KAUNAS UNIVERSITY OF MEDICINE
FACULTY OF PHARMACY,
CHARLES UNIVERSITY IN PRAGUE
FACULTY OF PHARMACY IN HRADEC KRALOVE
Analysis of flavonoids and phenolic acids in grass of
Desmodium canadense (L) DC
Master’s work
Supervisors of the work: Dr. R.Sladkovsky Dr. G. Puodziuniene Hab. Dr. V. Janulis
Work was performed by Mintautas Kamandulis, 5th year 2nd group student
Kaunas
Hradec Kralove
Content
Abbreviations
……….3Introduction
………4Theoretical part………....6
I. Desmodium canadense – description
………61.1 Definition……….6
1.2 Taxonomy………6
1.3 Morphology……….6
1.4 Uses in medicine………..7
1.5 Main phytochemical compounds……….……8
II. Phenolic compounds
………..92.1 Flavonoids………..9 2.1.1 Chemical structure……….9 2.1.2 Classification……….…..10 2.1.3 Distribution in nature………...12 2.1.4 Use of flavonoids……….13 2.2 Phenolic acids………...…13
III. HPLC – theory and instrumentation
………...……153.1 Theory………...……15
3.1.1 Types of HPLC………15
3.1.2 Retention mechanism………..18
3.1.3 Stationary phases (adsorbents)………18
3.1.4 Mobile phases………..19
3.2 Instrumentation………...…19
3.2.1 HPLC system……….…..19
3.2.2 Mobile phase reservoir, filtering……….20
3.2.3 Pumps………..20
3.2.4 Columns………...21
3.2.5 Detectors………..21
3.2.6 Data systems………22
Experimental part
………25I. Reagents and materials………..25
1.1 Object………...25
1.2 Chemicals………...25
1.3 Vessels, filters and sorbents……….26
1.4 Solutions………..27
1.5 Instrumentation and software………..27
II. Experiment………28
2.1 Extraction………....28
2.2 Filtration………..28
2.3 Solid phase extraction.………28
2.4 High performance liquid chromatography………..28
Results and discussion
………..291. Development of HPLC method………...29
2. Choosing best extraction conditions………30
3. Development of SPE method………..31
4. Identification of compounds in extract………...…32
5. Quantification……….34
Conclusions
………43References
………44Acknowledgements………46
Abbreviations
HPLC – High Performance Liquid Chromatography SPE – Solid Phase Extraction
TRIS - Tris[hydroxymethyl]aminomethane LC – liquid chromatography
UV – Ultra-Violet
Introduction
Today the largest part of drugs in pharmacy is of synthetic origin. Herbal drugs are not used very often and are not very popular. But this situation is changing – to the pharmacy come more and more herbal drugs. And we should take more attention to this part of drugs, because herbal drugs has less adverse effects, and are more safe then synthetic analogs. Plants have many useful substances – and one of them is flavonoids with other phenolic compounds. This is a very big group of biological active compounds. Flavonoids have been referred to as "nature's biological response modifiers" because of their ability to modify the body's reaction to other compounds such as allergens, viruses, and carcinogens. They show anti-allergic, anti-inflammatory, and anti-cancer activity. In addition, flavonoids act as powerful antioxidants, providing remarkable protection against oxidative and free radical damage. As a result, consumers and food manufacturers have become increasingly interested in flavonoids for their healthful properties, especially their potential beneficial role in the prevention of cancer and cardiovascular diseases. The beneficial effects of fruits and vegetables are now often attributed to flavonoid compounds rather than to known nutrients and vitamins (1-3). Phenolic acids are interesting of their protective role against oxidative damage diseases (coronary heart disease, stroke, and cancers)
One of possible sources of flavonoids and phenolic acids – Canadian thick-trefoil (showy trefoil) –
Desmodium canadense (L) DC. This plant is not researched as good as many other plants, but there
are some works on it. In Lithuania, KUM, there were some research works on Desmodium
canadense (L.) DC. In Lithuania was created 0.2% solution is used for ophtalmoherpes and
adenoviral infections.
Flavonoids are a large group of natural polyphenols. According to their chemical structure, there are several different classes of flavonoids: flavonols, flavones, flavanones, flavan-3-ols, isoflavones, and anthocyanidins. Amount of those compounds is very important, when we are talking about quality of our plant material. In this work for determination of flavonoids was used High Performance Liquid Chromatography (HPLC) as the best method for determination of phenolic compounds.
The aim of work:
The main aim of this work is to determine flavonoids and phenolic acids, and their amounts in plant material of Desmodium canadense (L) DC.
The tasks of work:
1. Identify flavonoids and phenolic acids in herb of Desmodium canadense (L).
2. Determine flavonoids and their amounts in plant material, collected in different phase of vegetation. Determine phenolic acids and their amounts in the same plant material.
3. Research the influence of fertilizing to amount of flavonoids in Desmodium canadense (L)
Theoretical part
I.Desmodium canadense - description
1.1 Definition
Desmodium canadense (L) DC, Fabaceae. Common names - Canadian thick-trefoil, showy
tick-trefoil, hoary tick clover; puapilipili at Hawaii.
Part used in medicine: Plant material – dried herb. According to folk medicine – use without fruits and seeds.
Geographical distribution – North America: Canada, north and north-east of USA. It is possible to grow in other region with similar climate properties.
1.2 Taxonomy
Kingdom Plantae -- Planta, plantes, plants,
Subkingdom Tracheobionta - vascular plants
Division Magnoliophyta - angiospermes,
angiosperms, flowering plants,
Class Magnoliopsida - dicots,
Subclass Rosidae
Order Fabales
Family Fabaceae
Genus Desmodium Desv.- perennial
legumes, tick trefoil, tick clover, tick trefoil
Species Desmodium canadense (L.) DC.-
Canada tick clover, showy tick-trefoil, and showy tick trefoil.
1.3
Morphology
This native perennial is about 90 cm tall and normally erect, although it sometimes sprawls along the ground. The green central stem has fine white hairs, sometimes turning brown in response to drought. There are usually a few flowering side stems in the upper half of a large mature plant. The compound leaves consist of three leaflets that are grayish green. Each compound leaf has a short petiole with a pair of small deciduous sheaths at its base. The leaflets are 5 – 8 cm long and less than half as wide. They are oblong or lanceolate in overall shape, but rounded at the tips rather than pointed. Their undersides have fine hooked hairs that cling to clothing or the fur of passing animals. Numerous pink flowers in an elongate raceme occur at
the end of one or more of the upper stems. The flowers are about 1,3 cm across (as measured vertically), consisting of an upper and a lower petal. These petals are initially folded and keel-like, but eventually open wide, beginning with the upper petal. There is a small patch of dark pink at the center of the flower, from which emerges an upwardly curved white tube containing the stigmas and pistil. There is no floral scent. Each flower occurs on a hairy red pedicel, and has a hairy greenish red calyx. The blooming period occurs during mid-summer and lasts about 3 weeks. The flat seedpods have 3 to 5 segments and are about 6,5 cm long. Like the undersides of the leaves, they are
covered with fine hooked hairs, and are distributed by passing animals. Usually, the lower side of a seedpod is more rounded than the upper side. Technically, these seedpods are called „loments”. The root system consists of a taproot that is long, slender, and brown (4).
1.4 Uses in medicine
Offers to use Desmodium canadense for:
1. Increasing immunity, treat viral diseases (5).
2. Helepin D, which is got from Desmodium canadense (L) DC, has antimicrobic, anti-inflammatory, antiviral, hipoazotemic properties (6).
3. In Ukraine from Desmodium canadense is produced antiviral ointment “Flavex”, which has anti-inflammatory, analgesic properties and increases healing of wounds (5).
4. 0.2% solution is used for ophtalmoherpes and adenoviral infections (7).
5. Folk medicine. Hawaiians use leaves, smoke them for asthma. In China for kidney and gall stones. One of the plants used for back pain, joint pain and muscle cramps. Caution - do not use seeds of plant (8).
1.5 Main phytochemical compounds
Main biological active compounds in Desmodium canadense (L.) are flavonoids. Ukrainian scientists N.V. Chernobrovaya and other researched some flavanoids of desmodium canadense. They proved this using thin layer chromatography and wrote UV spectra of each compound. There were C-glycosides of apigenin and liuteolin: vitexin, saponaretin, vicein, homoorientin. Also rutin was found (9, 10). There were 2 types of saponaretin – saponaretin-1 and saponaretin-2, and also 2 glycosides – homoadonivernit and desmodin. No data about presence of phenolic acids was found, there are only small amounts of them. So analysis of phenolic acids was carried out for the first time.
II. Phenolic compounds
There are several thousand known to be found in plants, and several hundred found in grapes and wines. They can be classificated into this groups:
a. Benzoic acid derivatives b. Cinnamic acid derivatives c. Flavonoid derivatives.
They all are represented further (11).
2.1 Flavonoids
2.1.1 Chemical structure
Flavonoids are water soluble polyphenolic molecules containing 15 carbon atoms. Flavonoids belong to the polyphenol family (12). Flavonoids can be visualized as two benzene rings which are joined together with a short three carbon chain.
Figure No 2: Sample of Flavonoid basis
The skeleton above can be represented as the C6 – C3 – C6 system (13). One of the carbons of the
short chain is always connected to a carbon of one of the benzene rings, either directly or through an oxygen bridge, thereby forming a third middle ring, which can be five or six-membered (12). The flavonoid variants are all related by a common synthetic pathway. The flavonoid initially formed in the biosynthesis is now thought to be a chalcone and all other forms are derived from this by a variety of routes. Further modification of the flavonoid may occur at various stages resulting in: hydroxylation, methylation, isoprenylation, methylenation, dimerization, and, the most important – glycosilation of the hydroxyl groups or of the flavonoid nucleus.
Flavonoid O-glycosides: Flavonoid mainly occur as flavonoid O-glycosides in which one or more
of the flavonoid hydroxyl groups is bound to a sugar or sugars by an acid-labile hemiacetal bond. The effect of glycosilation is to render the flavonoid less reactive and more water (sap) soluble, the
latter property permitting storage of the flavonoids in the cell vacuole. Hydroxyl groups in any position on the flavonoid nucleus may be glycosylated. Glucose is the sugar most commonly involved; although galactose, rhamnose, xylose and arabinose are not uncommon. Other sugars occasionally encountered include allose, mannose, fructose, appiose and glucuronic and galacturonic acids. Disaccharides are also often found in association with flavonoids, e.g. sophorose, gentiobiose, rutinose, neohesperidose, and occasionally tri- and even tetra-saccharides. O-glycosilation is one of the last stages of biosynthesis in plants. It is possible the other modification – acylation, and it is an ester bond.
Flavonoid C-glycosides: Sugars may also be C-linked to the flavonoid, and in this case they are
directly attached to the benzene nucleus by a carbon-carbon bound, which is acid resistant. C-linked sugars have been found only at 6- and 8- positions on flavonoid nuclei. The range of sugars involved is apparently very much smaller than in O-glycosides, and includes glucose most commonly, and also galactose, rhamnose, xylose and arabinose. The range of flavonoid aglycone types involved is also very restricted (1).
2.1.2 Classification
Over 5000 naturally occurring flavonoids have been characterized from various plants. They have been classified according to their chemical structure, and are usually subdivided into 6 main subgroups:
1. Flavonols: Quercetin, Kaempferol, Myricetin, Isorhamnetin (2). They have double joint at C2
-C3, and hydroxy-group at C3 (16).
Figure No 3: Base of Flavonols
2. Flavones: Luteolin, Apigenin (2). Almost the same as flavonols, but they don’t have
Figure No 4: Base of Flavones
3. Flavanones: Hesperetin, Naringenin, Eriodictyol (2). This group has dihydro-γ-pirone ring, which is easy to open, and then flavanones become chalcones. This group is often found together with chalcones. There are some flavanones with metoxy- groups (16).
Figure No 5: Base of Flavanones
4. Flavan-3-ols: (+)-Catechin, (+)-Gallocatechin, Epicatechin, *Epigallocatechin, (-)-Epicatechin 3-gallate, (-)-Epigallocatechin 3-gallate, Theaflavin, Theaflavin 3-gallate, Theaflavin 3'-gallate, Theaflavin 3,3' digallate, Thearubigins (2). They have one difference from
flavanones – they have hydroxy group at C3. They are very labile, so they are not found in big
amounts. In more cases they are found in free form, not in glycoside form (16).
5. Isoflavones: Genistein, Daidzein, Glycitein (2). They are different from other groups of
flavonoids, because they have their phenolic ring not in position at C2, but in position C3 (16).
Figure No 6: Base of isoflavones
6. Anthocyanidins: Cyanidin, Delphinidin, Malvidin, Pelargonidin, Peonidin, Petunidin (2). This group has free valence at O atom in pyrano-ring. So they can make salts with acids and alkali. They have colors – e.g. yellow – pelargonidin, blue – delfinidin, violet – cianidin. More hydroxyl groups give stronger blue color, and more metoxy groups give stronger red color. They are found in glycoside form, and they are called anthocians. More often are found
anthocians with Cyanidin. Pelargonidin in more cases is found in tropical plants, and delphinidin – in plant from the north (16).
Figure No 7: Base of Anthocyanidins
7. Other subgroups:
1. Chalcones and dihydrochalcones. Isoliquiretin. They are flavonoids with opened pirone ring. In acid environment they become flavonoids.
Figure No 8: Base of chalcones
2. Aurones. Sulphuretin, aureuzidin, leptozidin. They are yellow, orange or yellow orange pigments of plants. They are very rare, and in most cases in form of glycosides (16). Six-membered heterocyclic ring C is replaced by a five - Six-membered ring (15).
Figure No 9: Base of aurones
2.1.3 Distribution in nature
Flavonoids are characteristic constituents of green plants with the exception of algae and hornworts. They occur in virtually all parts of plants including leaves, roots, wood, bark, pollen, nectar, flowers, berries and seeds. In the few recorded cases of flavonoids being found in animals, for example in the beaver scent gland, propolis, and in butterfly wings, it is considered, that the flavonoids originate from the plants upon which the animals feed rather, then being biosynthesized
in situ. Flavonoids occurs in more highly evolved plant groups, such as Angiosperms, and older
plant groups are lacking flavonoids, e.g. Chlorophyta or cyanophyta. Another important feature of the distribution of flavonoids in plants is the strong tendency for taxonomically related plants to produce similar types of flavonoids (14).
2.1.4 Use of flavonoids
Flavonoids are becoming very popular because they have many health promoting effects. Some of the activities attributed to flavonoids include: allergic, cancer, antioxidant, anti-inflammatory and anti-viral. The flavonoids quercetin is known for its ability to relieve hay fever, eczema, sinusitis and asthma. Together with carotenes, flavanoids are also responsible for the coloring of fruits, vegetables and hernia. Flavonoids have antioxidant activity (12).
2.2 Phenolic acids
Phenolic acids are plant metabolites widely spread throughout the plant kingdom. Recent interest in phenolic acids stems from their potential protective role, through ingestion of fruits and vegetables, against oxidative damage diseases (coronary heart disease, stroke, and cancers). Phenolic compounds are essential for the growth and reproduction of plants, and are produced as a response for defending injured plants against pathogens. The importance of antioxidant activities of phenolic compounds and their possible usage in processed foods as a natural antioxidant have reached a new high in recent years.
Phenolics in Plants. Phenolic acid compounds seem to be universally distributed in plants. They
have been the subject of a great number of chemical, biological, agricultural, and medical studies. Phenolic acids form a diverse group that includes the widely distributed hydroxybenzoic and hydroxycinnamic acids.
Chemistry of Phenolics.
Plant phenolic compounds are diverse in structure but are characterised by hydroxylated aromatic rings (e.g. flavan-3-ols). They are categorised as secondary metabolites, and their function in plants is often poorly understood. Many plant phenolic compounds are polymerised into larger molecules such as the proanthocyanidins (PA; condensed tannins) and lignins (17).
Figure No 10: Benzoic acid derivatives (C6-C1) Figure No 11: Cinnamic acid derivatives (C6-C3)
compound R1 R2
p-Hydroxybenzoic acid H H
Vanillic acid H OCH3
Gallic acid OH OH
Syringic acid OCH3 OCH3
compound R1 R2
cinnamic acid H H
p-Coumaric acid H OH
Caffeic acid OH OH
Ferulic acid OCH3 OH
Furthermore, phenolic acids may occur in food plants as esters or glycosides conjugated with other natural compounds such as flavonoids, alcohols, hydroxyfatty acids, sterols, and glucosides. Hydroxycinnamic acid compounds occur most frequently as simple esters with hydroxy carboxylic acids or glucose. Hydroxybenzoic acid compounds are present mainly in the form of glucosides (11).
III. HPLC – theory and instrumentation
3.1 Theory
High Performance Liquid Chromatography (HPLC) is one mode of chromatography, the most widely used analytical technique. Chromatographic processes can be defined as separation techniques involving mass-transfer between stationary and mobile phases. HPLC utilizes a liquid mobile phase to separate the components of a mixture. These components (or analysts) are first
dissolved in a solvent, and then forced to flow through a chromatographic column under a high
pressure. In the column, the mixture is resolved into its components. The amount of resolution is important, and is dependent upon the extent of interaction between the solute components and the stationary phase. The stationary phase is defined as the immobile packing material in the column. The interaction of the solute with mobile and stationary phases can be manipulated through different choices of both solvents and stationary phases. As a result, HPLC acquires a high degree of versatility not found in other chromatographic systems and it has the ability to easily separate a wide variety of chemical mixtures.
Figure No 12: Fast and high-efficient separation of some aromatics. Hypersil-C8 (100x2) 3 mm, 60% MeOH in Water, 1.5 ml/min., 1 - Benzamide, 2 - Benzil Alcohol, 3 - Acetophenone, 4 - Methyl Benzoate, 5 - Phenetole, 6 - Naphthalene, 7 - Benzophenone 8 - Biphenyl.
3.1.1 Types of HPLC
There are many ways to classify liquid column chromatography. If this classification is based on the nature of the stationary phase and the separation process, three modes can be specified.
In adsorption chromatography the stationary phase is an adsorbent (like silica gel or any other silica based packing) and the separation is based on repeated adsorption-desorption steps.
In ion-exchange chromatography the stationary bed has an ionically charged surface of opposite charge to the sample ions. This technique is used almost exclusively with ionic or ionizable samples. The stronger the charge on the sample, the stronger it will be attracted to the ionic surface and thus, the longer it will take to elute. The mobile phase is an aqueous buffer, where both pH and ionic strength are used to control elution time.
In size exclusion chromatography the column is filled with material having precisely controlled pore sizes, and the sample is simply screened or filtered according to its solvated molecular size. Larger molecules are rapidly washed through the column; smaller molecules penetrate inside the porous of the packing particles and elute later. Mainly for historical reasons, this technique is also called gel filtration or gel permeation chromatography although, today, the stationary phase is not restricted to a "gel".
Concerning the first type, two modes are defined depending on the relative polarity of the two phases: normal and reversed-phase chromatography.
• In normal phase chromatography, the stationary bed is strongly polar in nature (e.g., silica
gel), and the mobile phase is nonpolar (such as n-hexane or tetrahydrofuran). Polar samples are thus retained on the polar surface of the column packing longer than less polar materials.
• Reversed-phase chromatography is the inverse of this. The stationary bed is nonpolar
(hydrophobic) in nature, while the mobile phase is a polar liquid, such as mixtures of water and methanol or acetonitrile. Here the more nonpolar the material is, the longer it will be retained.
Above mentioned types cover almost 90% of all chromatographic applications. Eluent polarity plays the highest role in all types of HPLC. There are two elution types: isocratic and gradient. In the first type constant eluent composition is pumped through the column during the whole analysis. In the second type, eluent composition (and strength) is steadily changed during the run.
Figure No 13: Overlay of the four components trace analysis chromatograms. (A) is the isocratic elution, (B) is the gradient elution, shadow line is the gradient profile from 30% acetonitrile in water to 65% acetonitrile.
HPLC as compared with the classical technique is characterized by:
• small diameter (2-5 mm), reusable stainless steel columns;
• column packing with very small (3, 5 and 10 mm) particles and the continual development
of new substances to be used as stationary phases;
• relatively high inlet pressures and controlled flow of the mobile phase;
• precise sample introduction without the need for large samples;
• special continuous flow detectors capable of handling small flow rates and detecting very
small amounts;
• automated standardized instruments;
• rapid analysis; and
• high resolution.
Initially, pressure was selected as the principal criterion of modern liquid chromatography and thus the name was "high pressure liquid chromatography" or HPLC. This was, however, an unfortunate term because it seems to indicate that the improved performance is primarily due to the high pressure. This is, however, not true. In fact high performance is the result of many factors: very small particles of narrow distribution range and uniform pore size and distribution, high pressure column slurry packing techniques, accurate low volume sample injectors, sensitive low volume detectors and of course, good pumping systems. Naturally, pressure is needed to permit a given flow rate of the mobile phase; otherwise, pressure is a negative factor not contributing to the improvement in separation. Recognizing this, most experienced chromatographers today, refer to
the technique as high performance liquid chromatography still permitting the use of the acronym HPLC.
3.1.2 Retention mechanism
In general, HPLC is a dynamic adsorption process. Analyte molecules, while moving through the porous packing bead, tend to interact with the surface adsorption sites. Depending on the HPLC mode, the different types of the adsorption forces may be included in the retention process:
Hydrophobic (non-specific) interactions are the main ones in reversed-phase separations. Dipole-dipole (polar) interactions are dominated in normal phase mode.
Ionic interactions are responsible for the retention in ion-exchange chromatography.
All these interactions are competitive. Analyte molecules are competing with the eluent molecules for the adsorption sites. So, the stronger analyte molecules interact with the surface, and the weaker the eluent interaction, the longer analyte will be retained on the surface.
SEC (size-exclusion chromatography) is a special case. It is the separation of the mixture by the molecular size of its components. In this mode any positive surface interactions should be avoided (eluent molecules should have much stronger interaction with the surface than analyte molecules). Basic principle of SEC separation is that the bigger the molecule, the less possibility for her to penetrate into the adsorbent pore space, so, the bigger the molecule the less it will be retained.
3.1.3 Stationary Phases (Adsorbents)
HPLC separations are based on the surface interactions, and depend on the types of the adsorption sites (surface chemistry). Modern HPLC adsorbents are the small rigid porous particles with high surface area.
Main adsorbent parameters are:
• Particle size: 3 to 10 µm
• Particle size distribution: as narrow as possible, usually within 10% of the mean;
• Pore size: 70 to 300 Ĺ;
• Surface area: 50 to 250 m2/g
The last parameter in the list represents an adsorbent surface chemistry. Depending on the type of
the ligand attached to the surface, the adsorbent could be normal phase (-OH, -NH2), or
reversed-phase (C8, C18, Phenyl), and even anion (NH4+), or cation (-COO-) exchangers.
3.1.4 Mobile phases
In HPLC type and composition of the mobile phase (eluent) is one of the variables influencing the separation. Despite of the large variety of solvents used in HPLC, there are several common properties:
• Purity
• Detector compatibility
• Solubility of the sample
• Low viscosity
• Chemical inertness
• Reasonable price
Each mode of HPLC has its own requirements. For normal phase mode solvents are mainly nonpolar, for reversed-phase eluents are usually a mixture of water with some polar organic solvent such as acetonitrile.
Size-exclusion HPLC has special requirements, SEC eluents has to dissolve polymers, but the most important is that SEC eluent has to suppress all possible interactions of the sample molecule with the surface of the packing material.
3.2 Instrumentation
3.2.1 HPLC system
Figure No 14: basic HPLC machine scheme
The most important parts of HPLC machine are: mobile phase reservoir, pump, and column, detectors, and data system (18). Only data system is not shown in this picture. In the scheme is possible to see those parts and the schematically process of working HPLC system.
3.2.2Mobile phase reservoir, filtering
The most common type of solvent reservoir is a glass bottle. Most of the manufacturers supply these bottles with the special caps, Teflon tubing and filters to connect to the pump inlet and to the purge gas (helium) used to remove dissolved air. Helium purging and storage of the solvent under helium was found not to be sufficient for degassing of aqueous solvents. It is useful to apply a vacuum for 5-10 min. and then keep the solvent under a helium atmosphere.
3.2.3Pumps
High pressure pumps are needed to force solvents through packed stationary phase beds. Smaller bed particles require higher pressures. There are many advantages to using smaller particles, but they may not be essential for all separations. The most important advantages are: higher resolution, faster analyses, and increased sample load capacity. However, only the most demanding separations require these advances in significant amounts. Many separation problems can be resolved with
larger particle packing that requires less pressure. Thus, if the user has only moderate needs and a restricted budget, his money need not be spent on a maximum pressure pump.
Flow rate stability is another important pump feature that distinguishes pumps. Very stable flow rates are usually not essential for analytical chromatography. However, if the user plans to use his system in size exclusion mode, then he has to have a pump which provides an extremely stable flow rate.
An additional pump feature found on the more elaborate pumps is external electronic control. Although it adds to the expense of the pump, external electronic control is a very desirable feature when automation or electronically controlled gradients are to be run. Alternatively, this becomes an undesirable feature (since it is an unnecessary expense) when using isocratic methods. The degree of flow control also varies with pump expense. More expensive pumps include such state-of-the-art technology as electronic feedback and multiheaded configurations.
Modern pumps have the following parameters:
• Flow rate range: 0.01 to 10 ml/min
• Flow rate stability: not more than 1% (short term)
• For SEC flow rate stability should be less than 0.2%
• Maximum pressure: up to 5000 psi
It is desirable to have an integrated degassing system, either helium purging, or better vacuum degassing.
3.2.4. Columns
Typical LC columns are 10, 15 and 25 cm in length and are fitted with extremely small diameter (3, 5 or 10 mm) particles. The internal diameter of the columns is usually 4 or 4.6 mm; this is considered the best compromise among sample capacity, mobile phase consumption, speed and resolution. However, if pure substances are to be collected (preparative scale), larger diameter columns may be needed
Packing of the column tubing with the small diameter particles requires high skill and specialized equipment. For this reason, it is generally recommended that all but the most experienced chromatographers purchase prepacked columns, since it is difficult to match the high performance of professionally packed LC columns without a large investment in time and equipment.
In general, LC columns are fairly durable and one can expect a long service life unless they are used in some manner which is intrinsically destructive, as for example, with highly acidic or basic eluents, or with continual injections of 'dirty' biological or crude samples. It is wise to inject some test mixture (under fixed conditions) into a column when new, and to retain the chromatogram. If questionable results are obtained later the test mixture can be injected again under specified conditions. The two chromatograms may be compared to establish whether or not the column is still useful (18).
3.2.5 Detectors
The detector for an HPLC is the component that emits a response due to the eluting sample compound and subsequently signals a peak on the chromatogram. It is positioned immediately posterior to the stationary phase in order to detect the compounds as they elute from the column. The bandwidth and height of the peaks may usually be adjusted using the coarse and fine tuning controls, and the detection and sensitivity parameters may also be controlled (in most cases). There are many types of detectors that can be used with HPLC. Some of the more common detectors include: Refractive Index (RI), Ultra-Violet (UV), Fluorescent, Radiochemical, Electrochemical, Near-Infra Red (Near-IR), Mass Spectroscopy (MS), Nuclear Magnetic Resonance (NMR), and Light Scattering (LS) (19).
3.2.6 Data systems
Since the detector signal is electronic, use of modern data systems can aid in the signal analysis. The main goal in using electronic data system is to increase analysis accuracy and precision. There are different types of data systems. In routine analysis, where no automation is needed, a pre-programmed computing integrator may be sufficient. If higher control levels are desired, a more intelligent device is necessary, such a data station or minicomputer. The advantages of intelligent processors in chromatographs are found in several areas: 1) additional automation options are easier to implement. 2) Complex data analysis becomes more feasible. Software safeguards can be designed to reduce accidental misuse of the system. User programmable systems are becoming less expensive and increasingly practical. Other more advanced features can also be applied to a chromatographic system, including computer controlled automatic injectors, multi-pump gradient controllers and sample fraction collectors (18).
IV. Solid phase extraction
Solid-phase extraction (SPE) is an extraction method that uses a solid phase and a liquid phase to isolate one, or one type, of analyte from a solution. It is usually used to clean up a sample before using a chromatographic or other analytical method to quantitate the amount of analyte(s) in the sample. The general procedure is to load a solution onto the SPE phase, wash away undesired components, and then wash off the desired analytes with another solvent into a collection tube (20).
Basic principles. Traditional liquid-liquid extraction procedures employ a serial extraction of an
aqueous sample with an organic solvent resulting in a relatively large volume of solvent that must be dried and concentrated prior to analysis. In SPE procedures, a solid sorbent material, typically an alkyl bonded silica, is packed into a cartridge or imbedded in a disk and performs essentially the same function as the organic solvent in liquid-liquid extraction. For example, reverse-phase SPE employed to extract non-polar compounds, pesticides for instance, from polar samples such as water generally utilize a solid sorbent containing non-polar functional groups such as octadecyl (C18) or octyl (C8) bonded silica’s. Aqueous samples are pumped or pulled through a cartridge or disk and organic compounds in samples interact with non-polar functional groups on the sorbent and are effectively extracted from the sample. Organic compounds in the original aqueous sample are eluted from the cartridge or disk with a small volume of organic solvent.
Procedure. In simplistic terms, pumping a sample through a disk or cartridge to trap organic
analytes and then eluting the organic analytes from the solid sorbent with organic solvent is all there is to SPE methods. In practice; however, SPE methods are not quite this simplistic. Several steps are required in a typical reversed-phase SPE, including:
1. Washing solid phase sorbent with organic solvent or mixture of solvents to remove potential interferents from SPE system.
2. Conditioning or activating the solid sorbent with organic solvents or mixture of organic solvents and reagent water.
3. Preparation of sample, typically by addition of methanol, followed by extraction of sample by passing sample through solid sorbent.
4. Drying solid sorbent by passing air or nitrogen through disk or cartridge.
5. Cleanup of sample extract to remove possible contaminants in sample trapped in sorbent. 6. Elution of organic analytes from solid sorbent with organic solvents or mixtures of solvents. 7. Drying sample eluate with sodium thiosulfate to remove any residual water.
8. Concentrating sample extract and solvent exchange if necessary.
Washing extraction system with organic solvents is necessary in order to minimize any contamination that may be present in the system and ultimately interfere with analysis. Activation
of the solid sorbent is critical in order to effectively extract organic analytes from the aqueous sample. If the disk or cartridge is not conditioned properly, the solid phase particles may not be solvated causing problems with sample flow through the disk or cartridge and can ultimately result in low recoveries of organic analytes. Proper solvation or activation of the sorbent is important in order to prevent the polar sample from simply flowing past hydrophobic solid phase particles which minimizes contact between organic analytes in the sample with sorbent, and the organic analytes in the sample are not retained by the solid phase. Prior to processing the sample through the sorbent it is very critical to maintain solvation of the solid phase by either allowing a layer of solvent, usually methanol, or reagent water to remain on the disk or at the top of the sorbent column in cartridge systems. With some methods an additional cleanup step may be added to remove interferences in the sample that are also trapped by the solid phase. It is also critical to dry the eluate prior to final concentration and/or solvent exchange because the eluate often contains a small amount of water from the sample (21).
Experimental part
I. Reagents and materials
1.1 Object
Object – dried herb of Desmodium canadense, which was collected in Botanical Garden of Vytautas Magnus University in Kaunas, Lithuania. Material was collected in different phases of vegetation, including butanisation, flowering and growing of fruits. The exact time of collecting was fixated. Also was collected plant material from fields with different type of fertilization – plants, fertilized with carbamide, super phosphate, potassium nitrate and with mixture of those fertilising materials. The last part of samples were samples of new-planted plants, which were collected when passed few time after planting, and later, when plant regrew after cutting. Dried material was pulverized into particles not bigger than 0,63 mm. Around 0,5 g of pulverized material were weighted on analytical weighting machine, in accuracy of four places after coma with exact weight. Samples were collected in this time:
Collection time Growing phase
2003 06 13 Beginning of butanization
2003 06 23 Flowering
2003 08 02 Growing fruits
2003 08 12 Growing fruits, end of flowering
2003 08 22 Immature fruits
2003 09 08 Fruits mature
2003 09 18 Fruits
2003 09 22 Fruits after first cold
Table No 2: Samples and their collection times
1.2 Chemicals
All chemicals were used for analytical purposes. Ultra pure water was used. Chemical substances: 1. Standards, which are written in the table.
3. Methanol CHROMASOLV for HPLC, produced by Sigma-Aldrich Chemie GmbH, Germany.
4. Ammonium chloride, produced by Lachema, Czech Republic.
5. Acetonitrile CHROMASOLV for HPLC, produced by Sigma-Aldrich Chemie Gmbh, Germany.
6. TRIZMA® Base (Tris[hydroxymethyl]aminomethane), produced by Sigma-Aldrich Chemie Gmbh, Germany.
7. Isopropyl alcohol, produced by PENTA, Czech Republic.
Standards Purity, % Manufacturer
Syringic acid >98 Sigma-Aldrich Chemie Gmbh, Germany
3,4-dihydroxycinnamic acid (Caffeic acid)
97 Sigma-Aldrich Chemie Gmbh, Germany
Vanillic acid 97 Sigma-Aldrich Chemie Gmbh, Germany
Trans-4-Hydroxy-3-methoxycinnamic acid (Ferulic acid)
99 Sigma-Aldrich Chemie Gmbh, Germany
3,4-Dihydroxybenzoic acid 97 Sigma-Aldrich Chemie Gmbh, Germany
Gallic acid 97 Sigma-Aldrich Chemie Gmbh, Germany
Chlorogenic acid 95 Aldrich Chem. Co.
4-hydroxycinnamic acid 98 Sigma-Aldrich Chemie Gmbh, Germany
Vitexin 94 Carl Roth GmbH Co.
Rutin trihydrate 95 Aldrich Chem. Co.
Luteolin 97 Sigma-Aldrich Chemie Gmbh, Germany
Apigenin 95 Sigma-Aldrich Chemie Gmbh, Germany
Table No 3: Standards used in work.
1.3 Vessels, filters and sorbents
In experiment were used various laboratory vessels. Pipettors of 10-100μl, 100-1000μl, 1000μl, μ,-5000 μl were used to take accurate volume of solutions. Volumetric flasks for making solutions of 20ml, 25 ml, 250ml, and 500ml volume were used. For sample solutions were used 1ml and 2ml HPLC vials. Paper filters Filtrax diameter 125mm were used for obtained plant extracts. Syringe filters Teknokroma PTFE with pore size of 0.45 μm (diameter of 25 mm) were used for plant
extracts before injecting to HPLC machine. Anion - exchanger sorbent cartridges Supelco Discovery DSC-SAX were used to perform SPE.
1.4 Solutions
There was used several solutions for SPE and for analysis of extracts on HPLC. As extraction solution were used 50 % methanol (for acid extraction) and 70 % ethanol (for flavanoid extraction). Standards were solved in methanol. For development of SPE method there was used 25mM Tris
solution (pH=7) and 1M NH4Cl (pH=2) solution. There was used some mixtures with those
solutions: 25mM Tris with methanol in ratio (by volume) 0.5:9.5, 1:9, 1.5:8.5 and 1:4 (Tris:
methanol); 1M NH4Cl with isopropyl alcohol in ratio (by volume) 1:1 and 7:3 (NH4Cl: isopropyl
alcohol). For HPLC was prepared 0.085% p.a. phosphoric acid. This solution was used as mobile phase together with methanol in ratio (by volume) 1.5:8.5 (methanol: 0.085 % p.a. phosphoric acid).
1.5 Instrumentation and software
For extracting and improving solubility of standards was used ultrasonic bath “Sonorex RK31”, made by Bandenil Electronics, Germany. For weighting pulverized plant material was used analytical balances “Sartorius ME”, and for weighting of small amounts of standards were used microbalances “Sartorius 4503 micro”, both made by Sartalex. Analyses of samples were performed on 2 different HPLC machines. The first one, Shimadzu SIL-HTA, was liquid chromatograph equipped with two pumps – A and B – (model LC-10AD), degasser (model DGU-14A), automatic gradient controller (model FCV-10AL), column oven (model CTO-10AC), fluorescence detector (model RF-10A XL) and diode array detector (model SPD-M 10A). Working parameters were: max pressure – 35 MPa, min pressure 0.0 MPa. Flow rate – 1.0 ml/min, injection volume – 10 μl. Start wavelength – 190 nm, end wavelength – 400nm. Calculations were performed at 210 nm wavelength. The second one machine was made by Waters company, and was equipped with auto sampler (model Waters 717 plus), pump (model LCP 4100) and tuneable absorbance detector (model Waters™ 486). Working parameters were: max pressure – 35 MPa, min – 0.0 MPa. Flow rate – 1.0 ml/min, injection volume – 10 μl. Detection was performed at 280 nm. For degassing eluent when working with this system, was used Helium He 4.6, made by Linde AG, Germany. Discovery C18 4.0x125, particles size 5μm was used to perform analysis. For creating method was used Shimadzu LCsolution program, connected with Shimadzu HPLC system; and Waters HPLC system was connected with Data Apex program station v.1.7. For data storage and analysis were
used personal computer with present programs and other programs, such as Microsoft Word and Microsoft Excel, Microsoft PowerPoint.
II. Experiment
2.1 Extraction
Weighted pulverized leaves (around half of gram) were put into 50ml flat bottomed flask, and poured with about 20 ml of extraction solution (50 % methanol or 70% ethanol). All solutions were made by volume. And this flask was put into ultrasonic bath with water and left for thirty minutes.
2.2 Filtration
After thirty minutes flask was removed from the ultrasonic bath, and extract was filtrated using paper filters to volumetric flask to separate plant material, and was added extraction solution till 20 ml. Before injecting to HPLC system extract was filtrated with Millipore Millex – LCR filters.
2.3 Solid phase extraction
Solid phase extraction was performed only for phenollic compounds. Steps:
1. Conditioning step. For washing and wetting the cartridge of SPE column, 1 ml of methanol was passed through. Then for equilibration step was added 2 ml of 25 mM TRIS solution.
2. Sample loading step. 1 ml of extract was diluted by 2 ml of 25 mM TRIS and 1 ml of this solution was loaded to SPE cartridge. Loading was performed very slowly.
3. Washing step. To wash loaded sample, was used 1ml of 25mM TRIS and methanol mixture in ratio 1:4 (by volume). This step was performed also very slowly.
4. Elution step. For elution was used 1 ml of 1M ammonias chloride and isopropyl alcohol mixture in ratio 7:3 (by volume). This step was performed very slowly.
Sorbent was washed by 2ml of methanol after each experiment. SPE method for flavonoid compounds was not performed of higher elution strength of ethanol, which was used as extraction solution
2.4 High performance liquid chromatography
Analyses were performed on two different machines. For Shimadzu machine, sample was loaded to 2m HPLC vial, and vial was put in trail of HPLC device. For Waters machine sample was loaded to 1ml HPLC vial, and vial was put in carousel of HPLC device. After starting machine, system was purged to degas system, and then left working pump A eluting system by mobile phase (methanol: 0.085% phosphoric acid in ratio (by volume) 1.5:8.5) for 30 minutes to reach system equilibration point. The device was programmed to elute solution made by methanol: 0.085% phosphoric acid in ratio 1.5:8.5 for three minutes (pump A). Then was switched on pump B, increasing concentration of methanol till 30 % for 20 minutes. After this, concentration of methanol was enlarged till 100 % in 3 minutes, after this pump B was switched off and pump A was working and eluting mobile phase for 19 minutes. Total time of 1 sample analysis is 45 minutes. Method was created on Shimadzu LCsolutions program and downloaded to HPLC device. After working day system was washed by acetonitrile, till the pressure decreased to constant – about 3.5 MPa. Then all devices were switched off.
Results and discussion
1. Development of HPLC method
According to flavanoids and phenolic acids properties, reverse – phase (RP) HPLC method was chosen. For the development of HPLC method was chosen different standards of phenolic acids – Vanillic, caffeic, chlorogenic, ferrulic, benzoic, syringic, dihydroxycinnamic and gallic. As the starting program was this: at the beginning pump A pumps methanol and 0.085 % phosphoric acid in ratio 1.5:8.5 for four minutes, after this pump B begins to pump methanol and in 16 minutes increases its concentration to 50 %. Using this method only four peaks were separated. So there was changed pump A working time in five minutes and three minutes. With five minutes situation was the same, but with three minutes the last three non-separated peaks (vanillic, coffeic and chlorogenic acids) were separated. All analysis lasts 20 minutes. This method was tested with extract, and some corrections were made to have separated peaks. So there was changed methanol concentration to 40 % and 30 % in 17 minutes, and the better separation was using 30 % methanol concentration, and then pump B working time was changed from 17 minutes to 20 minutes. The final result was: pump A pumping methanol with 0.085 % phosphoric acid for three minutes, and then switched on pump B increases pure methanol concentration till 30% in 20 minutes. As sample chromatogram is given chromatogram, obtained in one of steps of development of HPLC method.
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 min -25 0 25 50 75 100 125 150 175 200 225mAU280nm,4nm (1.00) /1 .1 9 5 /1 .3 5 4 G allic acid/ 1. 882 3, 4 dihydrobenzoic acid/ 3. 233 /4 .1 7 8 /6 .1 4 4/6 .3 1 5 /6 .6 1 5 Chlorogenic acid/ 7. 016 Vanillic acid/ 7. 406 C o ffe ic a c id /8 .1 3 4 4-hydroxycinnamic acid/10.244 /10. 805 F errulic acid/ 11. 230 /12. 093 /13. 594 /13. 882 /14. 520 /15. 000 /17. 276 /18. 395
Figure No 15: Chromatogram, obtained using 50 % of methanol, and pump B working time 17
minutes.
2. Choosing best extraction conditions.
At the first, the best extraction time was chosen. For this purpose were prepared three extracts with 70 % methanol, and they were left in ultrasonic bath for 30, 45 and 60 minutes. As target compounds were chosen rutin, vitexin and chlorogenic acid. Calculating was performed comparing peak areas. Results were: rutin, vitexin and chlorogenic acid in 30 minutes extract as 100 % (to be easier to compare recoveries when using other extraction condition, it does not mean 100% extraction of target compounds to our extraction mixture); in 45 minutes extract rutin – 97.17 %, vitexin 101.01 %, chlorogenic acid 102.53 %; in 60 minutes extract rutin – 92.72 %, vitexin – 92.11 %, chlorogenic acid – 93.89 %. So as the best time was chosen half an hour extraction time. Later was chosen best extracting mixture. For this purpose were made extracts with extraction solution of 50% and 70 % methanol, 50 % and 70 % ethanol. As target compound were chosen the same compounds – vitexin and rutin, chlorogenic acid, and also caffeic acid. Results: chlorogenic, caffeic acids, rutin and vitexin in 50 % methanol – as 100% (also the same situation as it was choosing best extraction time, it does not mean full 100% extraction of our target compounds from plant material). So there was chosen two extraction mixtures – for phenolic acids – 50 % methanol, and for flavonoid extraction – 70 % ethanol. Results are shown in the table No 4 below:
Caffeic acid Chlorogenic acid Rutin Vitexin
50 % methanol 100.00 % 100.00 % 100.00 % 100.00 %
70 % methanol 84.55 % 90.18 % 97.28 % 89.19 %
50 % ethanol 99.85 % 79.63 % 98.25 % 98.84 %
70 % ethanol 89.41 % 88.36% 116.11 % 98.06 %
Table No 4: choosing best extraction mixture
3. Development of SPE method
SPE method was chosen to clean extracts from impurities, and at obtained chromatograms see only target compounds. SPE method is fit for use, if recovery after all steps is not less than 90 %. For this method was used Discovery DSC-SAX sorbent (anion exchanger) to clean methanol extracts with phenolic acids. For development of SPE method chlorogenic and caffeic acids were chosen as target compounds. They were solved in the pure methanol. At the beginning stage for washing step was used 100 % of 25 mM TRIS, for eluting step was used 1M ammonia chloride with isopropyl alcohol in ratio 1:1. After elution step recoveries were very low – 32.12 % for chlorogenic acid, and 51.56 % for caffeic acid. So it was changed percentage of organic phase in the sample solution, diluting sample solution with 25mM TRIS in ratio 3:7 and 1:4. Also percentage of organic phase in elution solution was corrected – 1M ammonia chloride with isopropyl alcohol was changed in ratio 7:3. Repeating SPE procedure, recoveries were better. Using 30 % of organic phase in loaded sample, recovery of chlorogenic acid was 92 %, and recovery of caffeic acid was 91.56 %. Using 20 % of organic phase in loaded sample, recovery of chlorogenic acid was 99.6%; recovery of coffeic acid was 98.41 %. So solutions in those proportions were correct. It was necessary to add into washing solution some organic phase. There was made different solutions of 25 mM TRIS – 25 mM TRIS with 5 %, 10%, 15% and 20% of methanol. Recoveries are reported in the table:
Methanol and 25 mM TRIS ratio Acid
0.5:9.5 1:9 1.5:8.5 1:4
Chlorogenic 95 % 95.44 % 93.32 % 99.91 %
Caffeic 79.27 % 82.1 % 85.75 % 91.37 %
Table No 5: recoveries
As the best solutions were chosen for loading step – sample with 20 % of organic phase, for washing step – 25 mM TRIS with 20 % of organic phase, and for eluting step – 1M ammonia chloride with isopropyl alcohol in ratio 7:3.
For flavanoid SPE procedure was not performed, because ethanol has stronger elution power, and loading to SPE cartridge with C18 is problematic of the flavanoid compound lost in loading step.
4. Identification of compounds in extract
Determination of phenolic compounds and flavanoids was performed in conditions presented before. Identification was performed by comparing retention times of compounds in extract with retention times of standards, and then by adding small amounts of standard solution and comparing peak areas after adding and before adding. Also was UV spectra checked, but flavanoids of their similar chemical structure have very similar spectra, so it is very problematic thing. As the sample is given UV spectra of the rutin:
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 nm -25 0 25 50 75 100 125 150 175 200 225 250 275 300 325 350 375 400 425 450 475 500 525 550 575mAU 19.78/ 1.00 237 282 195 255 354
Other compounds were identified mainly by the retention times, presented below, and 2 different chromatograms – the first one was used for analysis of flavanoids; the second one was obtained after SPE method and was used for analysis of phenolic acids:
Standard Rutin vitexin Chlorogenic
acid Vanillic acid Caffeic acid 4-hydroxy-cinnamic Ferulic acid Retention time 19.783 16.916 6.516 6.957 7.779 11.563 13.401
Table No 6: Retention times
So those retention times were used when identifying target compounds. As sample chromatograms are at Figure No 17 and 18. Figure 17 – chromatogram, used for identify and calculation of amounts of flavonoids. And Figure No 18 – chromatogram used to identify and calculate amounts of phenolic acids. All target compounds peaks are marked at chromatogram.
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 min -25 0 25 50 75 100 125 150 175 mAU 280nm,4nm (1.00) /1.200 /1.414 /2.373 /3.243 /4.097 /5.119 /5.977 /6.720 /7.089 /7.438 /7.867 /8.742 /9.175 /9.710 /10.174 /10.754 /11.097 /11.682 /12.229 /12.521 /12.930 /13.680 /14.081 /14.800 /15.222 /15.584 /15.988 Vitexin/16.915 /17.719 /18.318 /18.782 /19.200 Rutin/19.783 /20.390 /21.107 /21.669 /22.094 /23.142 /23.588
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 min 0.0 2.5 5.0 7.5 10.0 mAU 280nm,4nm (1.00) /1.223 /1.291 /1.446 /1.850 /1.954 /2.108 /2.405 /3.182 /3.503 /3.985 /4.544 /5.035 /5.483 /5.870 Chlor
ogenic acid/6.516 Vanillic
ac id/6.957 Coffeic ac id/7.779 /8.699 /9.487 /10.082 /10.596 4-hydr oxycinnai/ 11. 563 /12.401 /12.891 Fer ru lic acid/13.401 /13.952 /14.240 /14.685 /15.103 /15.454 /15.872 /16.768 /17.397 /17.824 /18.207 /18.671 /18.987 /19.652 /20.268 /20.976 /22.738 /23.504
Figure No 17: Chromatogram, used for quantification of phenolic acids after SPE
5. Quantification
Amounts of flavonoids and phenolic acids were calculated according those equations: 1. Amounts of flavonoids: m A F C A C × × × × = Standard Standard Sample 20
2. Amounts of phenolic acids:
m A F C A C × × × × = Standard Standard Sample 60
Asample – peak area of sample
CStandard – concentration of standard, in mg/ml
F – purity factor of standard in percents AStandard – peak area of standard
m – Mass of pulverized plant material for preparing extract. 20 – Volume of prepared extract for analysis of flavonoids. 60 – Volume of prepared extract for analysis of phenolic acids. Amounts were calculated in percents.
Figures for vegetation phases 06 13 06 23 0802 08 12 0822 09 08 0918 09 22 Vitexin 0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 C, % Date
Distribution of rutin and vitexin
Vitexin Rutin
Figure No 18: Distribution of flavonoids in vegetation phases
06 13 06 23 08 02 08 12 08 22 09 08 09 18 0922 Chlorogenic acid Caffeic acid 0 0,005 0,01 0,015 0,02 0,025 0,03 C, % Date
Distribution of chlorogenic, vanillic, caffeic acids
Chlorogenic acid Vanillic acid Caffeic acid
06 13 06 23 08 02 08 12 08 22 09 08 09 18 09 22 4-hydroxycinnamic acid 0 0,001 0,002 0,003 0,004 0,005 0,006 C, % Date
Distribution of ferulic and 4- hydroxycinnamic acids
4-hydroxycinnamic acid Ferulic acid
Figure No 20: Distribution of phenolic acids in vegetation phases
Figures for new-planted plants
Distribution of flavonoids in new-planted plants
0 0,02 0,04 0,06 0,08 0,1 0,12 0,14 0,16 0,18 0,2 1 2 Flavonoids A m ount , %
Butanisation of new-planted plant new-planted plant after regrowth
Figure No 21: Distribution of flavonoids in new-planted plants
Distribution of phenolic acids in new-planted plants 0 0,005 0,01 0,015 0,02 0,025 0,03 0,035 0,04 1 2 3 4 5 Acids A m ount , %
Butanisation of new-planted plant new-planted plant after regrowth
Figure No 22: Distribution of phenolic acids in new-planted plants
Explanation of numbers 1-5 at Figure No 22 is in the table No 7 below:
Number 1 2 3 4 5
Name of acid Chlorogenic
acid Vanillic acid Caffeic acid
4-hydroxycinnamic acid
Ferulic acid
Table No 7: explanation of Figure No 22
Figures for fertilizing
All other figures represent data, aquired analyzing samples, collected from plants with different fertilization material. As it was said, there were five fields of plants for experiment. The first one is control area – no any fertilizing. The second one is fertilizing with carbamide. The third one is
fertilizing with superphosphas. The fourth one is fertilizing with KNO3. The fifth one is the mixture
of all three fertilizes. There were two samples from each field, data was very identical, and for figures was calculated arithmetic average.
Vitexin 0 0,005 0,01 0,015 0,02 0,025 0,03 0,035 0,04 0,045 0,05
Control area Carbamide Superphosphas Kalii nitras Mixture
Fertilising Am o u n t, %
Figure No 23: Influence of fertilizing for amounts of vitexin
Rutin 0,11 0,115 0,12 0,125 0,13 0,135 0,14 0,145 0,15 0,155
Control area Carbamide Superphosphas Kalii nitras Mixture
Fertilising Am o u n t, %
Chlorogenic acid 0 0,005 0,01 0,015 0,02 0,025 0,03
Control area Carbamide Superphosphas Kalii nitras Mixture
Fertilising
A
m
ount
, %
Figure No 25: Influence of fertilizing for amounts of chlorogenic acid
Vanillic acid 0 0,005 0,01 0,015 0,02 0,025
Control area Carbamide Superphosphas Kalii nitras Mixture
Fertilising
A
m
ount
, %
Caffeic acid 0 0,005 0,01 0,015 0,02 0,025 0,03 0,035 0,04 0,045
Control area Carbamide Superphosphas Kalii nitras Mixture
Fertilising
A
m
ount
, %
Figure No 27: Influence of fertilizing for amounts of caffeic acid
4-hydroksycinnamic acid 0,00072 0,00073 0,00074 0,00075 0,00076 0,00077 0,00078 0,00079 0,0008 0,00081
Control area Carbamide Superphosphas Kalii nitras Mixture
Fertilising A m o unt , %
Ferulic acid 0,0013 0,00135 0,0014 0,00145 0,0015 0,00155 0,0016 0,00165
Control area Carbamide Superphosphas Kalii nitras Mixture
Fertilising
A
m
ount
, %
Figure No 29: Influence of fertilizing for amounts of ferulic acid
Influence of vegetation phases. After calculations at pictures is possible to see all results. A big
increasing of rutin amounts is possible to see at flowering phase – amount is about 0.62 %. This percentage is even bigger than in butanisation phase. After flowering, when fruits began to grow, amounts of routine are decreasing, and the lowest point is 0.12 % when fruits are immature. Later is small increase – till 0.15%. But after first temperature decrease below zero is detected big decrease of rutin amounts – only 0.099 %. Analogical situation is also with other compounds – amounts of them are decreasing at influence of low temperature. Vegetation phases have not so big influence for amounts of vitexin. At the butanisation phase amount of vitexin is 0.059%. Concentration is decreasing till plant is growing fruits till 0.013 %. Later is increase till 0.036 % at phase of immature fruits, but after this is decrease again - till 0.0077 %. Then amounts are increasing till 0.15 % before colds and after cold concentration is the same. Biggest amounts of phenolic acids are also at different phases: Chlorogenic acid 0.012 % at flowering phase, vanillic acid 0.024 % at flowering phase, and caffeic acid 0.027 % at butanisation phase, 4-hydroxycinnamic acid 0.004 % at flowering phase, and ferulic acid 0.006 % at growing fruits phase. After this concentration is decreasing till fruits maturing phase: Chlorogenic acid 0.005 %, vanillic acid 0.012 %, and caffeic acid 0.004 %, 4-hydroxycinnamic acid 0.001 %, and ferulic acid 0.004 %. Later, before first cold, amounts are increasing till: Chlorogenic acid 0.009 %, vanillic acid 0.016 %, and caffeic acid 0.008 %, 4-hydroxycinnamic acid 0.001 %, except ferulic acid, amounts of which are decreasing till 0.002
%. After cold amounts of all acids decrease till very small amounts. When looking to samples new-planted plants, there are no significant increases of amounts of flavonoids – even first sample has more flavonoids than the regrowed one. But amounts of rutin in new-planted plant were significantly smaller to compare with plant in flowering phase. Exception – vitexin, amounts of which was difference from 0.0103% at fruit growing phase and in new-planted plants – 0.0551% - five time more. And for phenolic acids there was small increase for chlorogenic acid (from 0.0166% to 0.0201%), caffeic acid (from 0.0292% to 0.0362%) and ferulic acid (from0.0008% to 0.0017%). Other phenolic acids had decrease of amounts – 4-hydroxycinnamic acid (from 0.0027% to 0.0016%) and vanillic acid (from 0.026% to 0.018%).
Influence of fertilizing. Fertilizing was made by carbamide, super phosphates, potassium nitras and
mixture of those three compounds. Amounts of vitexin have increased till 0.047% using mixture of fertile to compare with control area 0.037 %. Other fertile haven’t big influence. Amounts of rutin have increased till 0.16% using mixture to compare with control area 0.14 %. Other fertile haven’t
big influence. Amounts of phenolic acids were enlarged using KNO3 as fertilize:
Chlorogenic acid
Vanillic acid Caffeic acid
4-hydroxy-cinnamic acid
Ferulic acid
Control area 0.015 % 0.013 % 0.024 % 0.0008 % 0.0014 %
KNO3 0.025 % 0.021 % 0.040% 0.0008 % 0.0016 %
Table No 8: fertilizing with KNO3
Using other fertilizing, amounts of phenolic acids were analogical to control area with difference +- 0.001 % (not in percents from percents) comparing with control area.
Conclusions
1. There were identified two of flavonoids – vitexin and rutin. Other identified compounds were five phenolic acids – caffeic acid, chlorogrnic acid, vanillic acid, 4-hydroxycinnamic acid and ferulic acid.
2. The best time to collect herb is flowering phase, in this phase are largest amounts of flavonoids - rutin – 0.62 % (when at the beginning of vegetation 0.17 %, at the end 0.099%); bigger amounts of vitexin are at butanization phase – 0.059 % (at the end – 0.016 %).
3. Analogic situation is with phenolic acids with small exceptions. At the flowering phase chlorogenic acid 0.012 % (at the begining of vegetation 0.012 %, at the end 0.001 %), vanillic acid 0.024 % (at the begining of vegetation 0.016 %, at the end 0.003 %), 4-hydroxycinnamic acid 0.041 % (at the begining 0.002 %, at the end 0.0006 %). Caffeic acid 0.017 % at the butanisation phase (at the end 0.003 %) Ferulic acid 0.0059 % at immature fruits phase (at the begining 0.002 %, at the end 0.004 %).
4. New-planted plants (butanisation phase) had more vitexin (0.0694 %), but less rutin (0.18%). Smaller amounts were after regrowth.
5. New-planted plants (after regrowth) had more phenolic acids to compare with flowering phase: chlorogenic acid 0,02 % to 0.012 % caffeic acid 0.036 % to 0.018 %. Exept those acids: vanillic acid 0.018 % to 0.024 %, 4-hydroxycinnamic acid 0.0016 % to 0.0041 % , ferulic acid 0.0017 % to 0.0039 %.
6. New-planted plants after regrowth had more chlorogenic (0.02 % compare to 0.016 %), caffeic (0.036 % compare to 0.029 %) and ferulic (0.0017 % compare to 0.0008 %)acids. Other new-planted plants had more vanillic (0.026 % compare to 0.016 %) and 4-hydroxycinnamic acids (0.0027 % compare to 0.0017 %).
7. For amounts of vitexin largest influence was when using mixture of fertilizes – 0.047 %, comparing with control area 0.041 %. For amounts of rutin largest influence was when used carbamide as fertilizing material was – amount 0.15 %, comparing with control area 0.14 %.
8. Only KNO3 has increased amounts of phenolic acids significantly: 0.025 % of chlorogenic
acid, 0.021 % of vanillic acid, 0.04 % of caffeic acid, 0.0008 % of 4-hydroxycinnamic acid, 0.0016 % of ferulic acid.
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Acknowledgements
This work was performed in Czech Republic, in Charles University in Prague, faculty of pharmacy in Hradec Kralove, at the department of analytical chemistry. I would like to express my gratitude for everyone, who helped me in working on this thesis. I gratitude for Kaunas University of Medicine for financial support, and thanks for Socrates Erasmus program. Thanks for Doc. P.Solich; for my supervisor Dr.Radek Sladkovsky, Czech PhD students for helping to understand devices, solving problems, and recommendations and advices.
