UNIVERSITY OF AGRICULTURAL SCIENCES AND VETERINARY MEDICINE

DOCTORAL SCHOOL

ZOOTECHNY AND BIOTEHNOLOGIES

Eng. SIMONA VICTORIA ILEA (ZĂVOI)

CHARACTERIZATION OF SOME ABORIGINAL HERBS AND OF SOME DERIVED BIOPRODUCTS

WITH HEPATOPROTECTIVE ACTION

SUMMARY OF THE PhD THESIS

SCIENTIFIC COORDINATOR Prof. Dr. Carmen SOCACIU

Cluj-Napoca 2011

  I

  II

SUMMARY

INTRODUCTION ...................................................................................................... IV

AIM AND OBJECTIVES OF PERSONAL RESERCHES ................................... VI

CHAPTER I.

HERBS WITH HEPATO-PROTECTIVE ACTION:

BOTANICAL CHARACTERISTICS AND GENERAL CHEMICAL

COPOSITION ............................................................................................................ VI

CHAPTER II.

BIOACTIVE COMPOUNDS WITH HEPATOPROTECTIVE PROPERTIES.VII

CHAPTER III.

COMPARATIVE CHARACTERIZATION OF HYDROALCOHOLIC,

HYDROGLICERIC AND METHANOLIC EXTRACTS COMPOSITION ...... VII

III.1. CHARACTERIZATION OF HYDROALCOHOLIC AND

HYDROGLICERIC EXTRACTS COMPOSITION. .................................. VIII

III. 1.1. Materials and methods ................................................................................... VIII

III.1.2. Results and discussions. ................................................................................. VIII

III.1.2.1. UV-VIS spectrometric analysis. ............................................................ VIII

III.1.2.2. FT-MIR spectrometric analysis ............................................................... XI

III.1.3. Conclusions. ................................................................................................... XIV

III.2. CHARACTERIZATION OF METHANOLIC EXTRACTS COMPOSITION.XV

III. 2.1. Materials and methods. ................................................................................... XV

III. 2.2. Results and discussions .................................................................................. XV

III.2.2.1. UV-VIS spectrometric analysis .............................................................. XV

III.2.2.2. Determination by spectrometry of the total phenols content from

methanolic extracts .................................................................................. XVI

III.2.2.3. FT-MIR spectrometric analysis ............................................................. XVI

III.2.2.4. HPLC-UV chromatographic analysis of methanolic extracts ............... XXI

III.2.3. Conclusions ................................................................................................. XXIV

CHAPTER IV.

 

 

III

ANALYSIS OF COMPOSITION VARIABILITY OF INVESTIGATED

PLANTS BY HPLC-UV CHROMATOGRAPHY AND FT-MIR

SPECTROMETRY, COUPLED WITH CHEMOMETRIC ANALYSIS

.................................................................................................................................. XXV

OBJECTIVES AND MEASUREMENT ................................................................. XXV

IV. 1. HPLC-UV CHROMATOGRAPHIC ANALYSIS ........................................ XXV

IV.1.1. Materials and methods ................................................................................. XXV

IV.1.2. Results and discussions. ............................................................................... XXV

IV.1.2.1. HPLC-UV chromatographic separation and identification of the specific

fingerprint of methanolic extracts from 4 sample variants from each plant

type ............................................................................................................. XXV

IV.1.2.2. Chemometric analysis in NT (nontarget) and T (target) system ........ XXVI

IV.1.3. Conclusions .................................................................................................. XXX

IV. 2. FT-MIR SPECTROMETRIC ANALYSI ...................................................... XXX

IV.2.1. FT-MIR spectrometric analysis and identification of plant specific

fingerprint ................................................................................................................. XXX

IV.2.2. Chemometric analysis in NT (nontarget) and T (target)system ........... XXXII

IV.2.3.Conclusions ......................................................................................... XXXIII

CHAPTER V

OBTAINMENT AND CHARACTERIZATION OF SOME BIOPRODUCTS

WITH HEPATOPROTECTIVE EFECTS ..................................................... XXXIV

AIM AND OBJECTIVES OF THE RESEARCHES ........................................... XXXIV

     V.1. Materials and methods ........................................................................... XXXIV

V.2. Results and discussions ............................................................................ XXXV

V.2.1. UV-Vis spectrometric analysis .......................................................... XXXVI

V.2.2. HPLC-UV chromatographic analysis .............................................. XXXVII

V.2.3. FT-MIR spectrometric analysis ...................................................... XXXVIII

        V.2.4. Conclusions ....................................................................................... XXXIX

GENERAL CONCLUSIONS .................................................................................. XLI

SELECTIVE BIBLIOGRAPHY .......................................................................... XLIII

 

 

IV

INTRODUCTION

From centuries, herbs demonstrated their benefic effects in some diseases

prophylaxis and therapy, without secondary effects, in comparison with synthesis

medicines. Their composition is dependent on ontogenic and phenotypic factors,

influenced by the environment, age, harvest period, drying and storage method, but

also on the type of the extraction solvent (Tămaş M., 1999, Tămaş M. and I. Oniga,

2000) .

Due to their natural heterogeneity, the quality of these plants from spontaneous

flora show fluctuations, thus it is necessary to standardize the extracts, to attest and

test the purity and the action (Yadav N.P. and Dixit, 2008, Hussain K. and col., 2009).

The fingerprint identification of these phytochemical compounds by chromatography

and spectroscopy can offer useful information about the qualitative and quantitative

composition of herbs, with recognition, by chemometric methods, of recognition

markers (Maloney V., 2004 , Bender D.A., 2005) and discrimination between

different plants or extract types, or standardized formula (McGuffin M. and col., 1997,

Liang Y.Z. and col., 2004 , Yadav N.P. and Dixit, 2008, Giri L. and col., 2010), in

agreement with WHO requierements (WHO, 2003).

The evaluation of herb products by their metabolimic fingerprint can be

achieved by advanced methods that include high performance liquid chromatography

(HPLC with UV detection (DAD), ELSD, MS) or chromatography gas with MS

detection (GC-MS), chromatography on thin layer with densitometric quantification

(HPTLC-densitometry), infrared vibrational spectrometry or Raman (FT-MIR, NIR,

Raman), magnetic resonance spectrometry NMR or a combination of these techniques

(Fan X.H. and col., 2006, Mattoli L. and col., 2006, Gong F. and col., 2005, Li S. And

col., 2008, Hashimoto A. and T. Kameoka, 2008, Gong F. and col., 2009, Giri L.

andcol., 2010).

UV-Vis spectroscopy is a simple, inexpensive and easy to realize technique,

useful in order to identify and quantify the main phytochemical compounds,

discriminating between lipophilic and hydrophilic compounds, correlated with the

polarity of the solvents used.

 

 

V

Infrared spectrometry technique with Fourier transformant (FTIR) is a rapid

and non-destructive investigation, easy to use in order to determine the fingerprint of

plants extracts or powders, less known that chromatography or classic

physicochemical methods (Li Y. and col., 2004b, Hussain K. And col., 2009, Liu H.

and col., 2006). The use of attenuanted total reflectance system (ATR) permits rapid

measurements by FTIR in liquids (oils and extracts) permitting the identification and

quantification of biomarkers from plants (Schultz H. and M. Baranska, 2007).

In general, herbs with hepatoprotective action are rich in polyphenols with

antioxidant potential (such as Thistle, Artichoke, Dandelion, Celandine, St. John’s

Wort), knowing that hepatic diseases assume cellular necrosis and oxidative stress

(Utrilla M.P., 1996, Negi A.S. and col., 2008).

Taking into account their intense antioxidant potential, the polyphenolic

compounds have a particular importance. Within polyphenolic compounds, flavonoids

family plays a special role, by the direct impact upon health (Harbone, J.B.and C.A.,

Williams, 2000).

In the PhD thesis entitled “Characterization of some aboriginal herbs and of

some derived bioproducts with hepatoprotective action”, we had as aim the

research and study of some plants known as hepatoprotective plants, in order to obtain

some bioproducts with hepatoprotective effect. This effect resulted after the synergic

action of the active principles from these plants. The thesis is divided in two parts.

The first part is bibliographic study and includes two chapters (1-2):

Chapter I. Herbs with hepatoprotective action : botanical characteristics and general

chemical composition.

Chapter II. Bioactive compounds that give hepatoprotective properties.

The second part includes the personal researches and has three chapters (3-5):

Chapter III. Comparative characterization of hydroalcoholic, hydrogliceric and

methanolic extracts composition.

Chapter IV. The analysis of composition variability of the investigated plants by

HPLC-UV chromatography and FT-MIR spectroscopy, coupled with chemometric

analysis.

 

 

VI

Chapter V. The obtainment and characterization of some bioproducts with

hepatoprotective effect.

AIM AND OBJECTIVES

The aim of the researches was the study of bioactive compounds of some

aboriginal plants known as having hepatoprotective potential, the characterization of

their composition in order to obtain some bioproducts with hepatoprotective effect, by

the synergic action of the active principles from these plants.

The investigated herbs were Dandelion, Artichoke, Celandine, Thistle, St.

John’s Wort, Wolf’s Claw and Sea buckthorn (leafs and fruits), 8 types in total, from

which we investigated different samples harvested from different locations.

The punctual objectives of the personal researches were:

1. The comparative characterization of extracts composition of hydroalcoholic,

hydrogliceric and methanolic type from the 8 investigated plants, using

advanced analytical methods (UV-Vis type spectrometry, Medium Infrared

spectrometry (FT-MIR) and HPLC with UV detection (HPLC-UV).

2. The analysis of composition variability of the investigated plants by HPLC-UV

chromatography and FT-MIR spectrometry, coupled with chemometry.

3. The application of the chemometric analysis and of ANOVA type statistic

analysis for the results interpretation.

4. The obtainment and characterization of two types of bioproducts with

hepatoprotective potential, that have as ingredient the studied plants (1-8).

CHAPTER I.

HERBS WITH HEPATOPROTECTIVE ACTION: BOTANICAL

CHARACTERISTICS AND GENERAL CHEMICAL COMPOSITION

The plants investigated in this study are the following: Dandelion, Artichoke,

Celandine, St. John’s Wort, Thistle, Wolf’s Claw, Sea buckthorn-leafs and Sea

buckthorn-fruits. Details regarding their botanical characteristics and chemical

composition are presented in the thesis.

 

 

VII

CHAPTER II.

BIOACTIVE COMPOUNDS THAT OFFER HEPATOPROTECTIVE

PROPERTIES

The bioactive compounds that enter in the composition of these plants are

presented in detail, highlighting those types of molecules that offer hepatoprotective

properties. Both chapters (chapter I and chapter II) include data obtained on the basis

of the study of literature.

CHAPTER III

COMPARATIVE CHARACTERIZATION OF HYDROALCOHOLIC,

HYDROGLICERIC AND METHANOLIC EXTRACTS COMPOSITION

OBJECTIVES AND EXPERIMENTS

In order to characterize the extraction capacity of the active components from

the 8 types of studied plants offered by different solvents and the specific composition

of these extracts dependent on the solvent type and polarity we followed two

objectives, namely:

I. the establishment of the role and importance of the used solvent regarding the

extraction effectiveness of the bioactive compounds from the investigated

plants

II. the establishment of optimum methods (of UV-Vis spectrometry, FTIR and

high performance liquid chromatography) for the correct evaluation of the

recognition „fingerprint” of the plants and extract type, of quantification of

bioactive compounds concentrations and of authenticity evaluation of these

extracts.

III. In order to accomplish these objectives we made two experimental modules,

each with well established objectives.

 

 

VIII

III.1. CHARACTERIZATION OF HYDROALCOHOLIC AND

HYDROGLICERIC EXTRACTS COMPOSITION

III. 1.1. Materials and methods

We used dry plants (s.u > 90%) from 1-8 categories, mentioned in the previous

chapters, two samples in parallel, which were extracted with solvents of hydrogliceric

(G) and hydroalcoholic types (E).

The methods used for identification and quantitative analysis were UV-Vis

spectrometry and IR spectrometry with Fourier transformant on 400-700 cm-1 (

Middle Infrared = MIR) domain, noted FTMIR.

III.1.2. Results and discussions

III.1.2.1. UV-VIS spectrometric analysis

We identified absorptions maximum at wavelengths specific to each compound, being

determined the intensities of these peaks (A).

According to specific absorptions, we identified the molecule groups, on domains:

212-214 nm domain is assigned to lipidic compounds (phytosterol, polar

lipids), to some vitamins (Vitamin C, E) and to terpenoids

275-290 nm domain is assigned to phenolic compounds (free phenolic acids or

derived of phenolic acids).

320-330 nm is assigned to free flavonoid compounds or to glycosylates.

390-420 nm domain is assigned to flavonoidic compounds and to quinones

derived by polyphenols oxidation.

Chemometric analysis of PCA and CA type

On the basis of the data obtained we made the chemometric analysis of PCA and CA

type of extracts in ethanol (E) and glycerine (G) of the 8 investigated plants.

A. PCA differentiation analysis of the plants, according to the data from UV-Vis spectrometric analysis

B. PCA differentiation analysis on the basis of absorption maximum of the spectra in extracts of glyceric (G) and ethanolic (E) type on 212-214 nm, 275-290 nm, 320-330 nm and 390-420 nm domains

 

 

IX

C. CA differentiation analysis, on 6 levels of the plants used in order to obtain the glyceric and ethanolic extracts

Fig. III.1. PCA and CA chemomentric analysis made for UV-Vis spectrometric

analysis of the extracts, made in ethanol (E) and glycerin (G)

The chemometric analysis emphasizes the following :

1. Dandelion differentiates singnificantly from Celandine-Artichoke and

Thistle-Wolf’s Claw, and associates by composition with the fruits of Sea

buckthorn and St. John’s Wort, especially due to a higher content of

compounds that absorb at wavelenghts major than 320 nm, namely

flavonoids.

2. Artichoke is richer in phenolic compounds (cinnamic acid derivatives) and

is similar to Celandine by terpenoids derivatives

3. Thistle seeds are richer in lipids (phenolic acid esters) that absorb at lower

wavelenghts (under 278 nm). Thistle associates with Wolf’s Claw due to

the higher content of lipidic compounds.

 

 

X

 

 

XI

4. Sea buckthorn leafs differentiates by a high content of lipids ( phenolic acid

esters, resembling with Thistle-Wolf’s Claw group), but has similarities

with Sea buckthorn fruits.

5. By integrating the data we can conclude that : St.John’s Wort-Sea

buckthorn group is dominant flavonoid, Artichoke-Celandine group is

characterized by phenolic acid and tannic derivatives and Thistle-Wolf’s

Claw group, but also Sea buckthorn leafs have lipidic compounds and

terpenoids. Dandelion is delimited by these groups, being very rich in polar

compounds of phenolic acid type, especially coumaric acid and coumarins.

For the first categories, the extraction in glycerin is beneficial, while for the

last category, the extraction in ethanol is superior.

III.1.2.2. FT-MIR spectrometric analysis

From the comparative analysis of FTIR spectra aspect we observe that :

In area 8 (3000-3350 cm-1 ) there are differences that identify the water content

of the samples, respectively we can recognize the evaporated samples by a

lower intensity and a smaller area of the signals, significantly in the case of

ethanolic extract. At the glyceric extract the differences are smaller.

In area 7 (2800-3000 cm-1) 2 signals appear characteristic to glyceric extract,

that can be assigned to methyl groups from metoxilat compounds or alkyl

chains from lipidic compounds, and also to the presence of some aldehydes.

In area 6 (2800-3000 cm-1) there is a prominent signal in the case of both

extract types (E or G), very intense in the case of Artichoke extract and

separately in two signals in the case of evaporated extract E and G from Sea

buckthorn fruits. This signal can be assigned to ester bounds but also to C=O

bounds from aldehyde and ketone, and to N-O bounds from aminoacids or

peptides.

Areas 4 (1300-1440 cm-1), 3 (1170-1230 cm-1) and 2 (1000-1100 cm-1)

differentiate the most these extracts, both in evaporated and non evaporated

form. They represent the fingerprint region of the extracts from these plants.

They correspond to elongation vibrations of C-C, C-O bounds, of esteric

bounds and respectively of simple or complex glucydic compounds that are

soluble in the solvents used. Generally these areas are better represented and

more intense at the extracts in glycerin (G). In the case of Artichoke, area 2-4 is

more intense (extracts E and G), and also for St. John’s Wort-G, Thistle-G,

Wolf’s Claw-G, Celandine-G. In the case of Sea buckthorn, the fruits have

evaporated E extract very rich in areas 2-4, richer than in leafs, instead in

extract G the fingerprint in 2-4 areas is similar.

Chemometric analysis of differentiation between FTIR absorption areas

Fig.III.2. include reprezentarea analizei chemometrice de diferenţiere între zonele de

absorbţie FTIR ale extractelor neevaporate de tip E şi G, ale celor 8 plante analizate.

Fig.III.3 includes the representation of chemometric differentiation analysis between

the FTIR absorption areas of non evaporated extracts of E nad G type, of the 8

analyzed plants.

PCA differentiation analysis according to the solvent and the FTIR absorption area

 

 

XII

PCA differentiation analysis between the plants, on the basis of FTIR fingerprint

CA differentiation analysis between the plants, on the basis of FTIR fingerprint.

Fig. III. 2. PCA and CA chemometric analysis made for FTIR absorptions of nonevaporated extracts from the 8 investigated plants, made in ethanol (E) and

glycerin (G)

 

 

XIII

 

 

XIV

We established a good correlation between PCA and CA analysis.

III.1.3. Conclusions

FTIR spectrometry has allowed the specific „fingerprint” highlighting of

ethanolic and glyceric extracts of the investigated plants, emphasizing the specific

areas with intensities that allow the comparative evaluation of specific functional

groups from each plant.

Generally, the plants richer in lipidic compounds and more soluble in ethanol,

Sea buckthorn fruits, Thistle, had bigger FTIR absorptions in areas 7, 6, 4, 3

(fingerprint areas similar to oils) while the plants rich in flavonoids had area 2 more

intense.

PCA chemometric analysis was useful in order to differentiate the extract types

(G and E), the glyceric extract determining better extractions of the compounds that

absorb in 2-3 areas, while the ethanol facilitated the extraction of the compounds that

absorb in areas 3 and 6. Insignificantly differentiated according to the solvent were

areas 7, 8 and 4.

The chemometric analysis made on the basis of the data obtained (by PCA test)

from evaporated extracts samples point out that the solvent and the areas that

significantly differentiate the samples are : glycerin (areas 2 and 3), ethanol (areas 3

and 6). Areas 7-8 and 4 are insignificantly differentiated.

The cluster comparative analysis (CA) according to the FTIR spectra aspect

emphasizes that Wolf’s Claw and Artichoke form a group (I) delimited by Sea

buckthorn (leaf-fruit) and St. John’s Wort (II) and Celandine-Thistle (III). Also in this

case Dandelion is differentiated by the other plants, similar to what we observed at the

UV-Vis spectrometric analysis.

Sea buckthorn (leafs and fruits) and St. John’s Wort can group, being very well

correlated by their properties. Celandine and Dandelion are the third group,

characterized by polar lipidic compounds.

FTIR spectrometric analysis was repeated on a bigger number of samples of

each type in order to establish if these conclusions are valid. The results are presented

in Chapter IV.

 

 

XV

III.2. THE CHARACTERIZATION OF METHANOLIC EXTRACTS COMPOSITION

III. 2.1. Materials and methods We used dry plants (s.u > 90%) from 1-8 categories, 2 samples of 15 g each in

parallel, that were extracted with 85% acidified methanol (90% methanol, acidified

with 1% hydrochloric acid).

UV-Vis spectrometric analysis

On the basis of the measurements we calculated the extraction factor in methanol (M)

which was then compared with the values recorded at the other solvents (E and G).

The determination of total polyphenols content

The detremination of total polyphenols content was made by FOLIN-CIOCÂLTEU

Method.

HPLC-UV chromatographic analysis

HPLC chromatographic separation of the phenolic compounds from herbs methanolic

extracts was made in a HPLC Agilent 1200 with UV-Vis detector, using a gradient

system with two mobile phases (Table III.5.) with a flow of 1 ml/min.

FT-MIR spectrometric analysis

The measurements were made only on non evaporated extracts.

III. 2.2. Results and discussions

III.2.2.1. UV-Vis spectrometric analysis

In order to have a comparative image regarding the extraction factors of

different herbs (plants 1-6) according to the solvent and the extracted substances

concentration, Fig. III. 3 represents the EF medium values on �max. = 270-290 nm

interval (specific for phenolic derivatives) extracted in E, G, M (EFE1, EFG1, EFM1)

and on �max. = 317-340 nm interval (for flavonoidic derivatives) (EFE2, EFG2,

EFM2).

Fig. III. 3. Comparative medium values of extraction factors (EF) in different solvents

(E, G, M) at 270-290 nm (corresponding to phenolic compounds) (EFE1, EFG1,

EFM1) and 317-340 nm (corresponding to flavonoidic derivatives) (EFE2, EFG2,

EFM2) for plants 1-6

According to the data presented, we observe that EF values of methanolic extracts (M)

are superior to the EF values for G and E, especially in the domain specific to the

phenolic acids absorption (278 nm) (EFE1, EFG1, EFM1) in comparison with the

values of EF specific to flavonoidic compounds (EFE2, EFG2, EFM2).

III.2.2.2. The determination by spectrometry of the total phenols content from methanolic extracts The richest sources of polyphenols are St. John’s Wort and Sea buckthorn leafs,

followed by Dandelion, Sea buckthorn fruits and Celandine.

III.2.2.3. FT-MIR spectrometric analysis

Calibration of the method with galic acid and mixture of galic acid with

Dandelion and respectively St. Jonh’s Wort

In order ro highlight in FT-MIR spectra the absorptions specific to phenolic

compounds, we made measurements on a set of 5 concentrations of galic acid.

Figure III. 4. represents the aspects of FT-MIR spectra on the fingerprint

domain 750-2000 cm-1.

 

 

XVI

2 0 0 0 1 8 0 0 1 6 0 0 1 4 0 0 1 2 0 0 1 0 0 0 8 0 00 ,0

0 ,1

0 ,2

0 ,3

0 ,4

0 ,5

0 ,6

0 ,7

0 ,8

Abso

rban

ce a

.u.

W a v e n u m b e r ( c m -1 )

0 ,0 6 2 5 m g /m l 0 ,1 2 5 m g /m l 0 ,2 5 m g /m l 0 ,5 0 m g /m l 1 m g /m l

Fig. III. 4. Calibration curve of galic acid by FT-MIR spectra registration (750-2000 cm-1)

We observe that there are 4 sets of significant signals, from which those from 1614 and 1743 cm-1 present a good correlation concentration – signal intensity (Fig.III.5.).

A.

00,05

0,10,15

0,20,25

0,30,35

0,40,45

0,0625 0,125 0,25 0,5 1

intensitate

conc. ac.galic (mg/ml)

1614

1743

 

 

XVII

B.

02468

1012141618

0,0625 0,125 0,25 0,5 1

aria

conc.ac.galic (mg/ml)

16141743

Fig. III. 5. Correlations between the concentration of galic acid and the intensity (A) or the area (B) of IR absorption signals from 1614 and 1740 cm-1

For the signal from 1743 cm-1, on the curve represented in Fig. III. 24 we calculated

the correlation coefficient R2, having the value of 0,9972.

y = 0,0805x + 0,0186R2 = 0,9972

0

0,02

0,04

0,06

0,08

0,1

0,12

0 0,2 0,4 0,6 0,8 1 1,2

concentratia ac.galic (mg/ml)

inte

nsita

tea

la 1

743

1/cm

Fig. III. 6. Calibration curve obtained for the galic acid (5 concentrations) according to the intensity of the signal from 1743 cm-1

 

 

XVIII

In order to emphasize these signals in the samples, we used St. John’s Wort and

Dandelion extracts at which we added 1 mg/ml galic acid (Fig. III. 7.). FT-MIR

spectra were then compared.

2000 1800 1600 1400 1200 1000 800

EM Sunatoare cu

ac.galic 1 m g/m l

W avenum ber (cm -1)

Ac.galic 0,5m g/m l

EM Sunatoare

EM Sunatoare cu

ac.galic 0,5m g/m l

2000 1800 1600 1400 1200 1000 800

EM Papadie cu

ac.galic 1mg/ml

EM Papadie cu

ac.galic 0,5mg/ml

Wavenumber (cm-1)

Ac.Galic 0,5 mg/ml

EM Papadie dil 1:10

Fig.III.7. Comparative spectra of methanolic extracts of Dandelion and St. John’s Wort, with controled addition of galic acid (1mg/ml)

We observe that there are differences at signals from 1743, at 1614, 1310 and at 1035

cm -1. Thus, in the methanolic extract with Sea buckthorn leafs we identified the

highest concentration of phenolic compounds, by both methods, and in Thistle and

Wolf’s Claw the lowest concentrations.

Therefore we demonstrate that FTIR method can be useful in the evaluation of

phenolic compounds concentration, provided that it is considered as an adjustement

factor by division that has the value 8.

FT-MIR spectrometric analysis of methanolic extracts (ME) from the

8investigated plants

Fig. III. 8 presents comparatively FT-MIR spectra of the 8 plant extracts and of a

mixture of phenolic compounds (used for HPLC-UV chromatographic analysis)

 

 

XIX

3 4 0 0 3 2 0 0 3 0 0 0 2 8 0 0 2 6 0 0 2 4 0 0 2 2 0 0 2 0 0 0 1 8 0 0 1 6 0 0 1 4 0 0 1 2 0 0 1 0 0 0 8 0 0- 1

0

1

2

3

4

5

6

E M F r u n z e c a t i n a

E M P e d i c u t aE M A r m u r a r i u

E M R o s t o p a s c a

E M A n g h i n a r e

Abs

orba

nce

a.u.

W a v e n u m b e r c m - 1

E M P a p a d i e

E M C a t i n a f r u c t e

E M A m e s t e c a c . f e n o l i c i

E M S u n a t o a r e

1236

7

8 5 4

Fig.III.8. General image of FTIR spectra registered in the methanolic extracts (FT-

MIR fingerprint) of the 8 investigated plants, in comparison with the mixture of

phenolic compounds used as standard at HPLC-UV analysis

The 8 areas specific to a FTIR spectra are emphasized. There are also emphasized

differences of the fingerprint in the fingerprint area (areas 2-6). Functional groups

identification was based on the assignement of stretching and deformation signals

from FTIR spectra. We identified 8 areas (marked 1 – 8) in MIR domain (4000-600

cm-1) and the “fingerprint” area was located between 900 and 1500 cm-1 (regions 1-4).

Fig. III. 9 presents a synthetic image of FTMIR specific areas 1, 2, 4, 6, 7 in which the

three extract types (E, G and M) present characteristic signals for plants 1-6.

Fig. III. 9. Synthetic representation of FTMIR specific areas 1, 2, 4, 6, 7 in which the

three extract types (E, G and M) present characteristic signals for plants 1-6

 

 

XX

When we compared the phenolic compounds concentrations, from FTIR

signals area, the signal from wave number 1743 cm-1 (I1743) and the concentration of

total phenolic compounds calculated by FC (Folin-Ciocâlteu) method were

significantly correlated (p

PCA analysis of the scores according to the type of phenolic compounds that can

differentiate the investigated plants

PCA analysis of the scores according to the type of the investigated plant and the

diffrentiation in phenolic compounds

 

 

XXII

CA analysis of the differentiation and grouping levels of the plants according to

their composition in phenolic compounds

Fig. III. 11. PCA and CA chemometric analysis made for the concentration values of

phenolic compounds separated by HPLC, of the methanolic extracts from the 8

investigates herbs

The chemometric analysis made for the concentration values of phenolic

compounds separated from the methanolic extract indicate that the ellagic acid,

catechins, protocatecuic acid and para-hydroxy benzoic can differentiate these herbs.

PCA analysis that differentiate the plants, emphasizes a net separation of Sea

buckthorn leafs (7) from the other plants, Dandelion (1), having a different

composition, followed by the association of sample 2, 3, 6 and 8 (Artichoke and

Wolf’s Claw, Sea buckthorn fruit) and Dandelion (1), Thistle (5) and St. John’s Wort

(4).

The analysis of the clusters (CA) points out a net differentiation of samples 7

(Sea buckthorn leafs) and 1 (Dandelion), a good association between samples 2

 

 

XXIII

 

 

XXIV

(Artichoke), 3 (Celandine), 6 (Wolf’s Claw) and 8 (Sea buckthorn fruit), in agreement

with those from PCA analysis. Samples 5 (Thistle) and 4 (St. John’s Wort) can not

group, but they are correlated by composition with samples 1 (Dandelion) and

respectively 2 (Artichoke).

III.2.3. Conclusions

As conclusions to the data presented in this chapter we mention the following:

1. UV-Vis and FT-MIR spectrometric methods are adequate to make the specific

and comparative fingerprint of the investigated plants. These methods have

value.

2. The values of the extraction factors EF in the three solvents (methanol, in

comparison with ethanol and glycerin) permited the adequate identification of

the types of bioactive compounds from these herbs and their polarity.

3. Significant and positive correlations were emphasized statistically between the

concentrations of phenolic compounds calculated by spectrometric methods

UV-Vis type and FTIR methods. Even if in absolute value these values are not

superimposable, these are statistically proportional to a reduction factor 8 that

must be applied for the methods based on FTIR spectrometry. The value of

MIR signal intensity from 1743 cm-1 can be considered a good indicator of

phenolic compounds concenatrations from these extracts.

4. FTIR data were correlated to the results of HPLC-UV chromatographic

analyses. Thus we estimated the phenolic compounds that offer antioxidant and

hepatotoxic character and how we can evaluate them by rapid methods (FTIR

and UG-Vis spectrometry), by validation in relation to accuracy methods as

HPLC. These data are extremely useful in order to evaluate the authenticity

biomarkers of these herbs.

 

 

XXV

CHAPTER IV

ANALYSIS OF COMPOSITION VARIABILITY OF INVESTIGATED

PLANTS BY HPLC-UV CHROMATOGRAPHY AND FT-MIR

SPECTROMETRY, COUPLED WITH CHEMOMETRIC ANALYSIS

On the basis of the data obtained on individual plants, using different solvents

and investigation methods, we selected the extraction method in methanol as having

the highest efficiency of extraction of the bioactive compounds from polyphenol

group and of some flavone glycosides, or other groups responsible for the

hepatoprotective action.

OBJECTIVES AND MEASUREMENTS

In order to characterize and verify the data validity and the composition

variability of the plants from the 8 investigated classes we intented to:

I. Establish the HPLC chromatographic fingerprint and characterize the

variability of extracts composition by chemometric analysis target and

nontarget.

II. Establish FT-MIR spectrometric fingerprint and the variability of

extracts composition by chemometric analysis target and nontarget.

HPLC-UV CHROMATOGRAFIC ANALYSIS

IV.1.1. Materials and methods

For the analysis we used parallel samples from the 8 types of investigated plants (4

samples from each plant type).

As methods we used : HPLC chromatographic analysis, FT-MIR spectrometric

analysis, chemometric analysis.

IV.1.2. Results and discussions

IV.1.2.1. HPLC-UV chromatographic separation and identification of the specific

fingerprint of methanolic extracts from 4 sample variants from each plant type

HPLC fingerprint specific to methanolic extracts of the 4 variants/plant points out a

different relation between the polar compounds, with small molecules (phenolic acids

 

 

XXVI

and derivatives that have retention time up to 25 min) (area 1) and complex

compounds (flavonoid glycosides or other big molecules) that have bigger retention

time up to 40 min (area 2).

In order to interpret adequately the variability and the similarities, respectively the

differences that authentify these plants, we applied the chemometry on the basis of the

data obtained by HPLC chromatography.

IV.1.2.2. Chemometric analysis in NT (nontarget) and T (target) system

On the basis of the data collected from HPLC analysis we applied NT and T system in

order to evaluate the variability and to authentify and differentiate the obtained

extracts.

Nontarget type analysis (NT)

In order to characterize the composition variability in polyphenols of the 8

investigated plants, in NT-nontarget system we used the data collected from HPLC

chromatograms in which the individual compounds were not identified, but we

considered all the signals during separation period (0-55 min). They were expressed in

areas of the signals that were introduced in the specific soft of PCA analysis.

PCA (Principal Component Analysis) method application

In NT ”influence” system that emphasizes the variability we considered the first 7

separate components from the mixture, and we obtained the graphic from Fig. IV. 1.

 

 

XXVII

Fig. IV.1. Representation of composition variability of the samples, according to the

main 7 separate components from HPLC chromatogram (on 0-55 min.

interval) in NT system

We observe that in the case of the samples of Sea buckthorn leafs the most significant

composition differences appear, and also in the case of Thistle. The rest of extracts,

especially those of Celandine, Sea buckthorn fruits, Wolf’s Claw, St. John’s Wort

were grouped compactly.

Fig. IV. 2 represents the scores for this PCA analysis, NT system.

Fig. IV. 2. Representation of composition variability by representing the sample

scores, by PCA analysis according to the main 2 components (on HPLC separation

interval 0-55 min.) in NT system

Leverage (PC-7)0 0.1  0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1

R es idual  X- v ar ian c e 

( PC - 7) 

Influence

0

200 

400 

600 

800 

1000

1200

1400

1600Armurariu

Anghinare

Armurariu Armurariu

Anghinare Anghinare Armurariu Catina fru

Catina fruCatina fru

PedicutaRostopa

Sunatoare

Sunatoare

Anghinare Catina fruedicuta 1 Catina fru

Catina fru

Catina fru

Catina fruPapadie 1

Papadie 2 Sunatoare

Papadie 3 Papadie 4PPedicuta 2

Rostopasca

3 sca

Pedicuta 4RostopascaRostopasca

Sunatoare

CA (Cluster Analysis) method application

The analysis of cluster type (CA) correlations based on HPLC-NT analysis.

Fig. IV. 3 includes the graphic representation of cluster type groups on several

correlation levels.

Fig. IV. 3. Cluster type correlations (CA) – average clustering using Bray-Curtis

distances and 8 clustering domains. These data are based on HPLC-NT

analysis, consifering the whole interval (0-55 min)

From the analysis of this graphic we emphasize the following:

• Except Artichoke and Dandelion, where the four samples are not homogeneous as

composition (they appear in 2 clusters differently situated), the other plants have

homogeneous composition (eg. St John’s Wort, Thistle, Celandine, Sea buckthorn

leafs).

• Similarities between the fingerprints, based on the opposition of the four plant types

from “Sea buckthorn leaf” group, the composition of Sea buckthorn leafs is the less

correlated with the composition of herbs.

PCA target (analysis T)

 

 

XXVIII

 

 

XXIX

by HPLC chromatography and according to the values of signals areas we

compared the extract samples. Fig. IV.4 represents the results obtained by PCA-T

analysis.

In order to establish if some phenolic compounds are responsible for variability and in

order to differentiate the plants, we considered a priori six phenolic derivatives

separated

Fig. IV. 4. Re e

separation interval HPLC , considering 6 major

phenolic acids: galic acid, protocatecuic, chlorogenic, para-coumaric, trans-

ociated two by two, reproducible between them. The rest of

xtracts, especially those of Celandine, Sea buckthorn fruits, Wolf’s Claw, were

rouped compactly.

presentation of composition variability by representing the scores for th

samples, by PCA analysis according to the main 2 components (on

0-55min.) in T system

cinnamic and prune

In the case of St. John’s Wort, we identified only one phenolic acid, para-coumaric

acid, therefore in PCA analysis we could not consider two components. In this case

also, the extracts of Sea buckthorn appear significantly different from the other

extracts, they are ass

e

g

 

 

XXX

A analysis in T system. By applying CA method we obtained

IV.2.1. FT-MIR spectrometric analysis and the identification of plants specific

d FTIR spectra by the combinations presented in Fig. IV. 5.

Dandelion-Artichoke extracts

IV.1.3. Conclusions

From the methanolic extracts of the studied plants, the extract from Sea buckthorn

leafs differentiates significantly from the point of view of the composition, from the

other plants, fact resulted by applying PCA method in NT system, in order to

evidentiate the variability of the main seven components separated in HPLC

chromatograms, on 0-55 min interval. The same thing reveals from the representation

of the scores for PC

similar results.

IV. 2. FT-MIR SPECTROMETRIC ANALYSIS

fingerprint

Results and discussions

In order to identify the qualitative and quantitative differences between the plants, we

overlappe

1. FTIR spectra overlapped for Wolf’s

Claw-Celandine extracts

2. FTIR spectra overlapped for

3500 3000 2500 2000 1500 10000,0

0,2

0,4

0,6

0,8

1,0

1,2

1,4

Abs

orba

nce

a.u.

Wavenumber (cm)-1

EM Rostopasca EM Pedicuta

1

2

3

4

5

67

8

3500 3000 2500 2000 1500 10000,0

0,2

0,4

0,6

0,8

1,0

1,2

1,4

Abs

orba

nce

a.u.

Wavenumber (cm-1)

EM papadie EM Anginare

1

3

4

5

678

2

 

 

XXXI

2. FTIR spectra overlapped for Thistle-St.

John’s Wort extracts

2. FTIR spectra overlapped for Sea

buckthorn leaf-fruit extracts

3500 3000 2500 2000 1500 10000,0

0,2

0,4

0,6

0,8

1,0

1,2

1,4

1,6

Abso

rban

ce a

.u.

Wavenumber (cm-1)

EM Sunatoare EM Armurariu

1

2

3500 3000 2500 2000 1500 10000,0

0,2

0,4

0,6

0,8

1,0

1,2

1,4

1,6

1,8

Abs

orba

nce

a.u.

Wavenumber (cm)-1

EM catina fruct EM catina frunze

1

2

3

4

6

5

78

3

5

467

8

Fig. IV. 5. The aspect of FTIR spectra overlapped for the extracts of the used plants,

taking two apparently similar plants

mains,

calculating the areas and the standard deviations corresponding for each plant variant.

The graphic representation of this variability in visible in Fig. IV. 6 and IV. 7.

Variability analysis by statistic analysis based on signal areas from FTIR spectra

In oder to characterize the composition variability of the investigated plants on

the basis of FTIR spectra we considered 700-1800 cm-1 and 1800-4000 cm-1 do

Fig. I

leafs (7). Ped-Wolf’s Claw (6). Arm-Thistle (5). S-St. John’s Wort (4) Ro-

Celandine (3) Ang.-Artichoke (2) Cfr-Sea buckthorn fruits(8) P-Dandelion(1)

V.6. The comparative anlysis of composition variability on the basis of area

calculation from FTIR spectra domain 700-1800 cm-1, with medium values

and standard deviation (SD) representation. Abbreviations: CF-Sea buckthorn

Fig. IV. 7. The comparative anlysis of composition variability on the basis of area

calculation from FTIR spectra domain 1800-4000 cm-1, with medium values

and standard deviation (SD) representation. Abbreviations: CF-Sea buckthorn

leafs (7). Ped-Wolf’s Claw (6). Arm-Thistle (5). S-St. John’s Wort (4) Ro-

Celandine (3) Ang.-Artichoke (2) Cfr-Sea buckthorn fruits(8) P-Dandelion(1)

IV.2.2. Chemometric analysis in NT (nontarget) and T (target)system based on FTIR spectrometric analysis

Fig. IV. 8. PCA analysis on 700-900 cm-1 domain, the spectra being normalized for

PCA analysis

 

 

XXXII

Fig. IV. 9. PCA analysis for 700-3200 cm-1 area. A. Non normalized spectra. B.

Normalized spectra at 2200 cm-1

IV.2.3. Conclusions

1. In the case of statistic analysis of composition variability on the basis of area

calculation from FTIR spectra for 700-1800 cm-1 domain, from medium values

representations and standard deviations, we observe significant differentiations

within the same plant, taken from different batches.

2. FTIR analysis for 1800-4000 cm-1 domain has different results from 700-1800

domain, the differences between the variants of the same plant being less

significant. Therefore, dependent on the batch, in the case of the same plant, we

have different signals in FTIR areas corresponding to stretching vibrations of

different chemical bounds of C-H, C-O, C-C, N-H and C=O type.

3. From the PCA analysis on 700-900 cm-1 domain we observe significant

differences between the signals of Sea buckthorn fruits and the other plants, the

same for Thistle.

4. In the case of PCA analysis of scores type for 700-3200 cm-1 area, there are

significant differentiations from the point of view of FTIR signals. Sea buckthorn

fruits and Thistle do not present significant statistic differentiations, being grouped

compactly.

 

 

XXXIII

 

 

XXXIV

5. From PCA analysis on 700-3200 cm-1 and respectively 700-900 cm-1 domains we

observe the grouping of the samples from the same plant, and we also observe

differences between groups afferent to different plants (8 distinct groups). There

are no significant differences by normalize spectra analysis in comparison with the

non normalized ones.

FTIR analysis was very useful in order to group the plants analysed according to the

characteristic functional groups, being a rapid and economic method.

CHAPTER V

OBTAINMENT AND CHARACTERIZATION OF SOME BIOPRODUCTS

WITH HEPATOPROTECTIVE EFECT

AIM AND OBJECTIVES OF THE RESEARCHES

The aim of this chapter is to obtain and characterize two bioproducts with

hepatoprotective potential obtained from the studied plants. The objectives of the

researches from this stage included:

The obtainment of two bioproducts A1 and A2, powder that could be administrated as

capsules of 0,3-0,5 g.

1. UV-Vis spectrometric characterization of A1 and A2 products, determination of

total phenols content.

2. Characterization of A1 and A2 bioproducts by HPLC-UV chromatographic

analysis.

3. Characterization of A1 and A2 bioproducts by FT-MIR spectrometric analysis.

V.1. Materials and methods

We realized the mentioned mixtures in equal proportions, thus:

Bioproduct A1 is formed from the following: Artichoke, Thistle and Dandelion,

mixed in mass proportion 1:1:1.

Bioproduct A2 contains: Sea buckthorn-leafs, Wolf’s Claw, Celandine and St. John’s

Wort, mixed in mass proportion 1:1:1.

We prepared three types of extracts from bioproducts A1 and A2.

A quantity of 15 g A1 and A2 plant mixture was dissolved in 100 ml volume from

each of the three solvent types:

a. 5% solution ascorbic acid in distilled water heated at 45°C

b. distilled water heated at 45°C

c. methanol with 1% HCl concentrat

The extractions were made in a 48 hours interval, at room temperature, then they were

filtered and kept at 4 ºC until anlaysis.

The methods used at extracts analysis were:

UV-Vis spectrometric analysis with identification of the main bioactive compounds

(chapter II).

Separation, identification and quantitative determination of the phenolic compounds

by high performance liquid chromatography HPLC-PDA (chapter III)

The establishment of spectrometric fingerprint in Infrared by FTIR technique (chapter

IV).

V.2. Results and discussions

Fig.V.2.a and V.2.b presents the image of the bioproducts before and after

encapsulation.

a.

b.

Fig. V. 2. Image of the plant powders used to obtain A1 and A2 bioproducts before (a)

and after (b) encapsulation  

 

XXXV

 

 

XXXVI

V.2.1. UV-Vis spectrometric analysis

From UV-Vis analysis resulted the UV-Vis spectra registered on the extracts of 1°-1c

and 2°-2c type obtained from A1 and A2 bioproducts.

Concentration values of total polyphenols.

By Folin-Ciocâlteu spectrometric analysis we obtained the following values of

polyphenolic compounds:

Bioproduct A1=1850 mg polyphenols / 100 g s.u.

Bioproduct A2=1260 mg polyphenols / 100 g s.u.

Thus, we note a higher concentration of polyphenols in bioproduct A1 that contains

Artichoke, Dandelion and Thistle, data confirmed from individual determinations of

polyphenols, made for each plant.

The values obtained are in accordance with those reported by other authors (Socaciu,

C. And col., 2002, Socaciu, C., 2008).

On the basis of UV spectra, using DO270nm şi DO320nm absorptions and the dilutions

made at the absorption reading, we calculated the extraction factor (EF), for each

bioproduct (A1 and A2), in each used solvent (a-c), presented in table V.1.

Table V.1.

The values of absorptions and dilutions of extraction factors (EF)for each bioproduct

(A1 and A2), in each used solvent (a-c)

Biopreparat Solvent DO270 nm DO320nm Diluţia Factorul de

extracţie(FE)

A1

B 0,768 0,423 100 119,1

C 0,936 0,457 200 278,6

A2 B 0,330 0,157 100 48,7

C 0,556 0,398 200 190,8

We observe that the extraction factors are about 2.5-4 times biggre in methanol (c)

than in water (b).

 

 

XXXVII

Comparatively, bioproduct A1 has EF bigger than A2 in accordance with the higher

values of total polyphenols in this bioproduct.

In conclusion we can affirm that:

1. Bioproduct A1, that contains Artichoke, Thistle and Dandelion, in equivalent

mass ratio, has a bigger polyphenols concentration, 1850 mg / 100g , in

comparison with bioproduct A2, that contains Sea buckthorn leafs, Wolf’s

Claw, Celandine and St. John’s Wort and whose polyphenols concentration is

1260 mg / 100 g.

2. The solvent of type a, containing acidulated water with vitamin C did not

extract efficiently the phenolic products. The most efficient solvent was

acidulated methanol (according to II.2.3.) followed by distilled water (b).

V.2.2. HPLC-UV chromatographic analysis

Conclusions

The data obtained by HPLC-UV analysis point out dependence of the compounds

from the extracts on the solvent. Thus, the solvent of a type (acidulated water with

abscorbic acid) extracts preferential the polar phenolic compounds, especially galic,

protocatecuic and chlorogenic acid, at both bioproducts (A1 and A2).

Distilled water solvent (b) extracts the same compounds type as A1 bioproduct, but

more phenolic compounds in the case of A2 bioproduct, namely it extracts the vanilic,

para-benzoic, para-coumaric and syringic acid.

Acidulated methanol solvent (c) extracts qualitatively and quantitatively the biggest

variety and quantity of major phenolic compounds, 6 compounds being identified

from A1 bioproduct, at which 3 unidentified compounds are added, possibly, silybin,

silybinin, taxifolin (they were not available pure) and 11 compounds identified in A2

bioproduct, together with 2 unidentified major compounds (possibly hypericin and

pdeudohypericin).

The quantitative results for the compounds identified in the two bioproducts show the

following:

• Bioproduct A1 is the richest in phenolic acids, especially galic, protocatecuic

and chlorogenic acid.

 

 

XXXVIII

• Chlorogenic acid has values of 95,2-112,4 mg /100 g product A1, while its

values are more reduced , from 37,7 at 49,9 mg /100 g product A2. generally the

extract in water with ascorbic acid is richer in this acids, the lowest values being in the

variant with aqueous extract (b).

• A1 bioproduct has as major compound the elagic acid, in concentration of 330,

95 mg /100 g product, and only in methanolic extract (c). It can be considered as

recognition marker for this bioproduct, together with transcinamic acid. Also, the

presence of silymarin and silybin (from Thistle – ingredient in A1 bioproduct) is a

recognition marker. Caffeic and para-coumaric acid are also recognition markers.

• Bioproduct A2 has, together with galic and protocatecuic acid, in equivalent

quantities in aqueous extracts (2a and 2b), quercetin as major compound in methanolic

extract (74,85 mg /100 g product) together with kempherol. We observe that in this

product at the retention time specific to elagic acid a signal appears (evaluated as

having a concentration of 315,5 mg /100 g), that was marked NI accompanied by

another signal that could be attributed to the components from St. John’s Wort –

hypericin and pseudohypericin).

• We observe that the methanolic extract of bioproduct A2 (2c) is richer in

flavonoids (quercitin and kaemferol) even if overall ir poorer in polyphenolic

compounds.

V.2.3. FT-MIR spectrometric analysis

In the case of the extracts from A1 and A2 bioproducts we made correlations between

the type and the domain of IR absorption frequencies and their assignement to the

specific functional groups. In the case of bioproducts analysed 8 frequency areas

delineate, marked from 1 to 8. Usually, areas 2-6 are considered as belonging to

fingerprint domain. The domains used for area calculation are usually from the

fingerprint domain (900-1500 cm-1). The two bioproducts A1 and A2 were

characterized by FT-MIR (ATR) spectrometric fingerprint. We observed that these

fingerprints are dependent on the type and acidity of the solvent. A1 bioproduct has as

specific recognition area, area (6), whose frequency is between 1600 – 1700 cm-1,

while A2 has as specific area, area (2) with frequencies between 1000 – 1100 cm-1 .

 

 

XXXIX

Area (6) is specific to esters and peptidic compounds domination, while area (2)

points out the predominance of glucids and glycosides.

We can observe that bioproduct A1 can be authentified by a higher concentration of

peptidic compounds, polar lipids and esteric derivatives of phenolic acids (phenolic

acid esters and phytosterol) that explain the choleretic-cholagogue effect, while

bioproduct A2 is rich in phenolic compounds (flavonoids) in glycosylated form. These

forms are more polar, more bioavailable and explain the effect of antioxidant

protection of the hepatic cell and metabolic stimulation.

V.2.4. Conclusions

In relation with the objectives we can conclude the following:

1. We obtained two bioproducts A1 and A2, under the form of encapsulated

powder, each having specific compositions. The obtained powders were introduced in

capsules, weighting 0, 20 g (A1) and 0, 27 g (A2). Their composition is different and

has specific action. Thus:

• A1: Artichoke, Thistle and Dandelion (1:1:1). This bioproduct has choleretic

and cholagogue effect.

• A2: Sea buckthorn-leafs, Wolf’s Claw, Celandine and St. John’s Wort

(1:1:1:1). This bioproduct has aan effect of antioxidant protection of the hepatic cell.

2. The composition of these bioproducts is represented by a high content of

polyphenolic compounds that explain the hepatoprotective action. Thus:

Bioproduct A1, that contains Artichoke, Thistle and Dandelion, has higher

concentrations of polyphenols, especially derivatives of phenolic acids, in medium

concentration of 1850 mg/100g.

Bioproduct A2, that contains Sea buckthorn leafs, Wolf’s Claw, Celandine and

St John’s Wort has a concentration in polyphenols of 1260 mg/100g, that belong to

flavonoids and phenolic acids category.

3. For the UV-Vis spectrometric analysis we used 3 different solvents (acidulated

water with ascorbic acid – a, distilled water – b, and acidulated methanol – c) and we

calculated the extraction factor specific to each solvent type.

 

 

XL

Type a solvent, containing acidulated water with vitamin C din not extract

efficiently the phenolic products.

The most efficient solvent was acidulated methanol (according to II. 2. 3.)

followed by Distilled water (b).

4. The data obtained by HPLC-UV analysis points out a composition of the

extracts (of a, b or c type), dependent on the used solvent. Thus:

Type a solvent (acidulated water with ascorbic acid) extracts the polar phenolic

compounds, mainly galic, protocatecuic and chlorogenic acid, at both bioproducts A1

and A2.

Distilled water solvent (b) extracts the same type of compounds at A1

bioproduct, but more phenolic compounds in the case of A2 bioproduct, namely itb

extracts also vanilic, para-benzoic, para-coumaric and syringic acid.

Acidulated methanol solvent (c) extracts qualitatively and quantitatively the

highest variety and quantity of major phenolic compounds, 6 compounds being

identified from A1 bioproduct (at which 3 unidentified compounds add, possibly

sybilin, sylibilin, taxifolin) and 11 identified compounds in A2 bioproduct, together

with 2 unidentified major compounds (possibly hypericin and pseudohypericin).

5. The quantitative results from HPLC-UV analysis for the identified compounds

in the two bioproducts show the following:

• A2 bioproduct is richer in phenolic acids, especially galic, protocatecuic and

chlorogenic acids.

• Chlorogenic acid has values of 95,2-112,4 mg /100 g product A1, while its

values are more reduced, from 37,7 at 49,9 mg /100 g product A2. Generally, the

extract in water with ascorbic acid is richer in these acids, the lower values being

present in aqueous extract variant (b).

• Bioproduct A1 has as major compound elagic acid, in concentration of 330, 95

mg /100 g product, and only in methanolic extract (c). We can consider it as

recognition marker for this bioproduct, together with transcynamic acid. Also, the

presence of sylimarin and sylibin (from Artichoke – ingredient in A1 bioproduct) is a

recognition marker.

 

 

XLI

• Bioproduct A2 has, together with galic and protocatecuic acids, in equivalent

quantities in aqueous extracts (2a and 2b), quercitin as major compound in methanolic

extract (74, 85 mg /100 g product) together with kempherol. We observe that in this

byproduct also at the retention time specific to elagic acid a signal appears (evaluated

as having a concentration of 315,5 mg /100 g), that was marked NI accompanied by

another signal that could be assigned to the components from St. John’s Wort

(hypericin and pseudohypericin). The methanolic extract of bioproduct A2 (2c) is

richer in flavonoids (quercitin and kaemferol) even if overall is poorer in polyphenolic

compounds.

6. We characterized by FTIR (ATR) spectrometric fingerprint the two

bioproducts A1 and A2 by their extracts of a, b and c type. We observed that their

fingerprints are dependent on the type and acidity of the solvent. Thus:

• Bioproduct A1 has a specific recognition area, area (6), whose frequency in

beween 1600 – 1700 cm-1.

• Bioproduct A2 has as specific area, area (2) with frequencies 1000 – 1100 cm-1.

• Area (6) is specific to esters and peptidic compounds dominance, while area (2)

points out the prevalence of sugars and glycosides.

In conclusion we can affirm that bioproduct A1 can be authentified by a higher

concentration of peptidic compounds, polar lipids and esteric derivatives of phenolic

acid (phenolic acid esters and phytosterol) that explain the choleretic-cholagogue

effect, while A2 bioproduct is rich in phenolic compounds (flavonoids) in

glycosylated form. These forms are more polar, more available and explain the

antioxidant protection effect of the hepatic cell and of metabolic stimulation.

GENERAL CONCLUSIONS

According to the aim of the thesis, the experimental activity included in this

thesis was the study of bioactive compounds of some aboriginal herbs known as

having hepatoprotective potential, and the characterization of their composition in

oder to obtain some bioproducts with hepatoprotective effect.

 

 

XLII

The investigated plants were Dandelion, Artichoke, Celandine, Thistle, St.

John’s Wort, Wolf’s C;aw and Aea buckthorn (leafs and fruits), 8 types overall, from

which we investigated different samples recovered from different locations.

According to the punctual objectives of the personal researches we can mention

the following conclusions:

1. We characterized comparatively extracts of hydroalcoholic, hydrogliceric and

methanolic type from the 8 samples and 7 investigated plants (Dandelion,

Artichoke, Celandine, St John’s Wort, Thistle, Wolf’s Claw, Sea buckthorn-

leafs and Sea buckthorn-fruits). The efficiency of the extraction was maximum

in methanol, recommended for the study of chemical composition but not for

therapy, due to its toxic effect. For therapy the fresh aqueous extract is

preferred or the use of acsula with micronized solid product.

2. We established the specific profile of each extract using complementary

analysis methods, namely spectrometric methods UV-Vis type, Medium

Infrared spectrometry (FT-MIR) and HPLC chromatrographic methods with

UV detection (HPLC-UV). They were interpreted statistically using ANOVA

test and chemometric analysis of PCA and CA type.

3. We made the analysis of composition variability of different variants from each

plant type and we identified the composition reproducibility by HPLC-UV

chromatography and FT-MIR spectrometry, coupled with chemometry, by

PCA and CA analysis. Chemometric analysis confirms a significant

reproducibility of the investigated samples, with minimum oscillations of the

composition.

4. We obtained and characterized two types of bioproducts with hepatoprotective

potential, that have as ingredients the above studied plants (1-8).

A1 bioproduct included as ingredients Artichoke, Thistle and Dandelion,

mixed in relation of 1:1:1. A2 bioproduct contains Sea buckthorn-leafs, Wolf’s Claw,

Celandine and St. John’s Wort, mixed in relation 1:1:1:1.

 

 

XLIII

A1 bioproduct has a choleretic-cholagogue effect, due to the big concentration

of peptidic compounds, polar lipids and esteric derivatives of phenolic acids (phenolic

acid esters, especially galic, protocatecuic and chlorogenic acid).

Bioproduct A2, by its high concentration in phenolic compounds (flavonoids)

in glycosylate form, has a stimulation effect of the hepatic cell.

Both A1 Bioproduct and A2 Bioproduct, by their complementary

composition offer an antioxidant pretection of the hepatic cell and has stimulation

action of the metabolic processes in hepato-biliary segment.

The researches are a useful starting point for the in vitro or in vivo investigation of the action of these bioproducts.

The originality of the studies is offered by:

The detailed analysis by performant analytical methods of the plants composition, in three solvent types

The study of composition variability of the samples recovered from different locations

The achievement of some original bioproducts with directed functionality and synergic action

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