Claims:CLAIMS
1. An herbal composition for improving spermatogenic activity comprising extracts of Asparagus adscendens, Withania somnifera, Tribulus terrestris, Emblica officinalis, Asparagus racemosus and at least one pharmaceutically acceptable excipient.
2. The herbal composition as claimed in claim 1, wherein said composition comprises: a) Asparagus adscendens in an amount of 80 to 120 mg; b) Withania somnifera in an amount of 80 to 120 mg; c) Tribulus terrestris in an amount of 70 to 100 mg; d) Embilica officibalis in an amount of 50 to 100 mg; e) Asparagus racemosus in an amount of 80 to 120mg of the total weight of the composition and pharmaceutically acceptable excipients.
3. The herbal composition as claimed in claim 1, wherein said composition comprises: a) Asparagus adscendens in an amount of 110 mg; b) Withania somnifera in an amount of 100 mg; c) Tribulus terrestris in an amount of 90 mg; d) Emblica officinalis in an amount of 80 mg; e) Asparagus racemosus in an amount of 100 mg of the total weight of the composition and at least one pharmaceutically acceptable excipient.
4. The herbal composition as claimed in claim 1, wherein said composition acts as sperm count and sperm motility enhancer.
5. The herbal composition of claim 1, wherein said composition acts as serum total testosterone, serum luteinizing hormone and serum follicle stimulating hormone enhancer.
6. The herbal composition as claimed in claim 1, wherein said composition acts as erectile function, intercourse satisfaction, overall sexual satisfaction enhancer.
7. The herbal composition as claimed in claim 1, wherein said composition is formulated into tablet or capsule.
8. The herbal composition as claimed in claim 1, wherein said composition is in a tablet form.
9. The herbal composition as claimed in claim 1, wherein said pharmaceutically acceptable excipients are selected from binder, binder solution, glidants, lubricants, disintegrants, and coating agents.
10. The herbal composition as claimed in claim 6, wherein said pharmaceutically acceptable excipients are the disintegrants are selected from Crospovidone and Sodium starch glycolate; binder is Microcrystalline cellulose; binder solution is isopropyl alcohol; glidant is Syloid 24 FP IH; lubricants is Magnesium stearate; and at least one coating agent selected from hydroxypropyl methyl cellulose, polyethyelene glycol 4000, titanium dioxide, iron oxide red, iron oxide yellow, black oxide of iron.

Dated this on 6th day of August 2021

Asha P. Hole (Patent agent)
IN/PA 2269

, Description:TECHNICAL FIELD
This invention relates to a herbal pharmaceutical composition useful for improving male sexual dysfunction. More particularly, the present invention relates to a composition comprising a combination of extracts derived from natural sources preferably plants which are useful in improving male sexual dysfunction, specifically with persons with low spermatogenic activity.
BACKGROUND
Male infertility is a global population health concern, with large prevalence in developed and developing countries. Numerous factors are defined with unequivocal and harmful effect on male reproduction function, i.e., modern sedentary life style problems like obesity, smoking, caffeine, alcohol, and drug abuse; nutritional deficiencies including vitamin and mineral deficiencies and oxidative stress; genetic causes related to male infertility, etc. WHO reports that between 48 million couples 186 million individuals live with infertility globally. The concern for the condition is not recent. In 2010, The World Health Organization proposed a scheme for the diagnostic classification of male infertility, based upon a standardized approach to clinical assessment and to the assessment of semen quality. The organization continues developing guidelines on the prevention, diagnosis and treatment of male infertility, as part of the global norms and standards of quality care related to fertility care.
The pathogenesis of male infertility can be reflected in defective spermatogenesis. Taking this into account the above factors, therapy is typically aimed at improvement of spermatogenic activity: parameters of semen such as sperm concentration, semen volume, and sperm motility, serum hormones like testosterone, LH, and FSH, and so on.
While synthetic drugs and surgical treatments are available for treating male infertility, herbal or plant-based medicines are known to have more positive effects and exhibit little or no associated adverse effects and toxicities. Herbal medicines have also long been used in the management of general sexual dysfunction (Saxena et. al., International Journal of Green Pharmacy, 2012, 6(2), 109-117). Various herbs and their extracts can be used for the purpose treating various aspects of low spermatogenic activity. Herbs chosen specifically for the composition of the instant invention include Asparagus adscendens, Withania somnifera, Tribulus terrestris, Emblica officinalis, Asparagus racemosus. The combination effect of the aforementioned herbs makes the composition a balanced combination of different types of Aphrodisiacs (i.e. spermatogenesis enhancers, spermato-poetics, semen correctives, libido enhancers & erection-sustaining drugs) for effective management of low spermatogenic activity thus increasing the chances of fertilization in infertile men.
The individual extracts cited above of the present invention have been reported in the literature of traditional medicine for their aphrodisiac activity. Such extracts are even available for the management of one or more symptoms of spermatogenic activity. Higher therapeutic dose of single extract and lack of selectiveness/effectiveness against various aspects of spermatogenic activity are of major concern. Compositions have been reported in the prior art that have been made to prepare the combinations of such extracts as a remedy for overall sexual satisfaction.
Bansode et. al. (Bansode et. al., Pharm Biol, 2015, 53(2):192-200) reported Asparagus adscendens administration of 200 and 300 mg/kg doses exhibited a significant increase in anabolic, reproductive, and sexual activities.
Ambiye et. al. (Ambiye et. al., Evid Based Complement Alternat Med. 2013; 2013: 571420) reported Withania somnifera administration of 225 mg improved semen parameters and serum hormone levels.
Salgado et. al. (Salgado et. al., Andrologia, 2017, 49(5)) reported Tribulus terrestris administration of 250 mg improved semen parameters in subjects.
Ali et. al. (Ali et. al., Elixir Appl. Botany, 2011, 36, 3339-3342) reported Emblica officinalis administration of 100 mg/kg body wt & 150 mg/kg body wt mitigated fluctuation in sperm count.
It is evident that various combinations or individual compositions of the above cited herbal extracts are available for improving various parameters of spermatogenic activity. It appears that there is no consensus on the choice of specific extracts to be used in such a combination composition. Further, there is also no consensus on the dose of individual extracts required for such combination compositions. Furthermore, the doses cited in the literature are considerably higher thus adding to patient burden. There is therefore a need for a novel composition that is efficacious at lower towards improving the above cited aspects of spermatogenic activity and importantly, still demonstrates safety through preclinical and clinical studies.
Accordingly, the present inventors report a novel composition comprising of Asparagus adscendens, Withania somnifera, Tribulus terrestris, Emblica officinalis, Asparagus racemosus with the ability to improve spermatogenic activity. A composition comprising these herbal extracts, in their respective therapeutic amounts disclosed further for improving spermatogenic activity has not been disclosed before in the literature. The instant composition exhibits the ability to improve all of the aforementioned aspects of spermatogenic activity such as sperm count, sperm morphology, serum total testosterone level, serum LH level, serum FSH level, erectile function, intercourse satisfaction while maintaining drug tolerability and compliance with no adverse events related to the therapy.
Therefore, the objective of the present invention is to provide an herbal composition comprising Asparagus adscendens, Withania somnifera, Tribulus terrestris, Emblica officinalis, Asparagus racemosus present in specific therapeutic amounts, useful for the treatment of various parameters of spermatogenic activity such as sperm count, sperm morphology, serum total testosterone level, serum LH level, serum FSH level, erectile function, intercourse satisfaction.
SUMMARY OF THE INVENTION
In view of the foregoing, for overcoming the disadvantages of the prior art, the object of the invention is to provide a herbal composition comprising Asparagus adscendens, Withania somnifera, Tribulus terrestris, Emblica officinalis, Asparagus racemosus for the improvement of spermatogenic activity.
To achieve the above, the present invention includes a composition comprising Asparagus adscendens, Withania somnifera, Tribulus terrestris, Emblica officinalis, Asparagus racemosus and pharmaceutically acceptable excipients for improving spermatogenic activity in a subject in need thereof. The present invention can be administered in the dosage forms selected from capsule, tablet, granules, syrup and other suitable dosage forms. Preferably, the composition of the present invention can be administered in tablet or capsule dosage forms.
According to one embodiment, is provided a herbal composition comprising extracts of Asparagus adscendens, Withania somnifera, Tribulus terrestris, Emblica officinalis, Asparagus racemosus and pharmaceutically acceptable excipients, for improving spermatogenic activity.
According to one embodiment, is provided a herbal composition of claim 1, wherein said composition comprises: a) Asparagus adscendens in an amount of 80 to 120 mg; b) Withania somnifera in an amount of 80 to 120 mg; c) Tribulus terrestris in an amount of 70 to 100 mg; d) Embilica officibalis in an amount of 70 to 100 mg; e) Asparagus racemosus in an amount of 80 to 120mg of the total weight of the composition and pharmaceutically acceptable excipients.
According to one preferred embodiment, said herbal composition comprises: a) Asparagus adscendens in an amount of 110 mg; b) Withania somnifera in an amount of 100 mg; c) Tribulus terrestris in an amount of 90 mg; d) Emblica officinalis in an amount of 80 mg; e) Asparagus racemosus in an amount of 100 mg of the total weight of the composition and pharmaceutically acceptable excipients.
According to one embodiment, said herbal composition shall act as sperm count and sperm motility enhancer.
According to one embodiment, said herbal composition shall act as serum total testosterone, serum luteinizing hormone and serum follicle stimulating hormone enhancer.
According to one embodiment, said herbal composition shall act as erectile function, intercourse satisfaction, overall sexual satisfaction enhancer.
According to one embodiment, said herbal composition is formulated into tablet. According to an alternate embodiment, said herbal composition is formulated into capsule. In a preferred embodiment, said composition is in a tablet form.
According to one embodiment, said herbal table composition shall comprise of pharmaceutically acceptable excipients selected from binder, binder solution, glidants, lubricants, diluents, disintegrants, emulsifying agents, anti-caking agents, anti-adherents coating agents, and granulating agents. In a preferred embodiment, said composition shall comprise of pharmaceutically acceptable excipients such as disintegrant, binder, glidant, lubricants, and coating agents.
In a more preferred embodiment, said composition shall comprise of pharmaceutically acceptable excipients wherein the disintegrants are selected from Crospovidone and Sodium starch glycolate; binder is Microcrystalline cellulose; binder solution is isopropyl alcohol; glidant is Syloid 24 FP IH; lubricants is Magnesium stearate; and at least one coating agent selected from hydroxypropyl methyl cellulose, polyethyelene glycol 4000, titanium dioxide, iron oxide red, iron oxide yellow, black oxide of iron.
BRIEF DESCRIPTION OF DRAWINGS
The detailed description is described with reference to the accompanying figures. In the figures, the left-most digit(s) of a reference number identifies the figure in which the reference number first appears. The same numbers are used throughout the drawings to reference like features and components.
Figure 1 is a graphic representation of the HPLC chromatogram showing separation of Gallic acid, Ellagic acid, Rutin, Withanoside-IV, Withanolide-A at 227 nm.
Figure 2 is a graphic representation of the HPLC chromatogram showing separation of Gallic acid, Ellagic acid, Rutin, Withanoside-IV, Withanolide-A at 254 nm.
DETAILED DESCRIPTION
To clarify the above and other purposes, features, and advantages of this invention, specific embodiment of this invention is especially listed and described in detail with the examples as follows. The principal and mode of operation of this invention have been described and illustrated in its embodiment. At the outset, a person skilled in the art will appreciate that this invention may be practiced otherwise than is specifically described and illustrated. The invention should not be limited by the above-described embodiment, method, and examples, but by all embodiments and methods within the scope and spirit of the invention. Also, in the following description of the invention, certain terminology may be used for the purpose of reference only, and is not intended to be limiting.
Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value and/or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.
The present invention provides a herbal composition comprising Asparagus adscendens, Withania somnifera, Tribulus terrestris, Emblica officinalis, Asparagus racemosus for improving spermatogenic activity. The present invention can be administered in an oral dosage form, preferably a tablet or a capsule suitable dosage form.
Central to this invention is a composition comprising of herbal extracts for improving spermatogenic activity. Several herbs have been reported in the literature for their ability to treat or manage one or more aspects of sexual dysfunction in males. The individual ability of these extracts to improve spermatogenic activity has been well established in the literature. Albeit, the literature suggests that these herbs exhibit the ability to treat or manage the condition in varying degrees. Therefore, an important aspect of the invention is to select the most appropriate combination of herbs and their herbal extracts to achieve a composition that presents the best results for improving spermatogenic activity. While several compositions containing individual herbs or in different combinations have been reported in the literature, a composition comprising the individual extracts cited in the present invention has not been reported for improving spermatogenic activity. The combination effect of herbs should result in a composition that is a balanced combination of different types of Aphrodisiacs (i.e., spermatogenesis enhancers, spermato-poetics, semen correctives, libido enhancers & erection-sustaining drugs) for effective management of low spermatogenic activity thus increasing the chances of fertilization in infertile men. Accordingly, the present invention reports a composition comprising of a novel combination of Asparagus adscendens, Withania somnifera, Tribulus terrestris, Emblica officinalis, Asparagus racemosus for improving various parameters of spermatogenic activity. Asperagus adscendens is known to increase sperm count and is also used in spermatorrhoea. Withania somnifera has been described as powerful stimulator of the Spermatogenesis thus increasing the sperm count. Tribulus terrestris is a classic aphrodisiac. Emblica officinalis is known for antioxidant properties. Asperagus racemosus is used as Aphrodisiac and spermato-poietic drug.
All herbal extracts of the composition of the present invention are reported to be generally safe and well documented in literature. Plant extracts are extremely complex, multicomponent mixtures. The extraction and determination of quality and quantity of bioactive constituents from the most appropriate part of the plant is vital. It should be noted that herbs and plants need to be processed. Accordingly, a part of the plant may be used as a raw material, or a liquid and/or solid extract thereof. Suitable procedures include multiple steps, for example maceration, percolation, and extraction using supercritical fluids, liquefied gases or suitable solvents are used for the preparation of the material. The bioactive ingredients need to be extracted from the plant material such as roots, fruits, seeds etc., further purified and isolated in order to make it fit for the designated use in a composition.
The herbal extracts and their bioactive constituent used in the present composition are extracted using various parts of herbs, are described below:
Table 1: Bioactive ingredients of the composition

Extract Botanical Name Bioactive constituent
Gokshur Extract Tribulus terrestris Ellagic acid
Ashwagandha Extract Withania somnifera Withanoside-IV Withanolide-A
Shatavari Extract Asparagus racemosus Rutin
Musali Svet Extract Asparagus adscendens Diosgenin
Amalki Extract Emblica officinalis Gallic acid Rutin

The term "Tribulus terrestris" or “Gokshur Extract” disclosed herein comprises the cultivated or naturally grown plant and commercially available plant, but not intended to limit thereto herein. The term "Withania somnifera" or “Ashwagandha Extract” disclosed herein comprises the cultivated or naturally grown plant and commercially available plant, but not intended to limit thereto herein. The term "Asparagus racemosus" or “Shatavari Extract” disclosed herein comprises the cultivated or naturally grown plant and commercially available plant, but not intended to limit thereto herein. The term "Asparagus adscendens" or “Musali Svet Extract” disclosed herein comprises the cultivated or naturally grown plant and commercially available plant, but not intended to limit thereto herein. The term " Emblica officinalis" or “Amalki Extract” disclosed herein comprises the cultivated or naturally grown plant and commercially available plant, but not intended to limit thereto herein.
Another important aspect of the invention is selecting the therapeutic dose of each herbal extract. The existing literature reports varied amounts of each herbal extract, when viewed individually or in any combination. When reporting doses of herbal extract, it is in fact the amount of the bioactive constituent therein that is reported. It is evident that there is no linear correlation between therapeutic doses of each herbal extract when used in a combination composition such as that described in the present invention. Higher dose of each drug may present potential issues of efficacy. A person skilled in the art shall appreciate that the most appropriate therapeutic dose of each herbal extract is vital to the optimal function of the combination. In other words, the therapeutic dose of each herbal extract should work synergistically so as to achieve a composition that fulfils the object of the invention. In other words, the synergistic amounts of the bioactive constituents of the herbal extracts need to be determined so as to achieve the desired composition. This is in addition to maintaining the lowest possible therapeutic dose of each herbal extract. Therefore, even though the existing literature cites different doses of each herbal extract that may lead to a combination composition; the desired composition can be arrived at only after carefully selecting the therapeutic doses, and thus cannot be construed as a mere admixture of known extracts, with known effects on spermatogenic activity, in known therapeutic doses.
The present inventors report that a composition comprising combination of said individual extracts in specific therapeutic doses disclosed further in this application has not been reported in the literature for improving spermatogenic activity. Accordingly, the present invention also reports a composition comprising of a novel combination of Asparagus adscendens, Withania somnifera, Tribulus terrestris, Emblica officinalis, Asparagus racemosus in specific therapeutic doses for improving spermatogenic activity.
In an important embodiment, said herbal composition comprises: a) Asparagus adscendens in an amount of 80 to 120 mg; b) Withania somnifera in an amount of 80 to 120 mg; c) Tribulus terrestris in an amount of 70 to 100 mg; d) Emblica officinalis in an amount of 80 to 100 mg; e) Asparagus racemosus in an amount of 80 to 120mg of the total weight of the composition. In a preferred embodiment, said herbal composition comprises: a) Asparagus adscendens in an amount of 110 mg; b) Withania somnifera in an amount of 100 mg; c) Tribulus terrestris in an amount of 90 mg; d) Emblica officinalis in an amount of 80 mg; e) Asparagus racemosus in an amount of 100 mg of the total weight of the composition.
A person skilled in the art shall appreciate that a mere comparison of amounts of herbal extracts present in prior art compositions may not be the best representation. For a more accurate comparison, human equivalent doses (HED) must be correlated. In other words, proven efficacy at a reduced human equivalent dose is a truer representation of ingenuity of the composition. For example, the instant composition comprises of 90 mg of Tribulus terrestris extract per 767 mg tablet i.e., 3.96 mg/ person/day (HED). This is 5.3-folds less than 200 mg of Tribulus terrestris extract i.e., 21.28 mg/ person/day (HED) reported by prior art (Kumari and Singh, Afri. Health Sci. 2018;18(3): 645-652) for effect on fertility. Similarly, the instant composition comprises of 100 mg of Withania somnifera extract per 767 mg tablet comprising of 1.08 mg/person/day (HED) Withanolide-A and 4.92 mg/person/day (HED) Withanoside-IV. This is 1.25-fold less than 1.35 mg/ person/day (HED) Withanolide-A and 1.25-fold less than 6.15 mg/ person/day (HED) Withanoside-IV reported by prior art (Ziegenfuss et al., Nutrients 2018, 10, 1807). It is deliberated that this could be attributed to having optimum amounts of all extracts with similar properties working synergistically to improve spermatogenic activity.
An embodiment of the present invention provides a herbal composition comprising Asparagus adscendens, Withania somnifera, Tribulus terrestris, Emblica officinalis, Asparagus racemosus and pharmaceutically acceptable carriers or excipients, for improving spermatogenic activity wherein the said composition is ready for oral administration. The term "pharmaceutically acceptable carriers or excipients" defined herein comprises binder, binder solution, glidants, lubricants, disintegrants, and coating agents, and many more classifications used to prepare medications and which are well-known in the art or previous literature.
In an embodiment of the present invention, the herbal composition comprising Asparagus adscendens, Withania somnifera, Tribulus terrestris, Emblica officinalis, Asparagus racemosus may be prepared as an oral dosage form for example, powder, tablet, capsule, soft capsule, aqueous medicine, syrup, elixirs pill, powder, sachet, granules. In a preferred embodiment, the herbal composition of the present invention in an oral dosage shall be a tablet.
In another embodiment of the present invention, the herbal composition shall further comprise of pharmaceutically acceptable excipients such as binder, binder solution, glidants, lubricants, disintegrants, and coating agents. It should be noted that the choice of excipients is not limited to those cited herein. A person skilled in the art shall appreciate that the choice of excipients and their respective amounts required for an optimal composition is arrived at after a considerable research, and therefore, should not be deemed as a mere optimization of suitable excipients for the purpose of the composition. In a preferred embodiment, the disintegrants are selected from Crospovidone and Sodium starch glycolate; binder is Microcrystalline cellulose; binder solution is isopropyl alcohol; glidant is Syloid 24 FP IH; lubricants is Magnesium stearate; and at least one coating agent selected from hydroxypropyl methyl cellulose, polyethyelene glycol 4000, titanium dioxide, iron oxide red, iron oxide yellow, black oxide of iron.
In another important aspect of the invention, said composition shall be efficacious and safe for improving spermatogenic activity in subjects in need thereof. Spermatogenic activity can be evaluated by assessing the efficacy of the composition against various parameters such as sperm count, sperm morphology, serum total testosterone level, serum LH level, serum FSH level, erectile function, intercourse satisfaction. The present inventors report that the instant composition is safe and effective medicine for the treatment of patients with low sperm count and thereby improving their overall spermatogenic activity. Further, it is reported that the composition exhibited good drug compliance and an overall safety. Thus, affirming the positive effect of the instant herbal composition on improving spermatogenic activity in male subjects in need thereof.
INCORPORATION BY REFERENCE
The publications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.
EXAMPLES
The following examples are provided for illustrative purposes only, and are intended to be purely exemplary of the disclosure and are not intended to limit the scope of the claims provided herein.
Example 1. Preparation of tablet
All raw materials i.e., roots of Asparagus racemosus, roots of Asparagus adscendens, roots of Withania somnifera, fruits of Tribulus terrestris, and dried fruits (without seeds) of Emblica officinalis were procured from Supherb life science. Extracts from all raw materials were prepared in house.
The premix herbal extract of Table 2 was used to prepare tablets. Permix herbal blend was sifted separately through 20#. Microcrystalline cellulose, Crospovidone, sodium starch glycolate, Syloid 244 FP were sieved through 40# and magnesium stearate through 60#. All ingredients were dry mixed followed by addition of isopropyl alcohol in the Rapid Mixer Granulator. The mixture of kneaded until granule formation was observed. Granules were dried in a Fluid bed dryer at 40 – 45 °C. Dried granules were sifted through 16# using vibratory sifter. Milled granules were passed through 16# sieve. For lubrication, sifted granules were transferred in blend along with Crospovidone, sodium starch glycolate, microcrystalline starch, Syloid 244 FP. Magnesium stearate was mixed in. The lubricated blend was collected in a double-line polybag. Tablets were compressed using below mentioned parameters: Target weight: 750 mg; Hardness: 100-170 N; D.T: 15 Min; Thickness: 6±0.3 mm; Friability: NMT 1% w/w.
Tablets were coated using coating agents - hydroxypropyl methyl cellulose, polyethyelene glycol 4000, titanium dioxide, iron oxide red, iron oxide yellow, black oxide of iron.
The bioactive constituents in tablets were quantified by performing HPLC assay. Typically, Withanoside-A, Withanoside-IV are detected at 227nm; and Gallic acid, Rutin, Ellagic acid at 254 nm. Using a unique HPLC method designed by the present inventor, all the bioactive components are detected at both 227 nm (Fig. 1) and 254 nm (Fig. 2) further described in Table 4.

Table 2: Composition of each tablet

Extract Botanical Name Quantity of extract (mg/tablet)
Musali Svet Extract Asparagus adscendens 110
Ashwagandha Extract Withania somnifera 100
Gokshur Extract Tribulus terrestris 90
Amalki Extract Emblica officinalis 80
Shatavari Extract Asparagus racemosus 100

Table 3: Bioactive constituent in each tablet

Botanical Name Bioactive constituent in extract (% w/w) Bioactive constituent in product (% w/w)
Asparagus adscendens Not taken for estimation Not taken for estimation
Withania somnifera Withanoside-IV: 1.23
Withanolide-A: 0.27 Withanoside-IV: 0.46 Withanolide-A:0.04
Tribulus terrestris Ellagic acid-1.1 Ellagic acid- 0.22
Emblica officinalis Gallic acid: 5 Rutin: 0.85 Gallic acid: 0.5 Rutin: 0.10
Asparagus racemosus Not taken for estimation Not taken for estimation

Table 4: HPLC detection of Bioactive constituents
Bioactive constituent Retention time Area at 227 nm
(µAU*sec) Area at 254 nm
(µAU*sec)
Gallic Acid 5.912 2577243 1234865
Ellagic acid 16.537 50406 67945
Rutin 17.248 372101 1737293
Withanoside-IV 19.137 459781 257488
Withanolide-A 32.310 106556 13156

Example 2. Evaluating efficacy of composition
An exploratory clinical study was conducted to evaluate safety and efficacy of the composition of the present invention on Male Infertility, specifically various parameters of spermatogenic activity.
Methods: 60 sexually active and married males, 21 - 45 years of age, with sperm count of 1 million to less than 15 million/ml and with normal sperm morphology were recruited into the study. Subjects were either randomized to herbal composition or Placebo group in 1:1 ratio. Subjects administered with the herbal composition were categorized as Group A and those administered with Placebo were categorized in Group B. Subjects were advised to take given medication in a dose of 2 tablets twice daily orally after meals with water for 90 days. Following parameters of spermatogenic activity were evaluated before treatment (Day 0) and Day 90:
1. Total number of sperm cells per milliliter of seminal fluid from baseline to 3 months
2. Total motile sperms (sperm motility) from baseline to 3 months
3. Sperm cells with normal forms (Sperm morphology) from baseline to 3 months
4. Semen volume, semen parameters from baseline to 3 months
5. Serum total testosterone, LH, FSH from baseline to 3 months.
6. Erectile function domain of International Index of Erectile Function (IIEF) from baseline to 3 months
7. Overall satisfaction (IIEF-OS) domain score of International Index of Erectile Function from baseline to 3 months

For efficacy variables such as, mean changes in sperm cells from baseline to end of therapy for within group and between the groups was estimated by student t test. Other variables i.e., total motile sperms, sperm morphology, semen other variables, serum testosterone, LH, FSH level from baseline to end of 3 months treatment with in the group and comparison between groups were analyzed by using students paired and unpaired t test. In this study Erectile function domain of International Index of Erectile Function (IIEF), Overall satisfaction (IIEF-OS) domain score of International Index of Erectile Function were assessed by non-parametric test by Wilcoxon sign rank, Mann Whitney u test or by chi square test. In this study all P values were reported base on two-sided test and these statistical tests were interpreted at 5% level of significance.
Results: The clinical study concluded that the composition tablet is safe and effective medicine for the treatment of patients with low sperm count. Further, results obtained are discussed with the help of tables and drawings as below:
2.1 Changes in mean sperms cells between the groups:
Subjects administered with tablet composition (Group A) were significantly effective in increasing total number of sperm cells per milliliter of seminal fluid than Placebo tablet (Group B) in subjects with low sperm count.
Table 5: Changes in mean sperms cells
Days Mean Sperm Cells (Million/ml) (X ± SD)
Group A Group B
Baseline (0) 08.10 ± 3.28 07.67 ± 3.18
90 13.97 ± 2.20 08.33 ± 3.00
Mean diff (Baseline – 90)
(P value = 0.00) 05.87 ± 3.09 0.66 ± 1.54

2.2 Changes in the mean total motile sperm (sperm motility):
Herbal composition (Group A) was significantly better in improving total motile sperms (sperm motility) than Placebo tablet (Group B) in subjects with low sperm count.
Table 6: Changes in mean total motile sperm
Days Mean Total motile sperms (%) (X ± SD)
Group A Group B
Baseline (0) 28.74 ± 10.11 27.66 ± 11.41
90 32.89 ± 09.01 29.41 ± 09.53
Mean diff (Baseline – 90)
(P value = 0.002) 04.15 ± 6.36 01.76 ± 06.68

2.3 Changes in the mean serum total testosterone:
At the end of the study, increase in serum total testosterone was significantly more (23.3%) in Group A subjects than Group B subjects administered with placebo tablet group (3.9%).
Table 7: Changes in mean serum total testosterone
Days Mean Serum Total Testosterone (X ± SD)
Group A Group B
Baseline (0) 349.50 ± 120.46 316.11 ± 194.46
90 430.80 ± 123.22 328.51 ± 204.74
Mean diff (Baseline – 90)
(P value = 0.001) 81.30 ± 68.71 12.40 ± 67.33

2.4 Changes in the mean serum luteinizing hormone (LH):
Serum Luteinizing hormone (LH) was significantly increased in Group A subjects than Group B subjects.
Table 8: Changes in mean serum LH
Days Mean Serum LH (X ± SD)
Group A Group B
Baseline (0) 3.92 ± 1.36 3.74 ± 1.54
90 4.70 ± 2.34 3.67 ± 1.46
Mean diff (Baseline – 90)
(P value = 0.024) 0.78 ± 1.44 -0.07 ± 0.36

2.5 Changes in the mean serum Follicle Stimulating Hormone (FSH):
At the end of the study, increase in serum FSH was significantly more in Group A subjects than Group B subjects administered with placebo tablet group.
Table 9: Changes in mean serum FSH
Days Mean Serum FSH (X ± SD)
Group A Group B
Baseline (0) 5.65 ± 2.28 5.93 ± 3.41
90 4.52 ± 2.05 5.74 ± 3.27
Mean diff (Baseline – 90)
(P value = 0.001) -1.13 ± 0.92 -0.19 ± 0.66

2.6 Changes in the mean erectile function:
Group A subjects exhibited significant improvement in erectile function on Days 60 and 90 than Group B subjects.
Table 10: Changes in mean erectile function
Days Mean Serum FSH (X ± SD)
Group A Group B
Baseline (0) 13.97 ± 1.69 14.03 ± 2.53
60 16.83 ± 3.63 14.77 ± 1.57
90 20.33 ± 3.02 16.40 ± 2.25
Mean diff (Baseline – 60)
(P value = 0.003) 2.87 ± 3.20 0.73 ± 2.02
Mean diff (Baseline – 90)
(P value = 0.001) 6.37 ± 2.93 2.37 ± 2.93

2.7 Changes in the mean intercourse satisfaction:
Group A subjects exhibited significant improvement in mean intercourse satisfaction by Day 90 than Group B subjects.
Table 11: Changes in mean intercourse satisfaction
Days Mean Serum FSH (X ± SD)
Group A Group B
Baseline (0) 6.50 ± 0.90 6.47 ± 1.04
90 9.93 ± 2.08 7.73 ± 1.78
Mean diff (Baseline – 90)
(P value = 0.001) 3.43 ± 2.22 1.27 ± 2.05
2.8 Changes in the mean overall satisfaction:
Group A subjects exhibited significant improvement in mean overall satisfaction on Day 90 than Group B subjects.
Table 12: Changes in mean overall satisfaction
Days Mean Serum FSH (X ± SD)
Group A Group B
Baseline (0) 4.20 ± 0.61 4.27 ± 0.69
90 6.47 ± 1.46 4.97 ± 1.22
Mean diff (Baseline – 90)
(P value = 0.001) 2.27 ± 1.34 0.70 ± 1.34

The clinical study exhibited good drug compliance by subjects. Mild to moderate adverse events were reported in both the groups, which were resolved without any treatment. In all the cases, association of the events was unlikely to the study drugs.