosemary G. Clarke, Ian T. Johnson, and Nr Ua. 2004. Allyl Isothiocyanate Causes Mitotic Block, Loss of Cell Adhesion and Disrupted Cytoskeletal Structure in HT29 Cells. Song, Lijiang, John J. Morrison, Nigel P. Botting, and Paul J. Thornalley. 2005. “Analysis of Glucosinolates, Isothiocyanates, and Amine Degradation Products in Vegetable Extracts and Blood Plasma by LC-MS/MS.” 98 Analytical Biochemistry 347 (2): 234–43. doi:10.1016/j.ab.2005.09.040. Srivastava, Sanjay K, Dong Xiao, Karen L Lew, Pamela Hershberger, Demetrius M Kokkinakis, Candace S Johnson, Donald L Trump, and Shivendra V Singh. 2003. “Allyl Isothiocyanate, a Constituent of Cruciferous Vegetables, Inhibits Growth of PC-3 Human Prostate Cancer Xenografts in Vivo.” Carcinogenesis 24 (10): 1665–70. doi:10.1093/carcin/bgg123. Sultana, Tamanna (author), G. P. (author) Savage, D. L. (authorDNRE McNei, N. G. (author) Porter, and B. (author) Clark. 2003. “Comparison of Flavour Compounds in Wasabi and Horseradish.” http://agris.fao.org/agris-search/search.do?recordID=FI2016100219. Tagliaro, F, G Manetto, F Crivellente, and F. P Smith. 1998. “A Brief Introduction to Capillary Electrophoresis.” Forensic Science International 92 (2–3): 75–88. doi:10.1016/S0379-0738(98)00010-3. Tang, Li, and Yuesheng Zhang. 2004. “Dietary Isothiocyanates Inhibit the Growth of Human Bladder Carcinoma Cells.” The Journal of Nutrition 134 (8): 2004–10. ———. 2005. “Mitochondria Are the Primary Target in Isothiocyanate-Induced Apoptosis in Human Bladder Cancer Cells.” Molecular Cancer Therapeutics 4 (8): 1250–59. doi:10.1158/1535-7163.MCT-05-0041. Tsao, Rong, Qing Yu, John Potter, and Mikio Chiba. 2002. “Direct and Simultaneous Analysis of Sinigrin and Allyl Isothiocyanate in Mustard Samples by High-Performance Liquid Chromatography.” Journal of Agricultural and Food Chemistry 50 (17): 4749–53. Tunc, S., E. Chollet, P. Chalier, L. Preziosi-Belloy, and N. Gontard. 2007. “Combined Effect of Volatile Antimicrobial Agents on the Growth of Penicillium Notatum.” International Journal of Food Microbiology 113 99 (3): 263–70. doi:10.1016/j.ijfoodmicro.2006.07.004. Valgimigli, Luca, and Renato Iori. 2009. “Antioxidant and pro-Oxidant Capacities of ITCs.” Environmental and Molecular Mutagenesis 50 (3): 222–37. doi:10.1002/em.20468. Vastenhout, Kayla J., Ruthellen H. Tornberg, Amanda L. Johnson, Michael W. Amolins, and Jared R. Mays. 2014. “High-Performance Liquid Chromatography-Based Method to Evaluate Kinetics of Glucosinolate Hydrolysis by Sinapis Alba Myrosinase.” Analytical Biochemistry 465 (November): 105–13. doi:10.1016/j.ab.2014.07.017. Veitch, Nigel C. 2004. “Horseradish Peroxidase: A Modern View of a Classic Enzyme.” Phytochemistry 65 (3): 249–59. Wilkinson, A. P., M. J. Rhodes, and G. R. Fenwick. 1984. “Determination of Myrosinase (Thioglucoside Glucohydrolase) Activity by a Spectrophotometric Coupled Enzyme Assay.” Analytical Biochemistry 139 (2): 284–91. Xiao, Dong, Sanjay K. Srivastava, Karen L. Lew, Yan Zeng, Pamela Hershberger, Candace S. Johnson, Donald L. Trump, and Shivendra V. Singh. 2003. “Allyl Isothiocyanate, a Constituent of Cruciferous Vegetables, Inhibits Proliferation of Human Prostate Cancer Cells by Causing G2/M Arrest and Inducing Apoptosis.” Carcinogenesis 24 (5): 891–97. Xu, K, and P J Thornalley. 2001. “Signal Transduction Activated by the Cancer Chemopreventive Isothiocyanates: Cleavage of BID Protein, Tyrosine Phosphorylation and Activation of JNK.” British Journal of Cancer 84 (5): 670–73. doi:10.1054/bjoc.2000.1636. Yu, E. Y., I. J. Pickering, G. N. George, and R. C. Prince. 2001. “In Situ Observation of the Generation of Isothiocyanates from Sinigrin in 100 Horseradish and Wasabi.” Biochimica Et Biophysica Acta 1527 (3): 156–60. Yu, R, S Mandlekar, K J Harvey, D S Ucker, and A N Kong. 1998. “Chemopreventive Isothiocyanates Induce Apoptosis and Caspase-3-like Protease Activity.” Cancer Research 58 (3): 402–8. Zhang, Yuesheng. 2004. “Cancer-Preventive Isothiocyanates: Measurement of Human Exposure and Mechanism of Action.” Mutation Research 555 (1–2): 173–90. doi:10.1016/j.mrfmmm.2004.04.017. ———. 2010. “Allyl Isothiocyanate as a Cancer Chemopreventive Phytochemical.” Molecular Nutrition & Food Research 54 (1): 127–35. doi:10.1002/mnfr.200900323. Zhang, Yuesheng, Li Tang, and Veronica Gonzalez. 2003. “Selected Isothiocyanates Rapidly Induce Growth Inhibition of Cancer Cells.” Molecular Cancer Therapeutics 2 (10): 1045–52. Zhao, Dayun, Jian Tang, and Xiaolin Ding. 2007. “Analysis of Volatile Components during Potherb Mustard (Brassica Juncea, Coss.) Pickle Fermentation Using SPME–GC-MS.” LWT - Food Science and Technology 40 (3): 439–47. doi:10.1016/j.lwt.2005.12.002. Zi-Tao Jiang, Rong Li. 2006. “Pungent Components from Thioglucosides in Armoracia Rusticana Grown in China, Obtained by Enzymatic Hydrolysis.” Food Technology and Biotechnology 44 (1). Zsolnai, T. 1971. “[Antimicrobial effects of thiocyanates, isothiocyanates and potential isothiocyanate forming substances].” Arzneimittel-Forschung 21 (1): 121–27. 101 102 103