Contribuţii la studiul farmacognostic şi biologic al ... - Gr.T. Popa

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Contribuţii la studiul farmacognostic şi biologic al ... - Gr.T. Popa

UNIVERSITATY OF MEDICINE AND PHARMACY

„GR. T. POPA” IAŞI

FACULTY OF PHARMACY

Contribuţii la studiul farmacognostic şi

biologic al fructelor de fenicul şi a

uleiurilor volatile extrase din acestea

REZUMATUL TEZEI DE DOCTORAT

SCIENTIFIC COORDINATOR:

Prof. Univ. Dr. URSULA STĂNESCU

PhD STUDENT:

Farm. Drd. Mihai Alexandru Băjan

IAŞI - 2012

1


The THESIS presents the following structure:

General Part

Chapter I.......................................................................................5

Current knowledge about Foeniculum vulgare Mill. species

1.1. Systematic screening................................................................6

1.2.Botanical description of Foeniculum vulgare Mill

species.............................................................................................7

1.3. Fennel culture........................................................................10

1.4. Comercial varieties.................................................................12

1.5.Chemical Compozition............................................................14

1.6. Stability...................................................................................21

1.7 Pharmacological action...........................................................21

1.8. Uses.........................................................................................27

1.9. Bioavailability.........................................................................32

Personal Part

Thesis subject choice motivation, purpose and

objectives.......................................................................................33

Chapter II.....................................................................................37

Material, methods and work technics

2.1.Presentation of the studied plant material and its

origin..............................................................................................37

2.2 Macro- and microscopic pharmacognostic analysis of fennel

samples...........................................................................................41

2.2.1 Macroscopic pharmacognostic analysis

2.2.2 Microscopic pharmacognostic analysis

2.3 Extraction methods..................................................................42

2.3.1. Methanol/ethanol extraction

2.3.2. Obtaining tinctures

2.3.3. Extraction procedures from the delipidated material

2.3.4. Obtaining plant extracts for antioxidant tests

2.3.5. Obtaining infusions

2


2.3.6. Separation of volatile oils by steam distillation

2.4. Qualitative chemical analysis.....................................45

2.4.1. Thin layer chromatography for flavonoidic

derivatives

2.4.2. Chromatographic analysis of polyphenolic acids

2.4.3. Thin layer chromatographic analysis of volatile

compounds

2.4.4. Chromatography for triterpene compounds

2.5. Quantitative chemical analysis...................................47

2.5.1. Spectrophotometric determination of flavonoids

2.5.2. Spectrophotometric determination of

polyphenolcarboxylic acids

2.5.3. Spectrophotometric determination of total

polyphenols

2.5.4. High performance liquid chromatography (HPLC)

analysis for flavonoids and polyphenolcarboxylic acids

2.5.5. Quantitative determination of volatile oils

2.5.6. Analysis of volatile fractions by gas chromatography

coupled with mass spectrometry (GC / MS)

2.6. Biological activity determination..........................................60

2.6.1 Determination of antioxidant activity

2.6.2.Evaluation of antimicrobial activity of the essential

oils of separated fennel fruits

2.6.2.1. Determination of minimum inhibitory

concentration (MIC) and minimum bactericidal

concentration (MBC)

2.6.2.2. Testing synergism between the

antimicrobial activity of fennel oils and antibiotics

Chapter III.................................................................................65

Macro- and microscopic pharmacognostic analysis

3.1 Introduction .........................................................................65

3


3.2. Material and work method..................................................65

3.3. Rezults and discussions.......................................................65

3.3.1. Macroscopic pharmacognostic analysis

3.3.2. Microscopic pharmacognostic analysis

3.4.Conclusions..........................................................................70

Chapter IV................................................................................71

Phytochemical analysis of Foeniculi fructus samples for

pharmaceutical use

4.1 Introduction............................................................................71

4.2.Preliminary phytochemical analysis........................................72

4.2.1. Introduction

4.2.2. Material and method

4.2.3.Results and discussions

4.2.3.1. Volatile fraction study

4.2.3.2. Polyphenolic fraction study

4.2.4. Conclusions

4.3. Contributions to the phytochemical study of Foeniculi fructus

samples for pharmaceutical use...................................80

4.3.1. Introduction

4.3.2. Material and method

4.3.2.1. Polyphenolic fraction study from 4 fennel

samples of known provenance

4.3.2.2. Study of volatile and polyphenolic fraction

of fennel fruits sold wholesale

4.3.3. Results and discussions

4.3.3.1. Analysis of polyphenolic derivatives of 4

samples of known origin fennel

4.3.3.2. Phytochemical study of 7 samples of fennel

fruits for pharmaceutical use

4.3.4.Conclusions

4


4.4. Comparative chemical profile of the volatile fraction isolated

from fennel fruits sold on the Romanian pharmaceutical

market..........................................................................................93

4.4.1.Introduction

4.4.2 Material and method

4.4.3. Results şi discussions

4.4.4.Conclusions

4.5. Chemical composition of volatile oil separated from the fennel

fruit samples taken from plants grown in the Neamt

county......................................................................................... 98

4.5.1. Introduction

4.5.2. Material and method

4.5.2.1. Plant material taken from CCB Piatra

Neamt

4.5.2.2. Plant material from SCDA Secuieni

4.5.3. Results and discussions

4.5.3.1. Chemical analysis of separated oils from

the fennel fruits collected from CCB Piatra Neamt

4.5.3.2. Morphological and chemical study of fennel

fruits grown Secuieni

4.5.4.Conclusions

4.6. Comparative chemical study of the essential oil extracted from

two samples of fennel fruits grown in the Neamt

county.........................................................................................109

4.6.1. Introduction

4.6.2. Material and method

4.6.3. Results and discussions

4.6.4. Conclusions

4.7. Research on the chemical composition of fennel fruits sold

wholesale / retail by pharmaceutical network.............................118

4.7.1. Introduction

4.7.2. Material and method

4.7.3. Results and discussions

5


4.7.3.1. Study of separated volatile oil from samples

of fennel tea

4.7.3.2. Study of the samples of separated essential

oil from the fruit fennel purchased from wholesalers

4.7.4.Conclusions

4.8. Study the polyphenolic fractions of the fennel fruits.......137

4.8.1 Introduction

4.8.2. Material and method

4.8.3. Results and discussions

4.8.4. Conclusions

Chapter V................................................................................149

Contributions to the study of biological activity of extracts and

fractions isolated from Foeniculi fructus

5.1. In vitro evaluation of free radical scavenger capacity of

extracts obtained from fennel fruits.........................................149

5.1.1. Introduction

5.1.2. Material and method

5.1.3. Results and discussions

5.1.3.1. Evaluation of antioxidant capacity of

extracts of fennel tea, using the DPPH method

5.1.3.2. Investigation of antioxidant capacity of

extracts and fragments obtained from fruits and

dried fennel bulk

5.2. Determination of antimicrobial and antifungal action of two

essential oils of fennel...............................................................166

5.2.1 Introduction

5.2.2. Material and method

5.2.3 Results and discussions

5.3. Conclusions........................................................................170

Chapter VI..............................................................................171

6


Safety and benefit / risk evaluation of fennel infusion

administration

6.1. Introduction.........................................................................171

6.2.Material and method.............................................................171

6.3. Results and discussions........................................................172

6.4. Conclusions..........................................................................177

Chapter VII..............................................................................178

General Conclusions..................................................178

Original Contributions...............................................181

Future study perspectives...........................................181

Relevant bibliography.........................................................182

The PhD thesis is structured in two parts:

the general part, the chapter in which we present the actual

knowledge in regards to the chemical composition of fennel

fruits, the therapeutic relevance of fennel fruits and some of

the bioactive compounds contained by them, as well as the

peculiar aspects of safe use of phytopharmaceuticals derived

from these.

the personal part includes 6 chapters; Chapter II, Material,

methods and work tehnics presents the fennel fruits samples

studied over the years, as well as pharmacognostic,

phytochemical, biological analysis methods and technics.

Chapter III covers the farmacognostic analysis of macro-

and microscopic plan samples, which was led and held in

accordance with details in both the FRX, and Ph.Eur 6th.

Chapter IV deals with the phytochemical analysis of

Foeniculi fructus samples for pharmaceutical use; phytochemical

7


determinations were performed on 45 fennel fruits samples, for 35

of them, the determinations included separation and chemical

investigation of the volatile oil and for 19 samples we analyzed the

poliphenolic fraction.

We assessed the pharmaceutical and aromatherapy quality

of the analyzed volatile oils according to the current requirements

(GC-MS analysis, the poliphenolic fractions were investigated by

TLC, HPLC and spectrophotometric determination).

In chapter V, Contributions to the study of biological

activity, we tested 13 samples for antioxidant activity of plant

complexes separated from plant material by extraction with 70%

ethanol and by infusion; we also monitored the antimicrobial and

antifungal activity for two volatile oils separated by hydro

distilation.

Chapter VI evaluates the impact (theoretical) of the

current administration of the Romanian funnel infusion to the

infants from the health safety and benefits/risks standpoints.

The PhD thesis ends with a chapter of Final Conclusions

followed by bibliography and the list of published articles.

INTRODUCTION

Foeniculum vulgare Mill. provides the Foeniculi fructus

product, recommended as an infusion in pediatry as antispasmodic,

carminative or expectorant, but also as a digestive, carminative and

expectorant for adults (especially elderly and convalescents) and

lactating women having a gentle galactogogue effect.

The product, designed for obtaining infusion, falls under

food supplements category, like all medicinal teas.

There is an EFSA (European Food Safety Authority)

Scientific Committee directive from June 2004 that states the

following:

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the botanic ingredients need to be identified by the

binomial scientific name, in the majority of cases at the

subspecies level or lower;

an adequate characterization is necessary, a description

of the botanical parts and of the work methodology that

was used. The safety of a botanical ingredient can olny be

evaluated based on the long term use or other phyto

medicins in which it is added;

an adequate quality control is needed due to possible

counterfeits, confusions and need for replacement;

We aimed in our study to assess the quality of the fennel

fruits, consistently keeping in mind these specifications.

The Foeniculum genra is represented by a single species, F.

vulgare Mill., where we distinguish two kinds: bitter and sweet.

Romania is typically acknowledged as a bitter fennel harvester and

exportator in Europe, but now pharmacies and specialized

companies sell F. vulgare Mill., under the sweet anise name.

In this situation, we questioned the pharmaceutical quality

of fennel fruits marketed as sweet anise for infusions in Romania

and whether they are consistent with the requirements set out in

directive EFSA, especially Ph.Eur. 6th which considers Foeniculi

fructus only the fruits from F. vulgare ssp. vulgare var. sweet.

If macro- şi microscopic, there are no major differences

between the two genra, from the chemical standpoint the

differences are significant: the volatile oil of sweet anise contains

mainly (more than 80%) trans-anethol, while the bitter fennel (F.

vulgare ssp. vulgare var. vulgare) contains 50-75 % t-anethole and

3-20 % estragole (the latter proved to be carcinogenic şi

genotoxic).

In this case, the target and the objectives of our research

were the following:

morpho-anatomic description of Romanian fennel fruits,

9


chemical description of the volatile fraction and the

poliphenolic components,

monitoring the chemical compozition variability of the

volatile oil based on genetic and pedoclimatic factors,

assessment of the pharmaceutical and aromatherapy quality

of the sweet anise fruits marketed in Romania,

monitoring the chemical stability/variability of

polyphenolic fraction,

in vitro evaluation of the antioxidant potential by

determinaton of the scavenger capability of DPPH radicali,

antimicrobian and antifungic test for some volatile oils,

tracking sinergism of antimicrobial activity of fennel

essential oils in combination with amoxicilin and

tetracycline antibiotics,

safety evaluation of the repeated use use of fennel infusion.

45 samples were analyzed, samples identified ny the

producers as Foeniculum vulgare Mill. Sweet anise fruits,

representing:

Bulk fruits –Medplant, Fares, Stef Mar, Oprean, Ciprod,

Plafar Timişoara (2006-2010)

Tea fruits –Fares, Plafar Bucuresti, Trident, Dacia Plant,

Larix, Cyani

Fruits of known origin: CCB Piatra Neamţ, Vânători

Neamţ, Dărmăneşti, Costişa, SCDA Secuieni.

We proceeded with:

• macro-and microscopic pharmacognostic analysis,

• phytochemical analysis, using the CSS working methods,

spectrophotometric assay, gas chromatography coupled

with mass spectrometry, HPLC methods,

biological activity investigation (antioxidant,

antimicrobian, and antifungical activities).

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Before presenting the results, we wish to note that they

refer only to fruit samples to be analyzed, they can not be

extrapolated to the other batch of listed companies, as we shall see.

Performing macroscopic pharmacognostic analysis of

fennel fruit, we found that there are size, color, general appearance

differences, but all samples corresponded to the requirements set

by Ph.Eur. 6th on the macroscopic appearance (Fig. 1).

Fig. 1 Macroscopic aspect of fennel fruits included in study in

2006

Also, all samples presented the expected microscopic

product features: parquet cells, drops of oil, aleurona endosperm

and micro rosets of calcium oxalate, fragments of the secretion

channel, phloem cells (Fig.2).

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Fig.2. Microscopic features found in Foeniculi fructus powdered

samples

If macro-and microscopic analysis could not bring further

clarification on samples belonging to the sweet or bitter variety, we

hoped that phytochemical analysis, especially the volatile oil will

allow a more precise botanical classification. Therefore, in each

year we selected a number of samples purchased from wholesale

dealers, pharmacies or directly from the grower, samples for which

we determined the extraction yield by steam distillation of

essential oil, followed by GC analysis -MS.

In this summary we present only the results from two series

of chemical investigations on essential oil, namely: the one

separated from pharmaceutical fennel teas, and the one separated

from the fruits sold as bulk by firms producing herbal preparations.

Analyzing 7 teas purchased in pharmacies in early 2010, as

12


indicated by Ph. ed. 6th, we find that all exceed its requirement to

have a minimum of essential oil of 20 mL / kg. There is evidence

Larix, cyan and Perez, with amounts ranging from 101 to 71.10

mL/kg (Table1).

Table 1. Volatile oils extraction yield from commercial fennel

samples (2 hous extraction)

Samples Code Volatile oil mL /kg

Fares Orăștie CF1 71.10

Plafar București CF2 29.10

Trident Ilfov CF3 28.10

Dacia Plant 1 CF4 30.00

Dacia Plant 2 CF5 35.40

Larix Sovata CF6 101.00

Cyani Ploiești CF7 92.70

GC-MS analysis (Table 2) of the tea samples shows that

none has the t-anethole minimum content (80%) required by the

European Pharmacopoeia, but Larix, Perez and Cyan samples that

are rich in volatile oil also have the highest level of anethole

content.

Table 2. Chemical composition of essential oils of fennel tea

RT

(min)

Compound

%

CF1 CF2 CF3 CF4 CF5 CF6 CF7

5.474 -tuien 0.07 0.07 0.06 0.06 0.05 0.08 0.11

5.630 -pinen 5.90 4.76 4.36 4.72 4.21 7.38 6.25

5.892 Camfen 1.02 0.27 0.26 0.24 0.21 1.08 1.22

6.223 Sabinen 0.54 1.73 1.72 1.92 1.49 0.67 0.76

13


6.320 -pinen 0.67 0.49 0.46 0.49 0.40 0.89 0.82

6.427 mircen 2.60 1.64 1.77 1.76 1.48 2.78 3.00

6.748 -felandren 1.30 0.75 0.66 0.77 0.61 1.67 1.81

6.913 -terpinen 0.07 0.06 0.04 0.05 0.03 0.07 0.08

7.030 o-cimen 1.27 - - - - - -

7.117 limonen 6.24 17.32 17.88 18.36 16.41 7.74 7.78

7.321 t--ocimen 0.03 0.11 0.11 0.12 0.09 0.04 0.04

7.555 -terpinen 2.22 1.68 1.52 1.74 1.65 2.90 3.52

7.759 cis sabinen hidrat - 0.10 0.11 0.12 0.13 - -

7.827 t-sabinen hidrat 0.35 0.09 - - - 0.32 0.54

7.992 terpinolen - - - - - - 0.70

8.119 fenchone 18.30 7.75 7.86 7.34 9.44 17.81 15.90

8.401 1.3.8-p-mentatriena - 0.02 - 0.02 - - -

8.547 fenchol 0.13 - - - - 0.13 -

8.556 t-p-2.8-mentadien-1-ol - 0.45 0.49 - - - -

8.780 t-limonen oxid - 0.79 0.80 0.95 0.71 - -

9.004 camfor 1.92 0.70 0.64 0.59 0.65 1.48 1.86

9.461 4-terpineol 0.20 0.32 0.23 0.23 0.17 0.14 0.14

9.723 estragol 9.47 44.96 46.07 47.51 46.29 8.48 8.38

10.005 t-carveol 0.07 0.35 0.37 0.34 0.40 - 0.06

10.248 acetat de fenchil - 0.62 1.17 0.69 0.77 0.03 -

10.394 D-carvona 0.69 0.43 0.46 0.44 0.35 0.07 0.21

10.676 p-anisaldehida 1.53 0.32 0.19 0.19 0.47 0.55 0.63

11.026 t-anetol 44.08 12.12 9.43 7.15 10.11 42.68 42.24

11.677

acetat de exo-2hidroxicineol

- 0.09 0.10 0.16 - - -

12.096 t-izoeugenol - - 0.05 - - - -

12.105 eugenol - 0.05 - 0.05 0.06 - -

12.455 metileugenol 0.06 - - - - 0.25 0.10

12.251 -copaen - 0.15 0.22 0.20 0.16 - -

12.387 -cubeben - 0.18 0.22 0.22 0.21 - -

12.864 -cariofilen - 0.09 0.11 0.11 0.10 - 0.02

13.136 t--farnesen - 0.09 0.13 0.11 0.10 - -

13.194 1-m-anisil-1-propanona 0.05 - - - - 0.05 0.18

13.641 germacren D 0.25 0.42 0.53 0.60 0.45 - 0.76

13.680 anisil acetona - - - - - 0.77 -

13.787 -muurolen - - 0.05 0.11 - - -

13.797 -amorfen - 0.04 - - - - -

13.884 -bisabolen - 0.05 0.07 - - - 0.06

14.050 miristicina 0.14 - - - - 0.47 0.33

14.171 -cadinen - 0.17 0.23 0.23 0.20 - -

14.283 cis-izoelemicin - - - - - - 0.02

14.293 -longipinen - 0.01 - - 0.03 - -

14.390 izobutirat de geranil - - - - - - 0.01

14


14.730 3-metoxicinamaldehida - 0.03 0.06 0.08 0.05 - -

14.828 Spatulenol - - 0.07 0.08 0.07 - -

14.915 Hexadecan - - - 0.12 0.08 - -

15.217 Dilapiol - - 0.36 0.31 0.29 0.09 0.16

15.226 t-izodilapiol - 0.18 - - - - -

15.810 1-etil-dibenzotiofen - - - - - - 0.10

16.072 Heptadecan - - - 0.14 0.12 - -

16.335 miristat de metil - - - - 0.04 - -

17.608

6.10.14-trimetil-2pentadecanona

- 0.02 0.06 0.07 0.07 0.02 0.02

17.161 Octadecan - - - 0.11 0.07 - -

18.202 Nonadecan - - - 0.01 0.02 - -

18.454 palmitat de metil - - 0.02 0.02 0.12 - 0.04

20.156

metilesterul acidului 6octadecenoic

- 0.02 0.09 0.07 0.55 0.06 0.20

20.380

metilesterul acidului

16-metil heptadecanoic

- - - - 0.02 - -

27.419 Pentatriacontan - - - - - - 0.03

There are three major components of volatile oil for sweet

fennel: anethole, estragole and fenchone. While Ph.Eur. 6th

requires a minimum of 80% t-anethole, it also limits fenchone

content to 7.5% and the estragole content to 10%.

The Fig. 3 image shows graphically the t-anethole content

found in teas.

15


Fig. 3. The real content of t-anethole found in fennel volatile oil

samples (minimum requested 80 %)

Proceeding in a similar manner for the fenchone and

estragole recorded values in gas chromatographic analysis , we will

get the pictures presented in Fig.4.

Fig. 4. Fenchone and estragole content from fennel volatile oils

16


For 4 samples, we see an increase of over 45% in the

estragole isomer content, in the absence of anethole.

Only the same Larix, Cyani and Fares samples remain with

estragole levels within the limits , but in the same time, the

fenchone content is more than 15% while the maximum allowed

value is 7.5%.

If too much fenchone is neurotoxic and can cause the

occurrence of epileptiform convulsions or laryngeal spasm and

dyspnea in infants, when applied to the skin, it produces dermal

irritation. But fennel volatile oil is an important component in the

composition of expectorant action creams that are applied by

rubbing directly on the upper chest, or abdomen, for spasmolytic

effect in the treatment of menstrual pain or bloating.

On the other hand, high concentration of estragole brings a

high risk of carcinogenicity and genotoxicity in Plafar Bucharest,

Trident and Dacia Plant 1 and 2 samples.

Since the fennel fruits, by the composition of volatile oil do

not meet the pharmaceutical requirements, we turned our attention

to the aroma therapeutic qualities.

Summing for this purpose the components identified by gas

chromatography according to the chemical structural class, we

obtained the values shown in Table3.

Table 3. Fennel volatile oils composition according to their

chemical structure

Cathegory (%) Samples

Standard

CF1 CF2 CF3 CF4 CF5 CF6 CF7

Monoterpenes 20.66 28.90 28.84 30.25 26.63 25.30 26.09 15-30%

17


MonoterpenketoneMonoterpenalcoholsMonoterpenoxydes

Aromatic

compounds from

wich esthers

Aldehide and

ketones

20.91 8.88 8.96 8.37 10.44 19.36 17.97 0.5-5%

0.75 1.31 1.20 0.69 0.70 0.59 0.74 1-3%

- 0.79 0.80 0.95 0.71 - - max 4%

56.60

53.61

57.66

57.13

56.16

55.55

55.29

54.71

57.27

56.46

53.34

51.41

52.12

50.92

55-85%

1.58 0.35 0.25 0.27 0.52 1.37 0.81 0.5-1.5%

Transposing this data into the corresponding

mnemotechnique models (Fig. 5), we find that the volatile oils

differ from standard samples analyzed.

Fig 5. Mnemotechnique profiles of the analyzed fennel oils

compared to standard

Although apparently essential oils largely meet the

requirements, we must not lose sight of the presence of ketones in

the amount of 2-4 times higher than allowed quantity (max. 5%) in

aromatherapy. It is in fact, the fenchone, which administered in

18


larger quantities than those permitted, result in adverse actions

already mentioned.

Even worse is the fact that for 4 of 7 samples,the t-anethole

is replaced by its isomer, estragole. The two ethers have a common

biogenesis in the maturation of the fruit,also they can be directed

enzyme synthesis by anethole or estragole by the. As you can see

the Plafar Bucharest, Trident and Dacia Plant samples, the

enzymes that convert precursor prevail immediately estragole and

anethole not.

Another series of studies focused on the fruits offered as

bulk herbs by the suppliers; the samples from Ciprod, Oprean, Stef

Mar, Plafar, Medplant and Timisoara.

In this case, we followed the differences in chemical

composition depending on the time of distillation of the plant

material water: 2 hours according to European Pharmacopoeia ed.

6th or 3 hours under FR X.

Variation of the three major compounds is shown in

Figures 6-8, 4 samples have a the anethole content greater than 3

hours drive 2 and a sample is below 10% anethole. None of the

evidence has at least 80% anethole, sweet fennel fruit requested

but at least 50%, as required for bitter fennel.

In terms of anethole content, it seems that the

recommended distillation time should be 3 hours as required by FR

X in the Foeniculi fructus monograph,instead of 2 hours, as the

European Pharmacopoeia prescribes.

19


Oprean sample that has a reduced content level of anethole, proves

to be rich in estragole, this situation being similar to the situation

encountered for tea(Fig. 7).

20


As previously discussed for teas, in regards to the samples

with a higher but insufficient level of anethole, the amount

determined in this mono terpene ketone is high (over 7.5%), from

the fenchone content standpoint (Fig. 8). If prolonging the

distillation time is favorabile to the anethole extraction, the

anethole almost has no influence over the oil content level in

fenchone and estragole.

Turning to the presentation of polyphenolic fraction study we

focused on three samples from the Magurele variety fennel plants

grown in CCB Piatra Neamt, which distinguish between

themselves by the age of samples: 5, 6 and 10 years (Fig.

21


9).

In parallel, we also processed two samples from SCDA Secuieni,

for which the cultures were initiated in 2008. When we harvested

the fruits in August 2011, plants growing in both groups were in

the fourth vegetation year.The seeds for SCDA 1B sample came

from the Magurele variety via Piatra Neamt while the seeds for

SCDA 2B came from the Hestia variety, Fundulea (fig.

10).

22


Although CCB 2B and SCDA 1B samples had a common origin

(the seeds came from Magurele variety fruits) we find that the

macroscopic appearance of the fruits is slightly different; the fruits

for the CCB 2B sample (parental for SCDA 1B) are slightly bigger

than the fruits from the daughter sample obtained from Secuieni

cultures (fig. 11).

Extracting polyphenolic fraction with 70% ethanol and

analyzing the flavonoids components and polyphenol carboxylic

acids by thin layer chromatography, we obtained the following

images (Fig. 12).

As you can see, samples that contain a small amount of

derived polyphenolic flavonoids and more polyphenol acids, the

chromatographic arrays are similar to each other, there are no

qualitative differences, only some quantitative differences.

23


By spectrophotometrical determination of the content of total

polyphenols, flavonoids and polyphenolcarboxylic acids, using the

previously degreased plant material, we got the Table 4 values,

which show that these substances are poorly represented in the

fruits.

For HPLC analysis, polyphenolic fractions were separated

using for this purpose plant material previously degreased (Fig.

13).

24


Degreasing was done for a latter improved separation of the

chemical agents by HPLC. By degreasing however, a part of

aglicons flavonoids were also removed.

HPLC analysis confirmed the existence of a polyphenolic

fraction low in components. There were 5 polyphenolcarboxylic

acids (caffeic acid, ferulic, chlorogenic, p-coumaric and rosemary)

and 4 flavonoids (rutoside, cvercetolul, apigenol and apigenol-7glucoside)

identified, as shown in Table 5.

25


Table 5. Polyphenols concentration determined by HPLC in

ethanolic extracts processed from fennel fruits in Neamt

County

µg/mL

Componentă CCB CCB CCB SCDA SCDA

1B 2B 3B 1B 2B

ac. cafeic 5.602 4.438 5.418 4.883 5.494

ac.clorogenic 2.22 1.27 2.33 1.42 1.28

ac. p-cumaric 2.056 1.924 2.077 1.879 1.946

ac. ferulic 3.336 2.816 3.335 2.779 3.099

ac.

rosmarinic

1.238 0.912 1.318 0.988 1.325

rutozidă 4.036 3.927 3.926 3.909 3.902

apigenol-7- 1.168 0.843 1.128 0.975 1.252

glu

cvercetol 2.45 2.400 2.455 2.404 2.413

apigenol 2.143 2.085 2.087 2.150 2.116

Out of the five acids identified by HPLC in fennel extracts

there are 2 main ones: the caffeic acid and ferulic acid; neither of

them is present in larger quantities, quantities that exceed the order

of several tens of mg (Fig. 14).

As you can see, there is a slight quantitative difference

between the samples; let's not forget that the CCB samples have

different ages (5, 6 and 10 years), and the SCDA samples have

different origins: Magurele origin for SCDA 1B and Hestia origin

for SCDA 2B. In both cases the plants were 4 years old.

26


Out of all the flavonoids, 4 of them were identified and

quantified by HPLC, rutoside and cvercetol being the main ones

from the quantitative standpoint; their samples had very similar

amounts (fig. 15).

If we make a comparison between the spectrophotometric

determination of acid polyphenolcarboxylic and the HPLC

27


quantification of the 5 derivatives identified, just as shown in the

image, we find no significant differences, which means that the

extracts had quite few components of this type that could not be

identified due to lack of appropriate standards (fig. 16).

If we apply the same type of comparison to the fraction

flavonoids, we must conclude that the plant material are still at 50-

66% unidentified components, with significant differences

between the two types of determinations (Fig. 17).

28


Turning to the investigation of antioxidant action of

extracts and vaporized in low temperature infusions obtained from

dried fennel tea samples (for which we already analyzed and

discussed the composition of the volatile fraction), we first

examined the polyphenol composition (Fig. 18).

29


When we compare the graphic values we find that the dry

hydroalcoholic extracts and the low temperature vaporized

infusions contain similar amounts of total polyphenols. We must

not overlook the fact that while infusions are made from 6 g/100

mL water extracts were prepared from 3 g plant material / 100 mL

70% ethanol.

Investigating the DPPH radical scavenger capacity (Table

5) of dry extracts and low temperature vaporized infusions, we

found that for the same doses 10, 20 or 40 mg / mL, the activity is

higher for 3% hydroalcoholic extracts than for the activity of low

temperature vaporized infusions of 6 %.

Calculating the 50% inhibitory concentrations, we confirm

the observation that hydroalcoholic extracts have higher

antioxidant activity than infusions, even if the latter were obtained

from a double quantity (6 g%) of fruit. Trident sample has the most

powerful antioxidant activity while Cyan sample has the weakest

(fig.19).

30


Comparing IC50 with total polyphenols content we

determined in the corresponding extracts, we find that there is no

similarity between the two parameters. This could be explained by

the fact that the total polyphenols spectrophotometric

determination is capturing not only the and flavonoids

polyphenolcarboxylic acids, but all reducing components of

extracts, reducing components that may be qualitatively and

quantitatively different from sample to sample (skeletal supporting

structure and polyphenols components may vary).

Also, let's not forget that both at the infusion and at the

polar organic solvent extraction, a certain amount of volatile oil

which contributes to the antioxidant activity of the phytocomplex

is extracted. If we attempt to draw a parallel between the chemical

composition of essential oil of tea samples analyzed and the

antioxidant activity reflected by IC 50, we find that the samples

with a higher estragole content also show higher antioxidant

activity, namely: Plafar Bucharest, Trident, Dacia Plant 1 and 2.

We may conclude that the volatile components, especially

estragole might have some antioxidant activity.

Antioxidant activity was investigated in a similar manner

from bulk samples from Ciprod, Oprean, Stef Mar Medplant,

Plafar Timisoara, BCH and RS 2A 1 A companies in 2010.

Calculating the final 50% inhibitory concentrations for

hydroalcoholic extracts made from ungreased and degreased plant

material and also for low temperature vaporized infusions, we find

that for some samples (Ciprod, Oprean, Stef Mar Plafar Timisoara)

the low temperature vaporized infusions are more active than the

polar organic solvent extracts (Fig. 20).

However, there are very large activity variations between

samples, and the degreasing of fruits, even though it is beneficial

31


for the determination itself does not consistently lead to increased

efficiency. This maybe because the degreasing plant material is

removing the volatile fractions and flavonoid aglicons, that are

typically present in undegreased fruits.

Study of antimicrobial and antifungal effect was investigated for

volatile oils extracted from two samples of fennel fruits.

The two oils tested, F1 Sec and CCB (obtained from fruits

harvested in 2005), showed a good antimicrobial activity and

bactericidal activity against Staphylococcus aureus, E. coli and an

excellent antifungal activity against C. albicans. For the two oils

we established the minimum inhibitory concentration (MIC) and

the minimum bactericidal concentration (MBC) (Table

6).

When investigating in even further depth the synergistic

action of both oils with amoxicillin and tetracycline respectively,

the synergistic action could only be confirmed for the amoxicillin

+ volatile oil against Sarcina lutea and E. coli associations.

32


Getting back to the motivation of the research study we

questioned the safety and exposed risks for infants that repeatedly

ingested infusions prepared from the fennel fruits analyzed by us

that proved to have a high content of estragole.

Calculating (after a generally accepted bitter fennel

formula) the daily intake of estragole when ingesting an infusion

prepared from 4.5-7.5 g fruit, we obtain values that are placed

between 24.41 -67.66 mg estragole. If we consider a weight of 5

kg infant, that means that the kg / body daily consumption ratio

may reach values placed between 4882-8120 mg estragole, as

shown in Table 7.

When we go for the visual representation of estragole calculated

values of infusions prepared from 4.5 g fruit, the situation becomes

much clearer: while Oprean bulk sample has the highest values,

Plafar Bucharest has the smallest ones(Fig. 21).

33


Theoretical assessments made by by Speijers G. and 20

other co-workers of the estragole intake by eating of bitter fennel

infusion revealed that the ingestion of infusions prepared from 4.5

-7.5 g of fruit (average content in volatile oil = 5 mL%) with a

volatile oil extractibility by infusion of 25-30%, the amount of

methyl-chavicol administered falls in the 1.9-15.8 mg range, which

means a 60 kg adult dose of 33-263 mg / kg / day, which

represents a serious carcinogenic risk.

In case we would have administered an infusion of 4.5 g Romanian

fennel with 45% estragole Volatile oil to an infant weighing 5 kg

(as was the case in 7 samples out of 35 analyzed), using the same

calculation, estragole would have been 24.41 - 40.60 mg or 4882-

8120 mg / kg. These values are much higher than what Speijers G.

and collaborators have calculated to be putting the consumer at risk

because of the carcinogenic and genotoxic effects associated with

estragole.

34


6%

11%

ESTRAGOL

sub 10% 10-15%

15-20% peste 45%

20%

FENCONĂ

sub 7.5% 7.5-20% peste 20%

23%

63%

14%

63%

Fig. 22. Anethole, estragole and fenchone content distribution

identified in the 35 separated fruit oils

When analysing the anethole, we observe that none of the

samples did reach the limit of min. 80%. Only one sample had a

content exceeding 70%, while the majority of samples (63%) didn't

even reach 50% anethole concentration.

For estragole, 63% of samples fall within limits, the rest

exceeding the amount allowed by the pharmacopoeia by a large

35


margin, while for fenchone 86% of samples do not meet the

requirements.

While the anethole comes with good spasmolytic and

expectorant activities, the samples analysed by us will have a

diminished therapeutic effect, due to the lack of the minimum

quantity for this component; it is true that for most samples, we

might think that spasmolytic effect is provided by fenchone

surplus, but in reality, by going over the generally accepted limit,

the fenchone also comes with neurotoxicity and dermo irritation.

The estragole analysis emphasizes not only the lack of benefit, but

also the high risks of carcinogenicity and genotoxicity for more

than 37% of samples.

GENERAL CONCLUSIONS. DEGREE OF

ORIGINALITY. FUTURE RESEARCH PERSPECTIVES

Our study conducted on the fennel fruits for pharmaceutical use

during 2005-2011, allowed us to draw some general conclusions:

we analyzed 45 samples of sweet fennel fruits purchased

from suppliers of herbs, growers or producers of tea

according to European Pharmacopoeia 6th ;

botanical identification was poor;

macro- şi microscopic aspects are not conclusive;

GC-MS analysis of the essential oil is pivotal;

none of the samples is meeting the existing standards

required levels of t-anethole, fenchone and estragole for

sweet anise;

in terms of chemical composition shown by the essential

oils, the samples present similarities with the bitter fennel;

out of the 35 volatile oils we studied, all of them present a

level of anethol that is too small;

36


86 % of the samples have levels of fenchone content that

surpass the required limit;

37 % of the samples have estragol over the required limit;

The volatile oils do not meet the aroma therapy

requirements;

the polyphenolic fraction is poorly represented, but there

are insignificant variations between the samples,

we identified 5 polyphenolcarboxilic acids and 4

flavonoids;

antioxidant action investigated by a single test varies from

sample to sample and is rather weak;

the volatile oil has an antimicrobian activity towards E.

coli and S. aureus, the antifungic activity is obvious.

ORIGINAL CONTRIBUTIONS

The present paper:

represents the first systematic study of pharmaceutically

used fennel in România,

the investigation was conducted in accordance with the

requirements of Ph.Eur. 6th and Directive Guide EFSA

represents the first benefits/risks analysis for those that

ingest fennel infusions repeatedly.

comes with possible remedial action recommendations, for

farmers and companies selling/processing medicinal plants.

FUTURE RESEARCH PERSPECTIVES

We mention the following as opportunities for further study:

in a research project proposal that will include Romanian

fennel tea, that will focus on the quality and safety aspects

of using fennel tea as a food supplement,

similar topics can be addressed using the model of this

paper.

37


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LISTĂ LUCRĂRI

Cotate B+

47


in extenso

83. Băjan M, Aprotosoaie AC, Spac A, Stănescu U.:

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Romanian fennel fruits”. Rev Med Chir Soc Med Nat Iasi,

2011, 115 (2), 590-594

84. Băjan M., Aprotosoaie A. C., Cioancă O., Şpac A., Gille

E., Stănescu U., Hăncianu M.: Chemical composition of

volatile oils isolated from the fruits of two sweet fennel

organic culture variants, Medicine in Evolution Volume

XVII, No. 3, 2011, 150-155

in extenso în reviste cotate B (suplimente ale unor reviste cotate

B+)

1. Aprotosoaie AC., Băjan M., Şpac A., Dorneanu V., Bajan

E., Stănescu U. Profilul chimic comparativ al fracţiunii

volatile separate din fructe de fenicul (Foeniculum

vulgare Mill) comercializate pe piaţa farmaceutică

românească. Rev. Med. Chir. Soc. Med. Nat. Iaşi. 2008,

112 (2), 1/supl, 19-23

Lucrări publicate în Proceedings la Conferinţe Naţionale /

Internaţionale

1. Poiată A., Dănilă D., Tuchiluş C., Băjan M., Aprotosoaie

A. C., Hăncianu M., Evaluation of microbial level

contamination in dried plant material by the standard plate

count, European Journal of Drug Metabolism and

Pharmacokinetics, 2009, 34, 29-14 th PAN –HELLENIC

PHARMACEUTICAL CONGRESS, Atena, 2009 May 9-

11- CD

48


2. Băjan M., Aprotosoaie A.C., Cioancă O., Stănescu U.,

Preliminary investigations regarding the determination of

some polyphenolic derivates from fennel tinctures by means

of HPLC, Romanian Biological Sciences, 2007, V (1-2), 8-

10 Medicinal Plants Ed X –Present and Perspectives

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