UNIVERSITY OF AGRICULTURAL SCIENCES AND VETERINARY

MEDICINE CLUJ-NAPOCA

DOCTORAL SCHOOL

FACULTY OF ANIMAL HUSBANDRY AND BIOTECHNOLOGIES

Eng. LAVINIA IOANA BRNUIU (TOMO)

BIOLOGICAL PROPERTIES EVALUATION OF THE

QUALITY MARKERS FROM

ROYAL JELLY AND APILARNIL(SUMMARY OF PhD THESIS)

SCIENTIFIC COORDINATOR

Prof. Eng. Liviu alexandru MRGHITA, 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 (Ilieiu, 1991). First description

of apilarnil composition was presented by Stngaciu (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