[FONT="]Clinical evaluation of spermatogenic activity of processed Shilajit (PS) in oligospermia [/FONT]
Tuhin Kanti Biswas[SUP]1*[/SUP], Srikanta Pandit[SUP]1[/SUP], Samaresh Mondal[SUP]1[/SUP], Sunil Kumar Biswas[SUP]1[/SUP], Utpalendu Jana[SUP]1[/SUP], Tapan Ghosh[SUP]1[/SUP], Paresh Chandra Tripathi[SUP]2[/SUP], Pratip Kumar Debnath[SUP]3[/SUP] Runa Ghosh Auddy[SUP]4[/SUP] and Biswajit Auddy[SUP]4[/SUP] [SUP]1[/SUP] Research Unit, J. B. Roy State Ayurvedic Medical College and Hospital
170-172, Raja Dinendra Street, Kolkata-700004, India
e-mail:
[email protected]
Telephone: +91-33-25335019, Telefax: +91-33-23371910
[SUP]2[/SUP] Institute of Postgraduate Ayurvedic Education and Research at SVSP Hospital
294/3/1, Acharya Prafulla Chandra Road, Kolkata 700 009, India
e-mail:
[email protected]
Telephone: +91-33-25335019, Telefax: +91-33-23371910
[SUP]3[/SUP] Former Professor, Department of Kayachikitsa
, J. B. Roy State Ayurvedic Medical College and Hospital, 170-172, Raja Dinendra Street, Kolkata-700004, India
e-mail:
[email protected]
[FONT="] [/FONT]
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 ([FONT="]Haslett
et al., 2002)[/FONT]. Literally, oligospermia means insufficient number of spermatozoa; but scientifically it means a
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Invalid Link Removed 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 LD[SUB]50[/SUB] 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 CrudeShilajit 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 1/10[SUP]th[/SUP] of LD[SUB]50[/SUB] 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[FONT="]. [/FONT]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 10[SUP]5 [/SUP]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.
