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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
REZUMAT
29
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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30
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
REZUMAT
31
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.
REZUMAT
32
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.
REZUMAT
33
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.
REZUMAT
34
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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36
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
and apilarnil samples
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(%
)
REZUMAT
37
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
and apilarnil samples
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)
Apilarnil/ApilarnilLaptisor de matca/Royal Jelly
8
7
6
5
4
3
2
1
Cont
inut
delip
ide
(%)/
Lipi
dsco
nten
t(%)
REZUMAT
38
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
and apilarnil samples
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).
Apilarnil/ApilarnilLaptisor de matca/Royal Jelly
17.5
15.0
12.5
10.0
7.5
5.0
Proi
tene
tota
le(%
)/To
talp
rote
in(%
)
REZUMAT
39
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.
Apilarnil/ApilarnilLaptisor de matca/Royal Jelly
70
60
50
40
30
20
Polif
enol
itot
ali(
%)/
Tota
lpol
yphe
nols
(%)
REZUMAT
40
Figure 7 . Boxplot graph regarding antioxidant potential of royal jelly
and apilarnil samples
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.
Apilarnil/ApilarnilLaptisor de matca/Royal Jelly
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)
REZUMAT
41
Figure 8. Boxplot graph regarding antioxidant activity of royal jelly
and apilarnil samples
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.
Apilarnil/ApilarnilLaptisor de matca/Royal Jelly
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)
REZUMAT
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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
REZUMAT
43
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
)
Timp de retentie (min)Retention time (min)
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.
REZUMAT
44
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
REZUMAT
45
2 4 6 8 10 12
0
100000
200000
300000
400000
Abs
orba
nta (
mA
U)
Abso
rban
ce (m
AU)
Timp de retentie (min)Retention time (min)
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.
REZUMAT
46
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.
REZUMAT
47
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
REZUMAT
48
Figure 15. Histogram of Royal Jelly distribution according to antimicrobial activity
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
REZUMAT
49
Figure 16. Histogram of Royal Jelly distribution according to antimicrobial activity
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
REZUMAT
50
Figure 17. Histogram of Royal Jelly distribution according to antimicrobial activity
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.
Figure 18. Histogram of Royal Jelly distribution according to antimicrobial activity
against Candida albicans
10,09,59,08,58,0
9
8
7
6
5
4
3
2
1
0
Diametrul zonei de inhibitie (mm) / Inhibition zone diameter (mm)
Num
arde
prob
e/N
umbe
rofs
ampl
es
1211109876
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
REZUMAT
51
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.
REZUMAT
52
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.