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. Kéry Ágnes, 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. Ágnes Kéry

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

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.

Ph. D. doktori értekezés

A Taraxacum officinale és Morus nigra bioaktív anyagainak vizsgálata

Kristó Tita Szidónia

Témavezeto: Dr. Kéry Ágnes c. egyetemi tanár

Semmelweis Egyetem, Gyógyszerésztudományi Kar, Farmakognózia Intézet, 2002

Összefoglaló

A növényi eredetu, biológiailag aktív anyagok egyre szélesebb köru és aprólékosabb farmakológiai

vizsgálata az új, gyors, pontos és hatékony extrakciós és fitoanalitikai módszerek alkalmazását sürgeti. A jól

összehangolt kémiai-farmakológiai kutatások egyúttal adatokat szolgáltathatnak a szerkezet-hatás

összefüggésekkel kapcsolatban is.

Számos kísérleti adat támasztja alá, hogy az eddig nem hatóanyagnak tekintett növényi összetevok jelentosen

befolyásolhatják a gyógynövények és kivonataik aktivitását, illetve biztonságos felhasználásuk is

módosulhat.

A Taraxacum officinale és Morus nigra bioaktív anyagai közül a gyulladáscsökkento, immunmoduláns,

daganatellenes tulajdonságokkal rendelkezo triterpének és fitoszterolok tanulmányozásához az ezidáig foleg

illó komponensek eloállítására alkalmazott hatékony, környezetkímélo szuperkritikus extrakciót

alkalmaztuk. Optimalizáltuk a kísérleti paramétereket (nyomás, homérséklet) és megvalósítottuk a

hatóanyagok jelentos és szelektív dúsítását. Az elonyök és hátrányok mérlegelése céljából a kivonás

hatékonyságát összehasonlítottuk a hagyományos módszerekkel. Az extraktumok kvantitatív értékeléséhez

VRK-denzitometriás módszert dolgoztunk ki és megállapítottuk, hogy a szuperkritikus extraktumokban a

hatóanyagok esetenként 10%-ig is feldúsultak.

A Taraxacum officinale és a Morus nigra liofilizált kivonatai antioxidáns, szabadgyökfogó aktivitással

rendelkeznek, amit in vitro vizsgálatokkal bizonyítottunk. A hatásért felelos vegyületek feltárásához

kapillárelektroforézist alkalmaztunk, melynek hatékonyságát nagynyomású folyadék-kromatográfiás

módszerrel is összehasonlítottuk. Azonosítottuk a drogok fenoloid komponenseit (Taraxacum officinale:

apigenin-7-O-

-glükozid, luteolin-7-O-

-glükozid, klorogénsav; Morus nigra: kvercetin-3-O-

ramnoglükozid; kvercetin-3-O-glükozid, klorogénsav) és eltéro hatásmechanizmusuk (kelátképzo,

szuperoxid ion scavanger) alapján valószínusítettük különbözo betegségekben való alkalmazhatóságukat.

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 CO

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 A

-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

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.

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.

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,

Marosvásárhely, 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. 60

C, 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 (40-45

C) and used for TLC- densitometric

evaluation.

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 105

C 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 ,65

C) 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 : H

O – CH

COOH. 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.

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 Fe

transformation and

expressed in ASE/mg.

Total scavanger activity

The total scavanger activity was proved with chemiluminometric methods (Blázovics et al.) and

expressed in I

(

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

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 ß-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 ß-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 35

C) 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

C). 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, 100

The calibration curves of standard substances (

-amyrin,

-sitosterol) were between 2.5-5.5

ng/ml).

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 ß-amyrin and ß-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.

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 (I

= 1150

g Mori nigri folium; I

= 750

g Taraxaci radix;

=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 (I

= 155

g Taraxaci folium; I

= 210

g Taraxaci radix; Mori nigri folium I

270

g). The dandelion leaves extracts exhibited a strong membrane-protective action

(IC

=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

. Comparing the two technics it can be concluded that CE

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

?? ?

glucoside,

apigenin

?? ?

glucoside and chlorogenic acid in Taraxaci folium extracts. The HPLC

quantitative analysis showed the concentration of luteolin

???

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

?? ?

glucoside and apigenin

?? ?

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.

REFERENCES

Closely linked to the dissertation

Hagymási, K., Blázovics A., Kristó, T., Sz., Fehér, J., Kéry, Á.: Taraxacum officinale - az orvos
szemével.
Fitoterápia, 1999. 4, 51-56.

Kristó, T., Sz., Terdy, P., P., Simándi, B., Kéry, Á.: Taraxacum officinale (gyermekláncfu)
hatóanyagainak kivonása különbözo extrakciós módszerekkel.
Olaj, Szappan, Kozmetika, 2000. 49, 93-97.

Hagymási, K., Blázovics, A., Lugasi, A., Kristó, T., Sz., Fehér, J., Kéry, Á.: In vitro antioxidant
evaluation of dandelion (Taraxacum officinale Web.) water extracts.
Acta Alimentaria, 2000. 29, 1-7. (If: 0,246/2000)

Hagymási, K., Blázovics, A., Fehér, J., Lugasi, A., Kristó, T., Sz., Kéry, Á.: The in vitro effect of
dandelions antioxidants on microsomal lipid peroxidation.
Phytotherapy Research, 2000. 14, 43-44. (If:0,422/2000)

Prechl, A., Simándi, B., Kéry, Á., Lemberkovics, É., Kristó, T., Sz., Balázs, A., Deák, A.:
Materials with Supercritical Fluid Extraction using carbon dioxide.
Olaj, Szappan, Kozmetika, 2000. 49, 102-105.

Kristó, T., Sz., Terdy, P., P., Simándi, B., Szoke, É., Lemberkovics, É., Kéry, Á.: Szuperkritikus
extrakció hatékonysága a Taraxaci radix nem illó növényi terpenoidjainak eloállítására.
Acta Pharmaceutica Hungarica, 2001. 71, 318-324.

Kéry, Á., Blázovics, A., Fejes, Sz., Nagy, É., Lugasi, A., Kursinszki, L., Czinner, E., Kristó, T.,
Sz., Apáti, P., Balázs, A., Szoke, É.: Antioxidant activity of medicinal plants used in phytotheraphy.
International Journal of Horticultural Science, 2001. 7, 28-35.

Lemberkovics, É., Kéry, Á., Simándi, B., Kristó, T., Sz., Kakasy, A., Szoke, É.: 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., Apáti, P., Szoke, É., Kéry, Á.: Analysis of antioxidant flavonoids from
Asteraceae and Moraceae plants by Capillary Electrophoresis.
Chromatographia, 2002. 56, S121-S126. (If: 1,317/2001)

Simándi, B., Kristó, T., Sz., Kéry, Á., Selmeczi,L., K., Kmecz, I., Kemény, 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, É., Simándi, B., Kéry, Á.: Production and
caracterisation of Taraxacum officinale extracts prepared by supercritical and solvent extractions.
Acta Horticulturae, 2002. 597, 57-62.

Partly linked to the dissertation

Prechl, A., Simándi B., Kéry Á., Lemberkovics É., Kristó, T., Sz., Balázs, A., Deák, A., Keve, T.:
Illatos barátcserje szuperkritikus extrakciója.
Olaj Szappan Kozmetika, 2000. 49, 106-108.

Apáti, P., Szentmihályi, K., Balázs, A., Baumann, D., Hamburger, M., Kristó, T., Sz., Szoke, É.,
Kéry, Á.: HPLC Analysis of the Flavonoids in Pharmaceutical Preparations from Canadian
Goldenrod (Solidago canadensis)
Chromatographia, 56, 65-68. 2002. (If: 1,317/2001)

Apáti, P., Szentmihályi, K., Kristó, T., Sz., Papp, I., Vinkler, P., Szoke, É., Kéry, Á.: Herbal
remedies of Solidago. Correlations of phytochemical characteristics and antioxidant properties.
Journal of Pharmaceutical and Biomedical Analysis, 2002., Közlésre elfogadva
(If: 1,177)

Apáti, P., Szentmihályi, K., Kristó, T., Sz., Szoke, É., Vinkler, P., Kéry, Á.: Comprehensive
evaluation of different Solidaginis herba extracts.
Acta Horticulturae, 2002. 597, 69-74.

Fejes, Sz., Lemberkovics, É., Balázs, A., Apáti, P., Kristó, T., Sz., Szoke, É., Blázovics, A., Kéry,
Á.: Antioxidant activity of different components from Anthriscus cerefolium L. Hoffm.
Acta Horticulturae, 2002. 597, 191-198.

LECTURES, POSTERS

Abstracts published in periodicals

Kéry, Á., Fejes, Sz., Kristó, T., Sz., Hagymási, K., Blázovics, A., Czinner, E., Lemberkovics, É.,
Szoke, É.: 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.

Kéry, Á., Kristó, T., Sz., Simándi, B., Lemberkovics, É., Szoke, É.: 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., Hagymási, K., Blázovics, A., Kéry, Á., Szoke, É.: Morus alba L. levéldrog
hagyományos és szuperkritikus extrakcióval eloállított kivonatainak fitokémiai vizsgálata.
Pécsi Fitoterápiás Napok, Pécs, 1998. április 18-19.
Összefoglaló: Programfüzet (P11). 33.

Hagymási, K., Kristó, T., Sz., Lugasi, A., Kéry, Á., Fehér, J., Blázovics, A.: Gyógynövények
antioxidáns aktivitásának in vitro screenelése. Pécsi Fitoterápiás Napok, Pécs, 1998. április 18-19.
Összefoglaló: Programfüzet 10.

Kéry, Á., Fejes, Sz., Kristó, T., Sz., Hagymási, K., Blázovics, A., Czinner, E., Lemberkovics, É.,
Szoke, É.: 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., Simándi, B., Kéry, Á.: Szuperkritikus extrakció alkalmazása
Taraxacum officinale (gyermekláncfu) hatóanyagainak eloállítására.
Szuperkritikus oldószerek analitikai és muveleti alkalmazása. Konferencia, Budapest 1999. május
20. Összefoglaló: Eloadáskivonatok 29.

Kristó, T., Sz., Terdy, P., P., Simándi, B., Kéry, Á.:A szuperkritikus extrakció, mint lehetoség a
Taraxacum officinale hatóanyagainak eloállítására.
Congressus Pharmaceuticus Hungaricus XI., Gyógyszerészek Országos Kongresszusa XI. Siófok,
1999. október 6-10. Összefoglaló: Programfüzet, 1999. Eloadásösszefoglalók (E-54) 39.

Prechl, A., Simándi, B., Kéry, Á., Lemberkovics, É., Kristó, T., Sz., Balázs, A., Deák, A., Keve,
T.: Illatos barátcserje (Vitex agnus castus L.) szuperkritikus extrakciója. Szuperkritikus oldószerek
analitikai és muveleti alkalmazása. Konferencia, Budapest, 1999. május 20.
Összefoglaló: Eloadáskivonatok 32.

Kristó, T., Sz., Terdy ,P., P., Simándi, B., Balázs, A., Kéry, Á.: The preparation of bioactive
constituents from Taraxacum officinale by means of various extraction methods.
8th Semmelweis Symposium, Budapest, 1999. november 4-5.
Összefoglaló: Pant. Pressent and Future in Neuroendocrynology, 140. 2000.

Szoke, É., Blázovics, A., Hagymási, K., Kristó, T., Sz., Apáti, P., Czinner, E., Fejes, Sz., Fehér, J.,
Kéry, Á.: Antioxidant activity of medicinal plants with phytotherapeutical significance.
SFRR Europe Winter Meeting, Dinard, France 1999. december 2-5.

Kristó, T., Sz., Terdy, P., P., Simándi, B., Kéry, Á.: A szuperkritikus extrakció hatékony
alkalmazása nem illó terpenoidok eloállítására.
Ph.D. Tudományos Napok 2000 Semmelweis Egyetem, Budapest, 2000.február 16-17.

Kristó, T., Sz., Terdy, P., P., Simándi, B., Kéry, Á.: Fitogyógyszeralapanyagok eloállítása
szuperkritikus extrakcióval.
Erdélyi Múzeum Egyesület Orvostudományi és Gyógyszerészeti Szakosztály X. Tudományos
Ülésszaka, Székelyudvarhely, 2000. május 11-13.

Kristó, T., Sz., Apáti,,P., Hagymási,,K., Blázovics,,A., Szoke,,É., Ganzler,,K., Kéry, Á.: 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.

Apáti, P., Szentmihályi, K., Kristó, T., Sz., Szoke, É., Vinkler, P., Kéry, Á.: 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

Kristó, T., Sz., Apáti, P., Hagymási, K., Blázovics, A., Szoke, É., Ganzler, K., Kéry, Á.:
Antioxidáns hatású gyógynövénykivonatok vizsgálata kapillárelektroforézissel.
Semmelweis Egyetem Ph.D. Tudományos Napok, 2001. február 21-22.
Összefoglaló: Eloadáskivonatok, 63 (P 12).

Apáti, P., Szentmihályi, K., Kristó, T., Sz., Szoke, É., Kéry, Á.: Solidaginis herba kivonatok
értékelése az ásványi elemek és flavonoid glikozidok kioldódása alapján.
Semmelweis Egyetem Ph.D. Tudományos Napok, 2001. február 21-22.
Összefoglaló: Eloadáskivonatok, 27 (P 01).

Apáti, P., Szentmihályi, K., Kristó, T., Sz., Szoke, É., Vinkler, P., Kéry, Á.: 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., Simándi, B., Kéry, Á.: 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.

Apáti, P., Szentmihályi, K., Balázs, A., Baumann, D., Hamburger, M., Kristó, T., Sz., Szoke, É.,
Ganzler, K., Kéry, Á.: 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., Apáti, P., Szoke, É., Kéry, Á.: 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

Kéry, Á., Kristó, T., Sz., Simándi, B., Lemberkovics, É., Szoke, É.: 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.

Simándi, B., Kéry, Á., Kristó, T., Sz., András, Cs., Prechl, A., Fekete, J.: Supercritical fluid
extraction of non-volatile terpenoids from medicinal plants.
4

International Symposium on High Pressure Technology and Chemical Engineering, Venice, Italy

2002. szeptember 22-25

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