BIOLOGICAL PROPERTIES EVALUATION OF THE QUALITY … · REZUMAT 31 research. Research in this field...

UNIVERSITY OF AGRICULTURAL SCIENCES AND VETERINARY

MEDICINE CLUJ-NAPOCA

DOCTORAL SCHOOL

FACULTY OF ANIMAL HUSBANDRY AND BIOTECHNOLOGIES

Eng. LAVINIA IOANA BĂRNUŢIU (TOMOŞ)

BIOLOGICAL PROPERTIES EVALUATION OF THE

QUALITY MARKERS FROM

ROYAL JELLY AND APILARNIL(SUMMARY OF PhD THESIS)

SCIENTIFIC COORDINATOR

Prof. Eng. Liviu alexandru MĂRGHITAŞ, PhD

CLUJ-NAPOCA

2013

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CONTENTS

INTRODUCTION......................................................................................................... 30

OBJECTIVES ............................................................................................................... 32

CHAPTER I. ROYAL JELLY AND APILARNIL – GENERALITIES ........................ 33

1.1. IMPORTANCE OF ROYAL JELLY AND APILARNIL................................... 33

1.2. PHYSICO-CHEMICAL COMPOSITION OF ROYAL JELLY AND APILARNIL

................................................................................................................................... 33

CHAPTER II. QUALITY MARKERS OF ROYAL JELLY AND APILARNIL........... 33

CHAPTER III. THERAPEUTICAL EFFECTS OF ROYAL JELLY AND APILARNIL

...................................................................................................................................... 34

CHAPTER IV. METHODS FOR THE DETERMINATION OF ANTIOXIDANT AND

ANTIMICROBIAL CAPACITIES OF ROYAL JELLY AND APILARNIL ................ 34

CHAPTER V. MATERIAL AND METHOD................................................................ 34

5.1. BIOLOGICAL MATERIAL ............................................................................... 34

5.2. APPLIED EXPERIMENTAL METHODS.......................................................... 35

CHAPTER VI. RESULTS REGARDING PHYSICO – CHEMICAL ANALYSIS ....... 35

CHAPTER VII. RESULTS AND DISCUSSIONS REGARDING

SPECTROPHOTOMETRIC ANALYSIS ..................................................................... 38

CHAPTER VIII. RESULTS AND DISCUSSIONS REGARDING HPLC ANALISYS 41

CHAPTER IX. RESULTS AND DISCUSSIONS REGARDING ANTIMICROBIAL

CAPACITY OF ROYAL JELLY AND APILARNIL ................................................... 46

CHAPTER X. GENERAL CONCLUSSIONS .............................................................. 51

ORIGINAL ELEMENTS .............................................................................................. 53

RECOMMENDATIONS AND PERSPECTIVES ......................................................... 53

BIBLIOGRAPHIC REFERENCES............................................................................... 54

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INTRODUCTION

Royal Jelly and apilarnil are bee products with biotrophic and biologically active

properties, energizing, revitalizing, toning and psihotonice which help restore the

immune system. Although it is not a traditional product of the hive, like honey, pollen,

propolis and wax, apilarnil is distinguished by its organoleptic, physico-chemical and

microbiological properties, which recommends it as a genuine and valuable product of

the hive.

Royal Jelly has been accepted and often used as a substance that supports health.

Research shows that Royal Jelly proteins could have physiological functions,

immunostimulative suppression of allergic reactions, and antihypertensives, and

stimulate cell proliferation. All these findings have opened a new era in the uses of Royal

Jelly.

Given the organoleptic, physico-chemical and certain harvesting conditions,

apilarnil differs essentially from Royal Jelly. It is good that beekeepers take into account

these differences in the process and technology of harvesting, always respecting the

principle responsability, professional accuracy and fairness in their activity of

manufacturers.

Royal Jelly has biological and physiological exceptional qualities, which is why it

is marketed widely, being used in various industries such as pharmaceutical, food

(dietary supplements), and cosmetics. Due to the diversity and multitude of uses, in

places where the production is insufficient in relation to demand, Royal Jelly is being

imported in large quantities. Therefore, research in this area should be expanded and

developed to allow a correct qualitative and quantitative assessment of its various

components. On the other hand, research allows the implementation of quality

assessment tests of available products which include Royal Jelly like it is or as an

additive. Research in this area has an important role in optimizing the process of

identifying counterfeit Royal Jelly.

Knowing that in our country, and in most countries (except Brazil), there is no

standard for Royal Jelly quality, making such a standard or at least implementing reliable

methods for the determination of its functionality and quality, have a high interest for

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research. Research in this field will contribute to the development of new technologies

for determining the acid 10-hydroxy-2-decanoic which is related to the authenticity of

Royal Jelly and also to establish quality markers for Royal Jelly and apilarnil.

This PhD thesis aims to bring out an important contribution regarding the study of

Royal Jelly and apilarnil by means of new chemical analysis in order to investigate their

quality. The quality of these products is given by components with nutritional value

(carbohydrate, lipids and protein), the amount of biologically active compounds (10-

HDA, phenolic compounds and peptides), as well as antioxidant and antimicrobial

capacities.

Present paper “Biological properties evaluation of the quality markers from

Royal jelly and apilarnil” is structured in two parts. In the first part, entitled "Literature

review" (Chapters I-IV) is shown the present state of knowledge with regards to proposed

objectives and some aspects concerning chemical composition, antioxidant and

antimicrobial activities, used analytic methods.

In the second part called “Original research” (Chapters V - X) are shown the

biological material (14 samples of royal jelly and 14 samples apilarnil), applied

experimental methods (physico-chemical, spectrophotometric, chromatographic and

microbiological), and also the results obtained and conclusions of the present study.

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OBJECTIVES

The objectives of the present PhD thesis are the following:

Research regarding physico-chemical composition of Royal Jelly and apilarnil by

determining humidity, ash, acidity and lipids.

Research regarding optimisation of the method used for total protein

determination from Royal Jelly and apilarnil – Lowry method.

Research regarding identification and quantification of sugars content from Royal

Jelly and apilarnil by means of High Performance Liquid Cromatography

tehnique coupled with Refraction Index detector.

Research regarding identification and quantification of 10-hydroxy-2-decenoic-

acid content from Royal Jelly using High Performance Liquid Cromatography

technique coupled with Photo Diode Array detector.

Research regarding identification of polyphenols content from Royal Jelly using

High Performance Liquid Cromatography technique coupled with Photo Diode

Array detector.

Research regarding antioxidant capacity of Royal jelly and apilarnil (DPPH assay

and FRAP assay) evaluated in vitro by using classical spectrophotometic

methods.

Research regarding antimicrobial capacity of Royal Jelly and apilarnil by testing

on international bacterial strains.

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CHAPTER I. ROYAL JELLY AND APILARNIL – GENERALITIES

1.1. IMPORTANCE OF ROYAL JELLY AND APILARNIL

Knowledge of biological markers of fresh Royal Jelly and apilarnil responsible for

their properties is essential in order to characterize the composition and quality of the

products that are produced as a result of their incorporation into the structure of these

valuable products of the hive.

1.2. PHYSICO-CHEMICAL COMPOSITION OF ROYAL JELLY AND APILARNIL

Royal Jelly is the only known source of 10-HDA acid, a compound with

antitumoral and antibacterial potential. Chemical composition of Royal Jelly is a mixture

of vitamins and aminoacids, and also unidentified compoundts (2.8%) (Boselli et al.,

2003). Literature shows some of the important components of Royal Jelly as: proteins,

sugars, lipids (Takenaka, 1984; Pourtallier et al., 1990, Lerker et al, 2003).

Microscopic examination of apilarnil shows cellular remains and also pollen cells.

In order to obtain the standard substance, the filtered product is being subjected to

liophilization, the final product being used as a powder (Ilieşiu, 1991). First description

of apilarnil composition was presented by Stângaciu (1999): water 65-75%, lipids 5-8%,

total proteins 9-12%, total sugars 6-10%, dry matter content 25-35%, unidentified

substances 3%, ash 2%.

CHAPTER II. QUALITY MARKERS OF ROYAL JELLY AND APILARNIL

Literature shows as quality markers of Royal Jelly and apilarnil the following

classes of substances: proteins, carbohydrates, lipids, polyphenols, amino acids, vitamins,

minerals. Also, a particular interest is shown in the presence of 10-HDA acid found in

Royal Jelly composition, possesing antitumoral and antimicrobial properties.

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CHAPTER III. THERAPEUTICAL EFFECTS OF ROYAL JELLY AND

APILARNIL

Due to theirs various chemical composition, Royal Jelly and apilarnil, are used in

medicine, in pure state or lyofilized, and also in cosmetics, as additive to face creams and

ointments. Cosmetic treatments with creams based on Royal Jelly offer elasticity to skin,

flexibility, while hair products having Royal Jelly give a natural and shiny look.

CHAPTER IV. METHODS FOR THE DETERMINATION OF ANTIOXIDANT AND

ANTIMICROBIAL CAPACITIES OF ROYAL JELLY AND APILARNIL

Antioxidant activity is due to polyphenols, chemical compounds present in Royal

Jelly and apilarnil composition. Literature has few references in terms of antioxidant

capacity and chemical composition of Royal Jelly (Nagai şi colab., 2001).

Methods which describe ntioxidant capacity of Royal Jelly and apilarnil are:

radical scavengind activity (DPPH) (Liu et al., 2008) and total antoxidant potential

(FRAP) (Gao et al.,2011).

Antimicrobial capacity of Royal Jelly and apilarnil is determined by means of

diffusion methods and successive dilutions methods. There are many studies that proove

the antimicrobial activity of the different strains bacterial (Eshraghi and Seifollahi,

2003).

CHAPTER V. MATERIAL AND METHOD

5.1. BIOLOGICAL MATERIAL

Biological material used in the present study is constituted of 14 Royal Jelly

samples and 14 apilarnil samples. The samples were obtained directly from beekepers

during May 2011 – August 2012. All samples were kept in the freezer at - 20°C until

analysis.

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5.2. APPLIED EXPERIMENTAL METHODS

Experimental procedures were conducted using four types of analysis: physic-

chemical, spectrophotometric, chromatographic and microbiological.

CHAPTER VI. RESULTS REGARDING PHYSICO – CHEMICAL ANALYSIS

Physico-chemical analysis were used for the primary characterization of Royal

Jelly and apilarnil composition with regards to water content, acidity, ash content and

total lipids content.

As a first characterization of obtained data and in order to have a clear image of

water content from Royal Jelly and apilarnil, boxplot diagram was employed for both

studied matrices. Boxplot diagram (figure 1) is a graphic representation of the five

specific values of theirs distribution in each area (minimum, first quartile, median, third

quartile and maximum) and extreme values. The graphic shows for each boxplot

diagram, a central line which is the median of measured values for each area. Where the

median is closer to lower margin, the values distribution is left oriented, as for the

opposed case is right oriented. Maximum value of the median for Royal Jelly is 63.59,

and for apilarnil os 71.08. The boxplot diagram represents the distribution of 50% of the

values while its length is the results variability. Extreme values are found at the exterior

of the boxplot, being marked with *. For the parameter water content, both studied

matices had one extreme value represented by sample A9.

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Figure 1. Boxplot graph regarding water content of royal jelly

and apilarnil samples

Maximum value of the median for Royal Jelly was 1.11, while for apilarnil was

0.90. For the parameter ash content was foundonly one extreme value for Royl Jelly

matrix represented by sample L5 (figure 2).

Figure 2. Boxplot graph regarding ash content of royal jelly


Maximum median value for acidity was 3.76 for Royal Jelly and 3.18 for apilarnil.

For the mentiond parameter, both studied matices showed no extreme values.

Apilarnil/ApilarnilLaptisor de matca/Royal jelly

75

70

65

60

55

Cont

inut

ulde

apa

(%)/

Wat

erco

nten

t(%

)

Apilarnil/ApilarnilLaptisor de matca/Royal Jelly

1.6

1.5

1.4

1.3

1.2

1.1

1.0

0.9

0.8

0.7

Cenu

sa(%

)/A

sh(%

)

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Figure 3. Boxplot graph regarding acidity of royal jelly and apilarnil samples

Maximum median velue for total lipids content was 4.62 for Royal Jelly and 3.44

for apilarnil. No extreme values were found for acidity in the case of both studied

matices.

Figure 4. Boxplot graph regarding lipids content of royal jelly


Apilarnil/ApilarnilLaptisor de matca / Royal Jelly

6

5

4

3

2

1

Aci

dita

tea

(mlN

aOH

0.1N

)/A

cidi

ty(m

lNaO

H0.

1N)


8

7

6

5

4

3

2

1

Cont

inut

delip

ide

(%)/

Lipi

dsco

nten

t(%)

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CHAPTER VII. RESULTS AND DISCUSSIONS REGARDING

SPECTROPHOTOMETRIC ANALYSIS

Total protein content for studied Royal Jelly samples shows an average of 12.70 ±

0.53%. Sabatini et al. (2009) established for this parameter the range 9-18% as

international standard, so all studied samples are within the mentioned range.

Maximum median value for Royal Jelly is 12.79, and 6.61 for apilarnil. Total

protein content showed no extreme value for apilarnil, while for Royal Jelly sample L2

was the extreme.

Figure 5. Boxplot graph regarding protein content of royal jelly


Total polyphenols content varies within wide limits for apilarnil, values obtained

in the preset study being between 25.05±0.68 mg GAE/g (A9) and 65.72±0.93 mg

GAE/g (A11).

Total polyphenols content for royal jelly samples does not as much as in the case

of apilarnil, values obtained were between 20.78±0.33 (LM 13) mg GAE/g and

30.76±0.98 mg GAE/g (LM 1).


17.5

15.0

12.5

10.0

7.5

5.0

Proi

tene

tota

le(%

)/To

talp

rote

in(%

)

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Figure 6. Boxplot graph regarding total polyphenols content

of royal jelly and apilarnil samples

Maximum value for the median of polyphenols content for royal jelly was 23.35,

while for apilarnil was 42.05. No extreme values were registered for the studied matrices

(figure 7).

Literature shows an average for the total antioxidant potential of 8 μmoli Fe / g(FRAP method) for liophilyzed royal jelly (Ross, 2009). Using the conversion of fresh royal

jelly into dry royal jelly, it can be stated that the values from the present study are within

the range mentioned by literature.

Regarding antioxidant activity of apilarnil measured by FRAP method we can say

that it exhibited a strong activity, registered values being higher than the ones for royal

jelly.

Maximum values of the median of antioxidant activity measured by FRAP method

for royal jelly is 3.56, while for apilarnil is 15.74. The boxplot diagram represents the

distribution of 50% of the values while its length is the results variability. Extreme values

are found at the exterior of the boxplot, being marked with *. For the parameter

antioxidant activity, only apilarnil had an extreme value registered for sample A12.


70

60

50

40

30

20

Polif

enol

itot

ali(

%)/

Tota

lpol

yphe

nols

(%)

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Figure 7 . Boxplot graph regarding antioxidant potential of royal jelly


The lowest antioxidant capacity was registered for samples L1 and L3 (0.11

mmoli Trolox/g royal jelly), while the highest was for sample L6 (0.30 mmoli Trolox/g

royal jelly).

For apilarnil samples, values of antioxidant capacity were higher. Sample A4 had

the highest antioxidant capacity measured by means of DPPH assay (0.12±0.01 mmoli

Trolox/g), while sample A7 had the lowest one (0.78±0.06 mmoli Trolox/g).

Boxplot diagram (figure 8) is a graphic representation of the five specific values of

theirs distribution in each area (minimum, first quartile, median, third quartile and

maximum) and extreme values. Maximum value of the median for royal jelly samples

was found to be 0.19, while for apilarnil was 0.22. Antioxidant capacity measured by

means of DPPH assay showed no extreme values for royal jelly samples, while for

apilarnil samples A7 and A8 were the extremes.


40

30

20

10

0

Pote

ntia

lula

ntio

xida

nt(µ

mol

iFeI

I/g)/A

ntio

xida

ntpo

tent

ial(

µmol

iFeI

I/g)

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Figure 8. Boxplot graph regarding antioxidant activity of royal jelly


CHAPTER VIII. RESULTS AND DISCUSSIONS REGARDING HPLC ANALISYS

Identification and quantification of sugars represents an important step towards

determination of authenticity of the samples, both quantitatively and qualitatively

speaking. In many cases the monosaccharides, glucose and fructose represent together

80% of the available sugars in royal jelly, other carbohydrates are in much lower

quantities.

The results obtained are comparable with those of Sesta (2006), which determined

the content of major sugars royal jelly samples, like glucose, fructose and sucrose.


0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

Cap

acita

tea

antio

xida

nta

(mm

oliT

rolo

x/g)

/Ant

ioxi

dant

capa

city

(mm

oliT

rolo

x/g)

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5 10 15 20 25 30 35

0

10000

20000

30000

40000

zaharo

za / su

crose

Abso

rban

ta (m

AU)

Abso

rban

ce (m

AU)

Timp de retentie (min)Retention time (min)

Proba L1 / Sample L1

fructo

za / fr

uctose

glucoz

a /glu

cose

turano

za / tu

ranose

erloza

/ erlo

se

maltoza

/ malt

ose

trehal

oza / t

rehalo

se

Figure 9. HPLC – IR chromatogram of Royal Jelly samples (L1)

Sugars profile of sample L1 from Cluj county is shown in figure 9, where is can be

seen the retention time of each identified sugar. Chromatograms recorded for all other

studied samples of royal jelly and apilarnil are presented in Appendices 4 and 5.

Sugars profile is very important for royal jelly since the amount of each one could

be used to detect a possible adulteration with honey or pure sugar.

Literature is quite poor in offering information with regards to sugars content in

apilarnil. No literature study with regards to this matter shows the quantification of

individual sugars from apilarnil. In the present study sucrose was identified in sample A1

(0.14%), comparison of the results being possible only with royal jelly.

Fructose was present in higher amount than glucose in all royal jelly samples, but

apilarnil samples did not show the same uniformity. Ten apilarnil samples showed

fructose in amounts lower than 1%, while glucose was present in quantities above 2%;

one sample had glucose above 1%; 2 samples showed amounts of glucose and fructose

almost equal; while one sample had fructose in a larger amount than glucose.

Maximum value of the median for apilarnil samples was 0.56 for fructose and 3.40

for glucose. The fact that the median value was close to one of the extremities proves that

apilarnil samples are not homogeneous as chemical composition. Sucrose was identified

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only in one apilarnil sample, with a lower quantity than in royal jelly, which does not

provide a clear image with regards to this matter.

Figure 10 represents the sugars profile and retention time for each sugar identified

for apilarnil sample (A1) from Cluj county.

5 10 15 20 25

0

5000

10000

15000

20000Ab

sorb

anta

(mAU

)Ab

sorb

ance

(mAU

)


Proba A1 / Sample A1

glucoz

a /glu

cose

zaharo

za / su

crose

turano

za / tu

ranose

maltoza

/ malto

se

izomalto

za / is

omalto

sefru

ctoza

/ fruct

ose

Figure 10. HPLC – IR chromatogram of apilarnil samples (A1)

Results obtained after identification and quantification of 10-hydroxi-2-decenoic

acid are presented in figure 11. The results were registered with LC-solution software

based on the similarity of the UV-VIS spectrum and retention times.

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Figure 11. 10-hydroxy-2-decenoic-acid content of Royal Jelly samples

10-HAD acid content in royal jelly samples ranges between 0.75% and 3.80%.

Literature data shows only a few records about the presence of 10-HDA acid. Garcia

Amoedo and Almeida Muradian, (2007) obtained values for the 10-HDA acid between

1.58% and 3.39%; while Kim and Lee (2010) obtained an average of 2.02%, values

which are somehow higher than the mean values from the present paper (1.88%).

Comparative chromatogram of the reference compound with one sample of royal

jelly is showed in figure 12.

00.5

11.5

22.5

33.5

44.5

L1 L2 L3 L4 L5 L6 L7 L8 L9 L10 L11 L12 L13 L14

Con

tinut

ul d

e 10

-HD

A (%

)10

-HD

A co

nten

t (%

)

Probe laptisor de matcaRoyal Jelly samples

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2 4 6 8 10 12

0

100000

200000

300000

400000

Abs

orba

nta (

mA

U)

Abso

rban

ce (m

AU)


10-HDA - s

tandar

d10-HDA - p

roba L

1

Figure 12. Comparative chromatogram of 10-HDA standard and Royal Jelly

samples (L1)

Literature data shows that royal jelly is a strong antibacterial agent. Antibacterial

properties of other bee products are attributed to polyphenolic compounds. By means of

high performance liquid chromatography coupled with a photo diode array detector, in

the present paper was tried to separate other compounds than peptides which could be

responsible for antibacterial properties. Polyphenolic compounds extraction was realized

as described in “Material and methods” chapter.

Each phenolic compound has a characteristic UV-Vis absorption spectrum. All

royal jelly samples were subjected to HPLC-PDA analysis; chromatograms were

registered at 280nm and 340nm, wavelengths specific to polyphenols.

Figure 13 represents the characteristic spectrum of polyphenolic compounds from

royal jelly sample L2. For correct assignment of these signals it is required a more

detailed structural analysis like LC-MS technique. Based on these spectra and their

similarity to the reference compounds used some signals were associated to specific

compounds.

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0 10 20 30 40 50

0

2000

4000

6000

8000

10000

Abso

rban

ta (m

AU)

Abso

rban

ce (m

AU)

Timp de retentie (min)Retention tine (min)

Figure 13. HPLC – PDA chromatogram of Royal Jelly sample L2

CHAPTER IX. RESULTS AND DISCUSSIONS REGARDING ANTIMICROBIAL

CAPACITY OF ROYAL JELLY AND APILARNIL

The antimicrobial activity of the two matrices of interest was assessed on the basis

of inhibition zone diameter of 6 international reference strains, including 2 strains of

Gram-positive bacteria: Staphylococcus aureus ATCC 6538P, Bacillus cereus ATCC

14579, three Gram-negative strains: Escherichia coli ATCC 10536, Salmonella thyphi

ATCC 14028, ATCC 27853 and Pseudomonas aeruginoasa; and one yeast strain of

Candida albicans ATCC 90028.

Antimicrobial activity of royal jelly against Gram-positive bacteria

Staphylococcus aureus ATCC 6538P ranged between 6.00mm and 16.67mm; while only

one sample of apilarnil with the highest amount of glucose (7.27%) showed inhibition

against mentioned bacteria.

Histogram of royal jelly samples distribution regarding the inhibition zone

diameter against Gram-positive bacteria Staphylococcus aureus ATCC 6538P is a normal

one and shown in figure 14.

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Figure 14. Histogram of Royal Jelly distribution according to antimicrobial activity

against Stafilococcus aureus

Gram-positive bacteria Bacillus cereus ATCC 14579 was sensitive to apilarnil, 8

samples having inhibition diameters higher than royal jelly samples.

Histogram of royal jelly samples distribution according to inhibition zone

diameter of Gram positive bacteria Bacillus cereus ATCC 14579 is a normal one (Figure

15), with the exception of sample L14 which has the highest inhibition diameter of 11.67

mm.

161284

5

4

3

2

1

0

Diemetrul zonei de inhibitie (mm) / Inhibition zone diameter (mm)

Num

arde

prob

e/N

umbe

rofs

ampl

es

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against Bacillus cereus

Gram-negative bacteria Escherichia coli ATCC 10536, seems to be the most

resistan bacteria to all bee products, since it shows the lowest inhibition diameters for

royal jelly samples, while apilarnil shows no antimicrobial activity.

Average inhibition diameter of royal jelly samples against Gram -negative bacteria

Escherichia coli is 9.02 mm, while the literature presents an average diameter of 7.7 mm

(Garcia et al., 2010) of an 80% aqueous royal jelly extract.

1110987

3,0

2,5

2,0

1,5

1,0

0,5

0,0

Diametrul zonei de inhibitie (mm) / Inhibition zone diameter (mm)

Num

arde

prob

e/N

umbe

rofs

ampl

es

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against Escherichia coli

Histogram of royal jelly samples distribution regarding inhibition zone diameter of

Gram-negative bacteria Escherichia coli ATCC 10536 is normal and shown in figure 16,

with two exceptions, samples L9 (7.00 mm) and L14 (11.00 mm).

Royal jelly samples showed a higher inhibition zone diameter when were tested

against Gram-negative bacteria Pseudomonas aeruginosa, (values between 8.33 mm and

10.33 mm), while apilarnil samples registered values of the inhibition zone diameter

between 6.33 mm and 7.33 mm.

1110987

6

5

4

3

2

1

0

D iamet rul z onei de inhibit ie (mm) / Inhibit ion z one diameter (mm)

Num

arde

prob

e/N

umbe

rofs

ampl

es

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against Pseudomonas aeruginoasa

Five royal jelly samples had antimicrobial activity against the yeast Candida

albicans (Figure 18). Apilarnil samples had no antimicrobial activity against mentioned

yeast.


against Candida albicans

10,09,59,08,58,0

9

8

7

6

5

4

3

2

1

0


Num

arde

prob

e/N

umbe

rofs

ampl

es

1211109876

2.0

1.5

1.0

0.5

0.0


Num

arde

prob

e/N

umbe

rofs

ampl

es

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CHAPTER X. GENERAL CONCLUSSIONS

According to proposed objectives of the present PhD thesis entitled “Biological

properties evaluation of the quality markers from Royal jelly and apilarnil” the

following could be concluded:

1. Blind sampling showed a normal distribution which conducted to a general

characterization of royal jelly and apilarnil.

2. Physico-chemical composition of royal jelly and apilarnil was determined by

means of water content, ash, acidity and lipids content.

3. Quality markers of royal jelly and apilarnil were determined by total proteins

content, polyphenols and sugars profile assessment.

4. Humidity is a first indicator of royal jelly and apilarnil quality, due to the fact

that these bee products have the highest water content.

5. Proteins profile is a characteristic indicator of pollen consumed by bees and

also an indicator of floral origin.

6. HPLC-IR technique used for sugars characterisation shows the glucidic profile

of royal jelly and apilarnil.

7. Seven sugars were identified in royal jelly samples, most predominant being

glucose, fructose and sucrose. The last one was present in only one apilarnil

sample.

8. The most important quality marker of royal jelly is 10-HDA acid, responsable

for most therapeutical effects. The acid was not identified in apilarnil samples.

9. All royal jelly and apilarnil samples showed antioxidant activity assessed by

means of radical scavenging activity (DPPH method) and total antioxidant

potential (FRAP method).

10. Variability of total polyphenols content and antioxidant capacity of both

studied matrices resides in the specificity of each sample.

11. Totalpolyphenols content can not be considered a major criteria in assessing

antioxidant capacity. Vitamins and volatile compounds could also have a major

contribution.

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12. Polyphenloic profile study by means of high performance liquid

chromatography allowed identification of 11 compounds, p-coumaric acid and

t-cinnamic acid being most present.

13. All royal jelly samples showed in vitro antibacterial activity against all studied

Gram-positive bacteria, and selected activity against Gram-negative ones.

14. A few royal jelly samples (five) showed antimicrobial activity againt the yeast

Candida albicans, while apilarnil samples showed no activity against the

mentioned yeast.

15. 10-HDA acid is mostly responsible of antimicrobial activity of royal jelly

assessed in vitro.

16. Apilarnil samples showed selective antibacterial activity against Gram-positive

bacteria and almost none against Gram-negative ones.

17. Methods employed in the present PhD thesis are simple, cheap and offer

reliable results

18. Physico-chemical parameters of royal jelly and apilarnil is the main step in

establishing quality criteria of studied products.

REZUMAT

53

ORIGINAL ELEMENTS

1. Polyphenolic profile characterisation of Royal jelly and apilarnil by means of

spectophotometric methods and liquid chromatography technique (HPLC - PDA).

2. Identification and quantification of 10-hydroxi-2-decenoic acid from royal jelly

by means of high performance liquid chromatography technique (HPLC - PDA).

3. Antioxidant activity assessment of both studied matrices by means of radicals

scavenging activity (DPPH method) and total antioxidant potential (FRAP

method).

4. Antimicrobial capacity assessment of royal jelly and apilarnil against six

international bacterial strains.

5. Establishment of quality markers (water content, ash content, lipids content,

sugars content, polyphenols content, antioxidant activity, antimicrobial activity)

for royal jelly and apilarnil.

RECOMMENDATIONS AND PERSPECTIVES

1. Extension of the present study on country level and quality markers proposal for

royal jelly and apilarnil befor using them in cosmetic and pharmaceutical industry.

2. Identification of 10-hydroxy-2-decenoic acid from royal jelly is a quality marker

which needs to be analyzed always since it is responsible of therapeutical effects

of royal jelly.

3. Use of Royal jelly and apilarnil for therapeutical pouposses only after quality

control.

REZUMAT

54

BIBLIOGRAPHIC REFERENCES

1. Boselli E. , Maria Fiorenza Caboni, Anna Gloria Sabatini, G. L.

Marcazzan, G. Lercker, 2003, Determination and changes of free amino acids

in rozal jellz during storage, Apidologie 34, pag. 129 – 137;

2. Eshraghi S., F. Seifollahi, 2003, Antibacterial Effects of Royal Jelly on

Different Strains of Bacteria Iranian J Publ Health, Vol. 32, No. 1, pp.25-30

3. Gao H., N. Cheng, Q. Jia, B.N. Wang, J.J. Deng, W. Cao, (2011). In vitro

antioxidant activity of lyophilized rape royal Jelly. Food Science, 32(21), 52-55.

[only in Chinese]

4. García Mariana Celeste, Mónica Silvia Finola, and J. M. Marioli, 2010,

Antibacterial activity of Royal Jelly against bacteria capable of infecting

cutaneous wounds Journal of ApiProduct and ApiMedical Science 2 (3): 93 - 99

5. Garcia-Amoedo Luis Henrique and Ligia Bicudo de Almeida-Muradian,

2007, Physicochemical composition of pure and adulterated royal jelly, Quim.

Nova, 30, 2, 257-259.

6. Ilieşiu N. V., 1991, Apilarnil, Editura Apimondia, Bucureşti

7. Kim Joonyeong and Jongseok Lee, 2010, Quantitative analysis of trans–10-

hidroxy-2-decenoic acid in Royal Jelly products in USA by high performance

liquid cromatography, Jornal of Apicultural Science 54(1), 77-85.

8. Lercker G., E. Boselli, M. F. Caboni, Anna Gloria Sabatini, G. L.

Marcazzan, 2003, Determination and changes of free amino acids in royal jelly

during storage, Apidologie, 34, 129-137.

9. Liu J-R, Y. C. Yang, L. S. Shi, C. C. Peng, 2008, Antioxidant Properties of

Royal Jelly Associated with Larval Age and Time of Harvest. J Agric Food

Chem, 56: 11447–52.

10.Nagai T., M. Sakai, R. Inoue, H. Inoue, N. Suzuki, 2001, Antioxidative

activities of some commercially honeys, royal jelly, and propolis Food

Chemistry, 75, 2, 237-240(4).

REZUMAT

55

11.Pourtallier J., R. Davico, M. C. Rognone, 1990, Les analyses and le controle

de pureté de la geleé royale, L’Abeille de France, 753, 405-407.

12.Ross L., 2009, An Evaluation of the Antioxidant and Antimicrobial Properties

of Bee Products Commercially Available in the UK. Masters

Dissertasion,http://chesterrep.openrepository.com/cdr/bitstream/10034/94567/4/

chapter%203.pdf

13.Sabatini Anna Gloria, L. G. Marcazzan, M. F. Caboni, S. Bogdanov and

Ligia Bicudo de Almeida-Muradian, 2009, Quality and Standardisation of

Royal Jelly. Journal of ApiProduct and ApiMedical Science 1(1):1-6.

14.Sesta, G., 2006, Determination of sugars in royal jelly by HPLC. Apidologie

37: 84-90

15.Stângaciu S, 1999, Apiterapy course notes. Constanţa Apiterapy Research

Hospital, Bucuresti.

16.Takenaka, T., 1984, Studies on proteins and carboxilic acid in royal jelly. Bull.

Fac. Agr. Tamagawa Univ. 24: 101-149.

BIOLOGICAL PROPERTIES EVALUATION OF THE QUALITY … · REZUMAT 31 research. Research in this field...

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