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Subiect: HIRUDOLOGIA - tratament cu lipitori

  1. #31
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    Implicit Re: HIRUDOLOGIA - tratament cu lipitori

    Hirido-and apytherapy in urology
    Department of hirudo- and apytheraphy at Municipal clinic № 2 (Moscow region, Mitishi)
    Fomkina O.V.
    Chronic prostatitus, prostate adenoma, impotence and sterility are rather often diseases. Despite of plenty of medicinal preparations urological disease difficulty give in to treatment; percent of relapses is rather high.
    The most effective methods of treatment are apytherapy, hirudotherapy and their combination. These methods allow achieving positive results without negative side effects on an organism, which are provoked active antibioticotherapy.
    860 persons with this or that pathology (from them 247 men with urological diseases) namely have addressed to the clinic behind the help: acute and chronic prostatitis, prostate adenoma, and impotence. All of them were consulted and inspected. To the patients the analysis of prostate secret and ultrasonic research before and after treatment was conducted. At acute prostatitis were made 2-3 apytherapy courses by 15 sessions each, five patients - in a combination with hirudotherapy and one patient - two courses by10 sessions of hirudotherapy. The treatment was combined with phytotherapy, homeopathy, rectal suppositories and propolis. At all patients with an acute prostate inflammation already after the 1-st course the indexes of the analysis of a prostate secret were normalised.
    It is possible to approve that bee- and hirudotherapy as independent and in combination one another methods give proof positive results at treatment urological pathology without use of antibiotics

  2. #32
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    Implicit Re: HIRUDOLOGIA - tratament cu lipitori

    Role of hirudopharmacotherapy in complex treatment of patients
    Bondarevski I.I.
    State Clinic № 2 (Chelyabinsk)
    The rough progress in pharmacology in XX centuries second half has resulted in appearance of huge number of medicinal preparations. By selection of preparations the doctors first of all take into account their effectiveness, however, the operation of new and old medicinal means essentially does not differ.
    At treatment by the medicinal leeches it is necessary to take into account assignment of anticoagulants of a direct and indirect action, fibrinolityc preparations, antimetabolites (for example, metatrexate provoke to thrombopenia, anaemia).


  3. #33
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    Implicit Re: HIRUDOLOGIA - tratament cu lipitori

    Clinical examples of method iridotherapy application in combination with hirudotherapy
    " Medical center of hirudo- and iridotherapy" ( Korolev, Moscow region)
    Klimov N.I., Gnezdilova L.N., Filimonova T.I., Kiseleva P.S.
    Augmenting sessions of hirudotherapy in combination with iridotherapy we managed to reduce a reduction period of an organism. The clinical cases are resulted. It was shown the combination of methods gave positive results: hysteromyoma, glomerulonephritis and sarcoidosis.
    Conclusions:
    1. Iridotherapy which we used more than 13 years, demonstrates absence of phenomena of overdosages and negative collateral effects.
    2. Combined usage of methods irido- and hirudotherapy results in increase of efficiency of treatment and decrease of terms of its realization.

  4. #34
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    Implicit Re: HIRUDOLOGIA - tratament cu lipitori

    Hirudotherapy in complex treatment of psoriasis arthoropatia (case from practice)
    First hirudotherapy clinic in Republic (Belarusia, Minsk)
    Kuncevich L.L.
    Hirudotherapy has irreplaceable value in complex treatment of psoriasis arthropatia. Hirudotherapy realise by courses (4 times per one year) inclusive 7-10 sessions 2 times per one week. Therapeutically effect of hirudotherapy is stipulated by the following factors. Effect of biologically active substances produced by leeches on hit joints: improving of the microcirculation at the expense of prevention of development microthrombi (hirudin, destabilase complex) and antiinflammatory effect is provided with suppression of leukocytes proteases (eglins). Irritation (more long duration than at acupuncture) biological active points in time of bloodsucking and due to the leech saliva. It causes more high-power effect on hit joints.

  5. #35
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    Implicit Re: HIRUDOLOGIA - tratament cu lipitori

    Phyto-and hirudotherapy in rehabilitations of heart attack patients
    Ramenskoe, Moscow region
    Musina S.E.
    The high morbidity heart attack stipulates need of the improvement of methods of the reconstruction treatment. In this connection it is represented to perspective usage phyto-hirudothetapy. disturbance of microcirculation and the coagulative distresses of blood can be a first cause of many complications for ill heart attack and often are awesome complications at the given pathology. For improvement of microcirculation, periphery homodynamic, influencing on pathological changes of blood coagulation system is applied hirudotherapy, which one considerably improves condition of blood stream for myocardial infarction patients. Biologically active materials of leeches saliva (hirudin, destabilase, bdellins, eglins etc.) normalize a hemostasis, invoking increase of general anticoagulative and fibrinolytic activity of a blood, reducing thus intensity of an platelets aggregation and adhesion .

  6. #36
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    Implicit Re: HIRUDOLOGIA - tratament cu lipitori

    Effectiveness of hirudotherapy in combination with acupuncture at patiens with ischemia heart diseases at sanatorium stage of rehabilitation
    Medical prophylactic sanatorium "Podlipki" (Moscow region)
    Mituhina V.M., Voronova A.J.
    The hirudotherapy clinic exists 3 years. In a structure of the patients the majority suffers cardiovascular diseases - 70,3 % (213 patients). On the base of large practice of cardiologist and acupuncture we came to a conclusion that at hirudotherapy more effectively make leeching on biologically active points. It is not necessary completely to identify an action of leeches at hirudotherapy and needle at acupuncture, in spite of the fact that in both methods main is reflexive action. However invaluable action of hirudotherapy in comparison with acupuncture is the gamma of biologically of active substances, which are injected by the leech and which result in normalisation of pathological process (Baskova, Nikonov, 1991; Nikonov, 199.
    We have found to an expedient combination of both methods at treatment of the patients with various diseases. We mark high effectiveness of combinatory use of hirudotherapy and acupuncture at patients with arthrosis, arthritis and osteochondritis of backbone (21 patients) in comparison with application only of hirudotherapy methods.
    The group of the patients with ischemia heart disease received a course of treatments consisting of 6-8 hirudotherapy sessions and 9-10 acupuncture sessions. As a rule, the treatment began from acupuncture of distant BAP: Gi 11, МС 6, РР 4,6, Е 36, Tr 5 etc. At majority of patients it is marked expressed antianxiety effect. After 2-3 sessions the area of leeching there was a forward pectoral surface at the left in III-V intercost. As at acupuncture, and at hirudotherapy the following BAP frequently were used: Т 11,13,14, At 11,15,23, Gi 11, МС 6 etc. The combination of procedures per one day was following: in the beginning was made acupuncture and after 15-30 minutes was hirudotherapy.
    To the end of treatment course all patients marked increase of volume of physical activity: rate and duration of walking, transfer in-group of medical gymnastics with large loads. Thus, analysing our material, it is possible to make the conclusion about the greater effectiveness of methods combination - hirudotherapy and acupuncture in comparison with application only of hirudotherapy techniques. Both methods supplement and exponentiate one another.

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    Implicit Re: HIRUDOLOGIA - tratament cu lipitori

    The medical leech in treatment of vulva krauroz
    Regional veneral clinic (Ekaterinburg)
    Ribakova M.L.
    Krauroz of women sexual organs is a chronic disease with unexplored pathogenesis. Oncologists, gynaecologists and dermatologist exhibit the interest to this problem. Majority of the scientists consider that in the basis of disease the shifts in neuroendocrine system take place. The disease, as a rule, begins in climacteric period, menopause period, after undergo gynaecological operation, radial therapy. From existing methods of treatment any does not bring reassuring results and frequently (90,5 %) patients within the first year the aggravation comes.
    The complex treatment is conducted 13 patients in the age from 45 till 70 years, with duration of disease from 2 till 28 years. The leeching was conducted two times per one week till 4-5 pieces on a session on area of coccyx, sacrum and pubic area sequentially, and it is necessary: 1-2 leeches on area clitoris and leukoplakinal plaque or place greatest local itch. The treatment course consists of 5-7 procedures with corrective therapy of an appropriate pathology.
    Already after first - second sessions nine from thirteen patients have marked decreasing of itch; five-six procedures it completely has disappeared. The sensation of dryness in vulva area has decreased, the tissue has become more elastic, considerably has decreased hyperemia, and at four - completely has passed. Have smoothed out leukoplakinal stratification at four patients (and after the second course conducted with an interval in one month, completely have disappeared). The common condition of the patients was improved, the local comfort has appeared.
    Thus, despite of age of the patients, duration of disease, presence of an accompanying pathology, the hirudotherapy clinical effect allow us to forecast to improvement of quality of this patients category life.

  8. #38
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    Implicit Re: HIRUDOLOGIA - tratament cu lipitori

    HM1 OBTAINED FROM LEECHES Hirudo medicinalis IS TOOL FOR ANTITHROMBOTIC AND THROMBOLYTIC THERAPY
    ABSTRACT. To create effective medicinal tools aimed at normalisation of haemostasis biologically active substances produced by the medicinal leeches (Hirudo medicinalis) have been used. As a result of biochemical purification of homogenates of the whole leeches the preparation HM1 was obtained representing a balanced complex of natural active substances. The principal components of HM1 are hirudin, destabilase, blood plasma kallikrein inhibitor, similar to prostacyclin (PGI2) prostaglandins by the range of their physiological action, hyaluronidase, inhibitors of thrypsin, plasmin, chymothrypsin, elastase, catepsin G, analgetics etc. When orally administered to experimental animals HM1 has antithrombotic, thrombolytic, hypotensive, analgetic actions; improves reological properties of blood, causes DNA supermethylation effect. Multiple oral administration of HM1 into rats does not induce pathological changes in blood microcirculation. HM1 is practically a non-toxic substance.

    Clinical trials of HM1(356 patients) have confirmed the results of our experimental research. When orally administered HM1 has preventive antithrombotic, thrombolytic and hypotensive action.
    RESULTS
    1. Characterization of the HM1 preparation.
    HM1, the preparation obtained as a result of partial biochemical treatment of whole medicinal leeches, presents a balanced complex of BAS produced by the medicinal leeches.
    Testing of the preparation HM1 has shown (Table 1) that it contains hirudin, blood plasma kallikrein inhibitor, destabilase, leech’s prostaglandins, trypsin and chymotrypsin inhibitors.
    Table 1.
    Comparative characteristics the preparations isolated from medicinal leeches.
    parameters
    salivary gland secretion
    HM1
    Antithrombin activity AT NIH U mg-1)35,5±4,22,0±0,5
    Blood plasma recalcification time (APC U mg-1)140±2160±15
    Isopeptidase (destabilising) activity (mm2 mg-1)180±1525±5
    Amidolytic activity (L-g-Glu-pNA; mol mg-1 s-1)(12,3±1,3) 10-3(1,0±0,2) 10-3
    Inhibition of proteolytical activity of: - trypsin (S-2222; АТ U mg-1)14,3±2,16,5±1,0
    - chymotrypsin (Succinyl-L-Phe-pNA; ACT U mg-1)01,8±0,5
    - blood plasma kallikrein (S-2302; APC U mg-1)22,6±4,05,0±1,5
    Contains of 6-keto-PGF1a (rg mg-1)1250±340500±100
    Powerful inhibitory potential of HM1 gives us reasons to assume that when orally administered the active components of the preparation will be protected from destruction by proteases in the intestinal system.
    2. Experimental research.
    а) Antithrombotic action of HM1.
    Antithrombotic action of the preparation was analysed with the use of pathophysiological model of venous stasis. 1 ml of the HM1 solution (with the protein concentration of 35 mg. ml-1) was orally administered to a group of the experimental animals (15 rats). The control group of the animals (n=15) received the equal volume of 0,85% NaCl solution. After 4, 10 and 24 hour thromboformation was induced by intravenous injection of human serum activated by glass. The results of this experiment are: after 4 hour the thromboformation degree was 0%, after 10 hour - 15±5% and after 24 hour - 50±5% (in the control group this index was 95±5%; p<0,05). It can be seen that even at the maximum interval between the introduction of the HM1 solution and serum the blockade of thromboformation degree makes 60±5%.
    b) Thrombolytic action of HM1.
    In the experiments the model of venous stasis was also used. 24 hours after the thromboformation 1 ml of HM1 (with the protein concentration of 35 mg ml-1) was orally administered into rats (n=20). The control group of the animals received equal volume of 0,85% NaCl solution (п=20). After 4 and 7 days the condition of thrombus was analysed: the thrombolysis degree in the experimental animals was 40±5 and 90±10%, respectively, whereas that in the control group was 5±5 and 20±5%.
    In the following series of the experiments we studied the thrombolytic action of HM1 at multiple administration into animals. 1 ml of the HM1 solution daily (with the protein concentration of 5 mg ml-1) was orally administered into rats (п=32) during 3 or 7 days. The animals of the control group received the equal doze of 0,85% NaCl solution. From the results of this experiment it can be seen that multiple administration of small dozes of the preparation causes thrombus destruction, and the higher the introduced protein concentration the higher the degree of thrombolysis.
    c) Influence of HM1 on the functional condition of platelets and rheology of blood.
    As a preparation of comparison heparin was used (nonfractioned; 500 U per tube). In the study of rheological properties of rats blood it was shown that aggregation and mechanical stability of the eritrocites of blood stabilized by HM1 were lower and filtration ability of eritrocites was higher than same factors in the blood stabilized by heparin (p>0,1). Integrally all the differences mentioned were exhibited at lower viscosity of the blood stabilized by HM1 equal to 6,3±0,4 mPa (with relation to heparin 7,3±0,5 mPa; at identical shift voltage values). In the estimation of HM1 influence on the rheological properties of blood of the patients with the ishemic heart disease the same dependence is shown that is the tendency to improve the rheological properties of blood in the presence of HM1 is obvious.
    The spontaneous aggregation of platelets occurred only in the presence of heparin (55 %), whereas in none of the samples containing HM1 modifications of optical density were observed. In the study of the influence of HM1 and heparin as platelets aggregation “inductors” when they were added to plasma samples obtained from the blood of patients with the ishemic heart disease it was shown that heparin caused platelets aggregation in 6 cases of 8. The presence of HM1 did not result in a change in the optical density of platelets- rich blood plasma.
    d) Influence of HM1 on blood microcirculation at multiple introductions.
    The HM1 solution in the doze of 1 ml (with the protein concentration of 35 mg ml-1) was daily introduced into the experimental animals during 14 days. On the 15th day have carried out a histochemical analysis of the condition of blood microcirculation in the skeletal muscle and mesocolon of rats was made. It has been shown that prolonged administration of HM1 does not change the density of microvessels and does not affect the diameter of capillaries.
    The fact of rapid dissociation of papules in the presence of HM1 (4-5 minutes) in contrast to histamine or physiological solution (over 20 minutes) is of great significance. Duration and mode of Guridon administration actually do not affect the number of marked vessels and the degree of vessels permeability. Taking into account these observations, it can be assumed that under the action of HM1 the permeability of endothelial cells membranes increases which is to some extent demonstrates by the mechanism of penetration of the components of this preparation from the intestinal tract into blood (transmembrane transport).
    e) Penetration of HM1 into blood at oral administration.
    A group of animals (50 rats) was orally administered 1 ml of the HM1 solution (with the protein concentration of 50 mg ml-1). After 5, 15, 25, 35, 45, 55 and 65 min blood samples to determine antithrombin activity, blood plasma recalcification time and aggregation of platelets were taken. The control group of animals (п=15) received the equal volume of 0,85 % NaCl solution. It is interesting to note that changes in the above factors occurred in two stages. The first peak of blood coagulation time increase (by 80±7 %, р<0,05), blood plasma recalcification time increase (by 60±8 %, р<0,05) and platelets aggregative activity decrease (by 25±5 %, <0,01) occurred within the interval of 15 up to 25 minutes after HM1 solution administration into rats. The second peak occurred after 55 min and was more expressed in the absolute values of the analysed parameters, i.e. lengthening the time of fibrinogen coagulation by thrombin by 95±8% (р<0,05); an increase in blood plasma recalcification time by 78±7% (р<0,05); a drop in the aggregation ability of platelets by 38±5% (р<0,1). The results obtained also indirectly indicate the transmembrane transport of the HM1 components from the intestinal tract into blood.
    f) Hypotensive action of HM1.
    The animals of the experimental group (line SHR, i.e. spontaneously hypertensive rats; п=7) were orally administered 1 ml of HM1 solution (with the protein concentration of 35 mg ml-1). The animals of the control group (п=10) were administered the equal volume of 0,85 % NaCl solution. The arterial blood pressure in rats of both groups was registered daily immediately after the preparations administration during two weeks. The results of this experiment prove the pronounced hypotensive action of HM1. The effect of single HM1 administration is retained during 4 days. Repeated administration of the equal doze of the preparation to rats of the experimental group resulted in the same effect.
    g) Analgetic action of HM1.
    In the following series of the experiments HM1 influence of on rats sensitivity to pain caused by thermal irritation of the tail was analysed. The time of the tail response was registered after 30 minutes upon the single oral administration of 1 ml of the HM1 solution with the protein concentration 35 mg ml-1 (п=15); the animals of the control group (п=15) received the equal volume of 0,85% NaCl solution. Lengthening of this factor by 59±6% (р<0,01) proves the analgetic action of the preparation. This effect of HM1 is retained during 90 minutes.
    h) Analysis of the toxic action of HM1.
    The use of methods permitting to estimate the influence of the preparation on the induction of a response of the cell and immunity has shown that multiple oral injections of HM1 do not induce any modifications in the number of antibody made cells as well as the titres of antierythrocytal antibody in the serum of mice which is indicative of the absence of toxic action.
    The study of the toxic action was carried out on three species of animals that were continuously oral administered HM1 in the dozes of 15, 75 and 150 mg kg-1. During 6-months the integral factors and the condition of basic functional systems were studied using physiological and biochemical research methods. The results of this experiment have shown that HM1 does not change the condition of peripheral blood, cardiovascular and nervous systems, mineral exchange, etc. does not essentially affect the integral factors, the basic functions of liver, which characterizes the preparation as a non-toxic substance.
    Thus, revealed effectiveness of the HM1 preparation as a potential tool of antithrombotic and thrombolytic action indicates its combined action on the functions of a number of physiological systems of organism that favourably distinguishes it from the common preparations with the similar effect.
    3. Medicinal form of the HM1 preparation and its clinical trials.
    For the use in clinical practice the medicinal form of the HM1 preparation, i.e. “HM1 in capsules for oral administration” is offered. One capsule contains 150 mg of HM1 without any synthetic components.
    Clinical trials were carried out in independent medical centres of Ukraine:
    - Department of clinical pharmacology and pharmacotherapy of the Institute of Therapy of the Academy of Medical Sciences;
    - The Centre “Medicine” of the Academy of Medical Sciences;
    - Ukrainian Scientific and Medical association “Medicine of Transport”.
    Clinical trials were made on patients with thrombophlebitis and ishemic heart disease, 359 patients of both sexes in all aged from 21 to 70. The criteria of inclusion were the following factors: disorders of microcirculation, trophic ulcers, phenomena of thrombophlebitis. The preparation was assigned in the doze of 1 capsule 2-3 times a day during 10 days. The patients of control group received the traditional treatment.
    Upon the completion of ten-day time course of the preparation reception all patients felt a remarkable relief: pain, oedema, itching, obturating had gone. The results indicating clinical improvements agreed with the changes in the condition of hemostasis system (Table 2).
    Table 2.
    Some characteristics of hemostasis in patients with thrombophlebitis during treatment with HM1.
    Characteristics examinedPrior to the treatment7 days after treatment beginning
    Blood coagulation (min.)3,1±0,24,3±0,35 *
    Platelets (С. U.)289±30278±25
    Prothrombin index (%)115±898±8 *
    Thrombin time (s.)8,9±0,58,0±0,4
    Fibrinogen (mg dl-1)238±25250±15
    * - р<0,05.

    Changes in the coagulation and fibrinolytic systems of blood expressed by lengthening the blood coagulation time, a decrease in prothrombin index, a decrease in fibrinogen concentration have been detected. The most significant alterations occurred in fibrinolytic activity of blood: the time of euglobulin fraction lysis was reduced to 21 % as compared to the reference value (р<0,05).
    Thermographic studies have shown that oral administration of HM1 results in normalisation of microcirculation in the affected zone. Thermoasymmetry in the initial phase was 21±3 %, in a day (after administration of two capsules of the preparation) 21±2,5 %, in 7 days 9,2±1,2 %, in 10 days 2,5±0,3 %.
    The following trials were carried out on patients with the purpose of preventing maintenance post surgery thromboembolic complications (cholecistectomy, rupture). All the patients in the first day after surgery were injected heparin (5000 U; subcutaneously) three times. From the second day the patients received 1 capsule of HM1 two times a day. Patients of the control group received anticoagulants of indirect actions according to the conventional scheme. Positive clinical effect of HM1 was observed: all patients gave no indications of thromboformation, or complications of postsurgery wounds (haemorrhages, inflammations, suppuration). Positive clinical effect of HM1 administration can be ascribed to normalisation of the parameters of blood coagulation system (Table 3).
    Table 3.
    Influence of HM1 on some hemostasis indices in patients after surgeries.
    Investigated indexThrough 24 hours after operationIn 5 days after operation
    Prothrombin index (%)120±10,295±9,6 *
    Thrombin time (s.)8,3±0,78,0±0,9
    Fibrinogen (mg/dl)285±15,2288±16,5
    Blood coagulation (min.)3,5±0,34,5±0,2 *
    Platelets (convent.U)245±18,5230±16,2
    * - р<0,05.

    Toxic, allergic side effects or cases of idiosyncrasy were not observed. During research neither clinical, nor laboratory modifications, that could be considered as side effects of HM1 action were detected.
    The efficiency of prolonged HM1 administration for the prevention of postsurgical thromboformation or blockade of the progress of thromboformation is thus shown at thrombophlebitis.
    The HM1 preparation in capsules (0,15 g) for oral administration is an effective medicinal tool having antithrombotic and thrombolytic action.
    DISCUSSION. Positive effect of leeching is stipulated first of all by salivary gland secretion (Table 1) which is injected into blood by medicinal leeches during bloodsucking. BAS included in the structure of leech saliva present a balanced complex of active substances the effectiveness of which we have tried to retain in the form of the stable preparation obtained from whole leeches. The data obtained both in this research and earlier permit to single out the following peculiarities of HM1. In the case of oral administration HM1 exhibits thrombolytic action which is stipulated by destabilase. This enzyme ensures selective hydrolysis of isopeptide bonds in stabilized fibrin (Baskova and Nikonov 1991). Thus, the thrombolytic action of HM1 is fibrinospecific. Complete lysis of thrombus occurs within a rather long time. Physiologically this is justified as slow thrombolysis is accompanied by reparative processes in the vessel wall. And by the moment of complete thrombolysis this site ceases to represent athrombogenous surface.
    HM1 as well as destabilase (Baskova and Nikonov 1991) does not affect the fibrinolytic system; accordingly activation of plasminogen to plasmin does not occur (that is there is no system plasminemia).
    HM1 has pronounced antithrombotic action which is stipulated both by inhibition of the intrinsic mechanism of blood coagulation (Baskova et al. 1987) and by antithrombin activity of hirudin (Markwardt 1989).
    HM1 reduces arterial pressure, has analgetic effect, does not cause pathological changes in microcirculation, and improves rheological property of blood. HM1 is actually a non-toxic preparation. The presence of the penetration factor hyaluronidase and also inhibitors of proteases in its composition (Fritz et al. 1971, Baskova et al. 1991) ensures its integrity in the intestinal tract and penetration into blood in the case of oral administration.
    Thus, HM1 presenting a complex of natural biologically active substances meets the basic requirements applied to a hypothetical “ideal” thrombolytic. The results of clinical trials have confirmed our assumption about the effectiveness of HM1 as the preventive antithrombotic and thrombolytic remedy.

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    Implicit Re: HIRUDOLOGIA - tratament cu lipitori

    SKIN APPLICATION OF BIOLOGICALLY ACTIVE SUBSTANCES PRODUCED BY THE MEDICINAL LEECHES AS A NEW TREND IN COSMETOLOGY
    INTRODUCTION

    On the basis of the leech saliva a series of medicinal compounds have been developed which are successfully used in clinical practice. BAS completely retain the native leech activity being a structural part of the preparations.
    When the mechanisms of BAS action are analysed, it should be noted that the direction of physiological action varies depending on the doze of the preparation used. It has been shown that the minimum dozes of BAS are the most effective for normalisation of metabolism processes and for muscle relaxation. We have used the effect of small dozes in our research of BAS effectiveness in the composition of the compounds for medical cosmetology.
    RESULTS

    Performance of the analysed preparations in in vitro experiments.
    As a result of testing the HM1 preparation and the salivary gland secretion it has been shown (Table I) that they contain hirudin, blood plasma kallikrein inhibitor, destabilase, the leech prostaglandins, and thrypsin and chymothrypsin inhibitors.
    The distinctions in the content of this or that component are accounted by the fact that HM1 is a complex preparation prepared from the whole leeches homogenate. Besides the leeches saliva it contains the intestinal tract blood and a soluble fraction of the leeches muscles.
    Taking into consideration the literature evidence that the homogenates of the whole medicinal leeches contain hyaluronidase and the fact of the presence of the destabilase complex in the composition of the preparations in question (Nikonov, G.I., 1996), it can be assumed that BAS will be free to penetrate into skin and blood microcirculation when applied to skin either in aqueous solutions or as a structural part of ointment or creams.
    Analysis of toxicity of salivary gland secretion and HM1 when applied to skin.
    On the model of human epidermal cells it has been found that the preparations in the dozes 0,001-100 mg.ml-1 are not toxic and do not affect the cells adhesion. Low concentration (0,001-0,01 mg.ml-1) induced promotion of prolyferation processes which was manifested in an increase in DNA synthesis by 42 % (p<0,05) and in protein synthesis by 3 to 5 times.
    Bearing these data in mind, the following concentrations of the preparations were chosen for skin application: medicinal leeches salivary gland secretion - 0,01 % and 0,1 % (in H2O); HM1 - 0,0001 %, 0,01 % and 1 % (in H2O). Applications were made daily during 30 days.
    As a result of the research made it has been found that the preparations have no irritating and allergic effects on the skin of the experimental animals, they exert a negative effect on the morphological structures of skin and liver and do not have any toxic action on the organism of the experimental animals.
    Dermo-resorbtive action of the preparations has been studied in the experiments on white mice. There were no lethal outcomes; the general state of coat and skin did not differ from that of the control group, the appetite was good, general intoxication phenomena were not observed. No indications of irritation were detected, increase in the weight of the experienced animals did not differ from that in the control group.
    The influence of HM1 on biochemical indexes of skin condition.
    In the study of the influence of long skin applications of the preparation on the biochemical indexes of the experimental animals skin pronounced biological activity of HM1 has been revealed (Table 4). The content of total water soluble proteins has increased in the skin of guinea-pigs under the action of HM1 in the concentration 0,0001 % by 5,3 times, at 0,01 % concentration by 3,9 times, at 1 % concentration by 2,9 times. Summarised lipids have increased by 96,4 % in the experimental animals skin under the action of the preparation in the concentration of 0,01 % as compared to the control group.
    Table 4.
    Effect of skin application of HM1 aqueous solution on biochemical indicators of skin of the experimental animals.
    Parameter ControlHM1 aqueous solution at concentration
    0,85% NaCl0,0001%0,01%1%
    Concentration of summarised lipids (mg·ml-1)26,67±2,6-52,39±7,4-
    Concentration of total soluble proteins (mg·g-1)9,82±1,1451,87±5,038,68±5,728,56±3,0
    Content of maleic dialdehyde (mmol·g-1)45,11±2,6654,04±4,0851,73±3,5857,85±3,53
    Content of soluble collagen (g%)33,5±1,137,6±0,934,9±1,3-
    Proteolytical (by tyrosine) activity (mmol·m-1·g-1) ·10-22,81±0,18-3,62±0,36-
    1 % aqueous solution of HM1 increases the level of maleic dialdehyde in skin by 28,2 %; 0,01 % by 14,7 %, and 0,0001 % by 19,8 %, respectively, i.e. none of the concentrations of the preparation used has any antioxidant activity. The content of soluble collagen in the skin of guinea-pigs after long application of the HM1 solution does not vary statistically reliably. Proteolytical activity in the animals skin under the action of 0,01 % HM1 aqueous solution is increased by 28,8 % (p<0,01).
    When applied to skin, the preparation does not affect the hydration properties of skin. After application of the preparation in the analysed concentrations the percentage of water in the skin of the experimental animals did not differ from that of the control.
    Antiinflammatory action of HM1 has been found. After 3 hours upon intracutaneous formalin injection the degree of inflammation suppression was 26,4±2,5%.
    Influence of leeches salivary gland secretion on the biochemical indicators of the experimental animals skin condition.
    At multiple skin applications the leech salivary gland secretion has a similar action on the biochemical indicators of the skin condition (Table 5). The protein level increased by 21,2% in the animals of the experimental group as compared with the control one when the 0,01% leech saliva solution was used, whereas the 0,1% solution reduced the protein content by 21,3%. The content of summarised lipids was reduced after the action of the leech saliva in 0,01% concentration by 17% and in 0,1% concentration by 44,2%.
    Table 5.
    Effect of leeches salivary gland secretion on biochemical indicators of skin of the experimental animals.
    Parameter ControlLeech salivary gland secretion in concentration
    0,85% NaCl0,01%0,1%
    Concentration of summarised lipids (mg·ml-1)134,41±8,64111,57±9,8775,0±7,33
    Concentration of total soluble proteins (mg·g-1)33,18±3,1540,22±3,7426,15±4,08
    Level of maleic dialdehyde (mmol·g-1)79,61±3,8766,63±4,3577,31±2,71
    However, in contrast to HM1 the salivary gland secretion has the antioxidant activity when applied in concentration 0,01% (a decrease in the maleic dialdehyde level by 16,6% as compared to the control). When the leech saliva is used in the concentration of 0,1% this effect does not manifest itself.
    Results of clinical trials of medical-cosmetic creams containing HM1 or salivary gland secretion of leeches.
    Clinical trials of a cream containing HM1 were made on patients with dry, sensitive, fading facial skin at the age of 30 to 55 years (20 persons). The cream was applied to the facial skin daily during 30 days. The cream is applied easily and smoothly, it is quickly absorbed, without leaving a greasy film. The cream has no irritating and allergic effects, it suppresses irritation of skin in patients with dry facial skin.
    According to humidomety data taken before and after cream application skin hydration increases from 30,0±2,6% to 40,9±3,3% (i.e. by 42,4 %; p<0,01). According to the data of elastometry the skin elasticity increased from 14,2±0,7% (before cream application) up to 15,3±0,5%, i.e. by 6 %.
    Thus, Institute of Cosmetology of the Russian Ministry of Public Health care has approved the cream containing HM1 as a compound for dry, sensitive, fading facial skin care. The first cream from this series was named ANTONIA.
    The experimental data on the biological activity of salivary gland secretion are completely confirmed by the clinical trials (25 patients). According to the conclusion of Institute of Cosmetology of the Ministry of Public Health Care of Russia the creams on the basis of the leech salivary gland secretion are recognised as a medical-cosmetic agent with the geriatric, antiinflammatory and antistress action. The creams line from this series of preparations was named BA (bioenergy-antistress).
    DISCUSSION

    The leech saliva is a balanced complex of BAS (Table I) which determines the effectiveness of this compound when it acts upon a number of organism physiological systems. On the basis of the results of this research and those obtained earlier it is possible to make a conclusion that the leech saliva is effective not only at intravenous and orally administration to the experimental animals (Nikonov, G.I., 1995), but also in the form of skin application. The very fact of BAS penetration through the epidermis is of interest. It may be assumed that two mechanisms are involved in this process: 1- under the action of hyaluronidase contained in the leech saliva (Claude, A., 1937) BAS are carried along the intercell contacts; 2- the liposomal nature of the destabilase complex (Nikonov, G.I., 1996) ensures active transport of BAS directly through the cells membranes.
    However, the leech salivary gland secretion is an expensive preparation and its accumulation in sufficient quantity involves certain problems. Therefore, we used the preparation HM1 allowed for clinical use in oral administration (Seleznev, K.G., 1996) as a source of BAS.
    Similar to the leech saliva the preparation HM1 has no irritating and allergic effects on the skin of the experimental animals and people; it is not toxic. HM1 has pronounced biological activity: under the influence of long applications to the skin of the experimental animals the content of soluble proteins, summarised lipids, the activity of enzymes significantly increased. However, it is the pronounced antiinflammatory action of the preparation, stipulated by the presence of bdellins and eglins in its structure, i.e. the inhibitors of thrypsin, chymothrypsin, plasmin, akrosine, catepsin G and other neutral proteases of granulocytes, that is of primary importance.
    Nevertheless, in contrast to the leech saliva HM1 has no pronounced geriatric action which reduces its polyfunctionality to some extent. This can be accounted for, firstly, by the distinctions in the content of destabilase (Table I) causing the rupture of isopeptide bonds formed at the cells ageing (Kahn, D.R., Cohen I., 1981); and secondly, by weak antioxidant action.
    The use of the preparation HM1 as an active component in the composition of cosmetic creams gives positive results exhibited in normalisation of proteins and lipids exchange, in pronounced antiinflammatory activity.
    Medical-cosmetic compounds on the basis of the leech saliva (in small dozes; Table 5), according to the conclusion of Institute of Cosmetology of the Ministry of Public Health Care of Russia have pronounced antistressoric and geriatric effects, i.e. they may be used in a complex of measures aimed at rejuvenation of the organism.
    Thus, the therapeutical effectiveness of biologically active substances produced by the medicinal leeches is exhibited in skin applications as well when BAS make part of the agents of medical cosmetics.

  10. #40
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    Implicit Re: HIRUDOLOGIA - tratament cu lipitori

    BIOLOGICAL ACTIVITY AND PHARMACOLOGICAL PROPERTIES OF ANTIPROCOAGULANT COMPLEX OBTAINED FROM LEECHES Hirudo medicinalis
    ABSTRACT. APC ( antiprocoagulant complex ) is the product of biochemical purification of a powder of the lyophilised whole medical leeches. APC differs from the starting drug preparation with enrichment of hirudin, blood plasma kallikrein inhibitor and leeches prostaglandins that are similar in their physiological influence spectrum to prostacyclin. APC contains some tracers of destabilase. When an electrophoresis is fulfilled in a polyacrilamyde gel, APC is revealed as a dominating strip (MM about 6.5 kD) and mixed proteins with MM for about 12 kD.
    In the case of an intravenous application to experimental animals APC indicates reliable antithrombotic, thrombolytic and hypotensive actions. Effective antithrombotic action is ensured by blockade of plasmic (blood plasma kallikrein inhibitor), platelet-vascular (leech prostaglandin) links of the intrinsic mechanism of blood coagulation and inhibition of thrombin activity (hirudin); whereas thrombolytic and hypotensive actions are carried out by the leech prostaglandin. APC combined with HM1 (the source of hyaluronidase and digestion system proteolytical enzymes inhibitors) and being used perorally, also demonstrates its antithrombotic property. APC is planned to be the medicine of a selective anticoagulant action.
    INTRODUCTION

    In some cases connected with the blood coagulation system pathology the direct, purposeful action is to be necessary, without any influence on fibrinolysis system or complement. Therefore, we have taken an attempt to create the drug preparation of the purposeful antiprocoagulant action (first of all for preventive maintenance postsurgery thrombosis, and for ill with risk of thromboformation).
    RESULTS

    1. Obtaining and characteristics of antiprocoagulant complex (APC).
    As a source of APC "HM1" was used - the lyophilised powder made of dried whole leeches. The starting material was subjected to multistage crioprocessing for fuller destructuring of Aeromonas hydrophila - bacterium-symbiont of leeches. After the following processes: the correction of the starting HM1 aqueous extract pH, thermoprocession and gelfiltration through Sephadex-G75 - the fraction enriched with hirudin, blood plasma kallikrein inhibitor and prostaglandins was obtained. The comparative analyses between the starting materials and the obtained drug preparation - APC is given in the Table 10. APC contains hirudin in 6O times more, blood plasma kallikrein inhibitor in 9 times more, prostaglandin in 5,7 times more, then in the starting HM1 solution. It should be noted that there were present some tracers of destabilase and absent any tracers of others biologically active substances (BAS) produced by the medicinal leeches in APC. Since BAS being contained in APC had an ability to block different stages of a hemostatic process it was naturally to suppose that APC being used in experiments in vivo (in prothrombosis state) would block the thrombogenic process.
    Table 10.
    Comparative analyses of HM1 and antiprocoagulant complex (APC).
    IndexesHM1APC
    Protein concentration at maximum solubility (mg/ml)3035
    Hirudin activity (ATNIH U/mg) 2.5±0.518O.O±2.5
    Kallikrein inhibitor (APC U/mg)6O±1554O±24
    Content of 6-keto PGF1a (pg/mg)5OO±1OO2888±65
    Destabilase activity
    a) mm2·mg-1
    b) mol·mg-1·c-1
    25±5
    (1.O±O.2) 1O-11
    6±4
    (O.2±O.O5) 1O-11
    Bdelins and eglins
    a) AT U/mg
    b) AXT U/mg
    6.5±1.O
    1.8±O.5
    ?
    O
    Strips destribution on electrophorethogram (kD) from 4 to 8Ofrom 4 to 8
    Inhibition of platelets aggregation (ADF - inductor) (%)50100
    2. The prophylactic antithrombotic action of antiprocoagulant complex (APC) in experiments on animals.
    Antithrombogenic action of APC was studied in experiments on animals (bridles white rats ) with the use of vascular thrombosis model by Wessler. The aqueous solution of APC (data of drug preparation was demonstrated in Table 10) in dose of 3,5 mg/kg of body mass was injected intravenously into rats of the experimental group (n=1O ), while the control group ( n=9 ) received an equal volume of O,85% NaCl solution. After 12, 24, 36 and 48 hours thrombosis in a stasied section of the jugular vein was stimulated. The results of this experiment are given in Table 11. So, APC containing blood coagulation inhibitors has a powerful anticoagulant effect which denotes its pharmacy purposefulness.
    Table 11.
    The prophylactic antithrombotic action of APC in the case of an intravenous injection and its combination with HM1 in the case of a peroral use by the experimental animals.
    Analysed drug preparation and the method of administration.The level of thromboformation (%) at an interval between drug administration and vein stasing ( in hours ):
    12243648
    Intravenous injection
    1. The aqueous solution of APC in dose 3.5 mg/kg (n=10)
    2. 0.85% solution of NaCl in volume 0.2 ml (n=9)
    0
    9O±5
    1O±5
    95±5
    2O±5
    9O±1O
    35±5
    9O±5
    Peroral administration
    1. The APC aqueous solution in dose 3.5 mg/kg and 10 mg/kg of HM1 (n=10)
    2. 0.85% NaCl in volume 0.5 ml and 10 mg/kg of HM1 (n=9)
    1O±1O
    95±5
    3O±1O
    9O±1O
    45±59
    O±5
    65±59
    O±5
    3. Prophylactic antithrombotic action of antiprocoagulant complex APC ( in the combination with HM1) in case of a peroral application to experimental animals.
    We had an opinion that an ideal anticoagulant preparation should be one which preserves its effect being used perorally. APC that has the antithrombotic substances only, will anyhow be subjected to destructuring in the digestion system. As a source of the enzymes inhibitors HM1 was used. It manifests its biological activity with any method of administration. There were the series of experiments, where animals received APC composition perorally in the dose - 3.5 mg/kg with HM1 in dose - 1O mg/kg. This HM1 dose is not sufficient to reveal the antithrombotic effect but it is enough to protect APC components from proteolysis in the digestion system of rats. It also contains the amount of hyaluronidase enough to provide APC penetration into intercell contacts of tissue.
    The APC aqueous solution containing HM1 as a suspension was perorally given to the experimental animals ( n=1O ). The control group rats ( n=9 ) received an equal volume of the HM1 suspension in O.85% NaCl solution. After 12, 24, 36, 48 hours a thromboformation was stimulated with an intravenous injection of human blood serum that was activated with glass. Shown in Table 11 results demonstrated that peroral application of APC in the combination with HM1 - the source of hyaluronidase and ferments inhibitors also led to the thromboformation process blockade.
    So, APC should be regarded as a potential drug of the selected anticoagulant action, that effective with both methods of application: the intravenous usage as an independent drug and the peroral usage in combination with proteolytic enzyme inhibitors ( HM1 ).
    4. Other APC characteristics as a potential drug preparation.
    Taking into consideration the fact that APC had being planned to be a drug preparation, it seemed to be necessary to reveal the other characteristics of the complex depending on its ingredients: prostaglandins, hirudin and kallikrein inhibitors.
    4.1. The thrombolytic action of APC.
    As prostanoid produced by the medicinal leeches are similar to prostacyclin in the spectrum of its physiological activity it seemed to be interesting to find out APC thrombolytic influence, because prostacyclin (PGI2 ) working mutually with vascular wall receptors stimulates excretion of t-PA (Musial et al., 1986). As a result the fibrinolysis is activated causing thrombolysis.
    As it was shown earlier, the intravenous injection of leeches' prostaglandins causes increasing of t-PA level in blood (Nikonov, 1992), so, we should expect the increase of the fibrinolytic potential as the result of APC application. The vein thrombosis model by Wessler was used for to analyse APC action. 18 hours later of the rat’s jugular vein section stasing, animals of the experimental group ( n=3O ) received intravenously the APC aqueous solution in dose 5.5 mg/kg ( APC characteristics were given in Table 10 ), while animals of the control group ( n=3O ) got an equal volume of O.85 % NaCl solution. After 24, 36 and 48 hours the thrombolysis degree was analysed. The results of this experiment indicated in Table 12 demonstrates that the intravenous injection of APC into rats causes a partial lysis of thrombus: 48 hours interval performs the thrombolysis degree of 5O % compared with spontaneous thrombolysis that was revealed in the control group of animals. In the case of thrombolysis which is stimulated with APC, fibrinolytic activity of destabilase can be excluded because of its low level in APC, so it can not influence veritable changes of amidolytic activity (relatively to chromogenic substrate L-g-Glu-pNA ) of blood plasma of rats in the experimental and control groups.
    Table 12.
    APC thrombolysis action in the case of an intravenous injection to experimental animals.
    Time after analised drug injectionThe thrombolysis degree caused with injection of:
    0.85% NaClAPC
    (h)macromethod (%)weighting (mg)macromethod (%)weighting (mg)
    2451.9±O.2101.8±O.1
    36151.5 ±O.3361.1 ±O.2
    48201.3±O.350O.8±O.2
    4.2. APC hypotensive action.
    Polyfunctionality of the stable analogue of prostacyclin synthesised by the medical leeches provides a definite variability of APC physiological activity. Prostacyclin has a well-known characteristic of decreasing of vascular tonus (Herman et al., 1987). The leeches prostaglandins (Baskova and Nikonov, 1987; Nikonov, 1992) have the same characteristics also.
    In the following number of experiments we've taken an attempt to reveal the hypotensive action of leeches prostaglandin in APC content at the intravenous application to experimental animals ( SHR line rats - spontaneous hypertensive rats).
    The APC aqueous solution was injected in veins of the rats' group ( n=8 ) in dose of 4.3 mg/kg ( drug data were given in Table 10 ). The control group rats got the same volume of O.85% NaCl solution ( n=7 ). The blood pressure was registrated after 6, 12, 24 and 36 hours in a tail section of vein; one - time intravenous injection of the APC aqueous solution for SHR rats leads to reliable stable decrease of an arterial pressure (6 h. - decrease at 33%, 12 h. - 25%, 24 h. - 10%). This effect is preserved during 36 hours for doses given.

    DISCUSSION

    As a radiant for directed selection of substances capable to block thromboformation a preparation HM1 representing complete gang of biologically active substances producing by the medicinal leeches (Nikonov, 1992) was selected by us. In an outcome of biochemical handling of an initial material was obtained preparation APC (antiprocoagulant complex), possessing ability to block different stage of hemostatic process (prostaglandins - platelet-vascular stage, kallikrein inhibitor - plasmic stage of intrinsic mechanism, and hirudin - plasmic linkage on the later stage of blood coagulation). On our opinion the similar combination of substances is more effective for prevent of thromboformation because hirudin in a homogeneous condition inhibits only the thrombin activity that is calls blockade of activation of a blood coagulation system on late stages of its development. Inhibition of blood coagulation on early stages of its activation (intrinsic mechanism) reduces the generation of thrombin amounts that in turn reduces initial hirudin dosage. Therefore APC is more effective than cleared hirudin antithrombotic action of which was studied by Markwardt et al. (1982). Hirudin in a doze 10000 AT NIH U/kg of a rat body mass on 90 % inhibits the thromboformation if the time between injection of hirudin and human blood serum does not exceed 1 hour. This doze is not practically effective if the interval is enlarged till 3 hours. In our experiments a doze of hirudin (in APC composition) equal 630 AT NIH U/kg is used. Thus 90 % inhibition of thromboformation is observed at an interval equal 24 hours (table 11). Thus the use of natural hirudin (but not recombinant) for prevent of thromboformation and DIC in clinical practice becomes real.
    Magnification of hirudin content in 60 times, kallikrein inhibitor in 9 times and prostanoid in 5.7 times (table 10) was by the reason expressed preventive antithrombotic action at intravenous injection to experimental animal (table 11).
    However using HM1 as a radiant of proteases inhibitors (Titova and Nikonov, 1994) we were possible managed such relation of preparations concentration at which APC has exhibited its antithrombotic action at orally administration into rats (table 11). However, APC effectivity in this particular method of application was less expressed (for 5O % ) in comparison with the intravenous injection of APC. This was obviously because of partial decomposing with digestive enzymes.
    So, the research shows that the antiprocoagulant complex (APC ) has well expressed antithrombotic action depending on its components : hirudin, blood plasma kallikrein inhibitor and an analogue of prostacyclin. The APC effectivity is preserved either in the case of an intravenous application ( as a "pure" drug ) or in the case of a peroral usage combined with HM1. Besides of revealed prophylactic antithrombotic action APC has thrombolytic and hypotensive actions denoted first of all with the stable prostaglandin analogue produced by the medicinal leeches. In it leech’s prostanoid demonstrate the likeness with PGI2: similarly to prostacyclin it interact with receptors of a vessel wall calling ejection of t-PA (Musial et al., 1986); decrease vascular tonus (Herman et al., 1987). Such action of APC can not be regarded as a negative, subordinate one, because at the base of prethrombosic state the following factors take place as: increasing of a vascular wall tonus and of an arterial pressure, and local spasming of a vessel gap.

  11. #41
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    Implicit Re: HIRUDOLOGIA - tratament cu lipitori

    A STABLE PROSTACYCLIN-LIKE SUBSTANCE PRODUCED BY THE MEDICINAL LEECH Hirudo medicinalis
    ABSTRACT. The medicinal leech Hirudo medicinalis produces a low-molecular mass compound with properties similar to those of prostacyclin (PGI2). It extracted with organic solvent, had affinity to 6-keto-PGF1a antibodies, inhibited human platelet aggregation induced in vitro by thrombin (by 50% at 4 pg/ml), and caused hypotension and secretion of t-PA secretion into the blood stream of rats.

    A main distinction from PGI2 is stability of the substance due to covalent binding with the polypeptide chain of destabilase. Due to the high aggregability of destabilase, the molecules of the protein-lipid complex are organised into micelles that can change their spatial orientation depending on the nature of the solvent. Incorporation of hirudin and blood plasma kallikrein inhibitor into the micelle structure causes the formation of liposomes (with MM of the structural monomer 25 kD). This complex with polypeptides provides not only it stability but also rapid transmembrane penetration. The pure prostacyclin-like substance (PC-LS) has a MM 391 D, and can be produced upon destruction of the destabilase polypeptide chain.
    RESULTS

    1. Leech prostacyclin-like substance (PC-LS).
    Results are shown in Table 15. The amounts stated are 6-keto-PGF1a equivalents. Since any analysed preparation is suitable for purification, in the further work the powder of lyophilised whole dried leeches (HM1©) was utilised.
    HM1 aqueous solution was subjected to 7-times extraction at pH 3.5 (as prescribed by Amersham). The high concentration of PC-LS (measured as 6-keto-PGF1a) in the organic phase inhibited platelet aggregation stimulated by ADP (Table 16A). Some (what percentage?) PC-LS was also present in the aqueous phase. The presence in the liposoluble phase of hirudin, destabilase and blood plasma kallikrein inhibitor (IK) activities was unexpected.
    The total content of PC-LS was considerably higher than the index for the starting HM1 aqueous solution (Table 16A). Electrophoretic analysis showed material at 12.3, 25 and 50 kD, corresponding fully to the electrophoretogramme of destabilase made by affinity chromatography on lysine-Sepharose and preparative electrophoresis (see Table 16B for activity analysis). Every fraction contained 6-keto-PGF1a-like material and destabilase activity. Furthermore, fractions with MM of 25 and 5O kD also demonstrated activities of hirudin and blood plasma kallikrein inhibitor.
    Table 15.
    Concentration 6-keto-PGF1a-like substances in the preparations from medical leeches.
    preparationprotein concentration (mg/ml)PC-LS (ng/ml 6-keto-PGF1a)block of platelet aggregation
    (qualitative)
    Leech salivary gland secretion3.53.466±O.545+
    Homogenate of leeches head7.3O.351±0.025+
    Powder of lyophilised leeches (HM1)10.22.437±0.356+
    Blood from leech digestion system9.20.209±0.012+
    Culture of Aeromonas hidrophila (Leech bacteria symbiont)2.30.450±0.028+
    Table 16.
    Characteristics of HM1 solution and its fractions obtained during PC-LS purification.
    preparationprotein concentrationPC-LSactivity of:
    mg/ml(ng/ml)hirudin ATU/mgdestabilase
    mol·mg-1·s-1
    KI* U/mg
    A
    HM1 solution1.631.674551.6 10-1174
    aqueous phase0.380.9702362.2 10-1184
    organic phase0.09600.00092.0 10-117
    B (destabilase fractions)
    MM 12.3 kD0.8701511.2 10-110
    MM 25.0 kD1.87175234.7 10-11138
    MM 50.0 kD2.20190199.5 10-11186
    C (chromatography on Silasorb-C
    starting solution1.051.62952.1 10-11119
    unlinked part0.350.357503.4 10-1146
    methanol eluate0.05100.00927.0 10-1113
    D (affinity chromatography on antibodies to 6-keto-PGF1a)
    starting solution2.301.8840.9 10-11107
    eluate0.06120.8181.6 10-1121
    * - blood plasma kallikrein inhibitor (KI)
    To obtain the PC-LS, other well-known methods of prostaglandin isolation were used: affinity chromatography on antibodies to 6-keto-PGF1a and chromatography on a specific sorbent Silasorb C8 (according to the directions of Amersham). In both cases the eluate had a high concentration of PC-LS and contained destabilase, hirudin and kallikrein inhibitor (KI) (Tables 16C and 16D). There was strong bonding between destabilase and PC-LS which was not broken either with the above methods, by electrophoresis at the denaturing conditions, affinity chromatography of destabilase on lysine-Sepharose, extended dialysis through pores that pass substances with MM up to 1000 D, nor with warming at 80C during 20 min.
    The lipid component of destabilase contributes to the micelle properties, i.e. destabilase shows amidolytic activity in as aqueous and organic surroundings. The activity of PC-LS was markedly increased after the complex was transferred from aqueous to organic medium.
    Consequently, PC-LS seems to be a component of a stable protein-lipid complex which can aggregate into high-molecular structures with hydrophobic and hydrophilic constituents. Including the polypeptide chains of hirudin and KI in such a structure gives a stable liposome. PC-LS can be obtained pure only after disintegration of the protein part of destabilase.
    2. Activity PC-LS as component of liposomes.
    The strong bond between PC-LS and protein components provides stability not only for storage (in a lyophilised state or in aqueous solution) (Table 17), but also for increased life in blood after intravenous or oral administrations.
    One mg of destabilase containing of 0.85 ng of PC-LS was administrated orally (by gavage) to rats (n=, and after 10, 25, 60, and 180 min blood was taken for the determination of platelet aggregability and PC-LS content. The reduction of platelet aggregating activity showed one peak at 25 min, and another at 60 min after administration. The liposomal structure may protect the complex components from hydrolysis by proteolytic enzymes in the gastrointestinal tract, and allow their penetration into the blood. A trans-membrane transfer requires a hydrophobic nature. Increasing blood PC-LS content was accompanied by lower platelet aggregatability for 3 hr after oral administration of the preparation.
    Similar to prostacyclin, PC-LS was vasoactive as shown by a reduction of the initially high arterial pressure of the SHR. The experimental group received 0.5 ml orally (by gavage) of the destabilase preparation containing 0.64 ng of PC-LM whereas the control animals were given an equal volume of 0.85 % NaCl solution. Arterial pressure changes were successively
    Table 17.
    PC-LS in stored preparations from medicinal leeches.
    preparation storage before determinationPC-LS (ng/ml 6-keto-PGF1a)
    HM1 1 yr
    2 yr
    3 yr
    4 yr
    5 yr
    0.53
    0.77
    0.80
    0.43
    0.34
    Destabilase (affinity chromatography on lysine-Sepharose)1 mo
    6 mo
    1 yr
    1.5 yr
    2 yr
    3 yr
    1.20
    0.70
    0.85
    0.57
    0.18
    0.12
    Leech saliva14 days
    21 days
    28 days
    2.08
    1.45
    0.87
    recorded in both groups. The overall arterial pressure of the control rats (n=10) was 175±10 mm Hg over 7 days. In the experimental group (n=9) the results were: day 1 after giving the preparation, 135±10 mm Hg (p<0.05), day 2, 145±5 (p<0.05), day 3, 150±10 (p<0.5); subsequent days no statistically significant difference from the controls.
    Both, intravenous and oral (gavage) administrations of liposomes somewhat increased the blood t-PA and plasmin contents. Normotensive male Wistar rats weighing 250-300 g were injected intravenously (n=12) and orally (gavage; n= with the liposome preparation in a dose containing 0.24 ng PC-LM, and values of their fibrinolytic system were recorded. The results are given in Table 18.
    3. Characteristics of purified PC-LS.
    Pure PC-LS was obtained by organic solvent extraction after proteolytic hydrolysis (incubation of the starting material with trypsin and chymotrypsin, 24 h, 370C) of the liposomes. The MM of PC-LS corresponded to 391 D, as determined by mass spectrometry.
    Platelet aggregation was studied using 1 NIH unit of thrombin as the aggregator in 1 ml of platelet-rich plasma. The starting (original) solution contained 2.4 ng/ml of 6-keto-PGF1a -like substance. It was diluted until inhibition of platelet aggregation reached 50% (0.02 ml of PC-LS solution diluted 12 times, so that 4 pg of PC-LS inhibited thrombin-induced platelet aggregation by 50%).
    Table 18.
    Some indices of fibrinolytic system after intravenous or oral administration of liposomes containing 0.24 ng PC-LS
    administration time after administrationconcentration
    and animals groupshourst-PA mg/mlPlasmin ng/ml
    Intravenous
    experiment671.58±0.241.8±1.2
    control671.17±0.191.2±0.9
    Oral (gavage)
    experiment42
    92
    0.96±0.08
    1.18±0.19
    8.0±1.0
    5.2±3.5
    control42
    92
    0.95±0.17
    0.74±0.09
    (5.4±3.9) 10-3
    (5.4±3.9) 10-3
    Intravenous injection of PC-LS 540 ng/kg into rats reduced platelet aggregation activity over 3 h. At 30, 60, 120 and 180 min the aggregation was reduced by 45±5 (p<0.01), 35±7 (p<0.01), 23±6 (p<0.05) and 15±8% (p<0.5; n=2 respectively.
    Purified PC-LS retained activity on storage at 40C for a long time. We estimated that half the activity was lost in 6 months in the lyophilised state, but in only 10 days in an aqueous solvent (Tris-HCl buffer, pH 7.4), and in about a month in an organic solvent (methanol, ethyl acetate).
    DISCUSSION

    The medical leech is a source of a prostaglandin-like substance with biological activity similar to that of prostacyclin (PGI2), as it inhibited platelet aggregation stimulated by different inducers, had antithrombotic potential, caused hypotension, and stimulated t-PA release into blood. PC-LS is biochemically similar to prostacyclin since it extracts into ethyl acetate, and it can be separated by affinity chromatography with an antibody to 6-keto-PGF1a, and by chromatography on Silasorb C8. Compared to some known analogues of prostacyclin, PC-LS was very stable on storage. It appears to form a complex with the polypeptide chain of destabilase (the MM of the complex being 12.3 kD), apparently by forming covalent linkage. Such structure provides the micelle properties of aggregates formed during the complex formation.
    Probably in case of contact micelles with blood inside of leech intestinal tract liposome is formed. The liposome contains besides destabilase and prostaglandin also hirudin and blood plasma kallikrein inhibitor.
    With regard to the spatial liposome structure, it seems that in aqueous solvents most of the PC-LS hydrophobic units are directed internally; and that liposomes transferred to an organic solvent turn inside out. This may account for their preservation in the gastrointestinal tract, and rapid penetration into the blood presumably by transmembrane transfer. PC-LS can be obtained in the purified state only after disintegration of destabilase polypeptide chain, and this substantially reduces the stability of PC-LS.
    The medicinal leech Hirudo medicinalis therefore is the source of a stable prostacyclin-like substance with a MM of 391 D, but whose chemical structure remains to be fully identified.
    REFERENCES

    1. Baskova I.P., Khalil S., and Nikonov G.I. 1991. Inhibition of human blood plasma kallikrein by salivary gland secretion and extract of the medicinal leech. Biochem. Res. Commun. 103: 552-558.
    2. Baskova I.P. and Nikonov G.I. 1987. Detection of prostaglandin in preparations from the medicinal leech. Doclady of Academ. of Sci. of USSR. 292: 1492-1493.
    3. Baskova I.P. and Nikonov G.I. 1988. Thrombolytic agents of the preparation from the medicinal leeches. Folia Haematol., Leipzig 115: 166-170.
    4. Baskova I.P. and Nikonov G.I. 1991. Destabilase, the novel e-( g-Glu)-Lys isopeptidase with thrombolytic activity. Blood Coagulation and Fibrinolysis 2: 167-172.
    5. Baskova I.P., Nikonov G.I., Misselvitz F. and Leitin V.V. 1987. Mechanisms of inhibition of vascular-platelet hemostasis by salivary gland secretion of the medicinal leech Hirudo medicinalis. Biochemistry 9: 1461-1467.
    6. Baskova IP, Ucupova GI, Nikonov GI: Lipase and Cholesterol Esterase Activities of Medicinal Leech (Hirudo medicinalis) Salivary Gland Secretion. Biochimia (USSR) 1984; v. 49, 4:676-678.
    7. Baskova IP, Nikonov GI, Dolgov VV, Tertov VV, Orechov AN, Olferev AM, Perova NV, Bazazan GG, Sitina NP, Repin VS: The effect of medicinal leech preparations on atherogenesis. Cardiology (USSR) 1989; v. 29, 5:75-79.
    8. Born G.V., Cross H. Aggregation of blood platelets. J.Physiol. 168 (1963) 17.
    9. Claude, A. Spreading properties of leech extracts and formation of lumph. J. Exp. Med. 66. 353-56 (1937).
    10. Fritz H., Gebhard M., Meister R. and Fink E. Tripsin-plasmin inhibitors from leeches. Isolation, amino acid composition, inhibitory characteristics. Proceedings of the Int. Res. Conf. on Proteinase Inhibitors. 1971, Munich, 271-280.
    11. Herman F., Hadhazy P., Magyar K. In Vitro Selectivity between Hypotensive and Platelet Antiaggregation Actions of Iloprost and PGI2 in Beagle Dogs. Thromb. Haemost., 1987; 58: 179.
    12. Kahn, D.R., Cohen, I. Factor XIII-catalyzed coupling of structural proteins. Biochim. Biophys. Acta. 668.490-495 (1981).
    13. Markwardt F. 1989. Inhibition of thrombin. In Prothrombin and related coagulation factors. Edited by F. Markwardt. Leiden Univ. Press, pp 116-131.
    14. Markwardt F., Hauptmann J., Nowak G., Kleben Ch. and Walsman P. 1982. Pharmacological studies on the antithrombotic action of hirudin in experimental animals. Thromb. Haemost. 47: 226-229.
    15. Markwardt F., Kaiser B. and Nowak G. 1989. Studies on antithrombotic effects of recombinant hirudin. Thromb. Res. 54: 377-388.
    16. Martinek K., Klyachko N.L., Kabanov A.V., Khmelnitsky Y.L., Levashov A.V. Micellar enzymology: its relation to membranology. Biochim. Biophys.Acta 981 (1989) 161-172.
    17. Musial J., Wilozinska M., Sladek K. Fibrionolytic Activity of Prostacyclin and Iloprost in Patients with Peripheral Arterial Disease. Prostaglandins, 1986; 31: 51-61.
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    19. Lowry O.H., Rosenlough N.Y. and Farr A.L. 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193: 266-273.
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    23. Nikonov G.I., Baskova I.P. 1990. Antihemostatic, thrombolytic and antiatherosclerotic properties of the medicinal leech saliva. Proceeding of the Ш Int. Cong. of leech scientists. 1990, Israel, p. 45.
    24. Nikonov G., Romanenko E. and Vaniushin B. 1989. The preparations from leech Hirudo medicinalis enhance DNA methylation in the rat liver. Abstract Book of the 19-th FEBS Meeting 1989, p. 261.
    25. Nikonov G.I., Titova E.A Kovanova E.K. and Seleznev K.G. 1994. The effect of the preparation Pijavit on metabolic process. Asclepion (Moscow) 1: 23-26.
    26. Nikonov, G.I. Destabilase complex - natural liposome synthesised by medicinal leeches. In Hirudotherapy and Hirudopharmacotherapy (G.I. Nikonov, ed.), 63-72. Moscow, 4 FVI (1996).
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  12. #42
    vodalau2004 este offline Utilizator Samanta
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    Implicit Re: HIRUDOLOGIA - tratament cu lipitori

    Am postat rezultate ale studiului lipitorilor pentru ca sa nu se creada ca tratamentul cu lipitori este ceva invechit arhaic. tel. 0742152189. Sa va dea Dumnezeu sanatate. Informatia este necesara! - natura este la indemana dvs. si pentru sanatatea dvs.

  13. #43
    vodalau2004 este offline Utilizator Samanta
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    Implicit Re: HIRUDOLOGIA - tratament cu lipitori

    CASE ONE
    Summary: A 25-year-old male fell off a ladder sustaining a ring avulsion injury beginning at the junction of the distal one third, proximal two-third level of the proximal phalanx.
    Following failed venous reconstruction with vein grafts, leeches were applied and color improved dramatically. Application continued every 4 to 6 hours. 5 days postoperatively, active range of motion was begun. 8 days postoperatively, venous flow was sufficiently restored.

    3 weeks postoperatively, a skin graft was performed. 6 weeks postoperatively, following treatment with active and passive movement as well as coban taping, the finger has a TAM of 224 degrees.

    Procedure illustrated in case one was performed successfully by Dr. D.H. Lalonde, B.Sc, M.Sc.,M.D., FRCSC Plastic and Reconstructive Surgery, St.John, N.B., Canada.

  14. #44
    vodalau2004 este offline Utilizator Samanta
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    Implicit Re: HIRUDOLOGIA - tratament cu lipitori

    CASE TWO
    Summary: A 45-year-old, white male patient who suffered a complete avulsion of the upper two-thirds of his right ear. The ear was reattached by doing a microvascular anastomosis of a small artery anteriorly. No veins were available for anastomosis. Soon after anastomosis, there was obvious venous congestion. Medicinal leeches were used for treating venous congestion.
    48 Hours Postoperatively:
    3 leeches have been applied every 8 hours.
    Following the initial application, obvious improvement was noted.
    24 Hours Postoperatively:
    Photograph shows severe venous congestion in the reimplanted ear following complete avulsion.
    6 Days Postoperatively:
    The entire reimplanted ear was viable and healing well.
    72 Hours Postoperatively:
    3 leeches have been applied every 8 hours for periods of 15 to 30 minutes. Marked improvement in color with decreased swelling and congestion was noted.
    Two Months Postoperatively:
    Complete survival of re-attached ear.

    Precedures illustrated in cases two and three were performed susccessfully by Dr. Kulwant S. Bhangoo, M.D.,F.R.C.S., FACS, FICS, Chief of Plastic Surgery at Mercy Hospital, Buffalo

  15. #45
    vodalau2004 este offline Utilizator Samanta
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    Implicit Re: HIRUDOLOGIA - tratament cu lipitori

    CASE THREE
    Summary: A 22-year-old male patient who suffered a severe crush injury to his right ear. Injury was caused by a concrete mixer resulting in almost complete avulsion of his right ear with multiple and extensive contusion crush-type lacerations. The ear was reattached. About 10 hours postoperatively, obvious venous congestion was noted. It was felt that medicinal leeches were required to salvage the ear.
    16 Hours Postoperatively:
    Photograph shows severe venous congestion in the reimplanted ear following surgery.
    48 Hours Postoperatively:
    4 leeches have been applied every 8 hours. Definite improvement in venous
    congestion was noted.
    Two Months Postoperatively:
    The entire ear survived except for a small area around the lobule of the ear.
    72 Hours Postoperatively:
    3 leeches have been applied every 8 hours. The ear was less swollen and obviously viable with markedly improved venous drainage.

    Precedures illustrated in cases two and three were performed susccessfully by Dr. Kulwant S. Bhangoo, M.D.,F.R.C.S., FACS, FICS, Chief of Plastic Surgery at Mercy Hospital, Buffalo, New York.

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