Figure 5.A.1 DSC Scan of Sildenafil Citrate. Fig 5.A.2UV scan of Sildenafil Citrate.

Transcription

1 OBSERVATIONS & RESULTS STUDY-I (Transdermal Drug Delivery System) Characterization of Sildenafil: Description of Drug: White colored crystalline powder. Solubility: Drug was found insoluble in water, Soluble in Ethanol, Methanol. DSC analysis: DSC of pure drug Sildenafil Citrate in range of C showed endothermic peaks at C for Sildenafil citrate Fig 5.A.1. DSC mw Peak Onset Endset Heat xC xC xC xmJ Temp [C] Figure 5.A.1 DSC Scan of Sildenafil Citrate. Fig 5.A.2UV scan of Sildenafil Citrate. Drug has given a very sharp peak at which a quite closer value to the theoretically given value (190 O C) for the drug sildenafil. UV analysis: After scanning for λ max over a range of nm on a double beam U V Spectrophotometer, solution of Sildenafil citrate of 100 mcg/ml concentration showed maximum absorbance at 245 and 290 nm. Page 67

2 . FTIR: Sildenafil Citrate showed characteristic peaks at cm -1 (aromatic out of plane bending), cm -1, 1172 cm -1 (SO 2 symmetric streching), cm -1 (Asymmetric Stretching) cm -1 (C=0 stretching), 2692 cm -1 (CH 3 Asymmetric Fig 5.A.3 FTIR Scan of Sildenafil Citrate. Streching), cm - 1 (aliphatic CH stretching),3298 cm -1 (N-H Symmetric and Asymmetric Streching) Page 68

3 Characterization of Tadalafil: Description of Drug: White colored crystalline powder Solubility: Drug was found insoluble in water, Very Slightly Soluble in Ethanol, Methanol, Soluble in DMSO. Fig 5.A.4 DSC scan of Tadalafil Fig. 5.A.5 UV Scan of Tadalafil DSC Analysis: DSC of pure drug Tadalafil in range of C showed endothermic peaks at C and C for Tadalafil Fig 5.A.4, which a quite closer value to the theoretically given value (300 O C) for the drug Tadalafil. It is pertinent to mention here that Tadalafil exist as co-crystallised form and various crystal forms have m.p ranging from C, C, C UV analysis: After scanning for λ max over a range of nm on a double beam U V spectrophotometer, solution of Tadalafil of 100 mcg/ml concentration showed maximum absorbance at 280nm. Page 69

4 FTIR of Tadalafil: Prinicipal peaks at wave numbers IR (KBr film) cm -1 (N-H Streching-Secondary amine group), cm -1 (C-H Strech - Aliphatic methyl group), cm -1 (C=C,Aromatic groups), cm -1 (C-N strech), cm -1 (C-H Strech,Aromatic), cm -1 (C=O, Amide group), cm -1 (C-O-C Symmetic Strech), cm - 1 (Benzene) Fig. 5.A.5 FTIR Scan of Tadalafil Page 70

5 Due to practical insolubility in any of the aqueous solutions the drug was not considered for the formulation. However few formulations were tried (which are not discussed) which were without any meaningful results. Further all the studies were carried out using sildenafil as prototype drug for TDDS formulations. Drug - Polymer Interaction study: DSC scans: As it is evident from figure no 5.A.6,5.A.7,5.A.8,& 5.A.9 ;Melting temperatures of pure Sildenafil and Chitosan, Sildenafil and HPMC were found to be 78 C, o C and C respectively whereas DSC scans of physical mixture of both the drug and chitosan gives characteristics peaks corresponding to the melting temperatures of Chitosan and Sildenafil i.e 78 o C and o C. It is here worth stating that these both peaks values are closer to those of Chitosan and Sildenafil suggesting no interaction between the drugs and polymer. Fig 5.A.6 DSC scan for Pure Chitosan. Fig 5.A.7 DSC scan for Pure HPMC. Page 71

6 Fig. 5.A.8 DSC Scan of Physical mixture of Sildenafil and Chitosan. Fig. 5.A.9 DSC Scan of Physical mixture of Sildenafil and HPMC. Page 72

7 Furthermore, DSC scan of physical mixture of Sildenafil and HPMC was done as per the mentioned procedure earlier. But, as evident from Fig.5.A.9. there was a shift in melting point of HPMC as it shifted from 256 to C. This was indicative of a physical interaction between drug and film forming agent. Since HPMC showed interaction with sildenafil, therefore all the further studies were carried out using Chitosan as film forming agent. However, other film forming agents i.e. Acrylates and Eudragit were available but they are known to retard the drug release from the polymer matrix so as to enhance the duration of action. In case of sildenafil due to its larger molecular weight i.e our primary goal of the formulation was to permeate the maximum amount drug from the formulation into the skin. Fabrication of TDDS patch of sildenafil Citrate: Various formulations as per the table 5.A.1 were prepared by using solvent casting method explained earlier in section (Materials and Methods): Estimation of release of the drug from the formulation: HPLC method: Different concentrations ranging from 1.5 micrograms to 1.5 ng/ml were prepared in methanol. The solutions were filtered with 0.45µM syring filters (Millipore USA), 20 µl of each sample was injected into the HPLC(Shimadzhu-Japan) using flow rate, mobile phase and wavelength as per the method discussed in previous section. Six such injections were injected for each concentration, Area Under the Curve and retention time were recorded and a standard curve was prepared. The equation Y=180.74x , R 2 = is further used to estimate the amount of drug released from the samples. LOD and LOQ were calculated using the formula as per ICHQ 1 R 2 guidelines and found to be 16ng/ml and 56 ng/ml respectively. Page 73

8 Area under the curve Results Table 5.A.3 : Area Under the curve for Sildenafil HPLC data. Conc. (ng/ml) Area under the 295 nm Mean Std.Dev No peaks were detected at this concentration Standard curve of Sildenafil at 295nm y = x R² = Concentration (ng/ml) Figure 5.A.10: Graph Showing Standard curve of Sildenafil at 295 nm, prepared using HPLC. Page 74

9 One ml sample from the Franz diffusion cell were taken out at regular intervals of 0 min, 30 min, 1,2,4,8,12,16,32 and 40 hrs. Each removal of sample is replaced by one ml of 20: 80 Methanol: Phosphate buffer Saline. Initial samples upto 1 hr did not show any amount of drug released from the transdermal patch, futther the amount of drug released from the patch increased with passage of time. For practical cumulative amount of drug released from the patch at the end of 40 hrs are reported in Table 5.A.2. Since spectra of sildenafil exhibited lambda max at 245 and 295, the estimations were done at 295. Because the interference due solvent is minimum at higher wavelengths. This method is further validated as per the ICH guidelines and can further be used to estimate even the presence of Tadalafil in samples. Observations during fabrication of the patches: Selection of Drug concentration: A saturated solution of drug has to be incorporated into the transdermal formulation so as to maintain the release of drug thoughout the study, a low concentration of drug in the formulation has a probability that patch is exhausted of the drug during the full coarse of the study. Therefore it is essential that such a solution is taken into an account for the formulation which does not have high solubility so that saturation of the matrix is reached with a lower amount of drug and thus preventing the wastage of the drug. Moreover higher solubility of the drug in the matrix will allow the matrix to hold the drug and thereby make it difficult for the formulation to release the drug into the skin. Selection of Film forming Agent: Eudragit RL 100, Eudragit RS 100 were taken as option for film forming agents which were dissolved in 5 ml of ethanol which could not produce a matrix. HPMC has potential interaction with Drug as evident from the DSC data, moreover due to requirement of adding a saturated solution of the drug without wastage of drug,an aqueous polymer would have been the ideal choice for the fabricating the transdermal patch therefore chitosan in 4% lactic was taken as film forming agent. Chitosan polymerises in acidic environment provided by lactic acid., since sildenafil has low water solubility a Page 75

10 saturation was achieved in 5 ml patch with 25 mg of the drug. Therefore all the formulations used had a constant amount of drug load i.e. 25 mg. Fabrication process was started with chitosan polymer in different concentrations with 4% lactic acid in water. Optimization was done on the basis of consistency of the mixture obtained which should neither be too high not to allow adequate mixing of the drug into matrix nor too low to make the mixture difficult to dry. In this context Chitosan 125 mg was found most suitable one which we carried further for the research purpose. Fo1,Fo2 and Fo3 were prepared to check the consistency of the film forming agent. It was observed that the consistency obtained in case of Fo1 was fine, whereas consistency was moderate in case of Fo2, Fo3 patch prepared was coarse and thick. Transdermal formulations were simultaneously subject to preliminary release studies using modified Franz-Diffusion Cell and it was observed that 0.2,0.3 and mg of drug was released (Table no.5.a.2) for Fo1, Fo2 and Fo3 respectively. This release pattern further supported our selection of chitosan (125 mg ) for the further studies. Since the amount of drug released from the formulation was well below the required amount to be reached in plasma, our next aim was to enhance the amount of drug to be released from the formulation. It is worth mentioning here that PEG-400 (1%) was used as plasticizer in all the formulations, some formulations were tried using Glycerol (0.2 %) but due to its humectant nature those formulations appeared sticky and wet.(not mentioned in Table 5.A.1). Transdermal patches made without the use of plasticizer were brittle and cracks were observed in those patches. After selecting 125 mg Chitosan as an optimum quantity for incorporation into a patch, the next aim was that of searching for some suitable penetration enhancer Selection of Permeation enhacer:. The first candidate tried for, was Isopropyl myristate (IPM) firstly in the concentration of 15% v/v which was later reduced to 5 % v/v. The most important thing observed here was that the patches so formed with incorporation of IPM as a plasticizer were found to be slippery and non sticky leading us to a decision to drop this penetration enhancer in formulation of said Transdermal Patch. Page 76

11 Table 5.A.1: Various Combinations Tried during Formulating TDDS patch. Ingredients Film Forming Agent Plasticizer Permeation Enhancer Drug Content Base Formulation Chitosan PEG- 400 Span 60 Tween 80 Sildenafil Citrate Lactic Acid Fo % mg 4% Fo % mg 4% Fo % mg 4% Fo % 1% 25 mg 4% Fo % 2% 25 mg 4% Fo % 4% 25mg 4% Fo % 0.25% 25mg 4% F % 0.5% 25mg 4% Fo Solution A Solution of Sildenafil in 30% Methanol 25 mg 4% Fo % 0.08% - 25mg 4% Fo % 0.16% - 25mg 4% Fo % 0.32% - 25 mg 4% Fo % 0.48% - 25 mg 4% Fo % 1% - 25 mg 4% Fo % 1.5% - 25 mg 4% Page 77

12 This was further extended to our choice of permeation ehancer to Tween 80 ( an aqueous surfactant) three different patch formulations i.e. Fo4,Fo5, Fo6 were tried using 1,2 and 4% of Tween 80 with an expectation of increase in amount of drug released from the formulation. But contrary to our expectation the amount of drug released drastically reduced in these patches with increase in Tween 80 concentration Table (5.A.2) 0.55,0.375 and 0.3 mg respectively, probably due to wrong selection of concentration of tween 80 or due to wrong selection of permeation enhancer. To rule this out Fo14 and Fo15 were selected with lower concentrations of Tween 80 i.e and 0.5% of Tween 80 and it was observed that release increased significantly and 1.3 mg respectively in Fo14 and Fo15. This indicates that optimum concentration of Tween 80 for better release of sildenafil is 0.5%, any increase in concentration of the tween 80 more than 1% leads to reduction of drug release from the formulation. Since the cumulative amount of drug released from Fo14 and Fo15 is still less than the amount required to produce therapeutic concentration. In order to rule out whether a formulation plays a key factor in releasing the amount of drug from the formulation, a solution of drug in methanol was kept in franz diffusion cell and release was estimated. This Fo-Solution released low amount of drug 0.68 mg, it is considered that a permeation enhancer in the form of matrix patch would be ideal to for better release of drug from the formulation. Our next aim was to increase the amount of drug released from the formulation,therefore a new permeation enhancer is selected i.e. SPAN 60 ( non-aqueous) soluble in mineral oil and alcohol. Here for the practical purpose SPAN- 60 was dissolved in 40% alcohol a 0.5% solution was prepared and added to the formulation in the concentration ranging fom % as per Table 5.A.1. It was further learnt that Fo12 which contained a 1% concentration of SPAN60 was release maximum amount of drug mg from the formulation. Although this formulation was found to be the more successful in terms of amount of drug release the amount released was also not sufficient to generate the required therapeutic concentration. Furtther this formulation was again fabricated into a batch of 6 such patches in order to check the release of the drug from each patch. Page 78

13 Table 5.A.2. Amount of drug released from different formulations. Ingredients Formulation Permeation Enhancer Span 60 Tween 80 Drug Content Sildenafil Citrate Amount of drug released from 15 cm 2 formulation % age of drug released from the formulation Fo mg Fo mg Fo mg Fo 4 1% 25 mg Fo 5 2% 25 mg Fo 6 4% 25mg Fo % 25mg F % 25mg Fo Solution A Solution of Sildenafil in 30% Methanol Fo8 0.08% 25 mg Fo9 0.16% 25 mg Fo % 25 mg Fo % 25mg Fo12 1% 25mg Fo13 1.5% 25mg Page 79

14 A Batch of formulation Fo12 consist of 12 patches was further prepared using the method described under the heading fabrication by solvent casting method and was analysed for the following studies: Physical characteristics of the prepared films: Physical Appearance: Table 5.A.4 shows the characteristics pertaining to physical appearance of the patches formed. Table no 5.A.4 Charectirsation of Physical appearance of the formulationfo12 Sr No Characteristics of appearance Fo12a Fo12b Fo12c Fo12d Fo12e 1 Transparency Smoothness Flexibility Stickiness Moist All the patches obtained were smooth,translucent, pale in colour appead moist, sticky and flexible. Thickness of the patch: Average of the thickness of five patches was calculated to be mm. Weight of films: 215.5, 214.0, 208, 201.9, 212 mg were the weights of five films. Folding endurance: The folding endurance value for five patches was found to be 406, 416, 437, 428, and 431 respectively. Percentage moisture absorption: Table no 5.A.5 gives the moisture absorption data obtained. Page 80

15 Table 5.A.5 : % moisture absorption data of the Batch of Fo12. Sr. No Formulation code Weight in mg Weight in mg after 24 hrs Increase in weight in mg % increase /% moisture absorption 1 Fo12a Fo12b Fo12c Fo12d Fo12e Flatness: is the the measure of ability of the film to get folded once kept flat,it is indicative of better cohersion rather than better adhesison. A good transdermal patch is the one which has complete flatness and minimum constriction. Table 5.A.6 gives the data on flatness. Table no 5.A..6 shows the different measurements justifying the flatness of the films. Sr No Formulation code Left side strip Centre strip Right side strip % constriction 1 Fo12a 6.0 cm 6.0 cm 6.0 cm 0 2 Fo12b 5.9cm 5.9cm 5.9cm 0 3 Fo12c 6.0 cm 6.0 cm 6.0 cm 0 4 Fo12d 6.0 cm 6.0 cm 6.0 cm 0 5 Fo12e 6.0 cm 6.0 cm 6.0 cm 0 Page 81

16 Drug content in patches: After adding the pieces of 1 cm 2 of the patch to the phosphate buffer saline we could not see any drug concentration in the solvent as no absorbance at 245 & 290 was seen. Therefore whole patch was dissolved in methanol and kept for shaking for half an hour the estimation done in the solution using a separate standard curve prepared by HPLC. Table 5.A.7 Drug Content in the patches. Sr. No. Patch no AUC at 290 nm Drug Content of Sildenafil in whole patch, (%) (98.4) (96) (97.2) (96.4) (95.6) In vitro Release Studies: Following the method described in chapter for in vitro release studies, firstly a study for patch was carried on using the Phosphate buffer saline + Methanol (80:20) in the receptor compartment. The study earlier was carried out on individual transdermal patch later extended to the five formulations, results were found to be consistent with earlier findings as average cumulative release of the drug from formulation Fo12 was found to be 12.2 % which yields 3.12 mg of drug in the receptor compartment. Page 82

17 Table : 5.A.8 : Invitro Release data Sr No Formulation code Amount of drug released % of drug released after 40 hrs after 40 hrs 1 Fo12a Fo12b Fo12c Fo12d Fo12e Skin Irritation Studies: Five Wistar albino rats were choosen for skin irritation studies, Transdermal patchers were applied on the dorsal region, with their hairs clipped and shaved. The patches were kept intact in their respective position for three days by an adhesive tape. Although their was no sign of itching or irritation seen among the rats, few rats were seen eating tape of their fellow animals. Irritation score was calculated by first tablulating the data for abraded or intact erythema or edema during 72 hrs. Primary irritation index was calculated :0/20 =0.0 Table 5.A.9. Page 83

18 Table 5.A.9. : Primary irritation studies Rat No Reaction 24 hrs 72 hrs Intact Abraded Intact Abraded 1 Erythema Edema Erythema Edema Erythema Edema Erythema Edema Erythema Edema Page 84

19 Observations and Results Study II Evaluation of herbal Aphrodisiacs Results of Protocol I: Sexual motivation test (Test for Libido) Fluoxetine treated rats were acclimatised in an open area for 5 mins and were then kept for 10 minutes, where they had free access to move either to a social stimuli (male rats) or sexual stimuli (female rats). Percentage time spent by fluoxetine treated rats in the proximity of female rats was calculated on 9 th, 18 th and 28 th day. ( Table 5.B.1) TABLE 5.B.1: Effect of four week treatment with terristeris and C.Cyminium on time Experimental Group spent in proximity of sexual stimuli. % Time spent with sexual incentive Day 9 % Time spent with Sexual incentive Day 18 % Time Spent with Sexual incentive Day 28 Control 40±2.5 38±2.9 42±4.3 Disease Control 41±3.2 20±1.9 9±3.2 (Flouxetine) Flouxetine + 38±5.6 23±2.4 13±2.5 Tribulus Terristeris Flouxetine + 40±4.1 32±1.6* 37±5.2* Cumminium Cyminium Flouxetine + Standard Drug Bupriprion 38±3.3 33±1.7* 35±2.4* Means values represent the mean of six rats (n=6). *=Significant difference values are exhibited at P<0.05 for comparison against diabetic control group by one way Anova-followed by Post Hoc Analysis. Flouxetine- a selective serotonin reuptake inhibitor (SSRI) is known to inhibit libido in male rats. Male wistar rats were subjected to dose of 10 mg/kg s.c of fluoxetine for 28 days and were allowed to stay in the open arena test, % time spent in the proximity of a female rat is an indicator of libido. An increase in % time spent with female rat (sexual stimuli) indicates increase in libido. A significant fall in % time spent with sexual stimuli (heated female rats) was observed in fluoxetine treated rats on Day 18 and 28 indicating the loss of desire for female rats on treatment of fluoxetine is established after a 18 days. The loss of desire by fluoxetine is more pronounced on 28 th day. On Day 9 so such decline in desire is observed. Page 85

20 % of time spent by treated rats with Sexual stimuli Results Tribulus terristeris at the dose of 100mg/kg b.w did not show any improvement in loss of desire caused due to fluoxetine. However, C.Cyminium at the dose of 150mg/kg showed significant improvement in increasing the sexual desire in fluoxetine treated rats. Effect of % time spent by low libido rat in proximity of sexual stimuli Control Disease Control (Flouxetine) Flouxetine +Tribulus Terristeris Flouxetine +Cumminium Cyminium *=Significant difference values are exhibited at P<0.05 for comparison against diabetic control group by one way Anova-followed by Post Hoc Analysis. Fig 5.B.1. Effect of pharmacological interventions on time spent in proximity of sexual stimuli. All values are expressed as mean of six rats. C.Cyminium extract was evident from 18 th day onwards and were more pronounced on 28 th day. Standard drug Bupriprion (anti-depressant) at the dose of 10 mg/kg was also found to exhibit a significant reversal of loss of desire caused by fluoxetine. C.Cyminium exhibited the increase in sexual desire which was comparable to Bupriprion. Flouxetine + Standard Drug Bupriprion Day 9 Day 18 Day 28 Page 86

21 Results of Protocol II: Erectile Function Rats were induced with diabetes as mentioned earlier and were evaluated for food intake, water intake, urine output and Blood glucose. The values shown here were are the mean values of six rats in each group. These parameters were observed throughout the study, however the table represent the values at the end of 8 week. Table 5.B.2. Effect of Tribulus terristeris & Cumminium cyminium on food/water intake, urine output and Blood glucose Groups Drug Treatment Water Intake in ml Group 1 Control (Male Adult wistar Rat, weighing gm) given normal saline Food Intake in gms Urine Output in ml Blood Glucose in mg/dl 15.1± ± ± ±9.8 Group 2 Diabetes induced (STZ-60mg/Kg I.P. 28.4± ± ± ±14.5 non treated for 8 weeks). Group 3 Diabetic rats + 100mg/kg Tribulus terristeris 29.3± ± ± ±12.1 * Group 4 Diabetes induced rats mg/kg 26.2±3.4 31± ± ±15.4 Cumm.cyminium Group 5 Diabetes Induced rats + Insulin S.C (4I.U/rat) 17.7±1.8* 20.2±2.8* 5.6±1.6* 128.5±8.7* Means values represent the mean of six rats (n=6). *=Significant difference values are exhibited at P<0.05 for comparison against diabetic control group by one way Anova-followed by Post Hoc Analysis. A significant increase in food intake was observed in diabetic rats as compared to normal rats. Further, both the extracts of Tribulus terristeris and Cumminium cyminium at the dose of 100mg/kg and 150 mg/kg b.w respectively didnot show any reduction in the increased food intake. However, a significant reduction of the enhanced food intake (due to diabetes) is observed in Insulin treated group A significant increase in water intake was observed in diabetic rats as compared to normal rats.further, both the extracts of Tribulus terristeris and Cumminium cyminium at the dose of 100mg/kg and 150 mg/kg b.w respectively didnot show any reduction in the increased water intake. However, a significant reduction in water intake (due to diabetes) is observed in Insulin treated group. Page 87

22 Units in ml/gm or mg/dl Results Effect of Tribulus terristeris & Cumminium cyminium on food/water intake, urine output and Blood glucose Water intake (ml) Food intake (gms) Urine Output(ml) Blood glucose(mg/dl) 0 Control Diabetic Diabetic +tt Diabetic +cc Diabetic + Insulin *=Significant difference values are exhibited at P<0.05 for comparison against diabetic control group by one way Anova-followed by Post Hoc Analysis Fig 5.B.2. Effect of pharmacological intervention on food intake,water intake, urine output and Blood Glucose. All values are expressed as mean of six rats.. A significant increase in urine output was observed in diabetic rats as compared to normal rats.further, both the extracts of Tribulus terristeris and Cumminium cyminium at the dose of 100mg/kg and 150 mg/kg b.w respectively didnot show any reduction in the increased urine output. However, a significant reduction in urine output (due to diabetes) is observed in Insulin treated group. Blood glucose levels were significantly increased in STZ treated animals. Elevated blood glucose levels were significantly reduced in Tribulus terresteris and insulin treated groups though the effect is more pronounced in case of insulin treated group. However the Cumm.cyminium at the dose of 150 mg/kg didnot show a significant reduction in blood glucose. Page 88

23 Table 5.B.2a Effect of Tribulus terristeris & Cumminium cyminium on maximum relaxation by Acetyl choline and its EC 50. Groups Drug Treatment Maximum Relaxation by Ach following precontraction with 100μM Phenyl Epherine Group 1 Control (Male Adult wistar Rat, weighing gm) given normal saline ±0.08 Ec 50 of Acetyl Choline Group 2 Diabetes induced (STZ-60mg/Kg I.P. non ±0.09 treated for 8 weeks). Group 3 Diabetic rats + 100mg/kg Tribulus terristeris 47.4* 5.9±0.42* Group 4 Diabetes induced rats mg/kg ±0.12 Cumm.cyminium Group 5 Diabetes Induced rats + Insulin S.C (4I.U/rat) 78.5* 5.4±0.13* Means values represent the mean of six rats (n=6). *=Significant difference values are exhibited at P<0.05 for comparison against diabeti control group by one way Anova-followed by Post Hoc Analysis Isolated Corpus Cavernosal tissue were mounted on organ bath filled with Kreb s Heinslet Solution. Cavernosal Strips were then subjected to 100µM Phenylepherine for precontraction followed by relaxation of the strips with Acetylcholine. Relaxation of corpus cavernosal tissue by acetyl choline is an indicator of penile erection. Diabetes is known to reduce the ability of the cavernosal tissue to relax. A mean contraction of 958±40.5 mg was obtained following 100 µm of phenylpherine. Precontracted tissue were subjected to relaxation with Acetylcholine The relaxtions obtained in Control animals were considered to be 100% and other groups were compared with control as reference. A significant reduction in relaxation of cavernosal tissue was observed in diabetic rats, Tribulus terristeris was able to significantly improve the maximum relaxation at the dose of 100mg/kg b.w, however there was no significant Page 89

24 improvement observed in reduced maximal relaxation in C.Cyminium treated group. Cavernosal strips from insulin treated diabetic group exhibited a maximum improvement. The improvement in % max. relaxation was more pronounced in Insulin treated group than among all treated groups. Table 5.B.2a. A Cummulative Dose Response Curve (DRC) of Acetyl choline was plotted for cavernosal strips excised from rats of each group at the end of 8 weeks, and from the DRC, EC 50 of the Acetylcholine was extrapolated using graphical method. Low values of Log of EC 50 signifies higher relaxation capability of the tissue at low dose of Acetylcholine. Insulin treated group and Tribulus terristeris treated group exhibited a significant difference than diabetic treated group- which showed the lowest capability to relax indicating setting up of erectile dysfunction. There was hardly any significant improvement observed in C.Cyminium treated animals. 120 Max % relaxation by Ach Max % relaxation by Ach. 0 Fig.5.B.2aEffect of Tribulus terristeris & Cumminium cyminium on maximum relaxtion by Ach. Means values represent the mean of six rats (n=6). *=Significant difference values are exhibited at P<0.05 for comparison against diabeti control group by one way Anova-followed by Post Hoc Analysis Page 90

25 Log Ec50 of Ach - Log Ec5 0 of Ach 0 Fig.5.B.2bEffect of Tribulus terristeris & Cumminium cyminium on EC 50. Of Ach. *=Significant difference values are exhibited at P<0.05 for comparison against diabeti control group by one way Anova-followed by Post Hoc Analysis Results of Protocol-III (Sperm Analysis): Sperm samples were collected from the same rats used for erection studies after sacrificing them. The collected sperm samples were duly cleaned/processesd and diluted and were subjected to different procedures as mentioned in protocol-iii earlier. Neubeaur Chamber was used for counting sperms. Table 5.B.3 Effect of Tribulus terristeris & Cumminium cyminium on Sperm parameters Control Diabetic Diabetic + T.Terristeris Groups Treatments Sperm Density millions/ml % Sperm Motility % Sperm viability Group 1 Control (Male Adult wistar Rat, weighing gm) given normal saline ± ± ± 3.72 Group 2 Diabetes induced (STZ- 60mg/Kg I.P. non treated for 8 weeks). Group 3 Diabetic rats + 100mg/kg Tribulus terristeris Group 4 Diabetes induced rats mg/kg Cumm.cyminium Group 5 Diabetes Induced rats + Insulin S.C (4I.U/rat) Diabetic + C.Cyminium Diabetic + Insulin 19.67± ± ± ± ± ± ± 3.39 * 55.43± 5.81 * 49.23± 5.84* 29.23± 5.84* 60.23± 4.27 * 62.34±4.49* Page 91

26 Units in Millions/ml or % Results Means values represent the mean of six rats (n=6). Effect of Tibulus terristeris and Cumminium cyminium on Sperm parameters Sperm Density % Sperm motility % Sperm Viablility Fig. 5.B.3 Effect of Tribulus terristeris & Cumminium cyminium on Sperm parameters. Means values represent the mean of six rats (n=6). *=Significant difference values are exhibited at P<0.05 for comparison against diabeti control group by one way Anova-followed by Post Hoc Analysis Diabetic rats has exhibited the significant reduction in Sperm density, % sperm motility and % sperm viability. Tribulus terristeris doesnot show any significant improvement in any of vital sperm parameters studied i.e. sperm density,% sperm motility or % sperm viability. However, a significant improvent improvement sperm density,% sperm motility and % sperm viability was observed in diabetic rats treated with Cumm.Cyminium and Insulin. Page 92