Apiacta 3, 2001

CHARACTERISTICS AND PROPERTIES OF THE APIS MELLIFERA APITOXIN AS THERAPEUTIC POTENTIAL; USE AND LIMITS

G. SALAMANCA GROSSO1; Carmen Rosa PEREZ FIGUEREDO2

1Departamento de Química – Facultad de Ciencias, Universidad de Tolima 2Escuela de Ciencias Químicas, Universidad Pedagógica y Tecnológica de Colombia,

Tunja Boyacá, COLOMBIA

Key words: apitoxin / apitherapy / Apis mellifera / therapy

Introduction There are many bee races in Colombia, among them being distinguished Apis mellifera mellifera L., 1758, or the European bee. It is a large bee, with a short tongue (5.7 to 6.4 mm), a wide abdomen, the quitine colour being very dark and uniform, partially with small yellow spots on the second and third tergite, but without yellow stripes. Its body is covered with long hairs. Generally, nervous in the open air, it quickly abandons the comb. It is frequently aggressive, but not always. Apis mellifera ligustica Spinola, 1806, or the Italian bee. Its size is something lesser than mellifera, its abdomen is thin, and the tongue relatively long (6.3 to 6.6 mm). The quitine colour on its abdomen is something lighter at the sternum level, as well as in the first two to four tergites (yellow stripes in the front part). Its quietness on the comb is variable, but generally good. Usually it is gentle. Apis mellifera carnica Pollmann, 1879, or the carniolan bee: generally, it’s very similar to the Italian (ligustica) one. It is thin, with a long tongue (6.4 to 6.8 mm). Its hairs are short and thick (the gray bee). Ac-cording to Brother ADAM (1966), it is the quietest and gentlest bee race: “Combs can be left outside of the hive for a long time, and if the strain is good, not even one will abandon the comb”. Apis mellifera caucasica Gorb, 1758: the shape and size of the body and hairs are very similar to those of the carniolan bee. The quitine is dark in colour, but frequently has brown spots on the first stripe on the abdomen. It is gentle and quiet on combs. Apis mellifera scutellata, 1804, or the African one: it is the newest race that came to Columbia, and it is characterized by its short tongue (5.9 to 6.24 mm), and its yellow stripes on the four front tergites. It shows an accented tendency to swarming , which is added to its defense capacity, that is increased by the facility with which it is excited; it is a good forager. The effect of the bees’ africanization is a result of that phenome-non’s expansion starting from Brazil. This hybrid shows yellowish tones; it is the smallest bee of its species, with short hairs and tongue. The defensiveness degree can be very variable in Africa. The hybridizations gave birth to a bee with high defensibility at F2, and the honey production is 30% higher. The study of the apitoxin therapeutic properties in our environment has not reached the necessary level among physicians, that is why the general properties of this product are still unknown, in spite of the fact that just in the tropical environment there was initiated the knowledge of some animals’ activity, if we are to refer to the origin, and, for this reason, the present paper will expose the general characteristics of these insects’ toxin, trying to render evident its virtues. This work illustrates a photographic sequence of 24 hours, that show the toxine effect generated by a sole bee. The sequence belongs to the author.

Composition and properties The apitoxin, produced by the worker bees, that the same inject through their sting when they are disturbed or see their hive in jeopardy, is composed of three different substances: one inflammatory, other convulsive, and the last one, paralysing. This apitoxin contains, among other elements, magnesium, phos-phorus, calcium, and proteins. There are well known the therapeutic properties of that apitoxine against artri-tis, ischias, lumbago, osteoarthrosis, eczema, and certain skin diseases. The treatment consists of the direct sting of a certain number of bees, or in apitoxine injections, obtained from the stings. The toxin of bees, also known as apitoxin, from the Latin apis, bee, and the Greek toxikon, poison, is a matter secreted by two glands, one of an acid nature, and other alkaline, incorporated in the abdomen of the worker bee, and which is introduced under the skin in proportion of 0.3 mg at each sting, through a very well known sting. The bee sting consists of a lancet 2 mm long, sharp at its tip, that grows by 0.1 mm in its diameter. The lancet has more small teeth, some of them 0.03 mm long. The teeth serve for retaining the liquid inside the object the bee has stung. The lancet penetrates the stung object up to half its length. Bee apitoxin is a translucid liquid, with a pronounced fragrance of honey and a bitter taste. The liquid can be considered a violent endothelial apitoxin, and a strong stimulant of the smooth muscles as well, but it also can be designated as a general protoplasmic apitoxin. Its main characteristics are the following: ap-pearance: a pale, transparent liquid, with a bitter taste and aromatic smell. Its properties are presented in Table 1.

Apiacta 3, 2001

Table I General properties of the Apis mellifera apitoxin

Parameter Characteristic Specific gravity 1.1316 pH Acid reaction Solubility In water. Reaction of insolubility in alcohol. Aqueous solutions are unstable, with

marked decomposition by effect of the bacterial activity. It is proven its solubility in glycerin.

Thermic effects Units ? with rapid drying at the environment temperature. It tolerates 100°C during 1 hour, or units ? during 10 days, without losing their capacity.

Chemical stability It shows units with reducing activity against potassium carbonate, potassium bichro-mate, bromine, chlorine, and hydrogen peroxyde. It is denaturated by ammonia, picric acid, and potassium bichromate.

Enzymatic stability Pepsines, pancreatine, renine, and the vegetable papain and papayotin dimish its activity.

Conventional units By an only sting, a bee brings in about 0.0010 mg of dried apitoxin, that is 0.25 to 0.35 mg of liquid apitoxin. That is called conventional unit.

The Apis mellifera toxin contains about 90% of humidity, there being also contained small proteins and molecules of histamine and melitine (50%), lysolecitine, and apamine (1 to 3%). The enzymes that are showing more interest are the A2 phospholipase (10 to 12%), and the hyaluronidase (1 to 3%). The environ-ment is an acid one, because of the formic acid levels, the presence of chlorides, and of the phosphate ions. There was demonstrated the presence of choline, a neurotransmitter, and of the tryptophane, of some mi-croelements, such as magnesium, iron, iodine, potassium, among others. It also contains secapine (0.5 to 2.0%), DCM peptide (1 to 2%), tertiapine (0.1%), procamine (1 to 2%), dopamine (0.2 to 1.0%), noradrenalin (0.1 to 0.5%), t-aminobutyric acid, glucose, fructose, phospholipids, and alerting pheromones. There was stated the therapeutic properties of apitoxin are due to the presence of magnesium and phosphate ions, that represent equivalent levels of 0.4% of dried matter.

Table II Characteristics of the main components of apitoxin

Components Observations Mellitin It is the fraction F1, formed by 13 amino-acids: leucin, glycin,

alanin, isoleucin, treonin, lysine, arginin, glutamic acid and other. The F1 fraction possesses hemolytic activity.

Phospholipase A and hyaluronidase It is the fraction F2, formed by 18 amino-acids, especially lysine and arginin. The F2 fraction possesses an indirect hemolytic effect.

Proteins Adolapin, apamin, catecholamin, dopamin, histamine, histidine, noradrenalin, 401 peptide, MDP peptide, secapin, and tetrapin.

Apis mellifera, in the tropical conditions, only attacks when it is provoked, or when it encounters physical stimuli produced by strange sounds, colours or smells. In case of severe accidents, the level of mul-tiple stings generally cause the death of the stung person, the outcome depending greatly of the sensitivity degree of the same; in the slight cases, usually allergic reactions appear, but those can also be severe, in function of age and sex of the stung persons. Pharmacodynamic activity Mellitin. Produces the most part of the general and local toxicity, attacks the neuro-muscular and ganglionary synapses, causes respiratory paralysis and hemolysis, is responsible for pain and inflammation, inhibits the colynesterase activity, and coagulates the fibrinogen, increases the capillary permeability, con-tracts the smooth muscles, frees the histamines, and diminishes the superficial activity. It is very thermosta-ble; its activity is not lost at 100°C, during 1 hour. The A2 phospholipase (A lecithinase) transforms the non-saturated fatty acids of the lecithin into lysolecithin. It prevents the coagulation of blood and induces the prostaglandines liberation. It produces the indirect hemolysis, inhibits the electrolytic transport, as well as the oxidative phosphorilation, and possesses antigenicity. Hyaluronidase. The hyaluronic acid in the conjunctive tissue facilitates the apitoxine penetration. It is a liberation factor, and also possesses antigenicity. Adolapin exerts a soothing effect on pain 80 times higher than morphine and opium. Apamin stimulates the secretion of heparin and produces systemic neuro-toxicity. Its activity is directed towards the central nervous system (CNS-neurotoxin). Recently there was demonstrated the vascular permeability of skin increases locally by intra-cutaneous application. Catecholamins, dopamins, and noradrenalin. They act upon the behaviour and physiology of insects, exert a strong influence upon the blood circulation, and increase the pulsation speed of the heart in the same. Given their low level, there is little probability they could affect the mammals. Histamine and sero-tonine induce pain, and cause dilatation and increasing of the blood vessels permeability, thus facilitating the

Apiacta 3, 2001

penetration of toxins into the tissue. The factors that are associated to the extensive dynamics at the organic level indicate the high levels of histamine determine the adrenaline secreting, which causes an excitation condition in mammals. The 401 peptide is a hundred times more active than the hydrocortisone. Finally, the MDP proteic unit (Mastocyte Degranulation Peptide) has a structure similar to that of apamin, that is it is af-fecting the mastocytes, frees the histamine, and increases the capillary permeability.

Therapeutic value The apitoxin potential can be validated by different effect types; in the literature there was reported

the emphasized stimulatory effect upon the immunologic system, usually manifested by the multinucleate, monocytes, macrophagous, lymphocytes T and B cells formation, beyond the fact it diminishes the protein content in the blood plasma by the variation of vessel permeability, as well the cardiac rhythm and the ar-terial pressure. Consequently, it possesses antiarhythmic properties, as suppresses the arhythmias caused by electric excitation and inoculation of strofantin. That liquid effectively influences the nervous system, blocking the transmission of stimuli to the peripheric and central synapses, improves the conduc-tion of the optic fiber impulses, and diminishes the demyelinization. During treating diseases, there is no antibodies formation against the apitoxin, and thus the human organism does not acquire habit to them, so the repeated stings or the apitoxin injections become more and more efficient in the organism. In doses close to the toxic ones, it can affect the normal regulations processes, it inhibits the descendent and as-cendent reticulary activity, and exerts a strong influence upon the higher regions of the central nervous system, especially upon the hemisphere cortex. Other properties of the product are shown in Table III.

Table III Therapeutic value of apitoxin

Activity Observations Dilatant Upon the capillaries; it accelerates and intensifies the circulation of blood. Anticoagulant Inactivation of plasmatic and tissue thromboplastine, and diminishing the thrombinic activity. Stimulatory Activates the production of endogen corticosteroids. It shows hemorrhagic effects, and influ-

ences the hipofisary system and the suprarenal cortex. It is hipotensive. Neurotropic Improves the metabolism of the central and peripheric nervous system. Suprarenal activity It suppresses the depression caused by the activity of the steroid hormones. Fibrinolytic activity It exerts a fibrinolytic activity, diminishes the prethrombotic and thromboflebitic condition. Other effects Bacteriostatic. Local anaesthetic. Antirheumatic. Activates upon the hipofiso-suprarenal system.

Dilatatory of the blood vessels. Inhibits the oedema. Stimulatory of the endorphin activity. The pharmacodynamic reaction of responding, is due to the accumulation of histamine, serotonin, and acetylcholin, with allergic reaction owed to the humoral antibodies against the allergenic factors presents in the saliva and the poison. In laboratory conditions, it is considered the lethal dose, on intravenous way, reaches 4.5 mg/kg for the rat, and 3.2 mg/kg for the rabbit. The poison is a hundred times more toxic in ver-tebrates than in the inferior animals, even if its power of activity is the same in both cases. Administering the venom, to both dog and cat, on endovenous way, generates a rapid diminishing of the arterial pressure, polypnea, intestinal hyperstaltism, and retardation in blood coagulation, attributed to the inhibition of the thromboquinase. From the above expressed it can be deducted the bee venom is a complex mix of chemical com-pounds with cito-toxic activity, such as phospholipase A, a polypeptide of the mellitine type, and the peptide apamine, among others. The phospholipase A is the main allergenic factor, that, together with the mellitine, represents 75% of the components. The apitoxin activity can be explained in virtue of its blocking effect of the respiratory system, and attaching the red corpuscles, thus causing hemolysis. There was demonstrated a mellitine injection (that is, a protein extracted from the bee poison), can entail a lowering of the arterial pres-sure, the hemolysis (that is, the destruction of the red corpuscles), a contraction of the striated and smooth muscular fibres, and suppresses the neuromuscular and ganglionary transmission. The hyaluronidase, an-other apitoxin component, also increases the permeability of the blood capillaries. The vessel permeability is definitive, as when it lowers, because of being perturbed the function of the capillary system, because of ageing or a morbid condition of the organism, it determines severe disturbs of the interchange conditions between the organs and the tissues. At present, there is well known the per-meability between the conjunctive tissue and the blood capillaries esentially depends on the fermentative activity of the hyaluronic acid, that forms part of the composition of the conjunctive tissue. (The substances that contain hyaluronidase are the following: bee venom, hirudine, ronidase, testicular extract, and spermin.) Very low doses determine a increase of that permeability. Apamin represents about 2% of the total apitoxin. It is less toxic than the phospholipase A, it be-haves like a neurotoxin with motor activity, and cardiac-stimulatory effects, similarly to adrenergic drugs, and with an anti-arhytmic effect. About 2% of the bee apitoxin could be identified as the MCD fraction (Mast Cell Degranulation), which could be very well interpreted as a degranulant factor of mastocites.

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Figure 1 - Photographic sequence, presenting the Apis mellifera apitoxin activity upon the face soft tissue, during a lapse of 24 hours.

Plates I to III, at 2 hours after the sting: local activity – moistening of the skin, localized, intense pain. Plates IV to VI: increase of the local temperature, anaesthetic activity, strong and localized endorphinic activity, 4 to 8 hours without evolution. Plates VI to IX, at 12 to 24 hours: levelling effect, diminishing of

the inflammation, localized itching (Photographs are belonging to the author).

There was noticed there are individuals that show immunity against apitoxin, and do not react to stings. It is the opposite case of the allergic ones, whose sensitivity makes to react excessively to apitoxin. There was proven that according to getting into age, the resistance is lesser. The arthritic and rheumatic pa-tients react less or at all to apitoxin. The pathologic immunity to apitoxin only is limited to rheumatism and real arthritis. It is known one of the best remedies for treating stings is drinking alcohol. Well, the alcoholics show a great resistance to apitoxin. There are four types of immunity, namely:

Congenital (very rare in humans), inherited from immune parents. Acquired (beekeepers), or active. Passive immunity, through blood and serum inoculation from an immunized man or animal. Pathologic immunity (rheumatic and arthritic patients). The rheumatic immunity is directly propor-

tional to the degree of the pathologic condition.

Therapeutic use The apitoxin application has to follow some special indications: firstly, the sting has to be removed,

taking into account the venom sac is still attached to the same. In the case of recent and slight stings, when there is no exaggerate reaction to the former, vinegar paints can be made, antihistaminic administered, and humid and cold compresses applied.

In case of stings in the distal parts of limbs, a garrot must be applied, for avoiding or delaying the toxin absorption (this should be made immediately after the sting), gluco-cortico-steroids creams can be ap-plied, as well as humid compresses with water and alcohol (1 : 1). In case of severe shock, 10 to 20 mg of calcium gluconate 20% are to be injected, on endovenous way, or the same quantity on intramuscular way. If necessary, these doses camn be repeated every 4 to 5 hours. They can be useful, as well as the adrenaline (from 0.5 to 1 mg intravenous or intramuscular), corticoids – intramuscular (25 mg of cortisone every 6 hours), as well as antihistaminic preparations – orally or on intramuscular way. Analeptics and vitamins C and K are a part of the adjuvant treatment. In most severe cases, the patient must be hydrated again, and

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oxygen has to be administered to him. The allergic reaction to the insect venom is reproductible, and repeats itself in successive situations, even aggravating itself.

The apitoxin therapy does not produce any collateral adverse effect, independently of the duration of the time for when it was applied. It is sure, effective, and only costs little money. Bees can be directly applied on the patient’s body, in order for them to sting him into the affected part of the body, or venom injections are to be used. The venom is obtained from bees, and it is kept under the form of powder in sterile containers, until it has to be dissolved. The main forms of the apitoxin application go from the direct sting by the bee, injection of standardized preparations, use of ultrasounds (by fonoforesis), ionization, mechanical friction, inhalation, and supralingual application. In conditions of treatment, direct administrations can be made, by both direct sting by the bee, and use of apitoxin through intradermic injections, or by administering ointments with apitoxin, inhalators, or tablets. The multiple sting syndrome appears in the conditions of a massive at-tack, that triggers complex reactions that can be resumed as they appear in the Figure 2.

Phospholipase

Liberation of

matters organic MCD

Peptides Diminishing of

anticholin-esterase

CNS depression

Mastolysis

Liberation of histamine

Sleepines Pain and itching

I

Peptides of the mellitin type

Anisimova l.M., Smirnov

diseases, in the XXXIVBanks, B., Possible TheraBasic I., Curic S., Tadic Z

mice, in the XXXIVth ABeaudet M., Les piqures dBeck F. Bodog, Bee VenoHealth Resources Press,

Utilización de la apitoxi

Activity onto themembrane

ntense hemolysis

Oedema

Insufficience respiratory

Cylinders in the urine appear

NTA*

IRA*

Figure 2 - Poisoning syndrome

R E F

A.A., Troshin V.D., Krilov V.N., Thth Apimondia Congress, 1995, Lausapeutic Use of a Peptide from Bee Ven., Orsolic N., Sulimanovic D., Antimepimondia Congress, 1995, Lausanne’abeille, en Revue Française d’Apicum Therapy. Bee Venom, Its Nature, a 1997 (under the name: The Biblena con fines terapéuticos, in I Bienal

Cutaneous lesions

Apamin

following multiple stings by Apis mellifera

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