Bees
Much research on bees is being done around the world, especially in the United States. Bees are
being studied both because of their ecological importance and the opportunities to benefit from them
commercially. In Finland, bee research concentrates on pollination and the protection of plants and
bees.
Figure 1. A bee on a purple coneflower (Finnish Beekeepers' Association).
Bee semiochemicals
Bees are social insects, so naturally their lives are regulated by a large and chemically diverse group
of semiochemicals. These include esters, carboxylic acids, terpenes, ketones and alcohols. Literature
on bee semiochemicals has been published since the 1950s. Over the past few years, however, it has
been observed that the chemical communication of bees is much more complicated than had been
thought. This complexity can be attributed to the synergistic effects of many pheromones and the use
of a single pheromone for several purposes at once. Additional research on chemical communication
in bees is needed.
Some of the semiochemicals used by honey bees, and the behavioral effects associated with them,
have been compiled in Table 1. Many of the semiochemicals that honey bees use as alarm
pheromones are esters and alcohols. Chemical communication in bees is largely centered around the
queen.
Table 1. Some honey bee semiochemicals and their behavioral effects.
Number
Chemical name
Effect
1
2
3
4
5
(E)-9-oxo-2-decenoic acid
(E)-9-hydroxy-2-decenoic acid
Methyl-4-hydroxybenzoate
4-hydroxy-3-methoxyphenylethanol
Queen mandibular pheromone (QMP)
Slows down the metamorphosis of a
worker bee from larva to adult and
decreases the amount of growth hormone
in the hemolymph
5
6
QMP
Linolenic acid
Decreases the fertilized queen bee's
attraction to the worker bees
1
(E)-9-oxo-2-decenoic acid
Inhibits growth hormone biosynthesis in
worker bees
5
QMP
Increases worker bees' hive-building
behavior
5
QMP
Attracts drones on mating flights
5
7
8
9
10
QMP
(Z,E)-3,7-dimethyl-2,6-octadienal
(E)-3,7-dimethyl-2,6-octadien-1-ol
Nerolic acid
Geranic acid
Attracts the swarm
at short or long distances
11
12
13
14
Methyl (Z,Z)-9,12-octadecadienoate
(Z,Z)-9,12-octadecadienoate
(Z)-9-octadecenoate
n-hexadecanoate
Causes worker bees to protect larval cells
15
16
Ethyl-(Z)-9-octadecenoate
Methyl hexadecanoate
Stimulates development of worker bees'
lower pharyngeal glands
16
Methyl hexadecanoate
Increases the weight of queen bee larvae
17
18
19
20
21
22
23
Isopentyl acetate
2-nonanol
Hexyl acetate
Butyl acetate
Benzyl acetate
Octyl acetate
1-butanol
Alarm pheromones that
1) alter flight activity
2) increase recruitment
3) assist with locating a site
Isopentyl acetate
Isopentyl acetate (C7H14O2) (see Figure 2) is one example of a pheromone. It is an acetate ester
pheromone and is one of dozens of honey bee pheromones (see Table 1). It has been known since
the 1960s that isopentyl acetate acts as an alarm pheromone for honey bees.
O
O
Figure 2. Molecular model and structural diagram of isopentyl acetate.
Chemical and physical properties of isopentyl acetate
Isopentyl acetate is a clear ester with a pleasant aroma. It is also called banana oil because of its
characteristic strong, banana-like odor. Isopentyl acetate has a molecular weight of 130.2 g/mol and
is a highly volatile compound. Volatility is typical of alarm pheromones. The boiling point of
isopentyl acetate is 142 °C. Other physical properties of the compound are shown in Table 2. The
compound is also known as isoamyl acetate and 3-methylbutyl acetate (FAO 1997, IPCS 2005).
Table 2. Chemical and physical properties of isopentyl acetate.
M
Boiling point Melting
(g/mol)
(°C)
point (°C)
130.2
142
-79
Density
(g/cm3)
0.876
Refractiv
e
index
1.4020
Solubility in water
(g/100 mL)
0.2
Chemical synthesis of isopentyl acetate
IPA can be synthesized in many different ways. The most common method is to synthesize it from
isoamyl alcohol and acetic acid using acid-catalyzed Fischer esterification, for example, which is an
important organic reaction. Figure 3 shows the chemical equation of the synthesis, and Figure 4
illustrates the reaction mechanism.
CH3
O
H3C
OH
CH3
O
+
H
+
Isoamyylialkoholi
Etikkahappo
CH3
O
H3C
OH
H3C
+
H2O
Isopentyyliasetaatti
Vesi
Figure 3. The chemical reaction equation.
+
OH
O
1
+
+
OH
H
OH
+
OH
HO
2
+
OH
OH
O
OH
+
H
HO
HO
OH
3
OH
+
O
O
+
+
H
+
H2O
+
H
H
HO
OH
+
4
O
+ HO
2
5
+
+ HO
2
OH
H
O
OH
OH+
O
O
OH+
6
O
O
O
Figure 4. The reaction mechanism.
+
There are six steps in the reaction (Figure 4). Steps 1, 3, 4 and 6 are acid-base reactions in which
rapid proton transfer takes place. In steps 2 and 5, bonds between carbon and oxygen are formed and
broken. The activation energies of steps 2 and 5 are much higher than those of the proton transfer
steps. Acid-catalyzed esterification has the advantage of being a simple process, but the high
temperature and strong acid catalyst required can make it an impossible reaction to carry out with
sensitive substrates. The synthesis has an 80% yield. Simple distillation in an oil bath at a boiling
point of 142 °C is sufficient to separate the product from the final mixture.
IR spectrum
The –C–CO2R stretch characteristic of an ester is visible in the isopentyl acetate IR spectrum (Figure
5) in the 1735–1745 cm-1 range. The –C–H stretches are visible just below 3000 cm-1, and the –C–O
and –CO2 stretches appear as several peaks in the 1050–1300 cm-1 range (Hase 1992).
Figure 5. IR spectrum of isopentyl acetate.
Isopentyl acetate occurrence in nature and applications
In addition to functioning as an alarm pheromone in honey bees, naturally occurring isopentyl
acetate also acts as a pheromone and kairomone in some flies and as a pheromone, kairomone and
allomone in some beetles.
Isopentyl acetate is also used as an artificial sweetener in foods and beer, for example, and as an
artificial perfume to mask unpleasant odors in products such as shoe polish.
Bee venom
The toxin produced by honey bees has long been of interest to humans. Bee stings are painful and
cause allergic reactions that can even be life-threatening in hypersensitive individuals. The body's
response to bee venom aroused interest in the possibility of medical applications for it. Bee venom
research began in 1897. According to the first study, the venom was secreted by the acid gland and
consisted primarily of formic acid. This erroneous information was printed in chemistry textbooks
for a long time, resulting in the widespread misconception about the venom's composition. This
section on bee venom introduces the chemical properties and composition of the venom produced by
honey bee workers and examines the mechanisms of its pharmacological effects on humans.
The properties and composition of bee venom
Honey bee venom is odorless, clear and water-soluble. The dried venom is light yellow in color,
although some commercial preparations are brown due to oxidation of some of the venom proteins.
A bee's venom is produced by the venom gland and Dufour's gland and stored in the venom sac. The
venom is 88% water. The remaining 12% contains enzymes, proteins, peptides, physiologically
active amines, amino acids, carbohydrates, phospholipids and volatile ingredients (see Table 3). Due
to the proteins it contains, the venom is known as a "protein venom." One bee yields 0.15–0.30 mg
of venom at a time.
Table 3. Composition of solid constituent of honey bee worker venom as percentages and
nmol/sting.
Class
(Crane 1990, 466)
Compound
(O'Connor & Peck 1980,
Crane 1990, 466)
Enzymes
Phospholipase A
Hyaluronidase
Acid phosphomonoesterase
Lysophospholipase
α-glucosidase
Proteins and peptides
Melittin (peptide consisting of 26
amino acids)
Apamin
MCD (mast cell degranulating)
peptides
Secapin
Procamine
Adolapin
Protease inhibitors
Tertiapin
Amines
Carbohydrates
Amino acids
Histamine
Dopamine
Norepinephrine
Glucose and fructose
t-aminobutyric acid
α-amino acids
Phospholipids
Volatile ingredients
%
(O'Connor &
Peck 1980,
Crane 1990,
466)
15–17
10–12
1–3
1.0
1.0
0.6
48–58
40–50
1–3
2
0.5–2
1–2
1.0
0.1, 0.8
0.1, 13–15
3
0.5–2.0
0.13–1.0
0.1–0.7
2
0.8–1.0
0.4, 0.5
1
5
4–8
nmol/sting
(Crane
1990, 466)
0.23
0.03
0.03
10–12
0.75
0.6
0.13
2
0.06
0.07
0.003
5–10
2.7–5.5
0.9–4.5
Pharmacological effects and uses of bee venom
The pharmacological effects of bee venom on humans have been surveyed by examining both the
complete venom and the individual and combined effects of components isolated from it. According
to one system of classification, the venom's effects on humans can be assigned to one of four levels
(see Table 4).
Table 4. Four levels of venom effects in humans (Fitzgerald & Flood 2006, O'Connor & Peck 1980,
Koulu & Tuomisto 2001, 277–278).
Level
Active substances
What happens?
1
MCD peptide (mast
cell degranulating
peptides)
Protease inhibitors protect hyaluronidase from
enzymatic destruction. Hyaluronidase breaks down
hyaluronic acid into polymers that function as
intracellular reinforcement. This enables the venom to
spread into tissues more easily. MCD peptides
degranulate dermal mast cells, which then release
histamine. Histamine, in turn, acts as a vasodilator and
increases the secretory activity of numerous glands.
Protease inhibitors
Small peptides
Hyaluronidase
2
Phospholipase A
Melittin (see Figure
6), the most
important active
substance in the
venom
As the venom infiltrates blood vessels to enter the
circulatory system, phospholipase A and melittin
cause red blood cells to disintegrate. Melittin destroys
a red blood cell by binding to the cell membrane. The
location of hemolysis in the body can usually be
identified unless a person has been stung in several
places. Phospholipase A and melittin can also cause
an allergic reaction.
3
Apamin
MCD peptide
Phospholipase A
Melittin
If someone has been stung several times, more of the
venom circulates in the body, leading to a more severe
toxic reaction. Apamin acts as a toxin on the central
nervous system, phospholipase A and melittin destroy
more red blood cells, and more histamine is released
by the action of MCD peptide.
4
Phospholipase A
Melittin
Hyaluronidase
Any of these venom components can cause an allergic
reaction in a hypersensitive individual, possibly
leading to anaphylactic shock.
Figure 6. Three-dimensional structure of melittin (Protein Data Bank, PF01372,
http://www.rcsb.org/pdb/explore.do?structureId=2MLT).
In humans, a fatal dose (LD50) is 2.8 mg of venom per kilogram of body weight. Mortality is also
affected by allergy and the location of stings on the body. The most common causes of death are
allergic reaction, cardiac arrest or suffocation. Stings in the mouth or on the neck are the most
dangerous.
Bee venom is used to manufacture lotions, balms, ointments and injected preparations. Good
treatment outcomes have been achieved in the use of bee venom for chronic pain, hearing disorders,
trauma, scars, multiple sclerosis, psoriasis and various rheumatic illnesses.
Honey
Honey was the first bee product to be used by humans. Honey was used just as a food source at first,
but many commercial interests have since developed around it. Aside from providing nourishment,
honey also has medicinal applications.
Honey is produced from dried nectar in the honey stomach of bees through the action of digestive
enzymes, glandular secretions and saliva. In the honey stomach, water evaporates from the nectar
and invertase enzyme transforms nectar sucrose into fructose and glucose.
The properties and composition of honey
Honey is a viscous fluid. Its viscosity is dependent on temperature and water content (see Table 5).
Honey varies in color from light to dark amber (see Figure 7). Not all of the compounds that
determine the color of honey are known, but they can be divided into water- and lipid-soluble
substances. Light-colored honey has fewer water-soluble compounds than lipid-soluble ones,
whereas dark-colored honey has fewer lipid-soluble compounds and is also darkened by the
oxidation of compounds. Tannins in honey, for example, precipitate and bind proteins when exposed
to air, thus altering the color. The color changes during storage, too, as reducing sugars react with
nitrogenous compounds or fructose decomposes in the acidic solution.
Figure 7. Colors of honey (Finnish Beekeepers' Association).
Table 5. Variation in honey viscosity depending on water content and temperature.
Water content, %
Temperature, °C
13.7
15.5
18.2
20.2
-
13.7
20.6
29.0
39.4
48.1
71.1
Viscosity, 1 P
(1 poise = 0.1 Ns/m2)
420
138
48
20
600
189.6
68.4
21.4
10.7
2.6
Density is another important physical variable of honey to consider because of its significance
during transport, for example. The density of honey is influenced by its water content (see Table 6).
Honey is also a strongly hygroscopic substance due to its high sugar concentration.
Table 6. Effect of water content on the density of honey (at 20 °C).
Water content, %
13.0
17.0
21.00
Density, kg/m3
1.4457
1.4237
1.3950
Honey is composed mainly of sugars, water, organic acids, minerals, nitrogenous compounds, ash
and enzymes (see Table 7). Sugars account for 90–95% of the dry weight of honey. Honey is used as
a food since it contains mostly fructose and glucose. Water content is an important consideration for
storage because honey can be stored without danger of fermentation if the water content is less than
18%. Organic acids, a side product of enzymatic digestion in the honey stomach of the bee, give
honey its characteristic flavor and an acidic pH. The pH of honey ranges from 3.42 to 6.10. There
are few trace elements in honey, potassium being the most abundant. Nitrogenous compounds and
enzymes (glucose oxidase) have an important role in honey formation. Due to pollen contamination,
honey also includes traces of proteins, amino acids and water-soluble vitamins.
The enzyme activity in honey is an important criterion for determining its quality. The freshness of
honey is represented by its diastase value. The diastase value indicates how many grams of starch
are hydrolyzed by the diastase enzymes in honey during one hour at 40 °C. The quality standards for
honey require a diastase value above eight.
Table 7. Honey composition as average percentages.
Compound
Average
Standard deviation
Range
Fructose
Glucose
Sucrose
Maltose
Higher sugars
Water
Free acids
(gluconic acid)
Lactones
Total acids
Ash
Nitrogen
38.2
31.3
2.3
7.3
1.5
17.0
0.43
2.1
3.0
0.9
2.1
1.0
1.5
0.16
27.2–44.3
22.0–40.7
0.2–7.6
2.7–16.0
0.1–8.5
13.4–26.6
0.13–0.92
0.14
0.57
0.169
0.041
0.07
0.20
0.15
0.026
0.0–0.37
0.17–1.17
0.020–1.028
0.000–0.133
Pharmacological effects of honey
Studies have found that honey inhibits bacterial growth. The antibacterial action of honey is
attributable to the effects of glucose oxidase enzyme, acidic pH, and hydrogen peroxide formed as a
side product of gluconic acid formation from glucose (see Figure 8). Glucose oxidase destroys
bacteria, acidic pH slows bacterial growth, and hydrogen peroxide has antiseptic properties.
Hydrogen peroxide oxidizes microorganisms and dissociates into water and oxygen. It is effective
against spores, fungi, viruses and bacteria. The germicidal effect of hydrogen peroxide is short-lived.
The hydrogen peroxide content of honey is approximately 1 mmol/L.
Glukoosi
+
H2O
+
O2
Glukonihappo
+
H2O2
Figure 8. Formation of gluconic acid and hydrogen peroxide from glucose.
Beeswax
Waxes are esters formed from long-chain carboxylic acids and long-chain alcohols. Hydrolysis of
beeswax (see Figure 9) yields straight-chain carboxylic acids of lengths C26 and C28 and straightchain alcohols of lengths C30 and C32 (Streitwieser et al. 1998, 543–544, and others).
O
C25-27 H51-55 CO C30-32 H61-65
Figure 9. Structural diagram of beeswax.
Beeswax is available in two forms, yellow (cera flava) and white (cera alba). Yellow beeswax is
made by melting honeycombs (see Figure 10) with hot water and filtering out impurities from the
melt. White beeswax is made by bleaching yellow beeswax with potassium permanganate, active
carbon or sunlight.
Figure 10. Honeycomb cross-section with eggs at the bottom (Finnish Beekeepers' Association).
The properties and composition of beeswax
Cera flava is yellow to brown in color, and cera alba is white to pale yellow. Both types of the wax
have similar physical properties (see Table 8). Beeswax pieces or slabs have a fine consistency and a
matte surface, do not crystallize, and are malleable when warmed. The waxes are insoluble in water,
highly soluble (90% v/v) in heated ethanol, and fully soluble in lipids and essential oils. The density
of both waxes is approximately 0.960 g/cm3. Beeswax has no taste, does not stick to teeth, and is not
perishable. Both of the waxes have the same characteristic smell. The waxes have a melting point of
61–66 °C, an acid value of 17–24, an ester value of 70–80 and a saponification value of 87–104.
Table 8. Properties of white and yellow beeswax.
Compound
Color
Solubility
Density, g/cm3
Melting point, °C
Acid value
Ester value
Saponification
value
Cera alba
White, pale yellow
Fully soluble in lipids and
essential oils
0.960
61–66
17–24
70–80
87–104
Cera flava
Yellow, brown
Fully soluble in lipids and
essential oils
0.960
61–66
17–22
70–80
87–102
Beeswax is a mixture comprising at least 284 compounds, of which more than 111 are volatile. Not
all of the compounds have been identified. Beeswax consists mainly of esters, alcohols and acids
(see Table 9).
Table 9. Composition of beeswax. The "Major" column shows the number of compounds that form
more than 1% of the fraction, and the "Minor" column shows the number of compounds that form
less than 1% of the fraction.
Number of components in the fraction
Compounds
Hydrocarbons
Monoesters
Diesters
Triesters
Hydroxy monoesters
Hydroxy polyesters
Acid esters
Acid polyesters
Free acids
Free alcohols
Unidentified
Total
% (fraction)
Major
Minor (below 1%)
14
35
14
3
4
8
1
2
12
1
6
100
10
10
6
5
6
5
7
5
8
5
7
73
66
10
24
20
20
20
20
20
10
?
?
>210
Applications of beeswax
Beeswax does not have direct physiological effects on humans. It is therefore used as an inactive
ingredient in medicinal and cosmetic products. In pharmaceutical preparations, a drug is mixed with
beeswax to help ensure a controlled rate of dissolution, storability and safety. In the cosmetic
industry, beeswax is used to produce stable emulsions of even consistency. Beeswax is also used in
the textile, information technology and other industries.
Propolis
In a beehive, propolis acts as chemical protection against pathogenic microorganisms (see Figure
11). The medical industry is interested in the chemistry of propolis because of its biological activity.
Its pharmacological properties have been studied for about 30 years. Propolis has been used as a
medicine in the treatment of wounds, burns and sore throats, for example.
Figure 11. Propolis in a beehive (Finnish Beekeepers' Association).
The properties and composition of propolis
Propolis occurs in the form of a wax-like resin. Its melting point is usually in the 60–70 °C range,
sometimes as high as 100 °C. Propolis consists mainly of resins (40–45%), waxes and fatty acids
(25–35%), essential oils (10%), pollen (5%), and organic compounds and minerals (5%) (see Table
10) (Krell 1996, 155–156). The composition varies according to geographic location and climate,
since vegetation varies with environmental conditions. Propolis from Brazil has attracted the most
interest because of its high biological activity.
Table 10. Classes of compounds found in propolis, by percentage.
Class of compounds
%, group of compounds
Resins
45–55%
- Flavonoids
- Phenolic acids and esters
Waxes and fatty acids
25–35%
- From beeswax or plant sources
Essential oils
10%
Pollen
5%
Organic compounds and minerals 5%
- 14 trace elements, of which Fe and Zn are
most abundant
- Ketones
- Lactones
- Quinones
- Steroids
- Benzoic acid and esters
- Vitamin B3
- Sugars
Pharmacological effects of propolis
Studies have found that compounds in propolis have antibacterial, anti-inflammatory,
anticarcinogenic, and antioxidant effects, and can help protect the liver and prevent allergies (see
Table 11). The pharmacological effects of propolis are significantly influenced by the flavonoids it
contains.
Table 11. Pharmacological effects of compounds found in propolis.
Effect
Antibacterial
Anti-inflammatory
Anticarcinogenic
Liver protection
Compounds
Flavanones, flavones, phenolic acids and
esters, prenylated p-coumaric acids,
diterpenes and prenylated
benzophenones
Flavanones, flavones, phenolic acids and
esters
Prenylated flavanones, caffeic acid,
prenylated p-coumaric acids, diterpenes,
prenylated benzophenones, benzofurans
and phenylethyl ester
Flavonoids, phenylethyl ester, caffeic
acid, prenylated p-coumaric acids,
caffeylquinic acids and ferulic acids
Antioxidant
Flavonoids, prenylated benzophenones,
phenolic acids and esters, prenylated pcoumaric acids and prenylated
flavanones
3,3-dimethylallyl caffeate
Allergy prevention
* Prenylated means there is a prenyl group bound to the molecule that facilitates
attachment to a cell membrane.
There are more than eight thousand known types of flavonoid compounds. Flavonoids are natural
secondary metabolites and act as water-soluble pigments in plants. In plants, flavonoids are found
mainly in the cell sap in the form of dissolved glycoside, i.e. flavonoid glycoside (see Figure 12), but
can also occur as free sulfates or phenols. In the flavonoid glycosides, there is a glycosidic –O– bond
between the sugar and flavonoid components.
OH
OH
O
HO
O R
OH
O
R = sokeriosa
Figure 12. Basic structure of flavonoid glycoside (Hiltunen & Holm 2000, 260).
Flavonoids have extensive pharmacological effects. The antioxidant effect of flavonoids is based on
preventing the formation of hydrogen peroxide and organic peroxides that oxidize cells. The lipid
peroxidation–inhibiting effect of flavonoids is enhanced by hydroxyls in the C-3´ and C-4´ positions
(see Figure 13). The lipid oxidation–inhibiting effect requires the flavonoid structure to have a
carbonyl in the C-4 position and hydroxyls in the C-3 and C-4´ positions, and the aryl group must be
free to rotate. In nearly 80% of flavonoids, there is a methoxy or hydroxyl group as a substituent in
the C-4´ position of the aryl group, and in approximately 50% in the C-3´ position.
hydroksyyli C-4 asemassa
OH
5
OH
6
O
HO
7
1
8
2
6
3
5
4
2
OH
4
hydroksyyli C-3 asemassa
O
OH
OH
3
1
karbonyyli C-4 asemassa
Figure 13. Myricetin.
In addition, the antioxidant activity of flavonoids influences their anti-inflammatory and liver
protectant effects. Flavonoids also suppress inflammation by inhibiting prostaglandin synthesis. In
prostaglandin synthesis (see Figure 14), arachidonic acid is transformed by 5-lipoxygenases and
prostaglandins into products that participate in inflammatory response.
5
CO2H
8
1
O
11
8
14
20
11
1
5
CO2H
O
14
20
OH
tromboheksaani
arakidonihappo
5-lipo-oksigenaasi
tulehdusta aiheuttavat aineet
Arakidonihappo
- esim. tromboheksaani
Prostaglandiini
HO
5
8
CO2H
1
11
HO
14
20
prostaglandiini
Figure 14. Simplified model of prostaglandin synthesis.