Characteristics And Properties Of Apitoxin

Apiacta 3, 2001

CHARACTERISTICS AND PROPERTIES OF THE APIS MELLIFERA APITOXIN AS THERAPEUTIC POTENTIAL; USE AND LIMITS 1

G. SALAMANCA GROSSO ; Carmen Rosa PEREZ FIGUEREDO

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Departamento de Química – Facultad de Ciencias, Universidad de Tolima Escuela de Ciencias Químicas, Universidad Pedagógica y Tecnológica de Colombia, Tunja Boyacá, COLOMBIA

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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). According 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 phenomenon’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, phosphorus, calcium, and proteins. There are well known the therapeutic properties of that apitoxine against artritis, 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: appearance: 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 pH Solubility

1.1316 Acid reaction 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. Units ? with rapid drying at the environment temperature. It tolerates 100°C during 1 hour, or units ? during 10 days, without losing their capacity. It shows units with reducing activity against potassium carbonate, potassium bichromate, bromine, chlorine, and hydrogen peroxyde. It is denaturated by ammonia, picric acid, and potassium bichromate. Pepsines, pancreatine, renine, and the vegetable papain and papayotin dimish its activity. 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.

Thermic effects Chemical stability

Enzymatic stability Conventional units

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 environment 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 microelements, 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

Mellitin

Phospholipase A and hyaluronidase

Proteins

Observations

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. It is the fraction F2, formed by 18 amino-acids, especially lysine and arginin. The F2 fraction possesses an indirect hemolytic effect. 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 multiple 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, contracts the smooth muscles, frees the histamines, and diminishes the superficial activity. It is very thermostable; its activity is not lost at 100°C, during 1 hour. The A2 phospholipase (A lecithinase) transforms the nonsaturated 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 neurotoxicity. 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 serotonine 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 affecting 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 arterial 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 conduction 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 ascendent 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

Dilatant Anticoagulant Stimulatory Neurotropic Suprarenal activity Fibrinolytic activity Other effects

Observations

Upon the capillaries; it accelerates and intensifies the circulation of blood. Inactivation of plasmatic and tissue thromboplastine, and diminishing the thrombinic activity. Activates the production of endogen corticosteroids. It shows hemorrhagic effects, and influences the hipofisary system and the suprarenal cortex. It is hipotensive. Improves the metabolism of the central and peripheric nervous system. It suppresses the depression caused by the activity of the steroid hormones. It exerts a fibrinolytic activity, diminishes the prethrombotic and thromboflebitic condition. 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 vertebrates 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 compounds 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 pressure, 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, another 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 permeability 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 behaves 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.

Apiacta 3, 2001

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 patients 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 proportional 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 applied, 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

Apiacta 3, 2001

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 attack, that triggers complex reactions that can be resumed as they appear in the Figure 2.

Peptides of the mellitin type

Phospholipase

Liberation of organic matters

MCD Peptides

Diminishing of anticholinesterase

CNS depression

Mastolysis Sleepines Activity onto the membrane

Liberation of histamine

Pain and itching

Intense hemolysis

Cylinders in the urine appear

Oedema Insufficience respiratory

Cutaneous lesions NTA*

IRA*

Apamin

Figure 2 - Poisoning syndrome following multiple stings by Apis mellifera

REFERENCIAS Anisimova l.M., Smirnov A.A., Troshin V.D., Krilov V.N., The use of Solapiven in reflexotherapy of humans with nervous system diseases, in the XXXIVth Apimondia Congress, 1995, Lausanne, Switzerland Banks, B., Possible Therapeutic Use of a Peptide from Bee Venom, in Bulletin de l’Institut Pasteur, 74 (1976), pp. 137-144 Basic I., Curic S., Tadic Z., Orsolic N., Sulimanovic D., Antimetastatic activity of bee venom and water soluble derivatives of propolis in mice, in the XXXIVth Apimondia Congress, 1995, Lausanne, Switzerland Beaudet M., Les piqures d’abeille, en Revue Française d’Apiculture, nov. (1990), p. 513-517 Beck F. Bodog, Bee Venom Therapy. Bee Venom, Its Nature, and its effect on Arthritic and Reumatoid Conditions, 1935, 1997 Health Resources Press, 1997 (under the name: The Bible of Bee Venom Therapy). Fierro Morales, Walter (1994) (Uruguay) – Utilización de la apitoxina con fines terapéuticos, in I Bienal de apicultura INTA H. Ascasubi. Abril

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Ford R.M., Honeybee venom immunotherapy. Letter, in Med J Aust, 1(10), (1980), p. 500 Forster K.A., Chemistry, pharmacology and therapeutically effectiveness of bee venom, in the XXIInd Apimondia Congress, 1969, München, Germany, p. 405-407 Gallesio Maria Teresa, Diagnosis and therapy of allergy to hymenoptera venom, in Apiacta (1986), No. 3 Krilov N.V., Veneno de abejas; propiedades, producción (en ruso). Nizhny Novgorod. Lobachevsky University 1995, 224 pp., illustrations; ISBN – 5-85746-076-X Masterov G.D., Apitherapy in the combined treatment of patients with pulmonary tuberculosis taking into account the hypophysealadrenal system indices, in Lik Sprava (1-2) (1995), pp. 120-2 Salamanca G.G., Efectos locales en la evolución de la apitoxína por acción inducida. Informe interno Departamento de Química Universidad del Tolima, 2000 Qiao Ju Han, Clinical treatment of systemis sclerosis by sting therapy, in the XXXVth Apimondia Congress 1997, Antwerp, Belgium Vick J., Warren Glenn B., Brooks Robert B., The effect of treatment with whole bee venom on cage activity and plasma cortisol levels in the arthritic dog, in Inflammation, Vol. 1, # 2 (1976), pp. 167-74 Vick J., Reid J., Beard G., Short T., Bredow Von J., Efficacite de la therapie au venin d’abeille dans les arthrites du chien, in the XXVIIIth Apimondia Congress 1981, Acapulco, Mexico, p.503 Warren D., Harman A., Garber T., Le venin d’abeille dans la recherche medicale, in the XXVIIIth Apimondia Congress 1981, Acapulco, Mexico, p. 504