Investigation of bioactive compounds

________________SEMMELWEIS UNIVERSITY______________
DOCTORAL SCHOOL OF PHARMACEUTICAL SCIENCES
Investigation of bioactive compounds from
Taraxacum officinale and Morus nigra
Dissertation (Ph. D. thesis)
Kristó Tita Szidónia
Tutor: Dr. K�ry �gnes, Ph. D.
Made: Semmelweis University, Faculty of Pharmacy, Department of Pharmacognosy
Budapest, 2002
Ph. D. Thesis
Investigation of bioactive compounds from Taraxacum officinale and Morus nigra
Kristó Tita Szidónia
Supervisor: Prof. Dr. �gnes K�ry
Department of Pharmachognosy, Faculty of Pharmacy, Semmelweis University, 2002
Summary
The results of the continously expanding and detailated pharmacological researches on bioactive plant
components call for new, accurate and efficient extraction and phytoanalitical methods to invent and identify
the pharmacologically active components. The syncronized biological-chemical studies can give new
answers to questions of structure-activity relationships too. It can be concluded from lots of experimental and
clinical data that some so far not bio-active considerated components may influence the efficacy and safety
of medical plant extracts through pharmacodynamic or pharmacokinetic way.
The antiinflammatory, immunomodulator triterpenes and phytosterols in Taraxacum officinale and Morus
nigra were studied in order to find optimal extraction technology. The environmentaly acceptable
supercritical fluid extraction have been choosen, which received reduced interest for the extraction of
medicinal plant metabolites except volatile and fixed oils. The optimal extraction parameters (pressure,
temperature) were determined, which resulted successfull enrichment of active components in supercritical
CO2 extracts. To consider the advantages and disadvantages of the supercritical fluid extraction, the results
were compared with the traditional extraction methods. For the quantive evaluation of the extracts a thin
layer chromatographic densitometric method was developed and it was demonstrated that some
supercritical extracts contained up to 10% (g/ 100 g) of active components.
It was proved that the liofilized aqueous and methanolic extracts of Taraxacum officinale and Morus nigra
exhibit significant antioxidant activity in in vitro models. To search for possible active components
responsible for scavanging activity, capillary electrophoresis was used providing good separation in a
relatively short time. Results were compared with high pressure liquid chromatography. Identifing the
fenoloid components (Taraxacum officinale: apigenin-7-O-?-glucosid, luteolin-7-O-?-glucosid, chlorogenic
acid; Morus nigra: quercetin-3-O rhamnoglucosyl, quercetin-3-O-glucosid, chlorogenic acid) we postulated
the possible traditional use, based on the antioxidant (chelating and superoxid scavanging) activity due to
their influence on pathological free radical reactions.
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Ph. D. doktori �rtekez�s
A Taraxacum officinale �s Morus nigra bioakt�v anyagainak vizsg�lata
Kristó Tita Szidónia
T�mavezeto: Dr. K�ry �gnes c. egyetemi tan�r
Semmelweis Egyetem, Gyógyszer�sztudom�nyi Kar, Farmakognózia Int�zet, 2002
�sszefoglaló
A n�v�nyi eredetu, biológiailag akt�v anyagok egyre sz�lesebb k�ru �s apról�kosabb farmakológiai
vizsg�lata az śj, gyors, pontos �s hat�kony extrakciós �s fitoanalitikai módszerek alkalmaz�s�t s�rgeti. A jól
�sszehangolt k�miai-farmakológiai kutat�sok egyśttal adatokat szolg�ltathatnak a szerkezet-hat�s
�sszef�gg�sekkel kapcsolatban is.
Sz�mos k�s�rleti adat t�masztja al�, hogy az eddig nem hatóanyagnak tekintett n�v�nyi �sszetevok jelentosen
befoly�solhatj�k a gyógyn�v�nyek �s kivonataik aktivit�s�t, illetve biztons�gos felhaszn�l�suk is
módosulhat.
A Taraxacum officinale �s Morus nigra bioakt�v anyagai k�z�l a gyullad�scs�kkento, immunmodul�ns,
daganatellenes tulajdons�gokkal rendelkezo triterp�nek �s fitoszterolok tanulm�nyoz�s�hoz az ezid�ig foleg
illó komponensek elo�ll�t�s�ra alkalmazott hat�kony, k�rnyezetk�m�lo szuperkritikus extrakciót
alkalmaztuk. Optimaliz�ltuk a k�s�rleti param�tereket (nyom�s, hom�rs�klet) �s megvalós�tottuk a
hatóanyagok jelentos �s szelekt�v dśs�t�s�t. Az elony�k �s h�tr�nyok m�rlegel�se c�lj�ból a kivon�s
hat�konys�g�t �sszehasonl�tottuk a hagyom�nyos módszerekkel. Az extraktumok kvantitat�v �rt�kel�s�hez
VRK-denzitometri�s módszert dolgoztunk ki �s meg�llap�tottuk, hogy a szuperkritikus extraktumokban a
hatóanyagok esetenk�nt 10%-ig is feldśsultak.
A Taraxacum officinale �s a Morus nigra liofiliz�lt kivonatai antioxid�ns, szabadgy�kfogó aktivit�ssal
rendelkeznek, amit in vitro vizsg�latokkal bizony�tottunk. A hat�s�rt felelos vegy�letek felt�r�s�hoz
kapill�relektrofor�zist alkalmaztunk, melynek hat�konys�g�t nagynyom�sś folyad�k-kromatogr�fi�s
módszerrel is �sszehasonl�tottuk. Azonos�tottuk a drogok fenoloid komponenseit (Taraxacum officinale:
apigenin-7-O-?-gl�kozid, luteolin-7-O-?-gl�kozid, klorog�nsav; Morus nigra: kvercetin-3-O-
ramnogl�kozid; kvercetin-3-O-gl�kozid, klorog�nsav) �s elt�ro hat�smechanizmusuk (kel�tk�pzo,
szuperoxid ion scavanger) alapj�n valósz�nus�tett�k k�l�nb�zo betegs�gekben való alkalmazhatós�gukat.
1. INTRODUCTION
The scientific investigation for bioactive components becomes increasing interest. By reevaluation
of some of the well known medical plants appears, that those components which were supposed to
be the bioactive parts may exhibit their activity in synergistic way or be activated by the presence of
other substances or even some other chemical structures may be responsible for the proposed
biological activity. The clinical efficacy of Calendula preparation for example is well documented
and the most relevant pharmaceutical activity for topical use appears to be anti-inflammatory one.
However, there was a high uncertainty in the characterization of other substances, such as saponins,
flavonoids that are known, in general to posses anti-inflammatory activity. This left the problem of
quality control of Calendula preparations unsolved: on which principles should they be
standardized? By means of bioassay-oriented fractionation of the CO2 extract of Calendula flowers,
the triterpenoids are shown as the most relevant principles of the drug [78-83].
So far only two major principles of antiphlogistic drug activity are available in the therapy. First:
Compounds inhibiting the synthesis of prostaglandins (salicylic acid),and glucocorticoids which
finally inhibit both the cyclo-oxygenase pathway and the lipoxygenase A2-reaction. The unwanted
effects of this class of anti-inflammatory agents are well known. What we need are novel
compounds inhibiting both branches of arachidonic acid cascade by a mechanism different from the
action of glucocorticosteroids. To achieve this goal in vitro screening methods have been introduced
which allows to study the mechanism of action of proposed chemical structures. There are many
reports on biological action of pentacyclic triterpenes (PTs), which could be relevant for
pharmacological effects including their anti-inflammatory properties and there are reports also on
highly interesting actions of PTs at reasonable concentrations in vitro. The low acute toxicity of
these natural compounds indicate, that PTs might provide a rich natural resource of lead compounds
for drug development.
The anti-inflammatory activity of some triterpenoids (like ? -amyrin, etc.) may depend on inhibition
of protein kinase C, without any involvement of neurogenic inflammatory mechanism [93]. Some
triterpenoids and sterols may be potential new lead compounds to find more potent tumor and HIV
inhibitors [85,86,87,90,92,109,111,112]. An average intake of food in west contains 250-500 mg
cholesterol and 200-400 mg non-cholesterol sterols, mainly phytosterols. Not only the ? -sitosterol
(BSS), campesterol and their glucosides (BSSG) present in blood-serum at the highest
concentration, but the phytosterols in general are very important to prevent cancer. In animals, BSS:
BSSG have been shown to exhibit anti-inflammatory , anti-neoplastic, anti-pyretic, and immuno-
modulating activity. A proprietary BSS:BSSG mixture has demonstrated promising results in a
number of studies, animal models, and human clinical trials. This phytosterol complex seems to
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target specific T-helper lymphocytes, the Th1 and Th2 cells, helping normalize their functioning
and resulting in improved T-lymphocyte and natural killer cell activity. A dampening effect on
overactive antibody responses has also been seen, as well as normalization of the DHEA:cortisol
ration [138,139,140].
Another class of active components, that has been targeting are the flavonoids. They possess a large
scale of activity: antiischaemic, anti-tumor, anti-inflammatory, anti-allergenic, gastro-protective
activity etc. Further more they are potential antioxidants, anti-radicals, chelating agents and inhibit
the lipid-peroxidation [141]. The science of today s searches for answers of relationship between
chemical structure and biological activity on biomolecular scales.
But a number of studies have found both anti-and prooxidant effects for many of these compounds,
depending on amount of intake. Shifts in proportions can cause diseases such as: diabetes mellitus,
colitus ulcerosa, Chrohn disease etc. [142]. The excessive consumption of triterpenes as citotoxic
agent can lead to recrudescence [94-95]. Sitosterolemia is a rare, recessively inherited disease
characterized clinically by accelerated atherosclerosis and xanthomas and biochemical by
hyperabsorbtion and retention of sitosterols and other plant sterols in tissues. The larger
accumulation of sterols and inadequate down regulation of HMG-CoA reductase in MDM may be
the mechanism for foam cell formation and explain, in the part, the increased risk of atherosclerosis
in sitosterolemia [125].
Therefore for proper use of drugs with different indications, to prevent or cure diseases, there are
increasing effort needed to standardize these drugs on chosen active principle(s). Taking these in
account the experts on finding new natural, compounds of plant origin emphasize the importance to
develop technics for rapid identification of active structures, on the other side to obtain these
structures selectively and at high concentration in extracts to enable in this way the production of
standardized drugs for chosen diseases.
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2. SETTING OF OBJECTIVES
With our experimental work we tried to furnish data to better understanding of problems sketched
in introduction with the study of the plants belonging to Asteraceae and Moraceae family.
The dandelion is well known since ancient times and is generally considered to have diuretic,
choleretic, tonic and laxative properties, among other. Traditional use for rheumatic conditions
could be related to observed anti-inflammatory activity [166,170,173,174,175]. The plant contains a
large variety of chemical structures: triterpenes, sterols, sesquiterpene lactones, phenoloids, inulin,
pectin, sugars, vitamins and others [168-176].
The black mulberry, not so widespread as the white one, is planted in gardens, because of their tasty
fruits. The leaf of black mulberry added to mixture of plants are indicated for blood-sugar
regulation, but has not any outstanding effect. The Chinese therapy uses for several diseases as anti-
inflammatory, anti-phlogistic etc. agent [193,194,195,197].
The aim of our work was to develop an environmental extraction method for the extraction of
triterpenes and phytosterols which are present in considering amount in both plants. For this we
chosen the supercritical fliud extraction (SFE). This method was used until now mainly to obtain
volatile components from various plants. We proposed to work out those optimal parameters among
them we can extract the non-volatile components from dandelion and black mulberry. To be
accessible on practical scale, we needed to interpret the physical-chemical processes. It is important
at the same time to compare to Soxhlet extraction, so being possible to evaluate the advantages and
disadvanteges of this procedure. This expensive method proper mainly for extraction of lipophilic
structures is used more and more by food- and pharmaceutical industry. Because it is an expensive
method it can only be justificated, if the choosen bioactive component can be selectively enriched
through the production of raw materials for medicine of plant origin. On the other hand there is an
urgent need for more experience using the method, as it was clearly declared on phytochemical
conferences.
This give the grounds to develop a method for the great number of samples obtained by
supercritical fluid extraction. For this we proposed to develop a thin layer- densitometric method
and wanted to analyze the influence of solvent system, reagent detector, detection time and
temperature to the reproducibility of results.
Having in view the use of Taraxacum officinale and Morus nigra as anti-inflammatory agents, and
considering that the flavonoids are regarded as potential bioactive components, we wanted to study
more detailed the antioxidant state of water extract of Taraxacum officinale and Morus nigra:
hidrogen donor activity, reducing capacity, total scavenger activity etc.
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To reveal the relationship between structure and activity we wanted to measure the poliphenolic,
flavonoid content of antioxidant plant extracts. Near by well known HPLC, we proposed the use of
capillary electrophoresis (CE), which becomes more attention lately, and wanted to enriches the
dates for usability, comparing to HPLC.
With experience in the CE analysis we wanted to contribute in solving the production and
standardization problems of phyto-medicines, having in view the efforts of harmonization in
Europe.
3. MATERIALS AND METHODS
3.1. Materials
The samples of Taraxacum officinale used for preliminary studies were collected from Budapest
1998 (herbs) and 1999 (autumn). The Taraxaci radix, Taraxaci folium used for supercritical
extraction were commercially avaible dried drugs collected from the field (delivered by Rózsahegyi
Ltd., Erdokeresztes, Hungary). For supercritical extraction we used sample of Morus nigra
originated from University of Medicine and Pharmacy, Institute of Pharmacognosy,
Marosv�s�rhely, Romania).
3.2. Methods
3.2.1. PRODUCTION OF SAMPLES
3.2.1.1. Production of lyophilized samples for antioxidative investigations
15 g drugs were used to make infuses and than they were lyophilized (shelf temp. 60oC, drying time
13 h, pressure 12-13 Pa).
3.2.1.2. Extraction of triterpenes and phytosterols with organic solvents in Soxhlet apparatus
The drugs were extracted with methanol and n-hexane. The dried extracts were redisolved in
methanol and used for TLC-densitometry.
3.2.1.3. Industrial Soxhlet extraction of triterpenes ans phytosterols
The amount of samples was 1200 g. As solvent was used 2650 ml n-hexane or 96% ethanol. The
dried extracts were redisolved in pethroleum-ether (40-45oC) and used for TLC- densitometric
evaluation.
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3.2.1.4. Supercritical extraction to obtain triterpen- and phytosterol rich fractions
The preparation of solvents and previous investigation
To measure the dried material content we measured on 105oC in oven until mass constancy. The
particle size was measured by sieve analyzes.
From Mori nigri folium with supercritical carbon dioxide
The amount of drug was 1348 g. The temperature changed between 39,8-43,9 the pressure 448-451
bar.
From Taraxaci radix and Taraxaci folium with carbon dioxide
The amount of drug was 1200g (Taraxaci radix) respective 980.32 g (Taraxaci folium). We changed
the pressure (150, 350, 450 bar) and the temperature (35, 50 ,65oC) according to a three level two
factorial experimental design using two separators.
3.2.1.5. Column-chromatography isolation for the identification of triterpenes and phytosterols
From the supercritical extracts we have isolated enriched fractions using silicagel as cartrig and n-
hexane ethyl-acetate, methanol as eluents.
3.2.2. QUALITATIVE AND QUANTITATIVE MEASURMENT
3.2.2.1. Determination of flavonoid content
The flavonoid content was measured after Deutsche Arznei Buch (DAB 10), and expressed in
hyperoside [202].
3.2.2.2. Determination of polifenol content
The measurment was made according to Ph. Hg. VII., which was based on reducing capacity of
poliphenols.
3.2.2.3. Capillary electrophoretic measurement for qualitative identification of phenoloids
We used a standard cartrig equipped Hewlett Packard instrument with UV detector. The
identification of peaks was made according to their mobility, UV spectra and with peak-addition
analysis.
3.2.2.4. HPLC measurements for quantitative determination of phenoloids
The analysis was made with ABL&EJASCO equipment with RHEODYNE (20? l) injector. We
used a mixture of two solvents: AcCN : H2O CH3COOH. The peaks were identified with
authenthic standards by their UV spectra and retention time.
3.2.2.5. Thin-layer and TLC-densitometric measurements
Preparation of samples
For the quantitative determination the unsaponified fractions of extracts were used according to the
Ph. Hg. VII.
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TLC-method development
Preinvestigations were carried out with three different solvent systems and reagent detectors. We
optimalized the reaction time and temperature. To densitometric evaluation we used CS-930
(Shimadzu Corporation, Kyoto, Japan) equipment. The calibration curves were made with standard
solution of ? -amyrin and ? -sitosterol, in the concentration of 2,5-5,5 ng.
3.2.2.6. HPLC investigation of fractions obtained by column chromatography
Standard substances were used. The components were identified on the base of their retention times
and/or UV spectra.
3.2.2.7. Investigations to prove the antioxidant activity
Hidrogen donor activity
1,1-diphenil-2-picrilhidrazin radical was used according to Hatano ?206?-Blois ?207? method. The
activity was expressed in 50% DPPH inhibition.
Reducing capacity
It was measured with Oyadzu ?208? method, investigating the Fe3+ ? Fe2+ transformation and
expressed in ASE/mg.
Total scavanger activity
The total scavanger activity was proved with chemiluminometric methods (Bl�zovics et al.) and
expressed in I50 (?g).
Lipid peroxidation
Enzymatically induced lipid peroxidation in liver microsomes was examined by the detection of
malonaldehyde (MDA) production. The protein concentration of rat liver microsomes was assayed
by the method of Lowry et al. NADPH cytochrome C reductase activity was measured by Jansson
and Schenkman.
RESULTS AND DISCUSSION
There are several in vitro and in vivo studies showing the importance of triterpenes and
phytosterols. The main aim of this Ph.D. thesis was to develop an extraction method to obtain and
enrich the triterpene and phytosterol content of Taraxaci folium, Taraxaci radix and Mori nigri
folium.
The supercritical extraction is an alternative technic to Soxhlet extraction. The main difference
between them that the products obtained by supercritical fluid extraction contain no solvent residue,
and the composition of extracts can be influenced by changing the method parameters. Dates about
equlibra content of systems with more components are very different to those obtained from biner
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systems. This could be explained, as the components influence each other solubility. There are only
few data about repartition between supercritical solvent and another phase ?fluid, solid? by systems
with several components.
During our experimental work, we have examined by comparing the supercritical fluid and Soxhlet
extraction the possibility to obtain triterpene and phytosterol rich fractions from three vegetable
drugs:
- The yield of �-amyrin in supercritical fluid extract from Mori nigri folium was three times
higher (2.082g/ 100g extract), than in the traditional extraction methods (0.063g/ 100g extract).
Two times higher concentration of �-sitosterol was found in SFE extracts (1.45g/ 100g extract)
from black mulberry leaves compared to those obtained by Soxhlet extraction. An unknown
substance (triterpene type) was also detected which yield was ten times higher in supercritical
extracts (6.11g/ 100g extract) than in Soxhlet extracts (0.642g/ 100g extract).
- Examining the yield of in the non-volatile terpene components SFE of Taraxaci radix, it can
concluded, that the supercritical fluid extraction (450 bar and 35oC) of ? -amyrin from
dandelion roots is more efficient (two times), than the ethanol extraction (24.54g/ 100g extract
and 13.01g/ 100g extract respectively). The extraction of phytosterols (those of free and
glicosydic type) can not be regarded as a more advantageous method to traditional extraction
methods.
- There is a similar tendency in the extraction dynamic of ? -amyrin from Taraxaci folium and
Taraxaci radix in different extracts (2.46g/ 100g): the SFE is also more efficient (450 bar and
35oC). Because the extraction with apolaric solvents (SFE, n-hexan) has a better yield, it proves
that the phytosterols are mainly present in free form.
For the qualitative-quantitative evaluation of the big number of samples, we an efficient and quick
thin layer chromatographic method combined with densitometry was developed. Having in view the
reproducibility of measurements, we proved the followings:
- Triterpenes and phytosterols are present in the unsaponified fraction.
- The solvent system used (n-hexan - etil-acetate, 6:2) is effective to separate the compounds in
question.
- The reagent used (cerium sulphate) is efficient for detection, the colour-intensity of the spots
considerably different from the background.
- The optimal time and temperature of detection where the measurements are reproducible was
determined as: 10 min, 100oC.
- The calibration curves of standard substances (? -amyrin, ? -sitosterol) were between 2.5-5.5
ng/ml).
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- The same spot was measured six times and the standard deviation was determined: 0.05-0.09%
which proved the reproducibility of measurements.
Having in view the economical aspect, we tried to characterize the tirterpene and phytosterol
content of different black mulberry and dandelion drugs:
- The triterpene and phytosterol content of Mori nigri folium (? -amyrin=0.052g/ 100g drug; ? -
sitosterole= 0.043g/ 100g drug) was remarkably lower (ten times) than the triterpene and
phytosterol content (? -amyrin=0.551g/ 100g drug; ? -sitosterole= 0.385g/ 100g drug) of
Taraxaci folium.
- Comparing the Taraxaci radix and Taraxaci folium drugs it was observed, that the leaves in
autumn are better triterpene source, while phytosterols are enriched in roots during spring.
- The distribution of bioactive compounds in individual plants resulted the following
observations: the leaves are rich in both active substances (? -amyrin=8.9%; ? -sitosterole=
14.1%), the roots are richer in triterpenes (? -amyrin=3.8%) and the flowers in phytosterols (? -
sitosterole= 5.8%).
These results give information to enable the economical choice: at which month and which part of
medical plants should be used to obtain the active substances economically.
The purified fractions obtained by column-chromatography were analyzed by HPLC and proved the
identity: of �-amyrin and �-sitosterole in dandelion leaves, roots and mulberryleaves. Due to the ? -
amyrin content the extracts can be used to inhibit inflammation processes (this may depend on
protein kinase C, without any other involvement of neurogenic inflammatory mechanism). The ? -
sitosterole content enables for extracts to be used as anti-inflammatory, anti-neoplastic, anti-pyretic
and immuno-modulating agents, because they seems to target specific T-helper lymphocytes, the
Th1 and Th2 cells, helping normalize their functioning and resulting in improved T-lymphocyte and
natural killer cell activity, as well as normalization of the DHEA: cortisol ratio.
New results and conclusion from studies on supercritical fluid extraction
? Supercritical carbon dioxide has been successfully employed as a solvent for the extraction of
non volatile triterpenes from medicinal plants. So the use of SFE can be substantially enlarged.
? Our experimental data show that the supercritical CO2 is suitable to obtain triterpene rich
fractions; finding the optimal parameters helps to influence the composition of fractions.
? The extraction at pressures and temperatures near to the optimum conditions gave products in
which the yield of active principles were several times (up to ten times) in extracts, the use of
SFE can be economical in pharmaceutical industry too.
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? The SFE satisfies the conditions put by the pharmaceutical industry: the active principles in
products, suffered no disintegration because of the mild conditions, they are selectively
enriched; the extracts are of good quality and they contain no solvent residue.
? SFE is recommended because it reduces or eliminates health, environmental and safety risks.
Our experimental data contribute to the formation of a database helping the quality control of
supercritical fluid extracts from medicinal plants.
Because the Taraxacum and Morus sp. are used in inflammatory processes and the flavonoids are
one of the chemical structures involved in these indications, we studied the antioxidant activity of
their extracts. Lyophilized samples from both parts of dandelion and mulberry leaves exerted
hydrogen donating ability in the presence of DPPH stable radical. The extracts from dandelion
leaves proved to be the most effective (I50= 1150?g Mori nigri folium; I50= 750?g Taraxaci radix;
I50=160?g Taraxaci folium). The reducing power property of extracts showed concentration
dependence (740 ASE/mg Taraxaci folium; 212 ASE/mg Taraxaci radix; 760ASE/mg Mori nigri
folium). The H2O2 scavanging activity of samples was determined by chemiluminometric method
in H2O2/.OH-microperoxidase-luminol system. The emitted light of this system was diminished by
the extracts in a concentration dependent manner; the extracts of Taraxaci folium proved to be the
most effective (I50= 155?g Taraxaci folium; I50= 210?g Taraxaci radix; Mori nigri folium I50=
270?g). The dandelion leaves extracts exhibited a strong membrane-protective action
(IC50=1,0mg/ml). These results show that the active principles of the studied vegetable drugs with
primary and secondary antioxidant as well as scavenging properties can preserve the structure of
membranes and protect against secondary lipid peroxidation. The Taraxaci folium and-radix
extracts can influence the microsomal NADPH/cytochrome P/450 reductase activity, they can
stimulate even without NADPH cofactor, but in a smaller rate. The dandelion leaves extracts proved
to be more effective in both systems. These medical plant extracts may serve as electrons to
cytochrom P/450 (in vitro) via a flavoprotein NADPH/cytochrom p/450 reductase in vivo, and
therefore may influence the drug metabolism, which can be regarded as some of the general
adaptative systems of the body. The aquous extracts of drugs antioxidant activity can be influenced
not only by the total poliphenol content (the aqueous extracts contain no triterpene or phytosterols),
but also from the structure of flavonoid.
To find something out about the chemical structure / pharmacological activity relationship we used
HPLC and CE analitical technics, which confirmed the literature dates, that HPLC is mainly
suitable for analyzing a small number of components, a CE should be used, when a big number of
theoretical plates (N) is required ?51?. Comparing the two technics it can be concluded that CE
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enables more analysis to be performed in the same time, because it is faster than HPLC. It does not
require solvent gradients or long column equilibration. CE does not consume organic solvents for
elution and the capillaries are much less expensive than the columns used for HPLC.
The CE qualitative analysis proved the presence of luteolin?7?O?? ?glucoside,
apigenin?7?O?? ?glucoside and chlorogenic acid in Taraxaci folium extracts. The HPLC
quantitative analysis showed the concentration of luteolin?7?O???glucoside in the plant material to
be 3.517 mg/g and chlorogenic acid 3.052 mg/g. The qualitative analysis of Mori nigri folium
extracts proved the presence of rutin, isoquercitrin and chlorogenic acid, no quercitrin was found.
The quantitative HPLC analysis showed the concentration of chlorogenic acid , rutin and
isoquercitrin in the plant material 1.836, 1.162 and 3.164 mg/g. Based on these results we have
postulated that the radical scavenging activity of Mori nigri folium is based on chelating and free
radical scavenging activity of rutin and other quercitrin glicosides (isoquercitrin). The antioxidative
activity of an extract from Taraxaci folium might be because of the superoxide anion scavanging
activitz of luteolin?7?O?? ?glucoside and apigenin?7?O?? ?glucoside.
New results and conclusions from studies on capillary electrophoresis and antioxidant activity
? The water extracts studied have showed antioxidant, free radical scavanging activity, which
was influenced not only by the total poliphenolic content, but by the composition and structure
of flavonoids. This partly explains their possible mechanism of action in organisms, contributes
to our understanding why so many plants can be used for successful treatment of different
diseases.
? Until now the CE was used for phytochemical analysis in a restricted way. Our results
contribute to the better understanding and usability of this analitical technic.
? The CE is a good choice for a fast analysis, and the simplicity is a special value when many
flavonoid-containing plants must be evaluated. This contributes at the same time to solve some
problems in connection with the design and standardization of phytomedicines.
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REFERENCES
Closely linked to the dissertation
Hagym�si, K., Bl�zovics A., Kristó, T., Sz., Feh�r, J., K�ry, �.: Taraxacum officinale - az orvos
szem�vel.
Fitoter�pia, 1999. 4, 51-56.
Kristó, T., Sz., Terdy, P., P., Sim�ndi, B., K�ry, �.: Taraxacum officinale (gyermekl�ncfu)
hatóanyagainak kivon�sa k�l�nb�zo extrakciós módszerekkel.
Olaj, Szappan, Kozmetika, 2000. 49, 93-97.
Hagym�si, K., Bl�zovics, A., Lugasi, A., Kristó, T., Sz., Feh�r, J., K�ry, �.: In vitro antioxidant
evaluation of dandelion (Taraxacum officinale Web.) water extracts.
Acta Alimentaria, 2000. 29, 1-7. (If: 0,246/2000)
Hagym�si, K., Bl�zovics, A., Feh�r, J., Lugasi, A., Kristó, T., Sz., K�ry, �.: The in vitro effect of
dandelions antioxidants on microsomal lipid peroxidation.
Phytotherapy Research, 2000. 14, 43-44. (If:0,422/2000)
Prechl, A., Sim�ndi, B., K�ry, �., Lemberkovics, �., Kristó, T., Sz., Bal�zs, A., De�k, A.:
Materials with Supercritical Fluid Extraction using carbon dioxide.
Olaj, Szappan, Kozmetika, 2000. 49, 102-105.
Kristó, T., Sz., Terdy, P., P., Sim�ndi, B., Szoke, �., Lemberkovics, �., K�ry, �.: Szuperkritikus
extrakció hat�konys�ga a Taraxaci radix nem illó n�v�nyi terpenoidjainak elo�ll�t�s�ra.
Acta Pharmaceutica Hungarica, 2001. 71, 318-324.
K�ry, �., Bl�zovics, A., Fejes, Sz., Nagy, �., Lugasi, A., Kursinszki, L., Czinner, E., Kristó, T.,
Sz., Ap�ti, P., Bal�zs, A., Szoke, �.: Antioxidant activity of medicinal plants used in phytotheraphy.
International Journal of Horticultural Science, 2001. 7, 28-35.
Lemberkovics, �., K�ry, �., Sim�ndi, B., Kristó, T., Sz., Kakasy, A., Szoke, �.: Evaluation of
supercritical plant extracts on volatile and non volatile biologically active lipophil components.
International Journal of Horticultural Science, 2001. 7, 78-83.
Kristó, T., Sz., Ganzler, K., Ap�ti, P., Szoke, �., K�ry, �.: Analysis of antioxidant flavonoids from
Asteraceae and Moraceae plants by Capillary Electrophoresis.
Chromatographia, 2002. 56, S121-S126. (If: 1,317/2001)
Sim�ndi, B., Kristó, T., Sz., K�ry, �., Selmeczi,L., K., Kmecz, I., Kem�ny, S.: Supercritical fluid
extraction of dandelion leaves.
Journal of Supercritical Fluids, 2002. 23, 135-142. (If: 1,975/2001)
Kristó, T., Sz., Terdy, P., P., Selmeczi, L., K., Szoke, �., Sim�ndi, B., K�ry, �.: Production and
caracterisation of Taraxacum officinale extracts prepared by supercritical and solvent extractions.
Acta Horticulturae, 2002. 597, 57-62.
14
Partly linked to the dissertation
Prechl, A., Sim�ndi B., K�ry �., Lemberkovics �., Kristó, T., Sz., Bal�zs, A., De�k, A., Keve, T.:
Illatos bar�tcserje szuperkritikus extrakciója.
Olaj Szappan Kozmetika, 2000. 49, 106-108.
Ap�ti, P., Szentmih�lyi, K., Bal�zs, A., Baumann, D., Hamburger, M., Kristó, T., Sz., Szoke, �.,
K�ry, �.: HPLC Analysis of the Flavonoids in Pharmaceutical Preparations from Canadian
Goldenrod (Solidago canadensis)
Chromatographia, 56, 65-68. 2002. (If: 1,317/2001)
Ap�ti, P., Szentmih�lyi, K., Kristó, T., Sz., Papp, I., Vinkler, P., Szoke, �., K�ry, �.: Herbal
remedies of Solidago. Correlations of phytochemical characteristics and antioxidant properties.
Journal of Pharmaceutical and Biomedical Analysis, 2002., K�zl�sre elfogadva
(If: 1,177)
Ap�ti, P., Szentmih�lyi, K., Kristó, T., Sz., Szoke, �., Vinkler, P., K�ry, �.: Comprehensive
evaluation of different Solidaginis herba extracts.
Acta Horticulturae, 2002. 597, 69-74.
Fejes, Sz., Lemberkovics, �., Bal�zs, A., Ap�ti, P., Kristó, T., Sz., Szoke, �., Bl�zovics, A., K�ry,
�.: Antioxidant activity of different components from Anthriscus cerefolium L. Hoffm.
Acta Horticulturae, 2002. 597, 191-198.
LECTURES, POSTERS
Abstracts published in periodicals
K�ry, �., Fejes, Sz., Kristó, T., Sz., Hagym�si, K., Bl�zovics, A., Czinner, E., Lemberkovics, �.,
Szoke, �.: Preventive value of some medicinal plants due to their antioxidant and free radical
scavenging activities.
First Congress of International Society of Prevention in Clinical Medicine, Budapest, 1998.
december 3-5.
Abstract: Journal of Prevention in Clinical Medicine, 1998. l, 16.
K�ry, �., Kristó, T., Sz., Sim�ndi, B., Lemberkovics, �., Szoke, �.: Quality of medicinal plant
products prepared by supercritical fluid extraction.
50th Annual Congress of Society for Medicinal Plant Research, Barcelona, September 8-12. 2002.
Abstract: Revista Fitoterapia 2002. 2 (1), 339. (B249).
Abstracts in Book of Abstracts
Kristó, T., Sz., Hagym�si, K., Bl�zovics, A., K�ry, �., Szoke, �.: Morus alba L. lev�ldrog
hagyom�nyos �s szuperkritikus extrakcióval elo�ll�tott kivonatainak fitok�miai vizsg�lata.
P�csi Fitoter�pi�s Napok, P�cs, 1998. �prilis 18-19.
�sszefoglaló: Programf�zet (P11). 33.
15
Hagym�si, K., Kristó, T., Sz., Lugasi, A., K�ry, �., Feh�r, J., Bl�zovics, A.: Gyógyn�v�nyek
antioxid�ns aktivit�s�nak in vitro screenel�se. P�csi Fitoter�pi�s Napok, P�cs, 1998. �prilis 18-19.
�sszefoglaló: Programf�zet 10.
K�ry, �., Fejes, Sz., Kristó, T., Sz., Hagym�si, K., Bl�zovics, A., Czinner, E., Lemberkovics, �.,
Szoke, �.: Screening of medicinal plants for free radical scavanging effects.
Society for Medicinal Plant Research, Vienna, Austria, August 31st - September 4th, 1998.
Abstract: in Abstracts of Plenary Lectures, Short Lectures and Posters: J. 44.
Kristó, T., Sz., Terdy, P., P., Sim�ndi, B., K�ry, �.: Szuperkritikus extrakció alkalmaz�sa
Taraxacum officinale (gyermekl�ncfu) hatóanyagainak elo�ll�t�s�ra.
Szuperkritikus oldószerek analitikai �s muveleti alkalmaz�sa. Konferencia, Budapest 1999. m�jus
20. �sszefoglaló: Eload�skivonatok 29.
Kristó, T., Sz., Terdy, P., P., Sim�ndi, B., K�ry, �.:A szuperkritikus extrakció, mint lehetos�g a
Taraxacum officinale hatóanyagainak elo�ll�t�s�ra.
Congressus Pharmaceuticus Hungaricus XI., Gyógyszer�szek Orsz�gos Kongresszusa XI. Siófok,
1999. október 6-10. �sszefoglaló: Programf�zet, 1999. Eload�s�sszefoglalók (E-54) 39.
Prechl, A., Sim�ndi, B., K�ry, �., Lemberkovics, �., Kristó, T., Sz., Bal�zs, A., De�k, A., Keve,
T.: Illatos bar�tcserje (Vitex agnus castus L.) szuperkritikus extrakciója. Szuperkritikus oldószerek
analitikai �s muveleti alkalmaz�sa. Konferencia, Budapest, 1999. m�jus 20.
�sszefoglaló: Eload�skivonatok 32.
Kristó, T., Sz., Terdy ,P., P., Sim�ndi, B., Bal�zs, A., K�ry, �.: The preparation of bioactive
constituents from Taraxacum officinale by means of various extraction methods.
8th Semmelweis Symposium, Budapest, 1999. november 4-5.
�sszefoglaló: Pant. Pressent and Future in Neuroendocrynology, 140. 2000.
Szoke, �., Bl�zovics, A., Hagym�si, K., Kristó, T., Sz., Ap�ti, P., Czinner, E., Fejes, Sz., Feh�r, J.,
K�ry, �.: Antioxidant activity of medicinal plants with phytotherapeutical significance.
SFRR Europe Winter Meeting, Dinard, France 1999. december 2-5.
Kristó, T., Sz., Terdy, P., P., Sim�ndi, B., K�ry, �.: A szuperkritikus extrakció hat�kony
alkalmaz�sa nem illó terpenoidok elo�ll�t�s�ra.
Ph.D. Tudom�nyos Napok 2000 Semmelweis Egyetem, Budapest, 2000.febru�r 16-17.
Kristó, T., Sz., Terdy, P., P., Sim�ndi, B., K�ry, �.: Fitogyógyszeralapanyagok elo�ll�t�sa
szuperkritikus extrakcióval.
Erd�lyi Mśzeum Egyes�let Orvostudom�nyi �s Gyógyszer�szeti Szakoszt�ly X. Tudom�nyos
�l�sszaka, Sz�kelyudvarhely, 2000. m�jus 11-13.
Kristó, T., Sz., Ap�ti,,P., Hagym�si,,K., Bl�zovics,,A., Szoke,,�., Ganzler,,K., K�ry, �.: Analysis
of antioxidant flavonoids by capillary electrophoresis (CE).
The 6th European Congress of Pharmaceutical Sciences EUFEPS 2000. Budapest, September 16-
19. Book of Abstracts: European Journal of Pharmaceutical Sciences, 2000. 11, S 274.
Ap�ti, P., Szentmih�lyi, K., Kristó, T., Sz., Szoke, �., Vinkler, P., K�ry, �.: Comprehensive
eveluation of different Solidaginis herba extracts.
The 6th European Congress of Pharmaceutical Sciences. EUFEPS 2000. Budapest, September 16-
19. Book of Abstracts: European Journal of Pharmaceutical Sciences, 2000. 11, S
16
Kristó, T., Sz., Ap�ti, P., Hagym�si, K., Bl�zovics, A., Szoke, �., Ganzler, K., K�ry, �.:
Antioxid�ns hat�sś gyógyn�v�nykivonatok vizsg�lata kapill�relektrofor�zissel.
Semmelweis Egyetem Ph.D. Tudom�nyos Napok, 2001. febru�r 21-22.
�sszefoglaló: Eload�skivonatok, 63 (P 12).
Ap�ti, P., Szentmih�lyi, K., Kristó, T., Sz., Szoke, �., K�ry, �.: Solidaginis herba kivonatok
�rt�kel�se az �sv�nyi elemek �s flavonoid glikozidok kioldód�sa alapj�n.
Semmelweis Egyetem Ph.D. Tudom�nyos Napok, 2001. febru�r 21-22.
�sszefoglaló: Eload�skivonatok, 27 (P 01).
Ap�ti, P., Szentmih�lyi, K., Kristó, T., Sz., Szoke, �., Vinkler, P., K�ry, �.: Comprehensive
evaluation of different Solodaginis herba extracts.
World conference on medicinal and aromatic plants. Possibilities and Limitations of Medicinal and
Aromatic Plants. Budapest, 2001. July 8-11.
Abstract: Abstracts - MAP Hungary, 2001. (P.IV.1.) 275.
Kristó, T., Sz., Terdy, P., P., Selmeczi, L., K., Sim�ndi, B., K�ry, �.: Production and
characterisation of Taraxacum officinale extracts prepared by supercritical fluid and solvent
extractions.
World conference on medicinal and aromatic plants. Possibilities and Limitations of Medicinal and
Aromatic Plants. Budapest, 2001. July 8-11.
Abstract: Abstracts - MAP Hungary, 2001. (P.IV.12.) 286.
Ap�ti, P., Szentmih�lyi, K., Bal�zs, A., Baumann, D., Hamburger, M., Kristó, T., Sz., Szoke, �.,
Ganzler, K., K�ry, �.: HPLC analysis of the flavonoids in pharmaceutical preparations from
Solidago canadensis.
Balaton Symposium'01 on High Performance Separation Methods. Siófok, 2001.September 2-4.
Abstract: Book of Abstracts,
Kristó, T., Sz., Ap�ti, P., Szoke, �., K�ry, �.: Analysis of antioxidant flavonoids from Asteraceae
and Moraceae by Capillary Electroforesis.
Balaton Symposium'01 on High-Performance Separation Methods. Siófok, 2001.September 2-4.
Abstract: Book of Abstracts, 18
K�ry, �., Kristó, T., Sz., Sim�ndi, B., Lemberkovics, �., Szoke, �.: Supercritical fluid extraction of
some non-volatile bioactive terpenoids.
International Congress and 49th Annual Meeting of the Society for Medicinal Plant Research.
Erlangen, September 2-6. 2001.
Abstract: Traits, Tracks and Traces. Abstract Book. 4.39., 172.
Sim�ndi, B., K�ry, �., Kristó, T., Sz., Andr�s, Cs., Prechl, A., Fekete, J.: Supercritical fluid
extraction of non-volatile terpenoids from medicinal plants.
4th International Symposium on High Pressure Technology and Chemical Engineering, Venice, Italy
2002. szeptember 22-25
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