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)[SUB]2[/SUB]DBP 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[FONT="] [/FONT]emergent[FONT="] [/FONT]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.
References
Acharya SB, Fortan MH, Goel RK, Tripathi SK, Das PK (1988) Pharmacological actions of Shilajit.
Ind J of Exp Biol 26(10): 775-777. Agarwal HSK, Kulkarni KS (2003) Efficacy and safety of speman in patients with
oligospermia: an open clinical study.
IndIan J Clinical Practice 14
(2): 29-31. Agarwal A, Saleh RA, Bedaiwy MA. Role of reactive oxygen species in the
pathophysiology of human reproduction.
Fertil Steril 2003; 79: 829-43. Al-Hamaidi AR, Umar M (2003) Safe use of Shilajit during pregnancy of female mice .
Online Journal of Biological Science 3(8):681- 684. Anisimov VE, Shakirzyanova RM (1982) Application of Mumie in therapeutic practice.
Kazan Med J 63: 65-68. Alvarez JG, Storey BT. Differential incorporation of fatty acids into and peroxidative loss of fatty acids from phospholipids of human spermatozoa.
Mol Reprod Dev 1995; 42:334-46. Berek JS, Adashi EY, Hillard PA (1988) Novak’s Gynecology. Baltimore: Williams and Wilkins; 933. Briggs C, Kunka S, Pennaneach C, Forbes L, Machin SJ (2003) Performance evaluation of a new compact hematology analyzer, the Sysmex, POCH-1001.
Laboratory hematology 9(4): 225-33. David F and Vasant L (2001)
The Yoga of Herbs. Twin Lakes, WI. Lotus Press; 250. Fortan MH, Acharya SB (1984) Pharmacological studies of Shilajit.
Indian J Parmacol 16, 45. Ghosal, S. Lal J, Sing SK Dasgupta G Bhaduri J, Mukhopadhyay M and Bhattacharya,S.K (1989) Mast cell protecting effects of shilajit and its constituents.
Phytotherapy Res 3: 249-252. Ghosal S (1994) The aroma principles of Gomutra and Karpuragandha Shilajit.
Indian J. Indg.Med 11(1): 11-14. Ghosal S (2002) Process for preparing purified Shilajit, composition from native
Shilajit, US Patent: 6,440,436, 1-14. Ghosal, S (2006)
Shilajit in Perspective. New Delhi: Narosa Publishing House 132-56. Gomez E, Irvine DS, Aitken RJ (1998) Evaluation of a spectrophotometric assay for the measurement of malondialdehyde and 4-hydroxyalkenals in human spermatozoa: relationships with semen quality and sperm function.
Int J Androl 21: 81-94. Griveau JF and Le Lannou D (1997) Reactive oxygen species and human spermatozoa: Physiology and pathology.
Int J Androl 20: 61-9. Halpern M (2003)
Principles of Ayurvedic Medicine. 5[SUP]th [/SUP]edition. Grass Valley, CA: California College of Ayurveda. Haslett C, Chilvers ER, Boon NA, Colledge NA (Eds.) (2002) Davidson’s Principles and Practice of Medicine. Edinburgh: Churchill Livingstone, 711. Jeong SP, Gee YK, Kun H (2006) The spermatogenic and ovogenic effects of chronically administered Shilajit to rats.
J Ethnopharmacol 107: 349-53. Kelginbaev NS, Sorokina VA, Stefandu AC, Ismailova VN (1973) Treatment of long tubular bone fractures with Mumie and preparation in experimental and clinical conditions.
Exp Surg Anesthes 18, 31-35. Kreuter J, Shamenkov D, Petrov V, Ramge P, Cychutek K, Koch-brandt C, Alyautdin R (2002) Apolipoprotein-mediated transport of nanoparticle-bound drugs across the blood-brain barrier.
J Drug Targetting 10:317. Kwon JT, Hwang SK, Hua J, Kim DS, Tehrani AM, Yoon HJ, Choi M, Yoon TJ, Han DY, Kang YW, Yoon B, Lee JK, Cho MH (2008) Body distribution of inhaled fluorescent magnetic nanoparticles in the mice.
J Occup Health 50:1-6. Maneesh M, Jayalakshmi H (2006) Role of reactive oxygen species and antioxidants on pathophysiology of male reproduction.
Ind J Clin Biochem
21 (2): 80-9. Mihara H and Uchiyama M (1978) Determination of malondialdehyde precursor in tissues by thiobarbituric acid test.
Anal Biochem 1: 271-78. Murawski M., Saczko J., Marcinkowska A., Chwikowska A, Grybooe M., Banaoe T (2007) Evaluation of superoxide dismutase activity and its impact on semen quality parameters of infertile men,
Follia Histochemica Et Cytobiologica 45 (1) :123-26. Oligospermia, from Wikipedia, the free encyclopedia,
http://en.wikipedia.org/
wiki/Oligospermia. Accession date: June 2, 2008. Pramanik D (2007)
Principles of Physiology. Kolkata, Academic Publishers
370-4. Sharma PV (1998).
Charaka Samhita. Chowkhambha Orientalia Chikitsasthana, Varanasi, Karaprchitiya RasayanaPada, 4th edition, vol 2, verse no.49-50. Sood, R (1999) Medical Laboratory technology: Methods and interpretation. New Delhi, JP Brothers. Schepetkin I, Khlebnikov A, Kwon BS (2002) Medical Drugs from Humus Matter: Focus on mumie.
Drug Development Research 57:140-159. 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.