MASARYK UNIVERSITY Faculty of Science Department of Chemistry

Application of separation techniques for analysis of phytochemical compounds Využití separačních metod v analýze fytochemických látek

Dissertation thesis

Author: Mgr. Vendula Roblová Supervisor: prof. RNDr. Vlastimil Kubáň, DrSc. Consultant: Mgr. Miroslava Bittová, Ph.D.

BRNO 2016

BIBLIOGRAFICKÝ ZÁZNAM Autor:

Mgr. Vendula Roblová Ústav chemie Přírodovědecká fakulta, Masarykova univerzita

Název práce:

Využití separačních metod v analýze fytochemických látek

Studijní program:

PřF D-CH4 ANAL

Studijní obor:

Analytická chemie

Vedoucí práce:

prof. RNDr. Vlastimil Kubáň, DrSc.

Konzultant:

Mgr. Miroslava Bittová, Ph.D.

Akademický rok:

2016

Počet stran:

84

Klíčová slova:

polyfenolické látky, extrakční techniky, příprava vzorků, kapilární elektroforéza, vysokoúčinná kapalinová chromatografie, antioxidační potenciál, máta peprná, rakytník řešetlákový, noni

BIBLIOGRAPHIC ENTRY Author:

Mgr. Vendula Roblová Department of Chemistry Faculty of Science, Masaryk University

Title of thesis:

Application of separation techniques for analysis of phytochemical compounds

Degree Program:

PřF D-CH4 ANAL

Field of Study:

Analytical Chemistry

Supervisor:

prof. RNDr. Vlastimil Kubáň, DrSc.

Tutor:

Mgr. Miroslava Bittová, Ph.D.

Academic Year:

2016

Number of Pages:

84

Keywords:

polyphenolic compounds, extraction techniques, sample treatment, capillary electrophoresis, high performance liquid chromatography, antioxidant potential, peppermint, sea buckthorn, noni

ABSTRACT This doctoral thesis comprises studies dealing with an application of separation techniques in the analysis of phytochemical compounds in various plant materials. The main goal of this work is the processing of plant samples and optimization of sample preparation through a combination of different extraction techniques. The studied analytes are representatives from various groups of polyphenols and the separation techniques HPLC and CZE were used for their determination. Different approaches were chosen for the determination of polyphenolic compounds and description of plant materials. The analyzed plant materials were parts of peppermint (Mentha x piperita), sea buckthorn (Hippophaë rhamnoides L.) and noni (Morinda citrifolia) plants. Another studied property describing the content of polyphenolic compounds in plants was antioxidant potential, which was expressed by the parameters total antioxidant activity and total polyphenol content. The work also includes a study focused on the analysis of sterols in vegetable oils. The preparation of oil samples for the determination of sterol content by RP-HPLC with offline SALDI MS detection is a presented part of study in this work.

ABSTRAKT Předkládaná disertační práce je souborem prací zabývajích se využitím separačních technik v analýze fytochemických látek v rozdílných rostlinných materiálech. Hlavní úlohou práce je především zpracování rostlinných vzorků a optimalizace přípravy vzorků kombinací rozdílných extrakčních technik. Sledovanými analyty jsou zástupci ze skupiny polyfenolických látek a k jejichž stanovení byly použity separační techniky HPLC a CZE. Pro různé rostlinné materiály byly voleny rozličné přístupy pro jejich stanovení. Studovanými rostlinnými materiály byly máta peprná (Mentha x piperita), rakytník řešetlákový (Hippophaë Rhamnoides L.) a noni (Morinda citrofolia). Dalším sledovaným parametrem popisujícím obsah polyfenolických látek v rostlinných materiálech je antioxidační potenciál, který je vyjádřen pomocí hodnot celkové antioxidanční kapacity a celkového obsahu polyfenolů. Součástí práce je také studie věnovaná analýze sterolů v rostlinných olejích, kde prezentovanou částí je příprava vzorků olejů pro stanovení sterolů pomocí RP-HPLC s offline SALDI MS detekcí.

ACKNOWLEDGEMENT First of all, I would like to thank my supervisor professor Vlastimil Kubáň for the opportunity to conduct my research under his supervision. My gratitude is also to doctor Miroslava Bittová and associated professor Petr Kubáň for their support, ideas and endless patience with me during my studies. Futher, I would like to thank to professor Manuel Valiente Malmagro and all of his co-workers for the opportunity to experience six wonderful months and be a part of his research group at Universitat Autonòma de Barcelona. I want to thank to my family and my dear friend Miroslav Hrstka for their support and understanding, encouragement and help when it was required. I am very grateful to all my colleagues in the Department of Chemistry, Masaryk University, and to all my friends, especially to Bára Lejsková, Pavel Klimeš, Eliška Kožuszniková and Adam Pruška for being here for me in good and bad times.

DECLARATION The work submitted in this dissertation is the results of my own investigation, except where stated otherwise.

Brno 2016

…………………………… Mgr. Vendula Roblová

© Vendula Roblová, Masaryk University, 2016

CONTENT 1

Preface

10

2

Theory

11

2.1

Polyphenolic compounds

11

2.2

Classification of phenolic compounds

12

2.2.1 Flavonoids

12

2.2.2 Non-flavonoids

16

2.3

Occurrence

17

2.4

Analysis of polyphenolic compounds

19

2.4.1 Sample treatment

20

2.4.2 Instrumental analysis

20

2.4.2.1 High performance liquid chromatography (HPLC) 21

2.5

2.4.2.2 Capillary zone electrophoresis (CZE)

22

2.4.2.3 Other techniques in analysis of polyphenols

23

Antioxidant potential of polyphenols

25

2.5.1 Total antioxidant activity

25

2.5.2 Total polyphenolic content

27

3

Aims of the study

28

4

The autor's contribution to the presented articles

29

5

Results

30

5.1

Capillary electrophoresis fingerprinting and antioxidant potential determination for Mentha products classification

30

5.2

HPLC monitoring of selected polyphenolic compounds in sea buckthorn (Hippophaë rhamnoides L.) 33

5.3

Characterization of noni (Morinda citrifolia) products by determination of organic acids and polyphenolic compounds 36

5.4

Samples preparation for determination of phytosterols in vegetable oils by RP-HPLC SALDI MS

38

Conclusion

39

6

8

7

List of abbreviations

41

8

References

43

9

List of publications

48

10

Curriculum vitae

50

Article I.

52

Article II.

64

Article III.

73

Article IV.

77

9

1 PREFACE Phytochemical compounds are natural substances in almost any plant, where they perform the role of secondary metabolites. The most commonly occurring phytochemicals are polyphenolic compounds which are further divided into many specific groups, based on their structure or properties, e.g. flavonoids, flavones, flavanols, isoflavones, stilbenes, lignans etc. [1]. Polyphenols representation in plants is varied due to their different functions, but many of these compounds have antioxidant activity. In organisms, they may act as antioxidants and limit oxidation processes. As mentioned in many previous studies [2], the polyphenolic compounds also have positive effects on human health. For example, salicin as a derivative of salicylic acid [3], is well known for pain-relieving and anti-inflammatory properties. Originally, salicin was extracted from the bark of the white willow tree and later was synthetically produced to become a drug called aspirin. Polyphenols also have positive effects for the prevention of cardio-vascular diseases. Currently, interest in phytochemicals as bioactive compound in food is increasing. Several studies have shown that polyphenols derived from plants are more effective antioxidants in vitro than vitamin E or C and that may significantly contribute to their protective effects in vivo.

10

2 THEORY 2.1 Polyphenolic compounds Plants synthetize a huge number of organic compounds that are typically categorized as primary or secondary metabolites. Primary metabolites include phytosterols, nucleotides, acyl lipids and organic acids, and have basic roles associated with

respiration,

photosynthesis

and

growth.

Phytochemicals

accumulated

in surprisingly high concentrations in some species are referred to as secondary metabolites. Lately, the function of secondary metabolites, especially phenolic compounds, has been attracting scientific attention as some appear to have a key role in protecting plants against microbial infection and acting as UV protectants, allelopathic agents, or signal molecules in the formation of nitrogen-fixing root nodules in legumes. They are also of interest because of their use as colorants, waxes, oils, fibres, flavouring agents, perfumes and drugs. This makes them potential sources of new natural drugs, herbicides or antibiotics [4, 5]. In literature, polyphenols are described as secondary metabolites that play the role of antioxidants in plants due to their antioxidant (radical-scavenging) activity. Up to date, more then 8 000 types of phenolic compounds have been identified in different plant species. Polyphenols are the most plenteous antioxidants in the human diet, the total dietary intake could be around 1g per day, that is highest amount of all classes of known dietary antioxidants in human diet. The dietary sources of these polyphenols are fruits, vegetables, cereals, beverages such as juices, coffee or tea and red vine. Despite the fact that the distribution of polyphenols in plants is wide their health effects have come to attention of researcher just recently. Studies strongly support the role of polyphenols in the prevention of degenerative diseases, cancers, osteoporosis or diabetes mellitus. There is also deep interest in polyphenols as possible inhibitors to neurodegenerative diseases such as Parkinson's and Alzheimer's disease. The mechanism of action is not fully understood but it is clear that it strongly depends on chemical structure [2, 6, 7]. Polyphenolics is a highly inclusive term that involves many different groups of flavonoids and phenolic acids. They can range from small compounds formed by a single aromatic ring to large polymeric structures; however, they are characterized 11

by having at least one aromatic ring with one or more hydroxyl group substituents (Fig. 1/a). They are commonly found conjugated to sugar or organic acids and can be classified based on their structure into two groups: 

flavonoids



non-flavonoids

2.2 Classification of phenolic compounds Polyphenols are characterized by having at least one aromatic ring with one or more hydroxyl groups attached to the structure. The variation of polyphenols is large, so for further classification they are subdivided into several families based on their structure, occurrence, properties or their biosynthetic pathway in the organism [4, 8, 9]. Flavonoids:

Non-flavonoids:



flavonols



phenolic acids



flavones



hydroxycinnamates



isoflavones



stilbenes



flavan-3-ols



lignans



flavanones



anthocyanidins

2.2.1 Flavonoids Flavonoids are qualitatively and quantitatively one of the largest groups of natural molecules known and they can by found throughout the plant kingdom. The highest concentration of flavonoids is found in the skin of fruits and the epidermis of leaves, where they play the role of secondary metabolites with significant antioxidant and chelating properties. They are involved in important processes such as UV protection, pigmentation, disease resistance and stimulation of nitrogen-fixing modules [4]. Flavonoids are benzo-γ-pyrone derivatives consisting of phenolic and pyrane rings and are further classified according to their substituents (Figure 1.). Dietary flavonoids differ based on the arrangment of hydroxyl, methnoxy and glycosidic side groups and conjugation between the aromatic rings.Most dietary flavonoids exist in food primarily 12

as 3-O-glycosides (commonly with glucose, galactose and rhamnose as glycosidic units) and polymers [10]. One of the most prominent and useful properties of many flavonoids is their ability to scavenge free radicals [11, 12]. These reactive species arise during many physiological processes, especially in the respiratory chain and in oxidations catalyzed by oxygenases or radiation. Moreover, flavonoids can also protect human low-density lipoprotein (LDL) against oxidation of its unsaturated fatty acid (FA) moieties [13].

Figure 1. General structures of flavonoids [4].

Another interesting property of flavonoids is that they can inhibit glutathione Stransferase, which can compromise the transport of amino acids across membranes in cells [14, 15]. The biochemical and medical significance of flavonoids is thoroughly described in review written by Havsteen [16]. The aforementioned properties imply that flavonoids play a significant role in many processes that attract the attention of scientists across various disciplines.

13

Flavonols Flavonols are likely the largest group of flavonoids and their structural variations are extensive and have been well documented. Commonly occuring flavonols, such as myricetin, quercetin, kaempferol or isorhamnetin, are mostly found as O-glycosides. Most frequently, the conjugation is at position 3 of the C ring, but substitutions can also be at positions 5,7,4´, 3´ and 5´of the carbon ring (Figure 1.). Even though the number of aglycones is limited, there are more than 200 different sugar conjugates just of kaempferol [4]. Flavonols, called glycosides, contain a sugar moiety, which can act as an important determinant of the absorption and bioavailability of the glycoside. Currently, the evidence does not yet allow a decision on the involvement of flavonols in the etiology of cardiovascular diseases or cancer. Common dietary sources of flavonols are broccoli, leeks, onions, lettuce, berries, olive oil, etc. Flavones Flavones, as opposed to flavonols, lack a hydroxyl group at the 3-position but they are also present in plants bound to sugars as O-glycosides or C-glycosides. Unlike flavonols, flavones are not widely distributed; the most abundant ones in plants are luteolin, apigenin and chrysin, and their occurrence has been reported in celery, parsley, buckwheat or red pepper [4]. Isoflavones Isoflavones differ from other flavonoids by having the B-ring attached at C3 position rather than the C2 (see Figure 1.). They are produced almost exclusively by leguminous plant and the highest concentrations occur in soybean. The isoflavones genistein and daizen have sufficient oestrogenic activity because their structure is closely related to phytoestrogens, and they appear to mimic the steroidal hormone oestradiol, which blocks ovulation and may seriously affect the reproduction of grazing animals (cows, sheep). On the other hand, human consumption of genistein and daidzein from soya products may reduce the occurrence of prostate and breast cancers [4].

14

Flavan-3-ols Flavan-3-ols (also referred as flavanols) are a wide subclass of flavonoids, ranging from simple monomers (e.g. izomers (+)/(−)-catechin) to oligomeric and polymeric proanthocyanidins, also known as tannins. Unlike flavones, flavonols, isoflavones or anthocyanidins, flavanols are not planar molecules because they have a saturated C3 position in the C-ring. Two chiral centres (at the C2 and C3 positions) produce four isomers for each level of hydroxylation of the B-ring. This may cause difficulties in their differentiation through the use of common separation techniques, such as high performance liquid chromatography (HPLC), as commonly used reverse phase columns are not able to resolve pairs of enantiomers. Catechins and their derivatives are naturally presented in cocoa, prune juice and green tea. There is a high occurrence of oligomeric proanthocyanidins consisting exclusively of (epi)catechin units, also called procyanidins, in apples, cinnamon and the seeds of red grapes [4]. Flavanones The flavanones are highly reactive easily undergo hydroxylation, glycosylation and O-methylation reactions. Flavanones, such as naringin, naringenin, hesperidin or eriodictyol, are found in especially high concentration in citrus fruits (orange, grapefruit, lemon). Hesperedin (hesperetin-7-O-rutinoside) is the most common flavanone glycoside and is found in citrus peel in significant amount. Some of these flavanones have an intensely bitter taste, but the related compound neohesperedin is used as a sweetener in non-alcoholic beers [4]. Anthocyanidins Anthocyanidins, also as their conjugated derivatives, anthocyanins, are widely spread throughout the plant kingdom. There are responsible for red, blue and purple colors of flowers, fruits, vegetables or others plant tissues such as leaves, stems, seeds or roots [17]. They participate in the protection of plants against excessive light by shading leaf mesophyll cells and also play a role in attracting pollinating insects. With a few exceptions, anthocyanins mostly occur in cells as glycosides. The most common sugar in glycosides is glucose, less common ones are arabinose, galactose or rhamnose. The most prevalent anthocyanidins in plant tissues are pelargonidin, cyanidin, petunidin and malvidin. In food they can be easily found in black currant, eggplant and in many types of berries. [18]. 15

2.2.2 Non-flavonoids Phenolic acids Phenolic acids are hydroxylated derivatives of benzoic acid (C6-C1) and they are common as independent molecules as well as combined esters or glycosides. One of the most common classes of polyphenols are phenolic acids containing a gallic acid as their base unit. The second class of phenolic acids contains hydroxycinnamates (Figure 2.), which are derivatives from cinnamic acid (C6-C3). They occur rarely in their free state and they are often esterified (e.g. caffeic acid, ferulic acid). Hydroxycinnamates, namely chlorogenic and quinic acids, are present in leaves of green mate and green coffee beans. The regular coffee drinker may have a daily intake of chlorogenic acid in excess of 1g [19].

Figure 2. Structure of hydroxycinnamates: a) gallic acid, b) cinnamic acid.

Stilbenes These compounds are well known for their antimicrobial activity and are synthesized in organisms after the entrance of a pathogen. They are produced by plants in response to attacks by fungal or bacterial pathogens. Resveratrol (trans-3,5,4´trihydroxystilbene), one of the most popular polyphenol, is a compound of interest to researchers because of its high concentration in grapes, nuts and vegetables. This has application in quality testing: several studies have determined the typical concentration of trans-resveratrol in wine between 0.1 and 10 mg/L, depending on the cultivar, origin and production [20]. Therefore, the quality of red wines based on trans-resveratrol concentration can be determined. Typical dietary sources of stilbenes are grapes, wine, peanuts and soy products [4, 21].

16

Lignans Lignans are compound naturally synthesized in plants during the metabolic pathway from cinnamic acid or p-coumaric acid. Their function was not clearly understood until recently, but studies indicate that they play an important role in the interactions between plants and fungi or plants and insects, e.g. enterodiol, enterolactone. Good sources of lignans for humans are fiber-rich foods, such as cereals, broccoli, soybeans or sesame seeds, because plant lignans are co-passengers of dietary fiber. Interest in lignans has been growing recently due to their potential applications in cancer chemotheraphy and other possible pharmacological uses [21].

2.3 Occurrence Apart from traditional vitamins and minerals, fruits and vegetables may contain a wide range of phytochemical compounds that may have biological effects in humans. As was mentioned before, polyphenols are also phytochemicals and they are found ubiquitously in various fruits, vegetables, tea leaves, olive oil, nuts and flowers. Common sources of polyphenols in the human diet are apples, berries, onions, jams, chocolates or beverages such as tea, coffee and wine. Mostly, interest in polyphenols has been related to their organoleptic properties, such as bitterness (flavanols), color (anthocyanins), or astringency [22]. The type of polyphenol is specific to particular fruits and vegetables, e.g. resveratrol is a stilbene typically occurring in grapes [23], soybeans are rich in the isoflavones daidzein and genistein (usually in several glycosylic form) [24], and the red color of some apples is caused by presence of anthocyanin and cyanidin-3-O-galactoside [25]. Because of this, the phytochemical compound content of the sample allows characterization not only of the general plant type, but also differentiates between different classes and taxons. For example, cranberries, elderberries and blackberries contain only one type of anthocyanin, but blueberries and blackcurrants contain a wide array of various anthocyanins [26]. In Table 1, the typical content of polyphenols in basic foodstuff is showed.

17

Table 1. The content of polyphenolic compounds in basic foodstuff [7]. foodstuff

content [mg/100g]

foodstuff

content [mg/l]

black currant

140 – 1200

orange juice

370 – 7100

apple

27 – 298

beer

60 –100

orange

50 – 100

red wine

2000 – 4000

barley

1200 – 1500

tea

750 – 1050

Herbs and spices are also commodities with a significant amount of phytochemicals, but a major contribution comes from the families Apiaceae and Lamiaceae. Frequently, they contain phytochemicals not found in other foodstuffs and they may play a role in herbal medicine. Herbs are a rich source of phenolic acids, especially derivates of hydroxybenzoic acids; for example, herbs from Lamiaceae are a significant dietary source of rosmarinic acid and caffeic acid conjugates [27]. Further, cereals contain characteristic phytochemicals that are not present in other commodities. Barley is a common cereal with significant proanthocyanidins and phenolic acid content: for instance, ~50 mg/kg bound ferulic acid and ~30 mg/kg pcoumaric acid are found in barley bran [28]. Likewise, a series of 24 caffeic and ferulic esters of glycerol and hydroxyl acids (~200–300 mg/kg of esterified ferulic acids) are present in oat meal. Beverages are another typical dietary source of polyphenols for humans. One of the most widely consumed beverages in the worlds is tea, consumed in one of three forms: green, oolong or black. Fresh tea leaves are unusually rich in polyphenols, comprising about 30 % of their dry weight, where the dominant polyphenols are flavan-3-ols. Typically, (−)-epigallocatechin gallate is the most commonly occurring one, followed by (−)-epicatechin gallate and (−)-epigallocatechin, (+)-catechin and (−)-epicatechin and esterified p-coumaric or caffeic acid. The content of polyphenols in tea depends on the processing of raw materials, mostly the drying and fermentation process that may be significant for final tea quality. This also applies to green coffee beans, which are one of the richest dietary sources of chlorogenic acids (6–10 % of the dry weight). There is a progressive destruction and transformation of chlorogenic acid during roasting, about 8–10 % loss for every 1 % loss of dry matter [29].

18

2.4 Analysis of polyphenolic compounds The attention devoted to polyphenols, especially to flavonoids, rapidly increased during the last five years. More than 300 papers were written on the analysis of flavonoids in plants, mainly to characterize and quantify selected constituents. Due to the fact that flavonoids occur in various conjugated forms or as aglycones, it is important to choose the right analytical strategy for analysis, based on type of sample, analytes and nature of the problem in question. The diversity of sample types and analytes gives a number of permutations and there is no one global strategy that will suffice for all situations. However, a typical analytical strategy involves the recovery of polyphenols from the sample matrix, followed by separation, identification and quantification. Usually, the recovery step involves solvent extraction using a range of solvents. Separation is commonly achieved using high performance liquid chromatography (HPLC), capillary electrophoresis (CE), or gas chromatography (GC) with a spectrophotometric detector or coupled with mass spectrometry (MS) to identify selected analytes [30, 31]. The scheme of procedures for the analysis of polyphenols is shown in Figure 3.

Figure 3. Scheme for the polyphenols determination in different type of samples [30].

19

2.4.1 Sample treatment Many sample pre-treatment methods for the determination of flavonoids in various samples have been developed. There are three main types of samples matrices: plants, food, and beverages/biological fluids. The solid samples are usually first homogenized by drying or freezing with liquid nitrogen, and dried materials can be pulverized. In the next step, analyte isolation is usually performed by solvent extraction (SE). The prepared liquid samples are filtered and/or centrifuged and can be directly injected into a separation system or further treated by solid-phase extraction (SPE) or liquid-liquid extraction (LLE) to isolate or concentrate the required analytes. SPE is the most widely used technique for sample pre-treatment because it is a simple technique with a wide-range of applicability [32]. The traditional LLE extraction is mostly used with solvents like ethyl acetate or diethyl ether for the extraction of polyphenols [33]. If aglycones are the target analytes, a small amount of acid (hydrochloric or formic acid) might be added to the organic extraction solvent. On the other hand, if the molecules of interest are intact flavonoid-glycosides, harsh extraction conditions and hydrolysis should be prevented. Also, Soxhlet extraction or reflux is used with aqueous methanol or acetonitrile solvents to isolate flavonoids typically from plant materials [34, 35]. Lately, the use of extraction techniques such as pressurized liquid extraction (PLE) [36], microwave-assisted extraction (MAE) [37] or supercritical fluid extraction (SFE) [38, 39] is increasing because of the demand for automatic techniques and reduced solvent consumption. More information about sample preparation is described in a paper by Robards [40]. 2.4.2 Instrumental analysis Formerly, the determination of polyphenolic compounds was performed by colorimetric measurements of the total polyphenols using one of a number of reagents with various selectivity. Because of the need for characterization and identification of individual phenols, traditional colorimetric methods were mostly replaced by separation techniques like HPLC, CE, GC, HTLC and others [9, 30]. For the purpose of this work, HPLC and CE combined with spectrophotometry or MS detection will be described in more detail.

20

2.4.2.1 High performance liquid chromatography (HPLC) One of the most suitable techniques for the separation and characterization of polyphenolic compounds is high performance liquid chromatography (HPLC). Separation is usually carried out in the reverse-phase mode (RP) with C8- or C18bonded silica columns [41]. Monolithic columns, which are filled with porous material, may also prove effective for separation in a short time: because of their low backpressure, they allow the use of higher mobile phase flow [42]. The determination of polyphenols using the monolithic column is described by Liazid A., et al. [43], who were able to successfully quantified 13 polyphenolic compounds in grapes in less than 8 minutes. The solvent system typically uses a gradient elution with aqueous phase containing acetate or formate buffer and an organic phase, mainly methanol or acetonitrile. LC is usually performed at room temperature, but sometimes a temperature up 40◦C may reduce analysis time and provide better repeatability of retention times. Further, ultra-performance liquid chromatography (UPLC) is an LC mode which enhances system efficiency and analysis time. However, run times of over one hour are sometimes required for the analysis of many compounds at once; for example, samples containing 30-50 compounds can be separated in single run, with many conjugates. In a study presented by Raczkowska J., et al. [44], the authors were able to determine five polyphenols in wine by UPLC six times faster than by HPLC, but for the pattern recognition analysis of isoflavones in soy sauces, the separation lasted over 340 minutes [45]. HPLC methods can be combined with many types of detectors, but in polyphenolic compounds research, UV spectrophotometry, fluorescent or electrochemical detectors have been widely used. More recently, multiple-wavelength or diode-array (DAD) detectors have become fully satisfactory tools for the determination and quantification of polyphenols. The weakness of these detection techniques is a lack of structural information and possible matrix peaks interference. Over last years, the coupling of HPLC with mass spectrometry detectors has notably improved the identification and structural

characterization

of

phenolic

compounds

[46].

In

most

cases,

the combination of MS and UV detection is used for confirming the identity of polyphenols in the sample and tandem mass spectrometry (MS/MS) is used for identifying unknown compounds [47].

21

2.4.2.2 Capillary zone electrophoresis (CZE) HPLC was the most popular technique for the analysis of dietary polyphenols for a long time, but recently capillary electrophoresis is becoming a suitable alternative. Small sample and solvent consumption, high separation efficiency and short analysis time are valuable qualities in polyphenols analysis. However, the drawbacks of electrokinetic methods are generally lower sensitivity and worse reproducibility compared to HPLC. Capillary zone electrophoresis is based on the different migration velocities of charged analytes in a capillary filled with background electrolyte (BGE) under a highvoltage electric field. Experimental variables influencing electrophoretical separation are voltage, temperature, injection time, buffer composition and capillary characteristics, the most important being the buffer pH. Acidity has an influence on the charge of polyphenols, which may affect their migration times. However, separation also depends on the electroosmotic flow (EOF) that originates from negatively charged silanolate groups of silica on the capillary wall. Cations from the electrolyte are attracted to the negatively charged wall and form a layer. The adjacent mobile layer is formed from free cations in the electrolyte, which migrate toward cathode while the bulk of the electrolyte co-migrates with them and induces a rise in the EOF. When the mobility of the EOF is higher than the electrophoretic mobility of the negatively charged solute, both cations and anions migrate in same direction and can be separated within one run. All neutral analytes are carried away with the rate of EOF together, unseparated. For neutral analyte separation, charged surfactants are used at a level higher than the critical micellar concentration. This mode of electromigration technique is called micellar electrokinetic chromatography (MEKC) and together with CZE is frequently used for the determination of polyphenolic compounds in various herbal material or foodstuff [48]. Moreover, CZE is suited for the analysis of samples with complex matrices, such as plants or food samples, because sample pre-concentration by large-volume sample electrokinetic stacking [49] or anion-selective exhausting injection sweeping [50] can improve the sensitivity of detection, as was reviewed by Simpson et al [51]. This, combined with speed, automation and low consumption of solvents gives CE advantages over other separation methods. Particularly, CE coupled with MS can serve as an important tool in polyphenols analysis because of the separation capabilities of 22

CE and the power of MS as detection and identification method. The other detections techniques used for CZE are similar to those used in HPLC, e.g. UV spectrophotometry, DAD, fluorescence or contactless conductivity detector. Coupling CE with MS is not an easy task and there are some problems that must be solved, such as the compatibility of solvents or values of applied voltages in CE. Firstly, the voltage applied to the separation in CE is not suitable for direct MS detection and an interface between the methods is required. The background electrolyte usually contains borate or phosphate ions that are not suitable for some ionizations techniques. On the other hand, appropriate buffers (like acetic acid, ammonium acetate or carbonate) for MS may not allow a good separation efficiency of polyphenols during CE measurements. A typical ionization technique for on-line coupling of CE with MS detection is electrospray ionization (ESI) in various modes because of the very low flow rates (nL/min) and no flow splitting is needed like with HPLC. Despite the problems mentioned above, the combination of high separation capabilities of CE and MS for confirmation and identification brings important advantages, not only for polyphenols analysis. CE is relatively new in food analysis, compared to HPLC or GC, but recently, large amounts of foodstuff were analyzed by this technique e.g. beverages, meats, fruits [52, 53, 54]. Nowadays, the analysis of polyphenols is not only focused on polyphenols in wine or food, but many studies focusing on the determination of polyphenols in natural products like honey [55], propolis [56] or olive oil [57] were published recently. CE offers simultaneous separation of a number of components with good resolution, as was described by Yanes et al. [58], who improved separation by modification of BGE. Also, the determination of the dissociation constants of flavonoids can be one of the many possible applications of capillary electrophoresis [59]. 2.4.2.3 Other techniques in analysis of polyphenols Traditional methods for polyphenolic analysis can be successfully replaced by counter-current chromatography (CCC) for fractionation and isolation from different types of samples [9]. CCC is a form of liquid-liquid chromatography in which two immiscible liquid phases are in contact without a solid support. One of the phases is pumped through a column filled with liquid stationary phase held in place by

23

centrifugal force. The normal operation mode is based on a stationary phase with a lower density than the mobile phase. Due to this arrangement, separation is set in descending mode, but ascending mode can also be applied [60]. Generally, it is recommended to optimize the solvent system for every new chemical compound source because a variety of solvents can significantly improve the sample fractionation and purification. A great overview on the application of CCC in polyphenols analysis was described in a paper by Berthod A., et al. [61]. Frequently, different modes of electrophoresis, such as micellar electrokinetic chromatography or isotachophoresis (ITP), might be useful for polyphenols determination. In most cases, ITP is employed as a pre-concentration technique in connection with CZE and has been used for the determination of antioxidants in different matrices. On-line coupling of these techniques allows the limit of detection to decrease and enables the removal of the matrix from a complex sample, e.g. wines or plant materials. The detection limits of electromigration methods can be constantly improved, for example by stacking (30-100 times), isotachophoretical sampling (201000 times) or by coupling SPE with CZE (50-7000 times). For on-line coupling of ITP and CZE, the column arrangement is an important factor because single column coupling improves just the limit of detection but use of two columns (ITP column with large inner diameter used as CE column) may also lead to an improvement of separation selectivity and efficiency [62]. Polyphenolc analysis can also be used as a control step during food processing, as was published by Zafra a., et al. [63]. The authors presented the use of gas chromatography (GC) as a tool for monitoring 21 polyphenols in wastewater olive oil. GC is not as widely used as HPLC because of the need for non-volatile polyphenols derivatization, traditionally by a silylation of free hydroxyl groups. It is possible to include this derivatization step in the sample preparation or in the injection-port [64]. GC allows the analysis of glycosylated and non-glycosylated polyphenols and when combined with SPE or liquid-liquid micro-extraction (LLME) as a clean-up method, relatively fast and sensitive separation of tens of compounds in real samples can be achieved [65].

24

2.5 Antioxidant potential of polyphenols Antioxidants, such as polyphenols, are present in plants in high concentrations and one of their major functions is to fight against hazardous oxidative damage in plant cells. Due to their redox-active properties, polyphenols significantly delay the oxidation of easily oxidizable substances. Antioxidant potential is a general term used for the description of antioxidants; two of the characteristics traditionally used for the description of herbal or food bioactivity are antioxidant capacity (AC) and antioxidant activity (AA). Antioxidant capacity expresses the total number of electrons in molecules which are converted per mole of antioxidant at full reaction under given conditions. This usually corresponds with the number of hydroxyl groups, though not always. The reaction rate is not considered, which means that slowly reacting antioxidants with many phenol groups have the highest contribution while fast reacting antioxidants with few phenol groups can be underestimated or overlooked. Antioxidant activity, on the other hand, is defined as the concentration of antioxidant required for the attainment of a specified rate or extent of reaction. Recently, several biochemical and chemical assays for the description of antioxidant potential of polyphenolic substances were developed. These assays are based on different reaction mechanisms, but the common principle is radical scavenging ability of natural compounds present in the sample. The antioxidant potential of polyphenols is a complex property and in this work, it will be described by the characteristics of total antioxidant activity (TAA)and total polyphenolic content (TPC). Assays for the determination of TAA and TPC in food or plant extracts mostly use colorimetry with different reagents [66]. 2.5.1 Total antioxidant activity The total antioxidant activity is the ability of compounds to inhibit the oxidative degradation of various compounds. Several assays using spectrophotometric methods have been applied for assessing of TAA in real samples of fruits, vegetable, herbs or spices. 

TEAC - Trolox Equivalent Antioxidant Capacity The TEAC assay is based on measurements of the scavenging of the stable free

ABTS•+

radical

(2,2'-azinobis(3-ethylbenzo-thiazoline-6-sulfonic

acid))

by 25

the compounds present in the sample. The wavelength maximum at 734 nm eliminates color interference from the sample, which makes this assay widely used for screening the antioxidant activity in plants extract, beverages, fruits, vegetable and foods. This method is applicable to determine hydrophilic and lipophilic antioxidants and their concentration is expressed in units called Trolox Equivalents (TE). Trolox (6-hydroxy2,5,7,8-tetramethylchroman-2-carboxylic acid) is utilized as a standard to antioxidant activity of a sample such as a complex mixture [67, 68]. 

DPPH method - 2,2-difenyl-1-picrylhydrazyl Monitoring the absorbance decrease of the synthetic radical DPPH• in the presence

of antioxidants is the basis of this method. DPPH (2,2-difenyl-1-picrylhydrazyl) is a stable free radical, caused by the delocalization of the free electron over the entire molecule, with a deep violet color and an absorption band around 520 nm. After the reagent is added to a sample that can donate a hydrogen atom, the loss of the violet color is observed as consequence of increasing DPPH-H reduced form (see Figure 4.).

Figure 4. Reaction scheme of free radical DPPH with an antioxidant [69].



ORAC – Oxygen Radical Absorbance Capacity Mostly, the ORAC method is used for antioxidant activity measurements in

biological samples in vitro. The assay measures the ability of a sample to reduce or stop generation of oxygen radicals, resulting in the loss of fluorescence. As the oxidative generation proceeds, the fluorescent intensity decreases and this intensity is measured for 30 minutes after the addition of free radical generator (azo-initiator compounds, e.g. fluorescein, β-phycoerythrin) to the sample.

26



FRAP – Ferric ion Reducing Antioxidant Power The basic principle of the FRAP assay is a reduction of ferric (III) ions to ferrous

(II) ions by antioxidants and the formation of colored ferrous-tripyridyltriazine complex at low pH. The values are obtained by comparing the absorbance changes of the reaction mixture at 593 nm. Trolox as a standard is traditionally used for TAA evaluation [70]. 2.5.2 Total polyphenolic content Phenols occurring in nature are of interest from many reasons (antioxidants, oxidation substrates, organoleptic properties, color) and their assessment as a group can be very informative. Separation techniques as HPLC or CZE are difficult to apply for such a varied group of compounds. Phenols are largely responsible for the oxygen capacity in most plants and food. TPC assays include the sum of all polyphenols, like monophenols, as well as analytes with more phenol groups. Considering the diversity of polyphenols, several methods have been designed for total polyphenolic determination using spectrophotometry. 

FCM – Folin-Ciocalteu method Folin-Ciocalteu's reagent is a mixture of complex polymeric ions formed from

phosphomolybdic and phosphotungstic heteropoly acids used for colorimetric in vitro assays of polyphenols. The reduction of heteropoly acids to blue Mo-W complexes and the oxidation of polyphenolates is the basic principle of this method. Absorbance of the blue complex is measured against a blank at 765 nm and the polyphenol content is expressed in unit called Gallic Acid Equivalent (GAE). Another commonly used standard for the evaluation of polyphenol content is catechin (for flavonoids) or tannic acid (for determination of polyphenols in wine) [71]. 

PBM – Price-Butler method The basis of this method is the concurrent oxidation of a phenolate anion to

a phenol radical and reduction of a hexacyanoferrite ion to hexacyanoferrate, forming Prussian blue. The sample color change from green to blue is measured against a blank at 720 nm and the resulting values are expressed as GAE [72].

27

3 AIMS OF THE STUDY The goal of the studies presented in this thesis was to characterize real samples (mostly plant material) based on their antioxidant properties and the content of phytochemical substances. Due to the differing composition of vegetal material, the optimization of sample preparation procedures, including extraction techniques and their combination, was done for various herbal samples. Selected polyphenolic compounds were determined by separation technique (CZE, HPLC) in vegetal materials. Different approaches for the determination and description of polyphenol composition and antioxidant properties were applied to the following plants: 

peppermint (Mentha x piperita)



sea buckthorn (Hippophaë rhamnoides L.)



noni (Morinda citrifolia) Antioxidant properties of the plant extracts were determined

using

spectrophotometric determination and described by the total polyphenolic content (TPC) and total antioxidant activity (TAA). Extraction techniques were also carried out for the preparation of oil samples before the analysis of sterols by HPLC with offline surface assisted laser desorption (SALDI) as detection.

28

4 THE AUTOR'S CONTRIBUTION TO THE PRESENTED ARTICLES The author's research presented in this thesis was carried out at the Department of Chemistry, Faculty of Science at Masaryk University. The following research articles are referred to in the text by their Roman numerals and attached at the end of this thesis: I.ROBLOVÁ, V., BITTOVÁ, M., KUBÁŇ, P., KUBÁŇ, V.; Capillary electrophoresis fingerprinting and spectrophotometric determination of antioxidant potential for classification of Mentha products. J. Sep. Sci., 2016, Submitted. Vendula Roblová designed and carried out all experiments. She wrote the manuscript. II.BITTOVÁ, M., KREJZOVÁ, E., ROBLOVÁ, V., KUBÁŇ, P., KUBÁŇ, V.; Monitoring of HPLC profiles of selected polyphenolic compounds in sea buckthorn (Hippophaë rhamnoides L.) plant parts during annual growth cycle and estimation of their antioxidant potential. Cen. Eur. J. Chem., 2014, 11, 1152-1161. Vendula Roblová designed sample treatment procedures and carried out parallel research with capillary zone electrophoresis. She participated in writing of the manuscript. III.BITTOVÁ, M., HLADŮVKOVÁ, D., ROBLOVÁ, V., KRÁČMAR, S., KUBÁŇ, P., KUBÁŇ, V.; Analysis of organic acids, deacetyl asperulosidic acid and polyphenolic compounds as a potential tool for characterization of noni (Morinda citrifolia) products. Nat. Prod. Commun., 2015, 11, 1817-1820. Vendula Roblová measured spectrophotometric determination. She participated in finishing of the manuscript. IV.VRBKOVÁ, B., ROBLOVÁ, V., YEUNG E.S., PREISLER, J.; Determination of sterols using liquid chromatography with off-line surface-assisted laser desorption/ionization mass spectrometry. J. Chrom. A, 2014, 1358, 102-109. Vendula Roblová carried out samples preparation and helped finalize the manuscript.

29

5 RESULTS 5.1 Capillary electrophoresis fingerprinting and antioxidant potential determination for Mentha products classification Mentha genus (see Figure 5.) are aromatic, perennial herbs, including a large number of cultivars that differ not only in plant physiology, odor and taste, but also in chemical composition. Herbs of the Mentha genus have significant antibacterial and antioxidant effects, which can also be related to the represented polyphenolic compounds [16]. The current study is aims to compare ten different Mentha varieties and twenty different samples of commercially available peppermint teas using CZE fingerprinting and spectrophotometric determination of antioxidant properties for sample characterization. The first group, Mentha varieties, consisted of dried and milled plant material containing leaves, stems and blossoms. For peppermint teas, the content of tea bags was mixed together and then the herbal material was used for sample preparation without any further treatment [73]. Several extraction techniques, such as Soxhlet extraction, methanolic reflux or infusion in ultrasonic bath, were used for sample preparation. Infusion in boiling water was chosen as the most suitable procedure for sample preparation for its simplicity and similarity to recommended peppermint tea preparation procedure from the producers [74]. kingdom: Plantae subkingdom: Trancheobionta phylum: Magnoliophyta class: Rosopsida order: Lamiales family: Lamiaceae tribe: Mentheae genus: Mentha Figure 5. Plant parts of peppermint Mentha x piperita and its botanical taxonomy. 30

Capillary zone electrophoresis with UV detection was used as tool for the analysis of polyphenolic compounds in aqueous infusions. In this study, traditional approaches, like identification and quantification of selected analytes, which is most commonly used for polyphenolic analysis, were replaced by a non-targeted approach without the necessity of analyte identification [75]. Because plant extracts contain large amounts of various compounds from the sample matrix and even a clean-up step with SPE or L-L extractions may not achieve the satisfactory results, using CE fingerprints may be an effective tool for sample composition characterization [76]. In this study, the traditional way of polyphenol determination by identification and quantification of selected analytes was replaced by a non-target approach without necessity for analytes identification. The 12 peak areas present in all obtained electrophoregrams from the 5th to the 25th minute were selected for CE fingerprinting of the individual sample. Two conditions were considered for peak selection: first, that the selected peak was common to all samples and second, that the relative standard deviation (RSD) of its migration time was lower than 5 %. While separation techniques are mostly used for the determination of particular analyte content, the spectrophotometric measurements provided general information about the antioxidant potential of sample. For the characterization of the antioxidant potential of the peppermint samples, spectrophotometric determination of TPC using Folin-Ciocalteu's reagent and TAA by cation radical DPPH were done for all samples and the results were expressed as gallic acid equivalent (mg GA/ of dry sample). The relatively strong correlation between TPC and TAA values for peppermint teas (r = 0.829) indicates that the polyphenols present in the tea are largely responsible for the antioxidant activity of the samples. The correlation of same parameters for the group of Mentha samples was even stronger (r = 0.946). The combination of the two different methods provided a data set that was used as the input for principal component analysis. PCA analysis combined the 12 selected peak areas from CE and values of TPC and TAA in the matrix with the two extracted components. The score plots showed the clusters of Mentha species and peppermint tea samples. This approach could be used for the assessment of the potential protective antioxidant effect of peppermint products.

31

These findings were published as Article I. “Capillary electrophoresis fingerprinting and spectrophotometric determination of antioxidant potential for classification of Mentha products.”

32

5.2 HPLC monitoring of selected polyphenolic compounds in sea buckthorn (Hippophaë rhamnoides L.) In traditional folk medicine of Mongolia, Siberia or China, the sea buckthorn (Hippophaë rhamnoides L., see Figure 6.) has been well known for its pulmonary, gastrointestinal and dermatological effects for more than twelve centuries. Due to the high concentrations of vitamin C in fruits (114-1500 mg per 100 g), sea buckthorn is one of the most enriched plants sources of vitamin C. Moreover, it is also a rich source of organic acids, saccharides, amino acids and polyphenolic compounds primarily flavonols and organic acids. Commercially available food supplements produced from sea buckthorn are teas, syrups, juices and oil [77]. kingdom: Plantae subkingdom: Trancheobionta phylum: Magnoliophyta class: Rosopsida order: Rosales family: Eleaegnaceae genus: Hippophaë L.

Figure 6. Plant parts of sea buckthorn (Hippophaë rhamnoides L.) and its botanical taxonomy.

CZE and HPLC methods were tested for the monitoring of polyphenols in sea buckthorn, but the vegetal matrix present in the samples caused difficulties with identification and quantification of selected analytes using capillary electrophoresis. Therefore, HPLC was chosen as suitable technique with sufficient sensitivity and good reproducibility

of measurements.

This

study

was

designed

to

determine

the concentration of eight selected polyphenolic compounds by HPLC during an annual growing cycle of sea buckthorn and compare with the antioxidant properties measured by spectrophotometric methods in order to suggest the optimal harvesting time of individual plant parts. There is an unambiguous relationship between the compound content present in the plant and the growing cycle of the plant. 33

The individual parts, leaves, shoots and fruits of sea buckthorn were collected from April to September, and the content of polyphenols as gallic acid, catechin, epicatechin, caffeic acid, p-coumaric acid, rutin and quercitrin was determined. Samples were collected from one plant at regular intervals. The plant material was dried to a constant weight at 40 ⁰C then cut, pulverized and used for extraction. Several extraction techniques were tested for sample treatment. The most suitable technique for treatment of individual plant parts was liquid extraction by methanol in an ultrasonic bath. After filtration, part of the sample was treated by L-L extraction with diethyl ether as the extraction solution. The organic phase was separated and then evaporated to dryness and the residue was re-dissolved in a small amount of methanol [78]. This combination of extraction procedures was sufficient for the elimination of the sample matrix and it facilitated the identification and quantification of selected analytes by reverse phase high performance liquid chromatography (RP-HPLC). The applied method was optimized from initial conditions for the determination of polyphenols in sea buckthorn as published by Zu, Y., et al. [79]. The separation was carried on a C18 column with a gradient elution of mobile phase (A: acetic acid, B: methanol/acetonitrile), which allowed the separation of eight analytes in 15 minutes. The polyphenols gallic acid, catechin, epicatechin, caffeic acid, p-coumaric acid, ferulic acid, rutin and quercitrin were identified and quantified in all type of samples. The highest polyphenolic content among part plants was observed in the leaves due to the high concentration of catechin, epicatechin, and gallic acid. While the content of catechin, epicatechin and gallic acid in leaves decreased significantly from April to September, the content of other observed analytes fluctuated with a rather decreasing tendency. The same analytes were also identified in young shoots and fruits. The catechin content in shoots increased more than twice from May to October; on the other hand, in fruits, the concentration of selected analytes was lower than in the other plant parts. An interesting observation was the significantly changing content of quercitrin in fruits from August to October, when concentration of quercitrin increased more than six times between the first and the last sample collection. Besides the polyphenolic monitoring of sea buckthorn, an assessment of the antioxidant potential by spectrophotometric methods was also a part of the study. Total antioxidant activity was determined by the reaction with cation radicals ABTS 34

and DPPH and the total polyphenolic content was determined using Folin-Ciocalteu's reagent [67, 80]. It is clear from the results that TPC values were decreasing in the order: leaves > shoots > fruits, and the highest values were found in leaf samples collected in May and June. A linear dependence (R2 =0.983) between TPC and TAA across all type of samples was determined. According to this observation, the total polyphenolic content contributes to the total antioxidant activity of studied samples. More information is provided in Article II. “Monitoring of HPLC profiles of selected polyphenolic compounds in sea buckthorn (Hippophaë rhamnoides L.) plant parts during annual growth cycle and estimation of their antioxidant potential.”

35

5.3 Characterization of noni (Morinda citrifolia) products by determination of organic acids and polyphenolic compounds Lately, several studies dealing with beneficial effects like antitumor activity [81] and analgesic or cancer preventive effects [82] of Morinda citrifolia were published. noni is a tree from the coffee family Rubiaceae with a wide utilization in folk medicine throughout Southeast Asia and Australasia. The plant bears fruits (see Figure 7.) throughout the year that are mostly used for noni juice production which is consumed primarily for its antiseptic effects. Just recently, the chemical composition of Noni was described. Besides proteins, saccharides and a number of phytochemicals noni also contains important nutrients like amino acids, essential fatty acids, selenium, roughage and high amounts of vitamin C. kingdom: Plantae subkingdom: Trancheobionta phylum: Magnoliophyta class: Rosopsida order: Gentianales family: Rubiaceae genus: Morinda

Figure 7. Plant parts of noni (Morinda citrifolia) and its botanical taxonomy.

The current study focuses on the analysis of organic acids, diacetyl asperulosidic acid (DAA) and polyphenolic compounds in various noni products by HPLC coupled with UV-Vis spectrophotometric detector and electrospray ionization time-of-flight mass spectrometer (ESI-TOF MS).

Two methods for reversed-phase high

performance liquid chromatography (RP-HPLC) were developed and used for analysis of DAA and four organic acids (citric, lactic, malic and succinic acid) in first case and for determination of seven selected polyphenols (caffeic acid, catechin, gallic acid, kaempferol, p-coumaric acid, quercitrin and rutin) in the second case. noni products, namely four dry fruit powders, two capsules filled with dry fruit powder and four juices, were characterized by the determination of two different groups of analytes 36

and significant differences between the products were found. The profile of analytes contained in the samples may point to differences between the samples which were not possible to see just from spectrophotometric methods. This is likely because TPC and TAA determination showed that those parameters separately are not efficient indicators for characterization of noni products. On the other hand, multi-analyte analysis may be a convenient tool for better characterization of noni products for consumers. Detailed findings were published in Article III. “Analysis of organic acids, diacetyl asperulosidic acid and polyphenolic compounds as potential tool for characterization of noni (Morinda citrifolia) products.”

37

5.4 Samples preparation for determination of phytosterols in vegetable oils by RP-HPLC SALDI MS Phytosterols, commonly known as plant sterols, play an essential role in the physiology of organisms. They form part of the cellular membrane in plant cells, where they have biological functions. The main goal of this study was to develop a method for the determination of phytosterols such as brassicaterol (BR), cholesterol (CH), stigmasterol (ST), campesterol (CA) and β-sitosterol (SI) in oil samples by reversed phase liquid chromatography with off-line surface-assisted laser desorption/ionization mass spectrometry (RPLC-SALDI-MS). The developed method was used for the determination of selected analytes in olive, sunflower and linseed oils. The total amount of sterols depends on many factors, but their content is one of the parameters for vegetable oil quality assessment. As always, sample preparation is the most crucial step in analysis. For extraction of sterols from vegetable oils, a modified extraction procedure described by Rocco A. and Fanali S. [83] was used. The sample preparation consisted of alkyne saponification followed by liquid-liquid extraction (L-L) of the unsaponificable fraction with diethyl ether without any further purification. The sterols recovery was not lower than 91 % with RSD less than 4 %. The isolated sterols were analyzed by HPLC in reversed mode with online UV detection. SALDI MS using silver nanoparticals as the SALDI matrix was applied as offline detection for the identification and quantification of phytosterols. The described technique represents a suitable alternative to gas chromatography for non-polar substance analysis. The findings are part of the Article IV. “Determination of sterols using liquid chromatography with off-line surface-assisted laser desorption/ionization mass spectrometry.”

38

6 CONCLUSION In this thesis, three distinct approaches for polyphenol analysis in plant material are presented. The combination of separation techniques and spectrophotometric determination of antioxidant properties provided complex information about the studied samples. In the first part, a non-targeted approach with no need for the identification of present compounds was used for assessment of various Mentha hybrids and peppermint products. Generally, 30 herbal samples were treated by different extraction techniques and water infusion was chosen as the most appropriate one. PCA was successfully employed for the classification of samples based on findings obtained from CZE fingerprinting and spectrophotometric determination. The proposed procedure might be useful for the assessment of potential protective antioxidant effects of peppermint teas. The second part is focused on the application of HPLC as suitable tool for the identification and quantification of selected groups of polyphenols in different plant parts of sea buckthorn during the annual growing cycle. The changing concentrations of eight polyphenols were investigated by HPLC from April to October together with the determination of total polyphenolic compound and total antioxidant activity by spectrophotometry. HPLC was found to be a better technique than capillary electrophoresis due to better reproducibility and sensitivity. The third study is devoted to the analysis of organic acids, DAA and polyphenols in noni products. Two different detection techniques, photodiode array and ESI-TOF MS, were used for analysis of DAA and organic acids. Significant dissimilarities in content of observed analytes were found in noni products. The antioxidant potential of the samples was determined as additional information about the polyphenols present in the samples. The last part presents an application of RPLC-SALDI MS for the determination of sterols in vegetable oils. Separation of five sterols was achieved in ten minutes using reverse phase LC. Besides on-line UV detection, an off-line SALDI MS method was employed as a detection technique with fmol detection limits. 39

A common part of all presented studies is sample preparation, which is one of the most crucial steps in the analysis of real samples. The optimization of sample treatment procedures like type and combination of extraction techniques is a time consuming but valuable step. The removal of matrices or isolation of analytes can significantly improve the selectivity and sensitivity of analysis. Besides the chemical principles, the simplicity, duration, amount of sample, recovery ratio and automatization should also be considered in the final treatment protocol. The combination of correct extraction techniques for sample treatment, separation methods for analysis and spectrophotometric methods for the determination of antioxidant properties can provide a powerful tool for polyphenolic analysis in real samples.

40

7 LIST OF ABBREVIATIONS UV

ultraviolet

LDL

low density lipoprotein

FA

fatty acid

LC

liquid chromatography

RP

reverse phase

HPLC

high performance liquid chromatography

UPLC

ultra performance liquid chromatography

TLC

thin layer chromatography

HTLC

high temperature liquid chromatography

MEKC

micellar electrokinetic chromatography

CCC

counter current chromatography

CE

capillary electrophoresis

CZE

capillary zone electrophoresis

BGE

background electrolyte

EOF

electroosmotic flow

ITP

isotachophoresis

GC

gas chromatography

MS

mass spectrometry

MS/MS

tandem mass spectrometry

TOF MS

time-of-flight mass spectrometry

ESI

electrospray ionization

Q-MS

quadrupole-mass spectrometry

IT MS

ion trap mass spectrometry

EB MS

electrostatic-magnetic mass spectrometry

LLE

liquid-liquid extraction

LLME

liquid-liquid micro-extraction

SE

solvent extraction

SPE

solid phase extraction

PLE

pressurized liquid extraction

MAE

microwave-assisted extraction

SFE

supercritical fluid extraction 41

MSPD

matrix solid-phase dispersion

SPME

solid-phase microextraction

FID

flame ionization detector

ECD

electron capture detector

NMR

nuclear magnetic resonance

DAD

diode-array detection

AC

antioxidant capacity

AA

antioxidant activity

TAA

total antioxidant activity

TPC

total polyphenolic content

TEAC

trolox equivalent antioxidant capacity

DPPH

2,2-difenyl-1-picrylhydrazyl

ORAC

oxygen radical absorbance capacity

FRAP

ferric ion reducing antioxidant power

FCM

Folin-Ciocalteu′s method

GAE

gallic acid equivalent

PBM

Price-Butler method

RSD

relative standard deviation

ABTS

2,2'-azinobis(3-ethylbenzo-thiazoline-6-sulfonic acid)

DAA

diacetyl asperulosidic acid

BR

brassicaterol

CH

cholesterol

ST

stigmasterol

CA

campesterol

SI

β-sitosterol

SALDI

surface-assisted laser desorption/ionization

ESI-TOF MS

electrospray ionization time-of-flight mass spectrometer

LC-SALDI MS

reversed phase liquid chromatography with offline surface-assisted

laser

desorption/ionization

mass

spectrometry

42

8 REFERENCES [1] [2] [3] [4] [5] [6] [7] [8] [9]

[10] [11] [12] [13] [14]

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9 LIST OF PUBLICATIONS Articles ROBLOVÁ, V., BITTOVÁ, M., KUBÁŇ, P., KUBÁŇ, V.; Capillary electrophoresis fingerprinting and spectrophotometric determination of antioxidant potential for classification of Mentha products. Journal of Separation Science, 2016, Submitted. 

BITTOVÁ, M., KREJZOVÁ, E., ROBLOVÁ, V., KUBÁŇ, P., KUBÁŇ, V.; Monitoring of HPLC profiles of selected polyphenolic compounds in sea buckthorn (Hippophaë rhamnoides L.) plant parts during annual growth cycle and estimation of their antioxidant potential. Central European Journal of Chemistry, 2014, 11, 11521161. 

BITTOVÁ, M., HLADŮVKOVÁ, D., ROBLOVÁ, V., KRÁČMAR, S., KUBÁŇ, P., KUBÁŇ, V.; Analysis of organic acids, deacetyl asperulosidic acid and polyphenolic compounds as a potential tool for characterization of noni (Morinda citrifolia) products. Natural Product Communications, 2015, 11, 1817-1820. 



VRBKOVÁ, B., ROBLOVÁ, V., YEUNG E.S., PREISLER, J.; Determination of sterols using liquid chromatography with off-line surface-assisted laser desorption/ionization mass spectrometry. Journal of Chromatography A, 2014, 1358, 102-109. Presentation ROBLOVÁ, V., BITTOVÁ, M., KUBÁŇ, P., KUBÁŇ, V.; Comparison of antioxidant properties of different Mentha x piperita species and commercial teas by capillary zone electrophoresis and spectroscopy. 11th International Interdisciplinary Meeting on Bioanalysis - CECE 2014. 2014. 



ROBLOVÁ, V.; Applications of separation methods for analysis of phytochemical compounds in plants. XD 107 Student Conference Spring 2016. Brno 2016. Posters ROBLOVÁ, Vendula, Miroslava BITTOVÁ, Petr KUBÁŇ a Vlastimil KUBÁŇ. Characterization of Mentha species and peppermint teas: Correlation between antioxidant properties and electrophoretic fingerprinting. In 22th International Symposium on Electro- and Liquid Phase-Separation Techniques (ITP2015). 2015. 

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ROBLOVÁ, Vendula, Miroslava BITTOVÁ, Petr KUBÁŇ a Vlastimil KUBÁŇ. Antioxidant properties and phytochemical composition of Mentha species and commercial peppermint teas. In 6th French - Czech "Vltava" Chemistry Meeting, Brno, 2015.2015. ISBN 978-80-210-7934-2. 



VRBKOVÁ, B., PREISLER, J., ROBLOVÁ, V., YEUNG, E.S.; Liquid chromatography with substrate-assisted laser desorption/ionization mass spectrometry for determination of sterols. International Mass Spectrometry Conference, 2014. Ženeva 2014. BITTOVÁ, M., HLADŮVKOVÁ, D., ROBLOVÁ,V., KRÁČMAR, S., KUBÁŇ, V.; Comparative study of antioxidant potential in products from noni (Morinda citrifolia L.). Acta Universitatis Palackianae Olomucensis Chemica 51S. Chiranal 2014, Palacký University, Olomouc, 2014. 

ROBLOVÁ, V., HODÁKOVÁ, J., BITTOVÁ, M., KUBÁŇ, V.; Phytochemical composition and antioxidant properties of Mentha species. Acta Universitatis Palackianae Olomucensis Chemica 51S. Chiranal 2014, Palacký University, Olomouc, 2014. 

KREJZOVÁ, E., BITTOVÁ, M., ROBLOVÁ, V., KUBÁŇ, V.; HPLC profiles of polyphenolic compounds and estimation of antioxidant potential in sea buckthorn plant parts. 20th International Symposium on Electro- and Liquid PhaseSeparation Techniques - ITP 2013, Tenerife 2013. 

ROBLOVÁ, V., HANZLÍKOVÁ, E., BITTOVÁ, M., KUBÁŇ, V.; Bioresidues from viticultural material - antioxidant potential of Vitis vinifera plant parts. Acta Universitatis Palackianae Olomucensis Chemica 50S. Chiranal 2012, Palacký University, Olomouc, 2012. 



BITTOVÁ, M., JELÍNKOVÁ K., ROBLOVÁ V., TRNKOVÁ, L.; Electrophoretic techniques in the analysis of polydeoxycytidylic acids. XII. Pracovní setkání fyzikálních chemiků a elektrochemiků. Brno: Mendelova univerzita v Brně, 2012. 

BITTOVÁ, M., ROBLOVÁ, V., MAJEROVÁ E., TRNKOVÁ L,; Polyethyleneimine coated capillary in nucleotide and short oligonucleotide analysis. 8th International Interdisciplinary Meeting on Bioanalysis - CECE 2011. 2011. 

ROBLOVÁ, V., TÁBORSKÝ P., TRNKOVÁ L., BITTOVÁ M.; Electrophoretical studies of interactions between oligonucleotides and chelerythrine. IX. pracovní setkání fyzikálních chemiků a elektrochemiků. Brno: Mendelova zemědělská a lesnická univerzita, 2009.

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10 CURRICULUM VITAE Name and surname:

Vendula Roblová

Title:

Mgr.

Date of birth:

7. 7. 1987, Hodonín

Address:

Na Dílech 220, Dubňany 696 03

Correspondence address:

Křížkovského 17, Brno 603 00

Phone number:

+420 723 211 719

e-mail:

[email protected]

Education 

2011 – now

Masaryk University, Faculty of Science, Brno Field: Analytical chemistry (Ph.D. program)



2009 – 2011

Masaryk University, Faculty of Science, Brno Field: Analytical chemistry (master program)



2006 – 2009

Masaryk University, Faculty of Science, Brno Field: Chemistry (bachelor program)



2002 – 2006

Secondary Technical School of Chemistry, Brno Field: Pharmaceutical substances

Work experience 

01/10/2009 – 28/02/2010 Eligo Brno – basic analysis of whey (control parameters and purity during processing)



01/09/2008 – 31/01/2009 Dacom Pharma s.r.o., Kyjov – country manager for Slovak Republic (presentation and sale of food supplements for warehouses and pharmacies)

Stays and internships 

05/02/2013 – 31/07/2013 Erasmus stay at Universitat Autónoma de Barcelona, Barcelona, Spain



02/07/2009 – 31/07/2009 Veterinary laboratories MVDr. Šotola s.r.o., Kroměříž – practice with HPLC, GC, AAS, ITP instruments, analysis of water and food samples

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Language skills 

English (advanced), Spanish (beginner)

Other skills and experience 

Computer skills: MS Office (Word, Excel, Powerpoint), ChemSketch, OriginPro, Simul (basics), Statistika (basics)



Driving license B



Knowledge of analytical chemistry, focused on separation methods



Teaching, consulting and assessment of bachelor and master students

Conferences and seminars 

16th School of Mass Spectrometry, Frymburk, 2015



22nd International Symposium on Electro- and Liquid Phase Separation Techniques (ITP 2015), Helsinki, 2015



6th French – Czech “Vltava” Chemistry Meeting, Brno, 2015



11th International Interdisciplinary Meeting on Bioanalysis, CECE, Brno, 2014



15th School of Mass Spectrometry, Frymburk, 2014



Scientific seminar – GC a CE in food, pharmaceutical and clinical analysis, Česká chromatografická škola, Praha, 2014



Advances in Chromatography and Electrophoresis and Chiranal. Olomouc, 2014



14th School of Mass Spectrometry, Jeseník, 2013



Advances in Chromatography and Electrophoresis and Chiranal, Olomouc, 2012



MendelNet, Proceedings of International Ph.D. Students Conference, Brno, 2011



8th International Interdisciplinary Meeting on Bioanalysis, CECE, Brno, 2011



Scientific seminar – Solid Phase Extraction Method Development, Phenomenex, Brno, 2010



IX. Workshop of physical chemistry and electrochemistry Brno, 2009

Other activities 

A member of student chamber of the Academic Senate, Faculty of Science, Masaryk University (09/2011 – 09/2014)



A tutor for foreign students in ISC organization (International student club)

Personal qualities 

Reliability, communication skills, organization skills, patience, independence



Hobbies: swimming, running, reading, skiing, volleyball, theater, music, traveling 51

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