Clinical Evaluation of Spermatogenic..

By: Tuhin Kanti Biswas1*, Srikanta Pandit1, Samaresh M

Clinical evaluation of spermatogenic activity of processed Shilajit (PS) in oligospermia

Tuhin Kanti Biswas1*, Srikanta Pandit1, Samaresh Mondal1, Sunil Kumar Biswas1, Utpalendu Jana1, Tapan Ghosh1, Paresh Chandra Tripathi2, Pratip Kumar Debnath3 Runa Ghosh Auddy4 and Biswajit Auddy4

1 Research Unit, J. B. Roy State Ayurvedic Medical College and Hospital,    170-172, Raja Dinendra Street, Kolkata-700004, India,e-mail: biswastuhin@rediffmail.com,Telephone: +91-33-25335019, Telefax: +91-33-23371910

 2 Institute of Postgraduate Ayurvedic Education and Research at SVSP Hospital,294/3/1, Acharya Prafulla Chandra Road, Kolkata 700 009, India,e-mail:  biswastuhin@rediffmail.com, Telephone: +91-33-25335019, Telefax: +91-33-23371910

3 Former Professor, Department of Kayachikitsa, J. B. Roy State Ayurvedic Medical College and Hospital, 170-172, Raja Dinendra Street, Kolkata-700004, India,  e-mail: pk_devnath@rediffmail.com

 Running title: Clinical evaluation of Shilajit in oligospermia

 Key words: Shilajit; oligospermia; testosterone, FSH, LH, Shilajit safety              

* Corresponding author

 

Summary

Safety and spermatogenic activity of processed Shilajit (PS) was evaluated in oligospermic patients. Initially sixty infertile male patients were assessed and the patients having total sperm count below 20 million/ml semen were considered oligospermic and enrolled in the study (n=35). 100 mg PS capsule was administered twice daily after major meals for 90 days. Total semenogram and serum testosterone, LH and FSH were estimated before and at the end of the treatment. Malondialdehyde (MDA), marker for oxidative stress, content of semen and biochemical parameters for safety were also evaluated. Twenty-eight patients who completed the treatment showed significant (p<0.001) improvement in spermia (+37.6%), total sperm count (+61.4%), motility (12.4 to 17.4 % after different time intervals), normal sperm count (+18.9%) with concomitant decrease in pus and epithelial cell count compared to baseline value. Significant decrease of semen MDA content (-18.7%) was observed. Also, serum testosterone (+23.5%; p<0.001) and FSH (+9.4%; p<0.05) levels significantly increased. HPLC chromatogram revealed inclusion of PS constituents in semen. Unaltered hepatic and renal profile of patients indicated that PS was safe at the given dose. The present findings provide further evidence of the spermatogenic nature of Shilajit, as attributed in Ayurvedic medicine, particularly when administered as processed Shilajit.

Introduction

The art of living depends upon several factors including maintenance of heredity with healthy progeny. Healthy progeny depends upon healthy gametes from both male and female partners of conjugated life style. Current epidemiological evidence suggests that 15% of couples experience infertility in the world and half remain untreated and/or unresolved. The distribution of the main diagnostic groups for infertility due to the male is 25%; ovulation 25%; tubal 20%; unexplained cause 25% and endometriosis 5% (Templeton, 1995). Among infertile couples, 40% are primarily due to the infertility of the male partner, while in 20% of these cases it is a combination of both male and female factors associated which lead to infertility (Agarwal & Kulkarni, 2003). Out of several causes of male infertility, in clinical practice oligospermia is considered one of the most prevalent causes (Haslett et al., 2002). 

Literally, oligospermia means insufficient number of spermatozoa; but scientifically it means a medical symptom characterized by less than 20 million spermatozoa per ml of ejaculate  (http://en.wikipedia.org/ wiki/Oligospermia, 2008).  The basic patho-physiology of oligospermia is still unknown but several hypotheses are considered to be responsible for oligospermia. Follicular stimulating hormone (FSH) and leutinising hormone (LH) play an important role in spermatogenesis. LH primarily stimulates Leydig cells to secrete testosterone; testosterone and FSH are the two hormones that act directly on Sertoli cells to promote spermatogenesis. Synthesis of androgen-binding protein (ABP) is a FSH dependent process and this protein binds testosterone and dihydrotestosterone and thus provides a local androgenic pool to support gametogenesis (Pramanik, 2007). Therefore, the levels of testosterone and FSH act as marker components and deficiency of which may result in oligospermia.

Partial mechanical obstruction may also result in oligospermia, which will lead to imminent surgical intervention (Berek et al., 1988). The excessive generation of reactive oxygen species (ROS) by abnormal spermatozoa and contaminating leukocytes has been defined as one of the important etiologies for male infertility. Generation and persistence of ROS in seminal fluid and sperm increase the rate of lipid peroxidation of sperm membrane which is manifested by a high MDA level (Maneesh & Jayalakshmi, 2006); a decrease in such high MDA levels of semen undeniably favors spermatogenesis.

There is no known specific drug for the management of oligospermia in modern medicine. Extensive clinical research is going on in oligospermia utilizing various natural sources of plants, mineral and animal origin as mentioned in different classical traditional texts throughout the world, including Ayurveda, the Indian ancient system of medicine. Shilajit is considered one of the wonder medicines of Ayurveda, which since ancient times, has been utilized for the management of male reproductive disorders.

Shilajit is a pale-brown to blackish-brown exudate that oozes from sedimentary rocks worldwide, largely in the Himalayas. Common people describe it from their knowledge as pahar-ki-pasina (sweat of mountains), pahar-ki-khoon (mountain blood), shilaras (rock-juice), asphalt, bitumen, etc. Shilajit is said to carry the healing power of these great mountains (           David & Vasant, 2001). It is an important drug of the ancient Ayurvedic materia medica and it is to this day used extensively by Ayurvedic physicians for a variety of diseases. Early Ayurvedic writings from the Charaka Samhita (Sharma PV, 1998) describe Shilajit as a cure for all disease as well as a Rasayana (rejuvenator) that promises to increase longevity. It is composed of rock humus, rock minerals and organic substances that have been compressed by layers of rock mixed with marine organisms and microbial metabolites (Ghosal, 1994). Several toxicological studies, both acute and sub-chronic, have already been performed by various scientists with Shilajit throughout the world. Per oral LD50 was found to be >2000mg/kg (Ghosal et al, 1989; Acharya et al, 1988) and Shilajit was found to be safe at  doses of 0.2g/kg and 1g/kg when used chronically (Kelginbaev et al, 1973; Anisimov et al, 1982; Fortan, 1984 and Al-Hamaidi et al, 2003).

Traditional uses of Shilajit primarily focus on diabetes and diseases of the urinary tract, but also include edema, tumors, muscle wasting, epilepsy and even insanity. Modern indications extend to all systems of the human body with a significant number of additions in the reproductive and nervous system. Clinical research confirms many of the properties for which Shilajit has been used (Talbert, 2004). In Ayurveda, Shilajit is employed for the management of male reproductive disorder, and in particular, under the parlance of Vrsya (an aphrodisiac with special reference to spermatogenesis) (Sharma PV, 1981).

However, in spite of its wide spectrum use as an aphrodisiac and spermatogenic activity by traditional Ayurvedic practitioners, no scientific report has so far been obtained for the spermatogenic activity of Shilajit in oligospermia in a clinical setting. The aim of the present research study is to explore the scientific evaluation of Shilajit for its spermaotogenic activity in oligospermic patients. In addition, evaluation of safety was carried out in the same Shilajit treated oligospermic patients.

Patients and methods

Preparation and analysis of the drug

Crude Shilajit rock was procured from Indian Herbs, Saharanpur, India and voucher specimen is kept in the laboratory with specific code number SJ/01/05. The processing of Shilajit was carried out by a patented procedure (Ghosal, 2002). According to this procedure, crude Shilajit rock was pulverized and passed through a 40 mesh screen. Powdered samples were extracted with hot (60°C) water, maintaining a solid: solvent ration of 1:6, for one hour. It was filtered and the process repeated once again with the marc. The final pooled filtrate was spray dried to make fine free-flowing powder, which is dark brown in color and designated as Processed Shilajit (PS). The powder was stored in desiccators at room temperature (24±2°C). 100 mg of PS powder was formulated in hard gelatin capsules. The standardization of PS involved application of various analytical and chemical methods like HPLC, HPTLC, FT-IR, 1H-NMR, UV-Vis Spectrophotometric examination, GC-MS, ESR spectroscopy. HPLC was carried out in a WATERS (USA) HPLC system with PDA detector and isocratic mobile phase consisting of acetonitrile: ortho-phsophoric acid: water (32:1:67) with a flow rate of 0.6 ml/minute using C-18 Novapak reverse phase column attached with a guard column for separation. The injection volume was 20 μl in water. The photodiode array detector wavelength was set at 240 nm. Particle size of the Shilajit in solution was analyzed using evaporative light scattering instrument.

Selection of the patient

The clinical trial was conducted between June 2006 and January 2008 at J. B. Roy State Ayurvedic Medical College and Hospital, Kolkata 700004, India after obtaining necessary permission from Institutional Ethics Committee (IEC) and the protocol was approved by the Scientific Review Committee (Ayurveda), Government of West Bengal. The IEC did not permit inclusion of any placebo group in the present study considering the grave nature of the disease. Patients who had a history of primary and secondary infertility for a period of 1-5 years were initially selected for the study. Thorough and relevant investigations were performed prior to inclusion and those matching inclusion and exclusion criteria were admitted to the study. Other relevant physical features were also considered. A total of 60 male patients aged between 30-45 years were initially included from the out patient department (OPD) of the same hospital. Out of a total of 60 patients, 21 patients were excluded as they could not meet up the inclusion criteria, 2 patients did not sign the consent form, and 2 patients did not start the treatment schedule. The rest 35 patients were enrolled for the treatment after obtaining consent according to the WHO-Helsinki protocol.

Inclusion/exclusion criteria

Eligibility was based on the following inclusion criteria: Infertile male patients aged between 30-45 years, irrespective of religion, occupation, income status, sperm count below 20 million sperm/ml, average motility <60 %, normal sperm <65% and  spermia <3.0 ml not currently receiving any other treatment from outside and willingness to give written informed consent for participation in the study. The exclusion criteria were any concomitant serious disorders of vital organs, receiving or having any infertility treatment for the past 1 month and any other treatment being received simultaneously that may influence the study. Patients of male infertility suffering from azoospermia, asthenospermia, necrospermia/necrozoospermia and teratospermia/teratozoospermia were also excluded from the present study. Patients with history of mumps, measles, small pox, chicken pox, tuberculosis or injury to genitalia in last 10 years were also excluded from the study.

 

Safety study

Safety evaluation was also carried out during the same study period, in the same group of patients with the same dose of PS (100 mg BID). Additional 2 ml of blood was collected for this purpose at the beginning and at the end of 90 days of PS treatment.   

Treatment schedule

All the patients were treated with PS 100 mg twice daily after principal meals, which were continued for 90 days to each patient. The present dose of PS (200 mg/day, p.o.) has been determined from the recommended dose of Shilajit in most of the debilitating states of health (Halpern, 2003) which was also simulating with 1/10th of LD50 dose in animals (Ghosal et al, 1989). Treatment of each patient was monitored weekly. No other treatment was advised to any patients that may influence the study. Each patient was closely observed for any adverse effects with PS. Subjects were given 15 days worth of capsules at each visit and compliance was monitored by traditional pill-count method at each follow-up visit and at the end of the study. A schematic representation of the study design is given in Figure 1.

Observation criteria

Semenogram is considered to be the direct method of analysis for the diagnosis and prognosis of the patients of oligospermia. This was performed by Giemsa and Leishman staining technique (Sood, 1999). Semen of each patient was collected for this purpose after a period of complete abstinence continued for not less than seven days. The features which were observed under the semenogram were the measurement of spermia, seminal pH, total sperm count (million/ml), liquefaction time (minutes), motility after 30, 60 and 120 minutes, spermatozoa morphology study (% of normal sperm), pus cells (per field), epithelia cells (per field) and RBC (per field). These were estimated on day ‘0’ and day ‘90’ interval. Hematologically, hemoglobin concentration (mg/dl), RBC (morphology), WBC (cmm), platelet count (x 105 per ml) and ESR (mm/hr) were measured following standard methodology (Briggs et al., 2003). These examinations were performed for the exclusion of any concomitant acute and chronic diseases, and were estimated at ‘0’ and ‘90’ days of treatment with the help of automated cell counter (Sysmex KX-21, Japan).

In the present study, the amount of malondialdehyde (MDA) in the total semen was measured as an index of lipid peroxidation. This was determined by the thiobarbituric acid (TBA) assay (Mihara & Uchiyama, 1978). Briefly, 100 μl of reconstituted lyophilized semen was added to 400 μl phosphate buffer (100 mM, pH 7.4) and 1 ml of TBA reagent containing 0.8% TBA (Sigma, USA), 15% tri-chloro acetic acid, 25% (1M) HCl and 0.4% butylated hydroxytoluene (BHT) in distilled water. The samples were heated in a boiling water bath for 30 minutes, cooled and centrifuged at 3000 rpm for 10 minutes, and absorbance of the supernatant was measured at 532 nm. All values were expressed as nmoles MDA/mg of dry semen.

In endocrinal investigations serum testosterone, LH and FSH levels were estimated on day ‘0’ and days ‘90’ of each patient to evaluate the role of the test drug on the hormonal level. These hormones were estimated using electrochemiluminescence immuno assay (ECLIA) using fully automated immunoanalyser, ELECSYS 1010 (Roche) and Beckman Coulter reagent kit (Germany).

The safety aspect of PS for the management of oligospermia was assessed on the basis of different biochemical parameters. The parameters evaluated in the present study were fasting blood sugar (mg/dl), urea (mg/dl), creatinine (mg/dl), uric acid (mg/dl), total bilirubin (mg/dl), total protein (gm/dl), albumin (gm/dl), globulin (gm/dl), SGOT (U/ml), SGPT (U/ml) and alkaline phosphatase (U/L). These parameters indicate the renal and hepatotoxicity of the test substances with specificity and systemic toxicity in general. Biochemical parameters were estimated using an automated analyzer (Hitachi-902/BS-300, Japan).

In the present study, chromatographic analysis of semen was carried out to assess the presence of PS bioactives in semen.  This innovative investigation helped us to identify another important marker for assessment of the effect of PS in spermatogenesis. Semen samples collected from each patient at the commencement and after 90 days of PS treatment were used for this purpose. The samples were lyophilized within an hour of ejaculation and stored at -20°C. The lyophilized semen samples were dissolved in double distilled water at a concentration of 5 mg/ml, sonicated for 10 minutes followed by centrifugation for 10 minutes at 4ºC at 4000 rpm. The chemical constituents of the supernatant were analyzed by HPLC using the conditions mentioned before. The processed Shilajit (0.5 mg) was similarly dissolved in 1 ml of water (with sonication) and was analyzed by HPLC.

Statistical analysis

Percentage changes for measures were expressed as the difference between means of the baseline and treatment phases divided by the mean of the baseline phase multiplied by 100. Paired ‘student’s t-test’ was carried out with the initial and final values and  p < 0.05 was considered statistically significant. Statistical analysis was performed using the computer statistical package SPSS/10.0 (SPSS, Chicago, IL, USA).

 Results

Standardization of Processed Shilajit (PS)

Standardized PS according to HPLC analysis (fig.2) was found to contain bioactive components like free and conjugated di-benzo-alpha-pyrones (DBPs), DBP-chromoproteins (DCPs) and fulvic acids and its equivalents within specified limits as mentioned in table 1.

Treatment efficacy

The objective variables were estimated on the basis of semenogram, hematological parameters and endocrinological investigations. Semenogram investigation revealed that there was a significant (p<0.001) increase of spermia (37.6%), total sperm count (61.4%), reduction of pus cell (-55.5%) and epithelial cells (-81.1%). Significant (p<0.001) gradual increase of sperm motility was also observed after 30 minutes (12.4%), 60 minutes (13.2%) and 120 minutes (17.4%) interval. Percent increment in normal sperm (18.9%) was also found to be significant (p<0.001) after 90 days of treatment with PS at a dose of 100 mg BID. Significant increment (p<0.001) of hemoglobin (5.2%) after 90 days of treatment was observed indicating normal maintenance of body physiology of patients. This was supplemented with significant increase (p<0.05) of WBC (6%). The most important biochemical markers for spermatogenesis like testosterone, LH and FSH were also estimated and highly significant increase (p<0.001) of testosterone (23.5%) and significant (p<0.05) increase of FSH (9.4%) were observed (Table 3). However, no significant alteration was observed in serum LH level of the PS treated patients. Significant decrease (-18.7%, p<0.001) in semen MDA content was also observed in patients received PS treatment.

HPLC analysis of semen

A distinct difference in HPLC chromatogram of the semen samples was observed before and after 90 days of treatment with PS (Fig.3a & 3b). Several small peaks denoting Shilajit constituents (3-OH DBP, 3,8(OH)2DBP and its amino acyl conjugates) were incorporated in the semen after treatment.

Safety

Investigations on safety study of PS with a dose of 100 mg BID revealed that there was no alteration in any of the objective features related to any systemic toxicities like serum urea (-0.9%), uric acid (-7.5%), serum bilirubin (9.6%), total protein (-1.3%), serum globulin (-6.7%), SGPT (7.5%), SGOT (-3.5%) and alkaline phsophatase (-6.7%). Significant lowering (p<0.05) of fasting blood sugar (-6.8%) supports the hypoglycemic effect of Shilajit (Trivedi et al., 2004) and serum creatinine (-7.8%) indicates that the drug does not show any adverse effect in renal profile even in normal state (table 2). Twenty eight patients completed the present study. There was seven drop-out cases. The main reasons for study withdrawals were protocol violations, follow-up link lost, and physician’s decision (Figure 2). No treatment emergent adverse events were reported to the doctors by any patients during treatment period.

Case study

In the present study four oligospermic patients informed about their fertility after 3 months of treatment with PS (100 mg BID) which has been presented as case studies.

Case 1: TKK, 37 years, a serviceman, complained of infertility after 3 years of married life. Investigations indicated that his wife had no problems to conceive. The overall health status of the patient was good . Semenogram of the patient showed low spermia (1.5 ml), low sperm count (15.5 million/ml), presence of pus cells in semen and serum testosterone level was found to be in the lower range (4.77 ng/ml). After 3 months of treatment with PS (100 mg BID) the spermia and sperm count increased to 2.5 ml (66.7%) and 22 million/ml (41.9%) respectively. No observable pus cell was detected in semen and serum testosterone level increased to 6.53ng/ml.  At the same visit he mentioned about his wife’s amenorrhoea for the previous 1 month and he confirmed the initiation of pregnancy with laboratory reports. 

Case 2: SS, a 30 year old farmer, complained of infertility after 5 years of married life. His wife maintained normal menstrual cycle. The patient had no history of complicated disease and possessed an overall healthy status. Semenogram of the patient showed low spermia (2.0 ml), low sperm count (17.6 million/ml), presence of pus cells and epithelial cells in semen and serum testosterone was found to be in the lower level (3.85 ng/ml). After 3 months of treatment with PS (100 mg BID) the spermia and sperm count increased to 3.0 ml and 22.5 million/ml respectively. No observable pus cell and epithelial cell was detected in semen. Serum testosterone level was increased to 4.71ng/ml.  He also mentioned about the amenorrhea condition of his wife for the last two cycles and in the follow up visit he confirmed his wife’s initiation of pregnancy along with laboratory reports. 

Case 3: TSG, a 36 year old businessman, complained of anxiety, hyperacidity, loss of strength, diminished sexual activity and mentioned that he had no issue after 3 years of married life. The patient possessed an overall good health. Semenogram of the patient showed low sperm count (13.5 million/ml), slightly lower sperm motility (65 after 1 hour) and presence of pus cells in semen. Serum testosterone level was initially 3.59ng/ml. After 3 months of treatment with PS (100 mg BID) distinct improvement in sexual activity was informed with concomitant decrease in anxiety and impending doom. The sperm count also increased to 27.7 million/ml. No observable pus cell was detected. Serum testosterone level was increased to 4.99ng/ml. He also mentioned about the amenorrhea condition of his wife for the last one cycle and in the follow up visit he confirmed his wife’s initiation of pregnancy along with laboratory reports. 

Case 4: SS, a 32 year old information technology professional, communicated with the principal investigator of the present study. The patient was suffering from infertility for 3 years. The patient possessed an overall health status and had no other history of complicated disease. Semenogram of the patient showed low sperm count (19.5 million/ml), slightly lower sperm motility (60% after 1 hour) and presence of abnormal forms of sperm in high concentration (50%). Pus cells were also observed in the semen. After 3 months of treatment with PS (100 mg BID) the sperm count increased to 37.1 million/ml. Sperm motility was increased to 80% after 1 hour and normal sperm cell count increased to 60%. Only a few pus cells were detected in semen. Serum testosterone level increased from 3.80 to 5.01ng/ml. His wife conceived and this was subsequently confirmed within one week of withdrawal of treatment indicating that the pregnancy ensued during the treatment period. 

 

Discussion

 

The male partner is considered a primary cause for infertility amongst couples and oligospermia is one of the most prevalent reasons for male infertility in clinical practice.  In most of the cases, functional deformity in spermatogenesis is the major reason for oligospermia, which involves either defective mechanism of testosterone, LH and FSH secretion, or there may be excess production of reactive oxygen specimen (ROS) or both. Reactive oxygen species can have beneficial or deleterious effects on sperm cells depending on their concentration (Murawski, 2007). Under normal physiological conditions, spermatozoa produce a small amount of ROS for proper capacitation and acrosomal reaction. ROS in semen are generated mainly by neutrophils and also by abnormal spermatozoa (Griveau & Le Lannou, 1997). Excessive production of free radicals or ROS can damage sperm because their plasma membrane and cytoplasm contain a large amount of polyunsaturated fatty acids (Agarwal et al., 2003). It has already been well documented that high levels of ROS in the semen (seminal plasma + spermatozoa) induced lipid peroxidation and were negatively correlated with the quality of sperm in the semen (Alvarez & Storey, 1995). MDA content of semen acts as a major marker for lipid peroxidation.

The present study clearly envisaged that PS could potentially control oxidative stress, which is reflected by lowered MDA levels and may stimulate spermatogenesis (Gomez et al., 1998; Jeong et al., 2006). This evidence is supplemented by significant increment of both testosterone and FSH, the two marker hormones that can directly induce spermatogenesis (table 3). At physiological level, testosterone did not produce any inhibition of FSH secretion, thus concomitant increase in serum levels of these hormones, observed in the present study, can promote spermatogenesis. Higher level of testosterone, in turn, inhibited LH secretion, which was also reflected in the present study. Apart from spermatogenesis, testosterone also controls the functional competence of the accessory sex organs, such as, prostate gland and seminal vesicles. Adequate seminal fluid, the secretions of seminal vesicles and prostate gland, is necessary for the survival and motility of spermatozoa. Hence, the probable reasons of the increased spermia, normal sperm count and motility in the present findings may be due the higher levels of testosterone. Spermatogenic activity of Shilajit is also reported pharmacologically in rats where the number of sperms in the epididymis and testes were significantly higher than that of the control animals. The serum testosterone level was also found to be significantly high in Shilajit treated rats. The changes of serum LH and FSH were negligible compared to that of the control animal (Jeong et al., 2006). 

One of the important constituents of PS is DBP; the ROS generated by sperm themselves and also by the phagocytes of the ejaculate can be put to balance or equilibrium by the reductants present in DBP-conjugates/DBPs (Ghosal, 2006). Other bioactives of PS, DCPs, fulvic acids, its equivalents, and amino acids can attenuate the deficiencies in oligospermic patients by captivating ROS and reactive nitrogen species (RNS), provide energy for sperm motility and therefore contribute to sperm health. Chromatographic analyses of semen after treatment with PS indicated inclusion of its constituents in semen. It was observed that PS in solution remains in a fine particle state with average diameter of the particle size being 954 nm. Being fine particulate state, ingredients of PS are quickly absorbed through intestinal tract and once in the systemic circulation, it can penetrate blood-brain (Kreuter, 2002) and blood-testis barrier (Kwon et al., 2008). Thus, PS constituents may be transmitted through blood-testis barrier to reach the target organelles, viz., seminiferous tubules and thereby exert their antioxidant actions. Hence, the observable effect of PS is probably due to the direct effect of its active constituents in the spermatogenesis process.

 The role of infection in male infertility is being increasingly recognized in the modern world. The decreased level of pus cells and epithelial cells after Shilajit treatment indicated that Shilajit had inhibited the growth of genital bacteria which are generally resistant to a number of currently recommended antibiotics. In literature, Shilajit has been recommended for genitor-urinary diseases (Schepetkin  et al., 2002).

 

Conclusion

The present findings, based on advanced diagnostic and clinical methodologies, support preclinical evidence about the effective medical management of oligospermia with Processed Shilajit (PS). Also, PS can be used therapeutically at a dose of 100 mg BID without any adverse side-effects.  HPLC analysis indicated that PS treatment for 90 days caused incorporation of some of its major constituents in semen. As clinical findings indicated a distinct improvement in the quality of semen of the treated oligospermic patients, it can be assumed that these improvements were partly due to inclusion of PS constituents in the semen. Regaining of fertility in four PS treated patients opens a window in the treatment of oligospermia with natural products. However, the exact role of these components on testicular function is yet to be established. This trend is expected to be more indicative if the PS treatment is continued for a longer period of time.

Acknowledgement

We acknowledge with thanks the suggestions and valuable comments of Prof. Shibnath Ghosal, former honorary advisor, Natreon Inc., Kolkata. Authors are indebted to Prof. Arup Mukherjee, Dept. of Pharmaceutical Technology, University of Calcutta, for estimation of particle size of Shilajit constituents. We also thank Natreon Inc, 2D Janine Place, New Brunswick, NJ 08901, USA for financial support to carry out the study.

 

 

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Talbert R (2004) Shilajit: A Materia Medica Monograph, A paper submitted in partial fulfillment of the requirements for the degree of Clinical Ayurvedic Specialist at California College of Ayurveda, 1117A East Main Street, Grass Valley, California.

Templeton A (1995) Infertility-epidemiology, aetiology and effective management. Health Bull (Edinb) 53(5): 294-8.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Legends of Figures

 

Fig. 1. Schematics of study design

 

Fig. 2. HPLC chromatogram of processed Shilajit by RP-C18 column

 

Fig. 3a. HPLC analysis of semen on day 0.

 

Fig. 3b. HPLC analysis of semen after 90 day treatment with processed Shilajit

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Table 1. Bioactive constituents with specified limits of processed Shilajit

 

Bioactive constituents                                                                   Specification              Actual content

                                                                                                                (%; w/w)             (%; w/w)

 

 

Free and conjugated                                                                                              ³ 0.30                                                  0.45

di-benzo-alpha-pyrones (DBPs)     

DBP-chromoproteins (DCPs)                                                        ³ 10.00                16.75

Fulvic acids and its equivalents                                         ³ 50.00                 54.80

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Table 2: Safety parameters at baseline and after 90 days of treatment with

 processed Shilajit (100mg BID) in oligospermic patients (n=28)

 

 

Parameters                                                     Processed Shilajit (100 mg BID)

                                                                       

                                                                        Baseline                                  90 days           % change  

                                   

FBS (mg/dl)                                                     95.6 (9.7)                                 89.1 (7.4)                                             -6.8                                                           

Urea (mg/dl)                                                    21.1 (5.0)                                 20.9 (3.8)                                             -0.9                                                           

Uric acid (mg/dl)                                             5.3 (1.4)                                   4.9 (1.5)                                               -7.5                                                           

Creatinin (mg/dl)                                            0.90 (0.14)                   0.83 (0.11)                               -7.8                                                   

Total bilirubin (mg/dl)                        1.04 (0.33)                   1.14 (0.39)                                9.6                                                                 

Total protein (gm/dl)                          7.6 (0.6)                                   7.5 (0.4)                                               -1.3                                                     

Albumin (gm/dl)                                              4.7 (0.4)                                   4.7 (0.3)                                                NC                                                           

Globulin (gm/dl)                                              3.0 (0.6)                                   2.8 (0.4)                                               -6.7                                                      

SGPT (U/ml)                                                   36.1 (14.2)                   38.8 (15.1)                               7.5                                                                 

SGOT (U/ml)                                                  34.1 (10.5)                   32.9 (8.5)                                             -3.5                                                     

Alkaline phosphatase (U/L)       129.8 (42.7)                 121.1 (41.0)                             -6.7     

 

 

Values represent Mean (SD); indicates p<0.05.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Table 3.  Objective variables at baseline and after 90 days of treatment with  

                processed Shilajit 100 mg BID) in oligospermic patients (n = 28)  

 

 

Parameters                                  Processed Shilajit (100 mg BID)

                                                                                   

                                                Baseline                                             90 days                                   % change

                                               

Semenogram

Spermia (ml)                                                   2.13 (0.72)                               2.93 (0.94)                               37.6*  

pH                                                                   7.90 (0.22)                               8.00 (0.22)                               1.27                             

Total sperm count                                          10.60 (4.34)                             17.11(8.97)                              61.4*  

(million/ml)            

Liquefaction time (min)                      38.71 (19.35)               39.33 (11.73)               1.60    

Motility

After 30 min                                                    64.07 (20.1)                             72.03 (22.7)                             12.4*                           

After 60 min                                                    59.64 (20.1)                             67.53 (21.8)              &nbs