Pharmacological, pharmacokinetic, toxicological and clinical studies on protodioscin

I. Viktorov, E. Bozadjieva, M. Protich, et al.
Higher Medical Institute; Medical Academy Institute of Endocrinology, Gerontology and Geriatrics; Medical Academy Institute of Obstetrics and Gynecology, Bulgaria
IIMS Therapeutic Focus (1994)

SUMMARY

Clinical investigations on a total of 212 males with disorders of sexual functions confirmed experimental data pointing at a pronounced stimulating effect on these sexual functions by the new phytochemical preparation of Tribulus terrestris extract. Administered in average daily doses of 1.5 g in the course of 30 to 40 days, it restores and improves libido sexualis in all forms of impotentia coeundi.

Studies on the acute, subchronic and chronic toxicities of protodioscin, the active ingredient of Tribulus terrestris extract, determined that the compound is to be classified as practically non-toxic substances. The harmlessness of the preparation deserves particular attention. No data about toxic manifestations were established under experimental conditions with acute, subchronic and chronic toxicities (behavioral, hematological, biochemical, functional and morphological studies). No data were established concerning carcinogenic and teratogenic effect.

The combined action of the preparation (stimulation of the sexual behavior and spermatogenesis) and the absence of adverse effects characterize the preparation as an original agent for the treatment of males with disorders in the sexual functions.

INTRODUCTION

The problem of stimulation of sexual function, and of spermatogenesis and ovogenesis in particular, is both of biological and medical significance, as it is associated with the problem of preservation of the sexual potential of male and female individuals. According to statistical data, 10 - 20% of all marriages are childless. In about 30 - 50% of the cases, male sterility is the cause. The diagnostics and treatment of male sterility is still a very difficult task.

At present, drugs on hormonal basis are predominantly used in the treatment of hypogonadism and genital anomalies. In fact, the substitutive hormonal therapy is sometimes ineffective and quite often it induces a lasting hypofunction of the hypothalamic-pituitary-gonadal axis. Therefore, regardless of the great number of new, highly effective and possibly less harmful drugs, studies in this field are very topical. The formulation of a non-hormonal preparation with sufficient activity and without harmful side effects, could contribute to overcoming of the therapeutic problems in the sphere of sexual functional disorders.

The plant Tribulus terrestris L., has long been known in folk medicine of Eastern countries and Bulgaria and has been used in the treatment of sexual deficiency. The present article is intended to acquaint the reader with the pharmacological, toxicological and clinical-therapeutic characteristics of the phytochemical preparation, marketed under the name Libilov™ and Tribestan®.

GENERAL INFORMATION

The active components of the plant Tribulus terrestris L. extract are non-steroid saponins of the furostanol type, termed protodioscin. A pronounced stimulating effect on the spermatogenesis is observed after the preparation is administered orally to sexually mature rats.

Increased numbers of spermatogonia, spermatocytes, spermatids and mature spermatozoa in the testes has been observed without widening of the diameter of the seminiferous tubules. In parallel, increased number of Sertoli cells is observed in a volume unit from rat testes. Oral administration stimulates the mitotic activity of spermatogonia in mature rats. Oral administration of the preparation leads to intensification of spermatogenesis and enhancement of the quality of spermatozoa in sexually mature rats. The percentage of motile spermatozoa increases, the characteristics of their motility improve, prolonging the period of their viability at the same time. Given orally to swine, protodioscin stimulates their sexual behavior. The clinical trials of the preparation have also confirmed the experimental data.

Protodioscin, administered to male subjects with disorders in the spermiogram, due to varicocele, increases the volume of ejaculated sperm by 1-2 ml, increases spermatozoid concentration by 30 million/ml, increases the percentage of motile spermatozoa by 30%. The preparation has a marked effect on the motility of spermatozoa in case of oligoasthenozoospermia, as revealed by the study of its therapeutic properties in men. The mean number of motile spermatozoa in the group studied was 29% prior to the study, and later it reached up to 36.6%. The motility rate of the spermatozoa prior to the treatment was 1.95 mm/sec and 3.76 mm/sec post treatment.

Of definite interest is the treatment of patients with unilateral and bilateral hypotrophy of the testes, combined with disorders of the spermiogram. The libido was enhanced after a 60-day protodioscin treatment and the characteristics of the spermiogram were improved. Recovery and enhanced libido were observed in patients with primary and secondary hypogonadism after administration, as well as prolonged erection.

Both experimental and clinical studies reveal that protodioscin is not toxic and induces no adverse effects.

CHEMICAL AND PHYSICAL PROPERTIES

Protodioscin is yellow-brown amorphous powder with a specific aroma and bitter taste; soluble in water, sparingly soluble in methyl alcohol and insoluble in chloroform.

PHARMACOLOGICAL STUDIES

Methods characterizing the stimulating effect on spermatogenesis

Spermatogenesis is a complicated process, covering proliferation of the spermatogonia, long-lasting process of the tissue meiosis and numerous changes in the spermatids during their preformation. The effect on the sexual cells can occur during the reproductive period - mitotic division of the spermatogonia or during the maturation of the spermatozoa. The effect on protodioscin on mitosis and maturation of the gonocytes has been studied using quantitative cytological methods. After oral administration of protodioscin in a single daily dose of 70 mg/kg body mass for 20 days, the testes of 8 rats were fixed in neutral formol-calcium and in Serra's solution, and later embedded in paraffin. The testes of 8 untreated animals were used as the control. The histological preparations from the testes were stained with hematoxylin (after Mayer) and fast-green (after Yordanov, 1976). Spermatogonia, spermatocytes and spermatids of 40 cross-sections through the seminiferous tubules were counted for each animal from both experimental and control groups (a total of 640), with identical diameter of the tubules (determined by eyepiece micrometer) in phase VII, according to the classification of Leblond and Clermon (1952).

Using light microscopy, the thickening of the layer of the spermatogenesis cells was observed in the cross-sections of the seminiferous tubules and a narrowing of their lumen in the treated animals (Fig 1). That resulted from the increased number of rows of sexual cells. The number of spermatogonia in the 8 experimental animals (i.e. in 320 sections of the seminiferous tubules) was 58 spermatogonia on the average per seminiferous tubule (between 48 and 63). The number of spermatogonia in one seminiferous tubule in the control animals was 36 (between 36 and 40 spermatogonia per tubule). The mean number of spermatocytes in a seminiferous tubules was identical to that of the spermatogonia. The number of spermatids in phase VII varied from 148 to 180 per seminiferous tubule in the treated animals (mean value 176). Their number in the control animals was between 112 and 125 (mean 119). The preparation significantly increased the number of spermatogonia, spermatocytes and spermatids in the testes of rats, with no other effect on the diameter of the seminiferous tubules.



Figure 1. Stimulation of spermatogenesis by protodioscin

Effect on DNA synthesis in gonocytes

The preparation's effect on DNA synthesis in the sexual cells has been studied by cytohistoradiography. The testes of rats treated with protodioscin (for 7 days) and with 3H-thymidine (every second day), and later with colchicine (3 hours prior to decapitation), were fixed in Serra's solution and embedded in paraffin. The sections were covered with Ilford liquid emulsion and left to stay for 25 days. A higher number of 3H-thymidine-labelled spermatogonia type "A" and "B" was found in the treated rats compared to the control animals (Fig. 2)



Figure 2. Effect of protodioscin on DNA synthesis.

The percentage of 3H-thymidine-labelled spermatogonia vs. total number

The mean number of spermatogonia per section from the seminiferous tubules was 56 in the treated animals, 41 of them labeled with radioisotopes. These numbers were 50 and 18 respectively, in the control animals. The increased number of spermatogonia, with 3H-thymidine included for the treated animals, suggested an intensified DNA synthesis under the effect of protodioscin, as well as an increased number of spermatogonia during the phase V of the cell cycle.

Effect on Leydig and Sertoli cells in the testes

It is well known that Leydig and Sertoli cells participate in the process of spermatogenesis. Quantitative cytological methods were used for the evaluation of the effect of the protodioscin on these cells. The results show that the number of Sertoli cells was increased in the seminiferous tubules of protodioscin-treated animals, compared to the controls (Fig. 3).



Figure 3. Effect of protodioscin on Leydig and Sertoli cells.

The mean number of Sertoli cells in a section of the seminiferous tubule in the treated animals was 29 versus 19.50 in the controls (increase by 40%). The cytological studies of the testes showed no differences in the number of Leydig cells between the experimental and control animals.

Effect on concentration, motility and survival of spermatozoa

The concentration, motility and viability of spermatozoa in the epididymis of rats treated for 30 days with protodioscin were studied immediately after decapitation. Sodium citrate was used as diluent. The mean spermatozoa number per ml was higher by two million in the treated animals, compared to the controls (Fig. 4).



Figure 4. Effect of protodioscin on the concentration and motility of rat spermatozoa.

The number of motile spermatozoa under the microscope was 8% higher in the treated animals. Furthermore, their spermatozoa were more viable. The loss of their advancing movements could be observed on the 75th minute, on the average, and in the control animal group - by the 45th minute (Fig. 5).



Figure 5. The effect of protodioscin on the viability of rat spermatozoa.

Effect on the sexual libido

The effect of protodioscin on the sexual behavior was studied on male pigs with confirmed lasting impotence. The preparation was administered orally and its effect on the sexual behavior and sexual reflexes was followed up daily. Individual animal reaction to the preparation was observed. The libido and sexual reflexes were restored in 71% of the animals with complete absence of libido, treated with a daily dose of 70 mg/kg for 10 days. In the animals with poor libido and long reflex period of sexual reflexes, recovery was recorded in 100% of the cases (Fig. 6).



Figure 6. Effect of protodioscin on animal libido.

Studies on serum concentration of the hormones from the hypophyseal-gonadal axis

The experiments were carried out on healthy subjects (8 male and 8 female), aged between 28 and 45 years (Milanov et al., 1981). The preparation was administered orally in a dose of one tablet, three times daily at 8-hour intervals for 5 days. The basal hormonal levels were determined before and after the intake of the protodioscin (at 8:00 am and at noon). The concentrations of the luteinizing (LH) and follicle-stimulating (FSH) hormones were determined by kits provided by Biodata (Italy). Serum testosterone was determined by the method of R.H.Williams (1967), serum estradiol - by the method of C.P.Orezyk (1974), using kits provided by the Sorin (Belgium). The results reveal that the drug elevated the level of the luteinizing hormone and testosterone in the orally treated healthy males, not affecting FSH (Fig. 7A).

In the females, the concentration of FSH and estradiol were increased under the effect of protodioscin, whereas the testosterone concentration was not significantly changed (Fig. 7B). The results show that the preparation has an effect on the hormones from the hypophyseal-gonadal axis, while at the same time not disturbing the hormonal balance in the body, thus enabling its administration as an agent stimulating the reproductive function.



Figure 7. Effect of protodioscin on the concentration of hormones in the hypophyseal-gonadal axis in the blood plasma of healthy males (A) and females (B).

Effect on the central nervous system

The screening system for neuro-pharmacological tests (R.Nikolov, 1980) was used in the studies. The following parameters of the treated animals were observed during the first stage of the screening: awareness, mood, motor activity, muscle tone and somatic reflexes.

The second stage of the screening covered the administration of many substances with an effect on the central nervous system, e.g. corazol, strychnine, nicotine, arecoline, phenamine, sodium hexobarbital, reserpine. The drug was applied intraperiotoneally to albino mice, H line, with a body mass of 18 - 22 g.

With a dose of 100 mg/kg body mass (1/4 of LD50), the drug had no effect on the behavior of the contact animals in the cage. During observations out of the cage, the animals became more excited, with enhanced reactivity. Their muscle tone was simultaneously reduced. In that dose, the drug inhibited moderately the corazol-induced convulsions, but the other reflexes were suppressed. The maximum tolerance dose - 300 mg/kg body mass - led to reduction of the motor activity, slight disturbance of gait and lower muscle tone of the limbs and stomach.

Effect on the cardiovascular system

The effect of the drug on the blood pressure values of cats under urethan narcosis was studied by the method of Ludwig Zyon (S.Vankov, 1981). The drug was injected intramuscularly and intraperitoneally as 10% aqueous solution. The intramuscular application of the drug in doses of 50, 100 and 150 mg/kg body mass had no significant effect on the blood pressure of the urethanized cats. A significant hypotensive effect was observed with the intraperitoneal application of the drug in a dose of 150 mg/kg body weight, advancing from the 5th to the 10th minute after application. The values of the arterial pressure decreased by 20% compared to the initial ones. The oral administration of protodioscin in a dose of 150 mg/kg on awake dogs had no effect on the blood pressure. The oral administration in doses of 50, 100 and 150 mg/kg body mass had no effect on the autonomic nervous system of the urethanized cats.

PHARMACOKINETIC STUDY

The experiments were carried out on albino, Wistar rats (180 - 200 g body mass) in 1981 by N. Dikova and V.Ognianova. The unchanged protodioscin in plasma, bile and urine was measured by thin-layer chromatography. Semi-quantitative measures were recorded and standardized by the precisely determined protodioscin concentrations. To determine the concentration of plasma protodioscin, the animals were intravenously injected with single doses of 50 and 200 mg/kg body mass. Citrate blood was withdrawn 2, 4, 10, 20, 30, 45, 60, 90, 120 and 180 min. after injection. To determine protodioscin excretion in the bile the animals were treated intravenously and orally with single doses of 50 and 200 mg/kg.

The bile was dynamically collected: up to the 6th hour, from the 6th to the 9th hour, from the 9th to the 24th hour after each application. Twenty-four-hour urine was collected. The results show that protodioscin was rapidly eliminated from the plasma and its concentrations were insignificant after the 180th minute. About 12 to 14% protodioscin were excreted in the bile and about 6 - 7 % in the urine within 24 hours after the intravenous administration of the doses of 50 and 200 mg/kg. Protodioscin from 2 to 4% were excreted with the bile after oral administration. No measurable concentration of unchanged protodioscin was found in 24-hour urine after oral administration.

TOXICOLOGICAL STUDY
(G.Tanev, S.Zarkova, 1980)

Acute toxicity

The acute toxicity of protodioscin was studied after intraperitoneal and oral application to albino mice, H line (18 - 20 mg body mass) and albino rats (160 - 180 g body mass). LD50 was also studied. It was concluded that the product can be included in the group of practically nontoxic substances. LD50 was 1942 mg/kg body mass with intraperitoneal application to mice and over 10,000 mg/kg body mass - with oral administration. The mean lethal dose of protodioscin with intraperitoneal application to rats was 750 (375 +/- 1,500 ) mg body mass, and after oral administration - over 10,000 mg/kg.

Subacute toxicity

The protodioscin was administered orally to albino Wistar rats for 30 and 90 days in the following doses: 75 mg/kg, 150 mg/kg, 225 mg/kg and 300 mg/kg body mass. No increased lethality was observed, nor a change in the behavior of the animals. No significant changes were observed in the routine clinical-laboratory and biochemical indices, nor morphological changes in the internal organs.

Chronic toxicity

Protodioscin was administered orally to albino rats for 6 months in doses of 75 mg/kg and 150 mg/kg body mass, as well as in 75 mg/kg body mass for 180 days to beagle dogs. The following toxic symptoms were looked for: changes in behavior, changes in the hematological, biochemical, functional and morphological parameters. No significant changes were found both in the behavior and in the reflexes of the animals. No increased lethality was observed. No pathological deviations from the physiological values were found in all hematological and clinical-chemical indices studied. No pathological changes in the structure of the internal organs, related to the toxic effect of the preparation, were detected.

Teratological and embryotoxic studies were simultaneously performed, as well as some experiments to follow the pre- and postnatal development (Z.Ilieva, 1980).

No teratogenic and embryotoxic action, nor deleterious effect on the development of the first generation after its littering, were found after the oral administration of the product in a dose of 750 mg/kg body mass to pregnant Wistar rats.

Studies were carried out to exclude the possible carcinogenic potential of protodioscin during a long-term treatment of rats (Gendzhev, 1981).

Increased incidence of neoplasms compared to the control animals was not observed with daily doses of 50 and 150 mg/kg body weight, administered orally for 23 months. No toxic damage was found morphologically in the rat organs.

DISCUSSION OF THE RESULTS

The experimental data on the biological activity of protodioscin show that its oral administration to rats significantly increased the number of spermatogonia, spermatocytes and spermatids, without any changes in the diameter of the seminiferous tubules. This fact is associated with the confirmed stimulating effect on spermatogenesis as a whole. It is well known that DNA synthesis occurs in the s-phase of the mitotic cycle. A fact of certain interest is that a significant increase of type A and B spermatogonia was found in the rats simultaneously treated with protodioscin and 3H-thymidine during the s-phase.

Hence, it can be concluded that the product intensifies the mitotic activity of spermatogonia. The cytologically detected increased incidence of Sertoli cells, caused by the product, presupposes that the mitosis of these cells has also been stimulated. The important role of Sertoli cells in the regulation of spermatogenesis is well known (Lacy, 1967; Kerr and Klester, 1974, Steinberger, 1971), hence the increased number of Sertoli cells during protodioscin treatment should be associated with the intensification of spermatogenesis. No changes were identified in the Leydig cells of the experimental animals, which suggests that the effect of the product on the spermatogenesis probably does not include these cells. The literature data show that the proliferation of spermatogonia in mammals and birds is FSH-stimulated (Stoinberger et al., 1964; Mancini et al., 1966; Ishiis and Furua, 1975; Krueger et al., 1974). The authors presume that the effect of FSH on spermatogenesis is due to Sertoli cells. The radioimmunological studies on healthy males showed no changes in the FSH-level under protodioscin effect, which suggests presence of a selective effect of the product on gonocytes. On the other hand, elevated LH-levels were found in protodioscin treated healthy males, which suggests the existence of central action.

The pharmacokinetic studies reveal no measurable concentrations of the product in the plasma after oral administration to rats, but spots unidentified so far were detected by the chromatographic methods.

The authors (Dikova and Ognyanova) presume a biotransformation of the product in the body. In such cases, some of the metabolites formed during the biotransformation can be expected to possess a stimulating effect at hypothalamic level.

The effect on the libido of the male pigs is clearly manifested. Protodioscin not only stimulates the libido, but also possesses a therapeutic effect as well in the cases of impotence, manifested in complete absence of libido. The effect of the product on the quality of the spermatozoa clearly shows that the spermatozoa of the treated animals are more viable and more resistant, suggesting a better fertility. Many researchers believe that the sexual behavior of the animals and the motility of the spermatozoa depend on testosterone levels. Other authors think that the sexual behavior is modulated by dihydrotestosterone. The problem of the mode of modulation of the sexual behavior remains debatable. If we assume that androgen-like factors are formed through biotransformation in the body, they would not induce changes in the interstitial cells.

Special attention should be paid to the harmlessness of the product. No evidence of acute, subacute and chronic toxicity has been found during the experimental behavioral, hematological, functional, biochemical and morphological studies. No data on carcinogenic and teratogenic effect are available.

The fact that the product has an effect on the hormonal balance in the body, without disordering its regulatory mechanisms, is of equal importance. The combined action of the drug (stimulation of sexual libido and spermatogenesis) and the absence of adverse effects, characterize it as an original agent for the treatment of males with disordered sexual function.

CLINICAL STUDIES

Materials and methods

The experimental data from protodioscin administration have been clinically confirmed so far by three groups of researchers: by the Higher Military Medical Institute under the guidance of Prof. I. Viktorov, Corresponding Member of the Bulgarian Academy of Sciences; by the Medical University of Sofia; State University Hospital of Endocrinology, Gerontology and Geriatry, under the guidance of Prof. E. Bozadzhieva, and by the National Institute of Obstetrics and Gynecology - under the guidance of Prof. M. Protich.

The studies were carried on 212 male individuals, aged between 14 and 60 years. The therapeutic properties of protodioscin were studied in patients with partial or complete impotence. Both the tolerance and the adverse effects of the product were studied. The studies were carried out by the method of simple blind experiment, using placebo. In nozological terms, the studies covered various types of male impotence: idiopathic oligoasthenozoospermia - 39 patients, resection of the left internal testicular vein in varicocele, with subsequent oligoasthenozoospermia - 50 patients; inflammatory process of the prostate with oligo- and azoospermia - 53 patients, primary and secondary male hypogonadism - 20 patients, impotentia coeundi - 50 patients. The product was individually administered to all patients who had not received hormonal agents for at least one month prior to the treatment. The duration of the treatment depended on acuteness of the disease - 30 - 60 days on the average (Bozadzhieva et al., and Protich et al.) and 90 days (Victorov et al.) the mean daily dose was 3 - 6 film tablets of 0.250g. Some of the patients were favorably affected by 3 tablets daily (Protich et al.), whereas the other research teams administered 6 tablets (3 x 2). The andrological state was used as the basis for the adequate evaluation of the reproductivity of the patients by all three teams. The basic parameters of sperm were observed, i.e. volume and pH of the ejaculate, concentration of spermatozoa (number/ml), percentage of motile spermatozoa, mean rate of population motility and percentage of pathological forms of the spermatozoa. Detailed case history on the sexual behavior of the patients was recorded prior to and after the therapeutic protodioscin course. The effect of the product on hair growth was observed in some patients. One of the teams (Bozadzhieva et al.) also observed the changes in the serum levels of gonadotropins, progesterone, testosterone, estradiol and cholesterol. The other team (Victorov et al.) followed up the changes in testosterone serum levels in the protodioscin treated patients. The hormonal levels were radioimmunologically determined using kits and reagents provided by the French-Italian-Belgian Association CEA-IRE-SORIN. The results from these studies were statistically assessed by variation analysis.

Results

Significant changes in spermatozoon motility were found after protodioscin intake for 60 days (with a daily dose of 3 film tablets) by males with idiopathic oligoasthenozoospermia. The number of spermatozoa with normal motility, as well as the mean motility rate, were increased.

The percentage of the motile spermatozoa was 29, on the average, prior to the treatment and reached up to 36.66 after the treatment (p < 0.005). The mean motility rate of spermatozoa prior to the treatment was 1.95 mm/sec, after the treatment - 3.63 respectively (p < 0.001). No changes were observed in the ejaculate volume (Fig. 8). In both cases (before and after treatment) the ejaculate volume was within the limits of the norm, about 4 ml on the average. The number of spermatozoa in the treated patients was higher by 3 ml per ml of ejaculate on the average. In some cases, normalization of the spermiogram occurred during repeated treatment with a daily dose of 6 tablets. In these cases, the improvement of the spermiogram (normalization of the increased viscosity, increased ejaculate volume, increased concentration and accelerated motility of the spermatozoa) was accompanied be elevation of the serum levels of the luteinizing hormone and testosterone, and reduction of the estradiol level.



Figure 8. Effect of protodioscin treatment on spermatozoa concentration and motility.

The patients with idiopathic azoospermia after 90-day treatment with a daily dose of 1.5g are of particular interest. The results were significant in three of all 7 patients treated. No spermatozoa were detected prior to the treatment. After the treatment, 3.5 million/ml ejaculate were recorded in one of the patients, 15 million/ml in the second and 28 million/ml in the third. The percentage of motile spermatozoa in the first patient was 10, and in the other two - between 25 and 30. The motility rate of the spermatozoa was about 5 mm/sec. In two of the patients, 30 - 40 spermatozoa per field were observed, and in the third - about 5 spermatozoa per field after the treatment, compared with the absence of any prior to the treatment (Fig. 9).



Figure 9. Effect of protodioscin on patients with idiopathic azoospermia.

One of the patients failed to be affected by the product. The studies on the patients from that nozological group continued with the administration of maintenance dose.

The clinical comparison of the results from the treatment with provirone of patients with idiopathic azoospermia and after protodioscin administration revealed a favorable effect on three of the patients (out of 6), unsuccessfully treated with provirone for a long period of time. The results in the patients with varicocele and oligoasthenozoospermia as regards the motility of the spermatozoa were identical in the reports of all research teams, regardless of the differences in the doses and duration of the treatment. Protich et al. found an average of 26.88% motile spermatozoa prior to the treatment and after a 60-day course with a dose of 1 tablet, three times daily - 39% (p < 0.02) with a mean motility rate of spermatozoa 2.06 mm/sec prior to the treatment and 4.44 mm/sec post treatment respectively (Fig. 10). No change in the ejaculate volume was found. The other team (Victorov at al.) observed more pronounced changes in the ejaculate volume after the 90-day treatment with a daily dose of 1.5 g (4.5 ml compared to 1 - 2 ml prior to the treatment, i.e. an average of 1.55 ml of ejaculate volume in all patients). The number of spermatozoa in 1 ml reached the values in 100% of the patients treated. The mean percentage of motile spermatozoa prior to the treatment was 2.06 and was increased to 33.09 (Tables 1,2,3).



Figure 10. Effect of protodioscin in patients with varicocele and oligoasthenozoospermia.