DESC:FIELD OF THE INVENTION
The present invention relates to a method of isolating active compound and a pharmaceutical composition thereof for improving male sexual dysfunction. More particularly, the present invention relates to a pharmaceutical constituent extraction, isolation and purification from the extracts prepared from Turnera diffusa, also called Damiana.

BACKGROUND OF THE INVENTION
Impotence is generally characterized by an inability to maintain a penile erection and is often referred to as erectile dysfunction. Erectile dysfunction affects men, regardless of age, place of birth, or prior sexual experience.

Impotence is estimated to affect about 10 million men in the United States alone. Impotence results from disruption of any of numerous physiological or psychological factors which cause the blood flow to and from the penis to remain in balance thereby preventing retention of sufficient blood to cause rigid dilation of the corpus cavernosa and spongiosa.

The drugs most commonly used include a-blockers, such as phenoxybenzamine and phentolamine; smooth muscle relaxants such as papaverine; prostaglandins having a vasoactive function such as prostaglandin E1 and combinations of such drugs having different receptor effects to enhance therapy. Intracavernous injection doses of papaverine, phentolamine, and prostaglandin E1 are widely available in market.

However, patients often find the injections of vasoactive drugs psychologically disturbing, painful, traumatic, or inconvenient as evidenced by a high discontinuance rate. In addition, adverse side effects including priapism, corporeal nodules and diffuse fibrosis, drug tolerance, bruising and hematomas, swelling and ulceration of the penile skin at the injection site have also been reported.

Also, as many as 75% of diabetic men suffer from maintaining an erection adequate for sexual performance (Metro et al., 1999). Diabetes increases the risk of ED nearly two-fold and ED is considered as one of the first symptom of diabetes (McCulloch et al., 1984). Also, Tadalafil, a common commercially available drug has been found to be an effective anti-diabetic agent and improved ED although well tolerated, causes headache and dyspepsia.

In order to overcome the side effects of existing state of art, there arise a need for herbal composition for erectile dysfunction which has minimum or no side effects and also acceptable among the diabetic male population.

A variety of botanicals are known to have a potential effect on the sexual functions, supporting older claims and offering newer hopes. There has been growing interest for natural herbal supplements since they are available readily, relatively free of side effects, and are affordable.

Of the many listed herbals, Turnera diffusa Willd., popularly known as damiana, of Turneraceae family, is a small shrub that grows in tropical and subtropical parts of America is well documented herbal aphrodisiac (Kotta et al., 2013).

Accordingly, the present invention provides a novel method of isolating active herbal compound from Damiana and a pharmaceutical composition thereof for improving male sexual dysfunction. Said method of isolation provides a higher yield of the Damiana component and said pharmaceutical composition is several times more effective in improving the male sexual dysfunction conditions.

Present invention overcomes the drawbacks of state of art by providing an herbal composition for improving or mitigating erectile dysfunction which has minimum or no side effects. Also, the present invention provides a cost-effective treatment for erectile dysfunction.

OBJECT OF THE INVENTION
The main object of the present invention is to provide a method of isolating active compound and a pharmaceutical composition thereof for improving male sexual dysfunction.

Another object of the present invention is to provide a method of isolating Teuhetenone and a pharmaceutical composition thereof for improving male sexual dysfunction.

Yet another object of the present invention is to provide a method of isolating derivatives of Teuhetenone and a pharmaceutical composition thereof for improving male sexual dysfunction.

Yet another object of the present invention is to provide a method of isolating Teuhetenone which is economical and provides a better yield.

Yet another object of the present invention is to provide a pharmaceutical composition which is effective in improving erectile dysfunction in males.

Yet another object of the present invention is to provide a method of isolating derivative of Teuhetenone which is economical and provides a better yield.

Yet another object of the present invention is to provide a pharmaceutical composition comprising derivatives of Teuhetenone which is efficient and effective for improving erectile dysfunction in males.

Yet another object of the present invention is to provide cost effective treatment for erectile dysfunction.

Yet another object of the present invention is to provide an herbal pharmaceutical composition for erectile dysfunction which has minimum or no side effects.

SUMMARY OF THE INVENTION:
Accordingly, the present invention relates to a method of isolating active herbal compound and a pharmaceutical composition thereof for improving male sexual dysfunction.

Various constituents of Damiana, such as Acacetin, Arbutin, Thymol, etc. have been reported for improving the medical condition of Erectile Dysfunction (ED),

In a study by Zhao et al (2008), acacetin, one of the active constituents have been shown to be the most potent inhibitor of aromatase activity which in turn would be responsible for
maintaining testosterone levels. Arbutin has been shown to have estrogen like activity (Zeng et al., 2018) reflecting its role in maintaining hormonal levels.

Thymol and carvacrol administration have been reported to decrease the oxidative damage and increased the antioxidant levels and improved the sperm quality parameters in a study by Güvenç et al (2019) and Chikhoune et al., (2015), although its role in testosterone levels in rats was not positive (Luaibi, 2017).

Accordingly, present invention relates to isolation of active compound from damiana extract, which has potential for improving erectile dysfunction in men. Said extraction process resulted in a prominent peak of Teuhetenone, which was analyzed and confirmed by Mass spectroscopic studies.

The present invention relates to an extraction procedure with green chemistry solvent-water which resulted in several fold higher yield of Teuhetenone than the commercially available Damiana extract. Accordingly, the present invention provides a method of isolating higher yields of herbal compound Teuhetenone and its derivatives from Damiana species.

The present invention also relates to a pharmaceutical composition comprising Teuhetenone and its derivatives along with suitable excipients/adjuvants. Said pharmaceutical composition is efficacious and effective for improving erectile dysfunction in males.

Accordingly, present invention provides an herbal composition for erectile dysfunction which has minimum or no side effects and also acceptable among the diabetic male population.

The experimental data demonstrated the efficacy of pharmaceutical composition of Teuhetenone in addressing Erectile Dysfunction in animals which can be postulated to human beings as well.

BRIEF DESCRIPTION OF DRAWINGS
Figure 1 displays the Damiana Extract Chromatogram.
Figure 2 (a) displays the Isolated compound chromatogram.
Figure 3 displays the Mass Spectroscopy results.
Figure 4(a) displays the NMR Spectroscopy of 1H NMR.
Figure 4(b) displays the NMR Spectroscopy of 13C NMR.
Figure 5 displays the Flash chromatograph result.
Figure 6 displays the chemical structure of Sildenafil and its ADMETlab 2.0 evaluation result.
Figure 7 displays the chemical structure of Teuhetenone and its ADMETlab 2.0 evaluation result.
Figure 8 displays the chemical structure of Tadalafil and its ADMETlab 2.0 evaluation result.
Figure 9 displays the Effect of SAVA 22 on Mounting Latency in Paroxetine induced Erectile Dysfunction.
Figure 10 displays the Effect of SAVA 22 on Mounting Frequency in Paroxetine induced Erectile Dysfunction.
Figure 11 displays the Effect of SAVA 22 on Intromission Latency in Paroxetine induced Erectile Dysfunction.
Figure 12 displays the Effect of SAVA 22 on Intromission Number in Paroxetine induced Erectile Dysfunction.
Figure 13 displays the Effect of SAVA 22 on penile tissue Nitric Oxide (mmol/mg of tissue) in Paroxetine induced Erectile Dysfunction.
Figure 14 displays the Effect of SAVA 22 on penile tissue Phosphodiesterase 5 activity (nmol/min/mg of protein) in Paroxetine induced Erectile Dysfunction.
Figure 15 displays the Effect of SAVA 22 on Blood Glucose Level (mg/dl) in Paroxetine induced Erectile Dysfunction.
Figure 16 displays the Effect of SAVA 22 on serum Testosterone (ng/dL) in Paroxetine induced Erectile Dysfunction.
Figure 17 displays the Effect of SAVA 22 on serum17ß-Hydroxysteroid dehydrogenase (Unit/min/mg protein) in Paroxetine induced Erectile Dysfunction.
Figure 18 displays the Effect of SAVA 22 on testes 17ß-Ketosteroid reductase (Unit/min/mg protein) in Paroxetine induced Erectile Dysfunction.
Figure 19 displays the Effect of SAVA 22 on testes 17ß-Ketosteroid reductase (Unit/min/mg protein) in Paroxetine induced Erectile Dysfunction.
Figure 20 displays the Effect of SAVA 22 on Systolic BP (mmHg) in Paroxetine induced Erectile Dysfunction.
Figure 21 displays the Effect of SAVA 22 on Diastolic BP (mmHg) in Paroxetine induced Erectile Dysfunction.
Figure 22 displays the Molecular docking of cGMP-specific 3',5'-cyclic phosphodiesterase(1XOZ) Binding Domain Complexed with Sildenafil showing 3D model of the interactions and the 2D interaction patterns and H-bond interaction.
Figure 23 displays the Molecular docking of cGMP-specific 3',5'-cyclic phosphodiesterase(1XOZ) Binding Domain Complexed with Teuhetenone A showing 3D model of the interactions and the 2D interaction patterns and H-bond interaction.
Figure 24 displays the Molecular docking of Protein (Estradiol 17 Beta-Dehydrogenase 1 (1EQU) Binding Domain Complexed with Tadalafil showing 3D model of the interactions and the 2D interaction patterns and H-bond interaction.
Figure 25 displays the Molecular docking of 17-Beta-Hydroxysteroid-Dehydrogenase (1FDT) Binding Domain Complexed with Tadalafil showing 3D model of the interactions and the 2D interaction patterns and H-bond interaction.
Figure 26 displays the Molecular docking of cGMP-specific 3',5'-cyclic phosphodiesterase (1XOZ) Binding Domain Complexed with Tadalafil showing 3D model of the interactions and the 2D interaction patterns and H-bond interaction.
Figure 27 displays the HPLC Analysis-Standard chromatogram.
Figure 28 displays the HPLC Analysis- Tablet chromatogram.
Figure 29 displays the Analytical method validation report for assay of Teuhetenone for Linearity.
DETAILED DESCRIPTION OF THE INVENTION ILLUSTRATIONS AND EXAMPLES
While the invention has been disclosed with reference to certain embodiments, it will be understood by those skilled in the art that various changes may be made, and equivalents may be substituted without departing from the scope of the invention. In addition, many modifications may be made to adapt to a particular situation or material to the teachings of the invention without departing from its scope.

Throughout the specification and claims, the following terms take the meanings explicitly associated herein unless the context clearly dictates otherwise. The meaning of “a,” “an”, and “the” include plural references. Additionally, a reference to the singular includes a reference to the plural unless otherwise stated or inconsistent with the disclosure herein.

The reference numerals as used in the invention is represented in Table 1 as below.

Table 1: Legend of Abbreviations used
S.no. Particulars Legend
1 High Performance Liquid Chromatography HPLC
2 Erectile dysfunction ED
3 Mass Spectrometry MS
4 Octadecyl-silica ODS
5 Nuclear magnetic resonance NMR
6 Demineralized water DM
7 Absorption, distribution, metabolism, excretion, and toxicity ADMET
8 Human Intestinal Absorption HIA
9 Blood Brain Barrier BBB
10 Phosphodiesterase isozymes PDE
11 Cyclic guanosine monophosphate cGMP
12 Protein data bank PDB
13 Not more than NMT
14 Relative humidity RH
15 International Council for Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use ICH
16 Reverse phase High performance liquid chromatography RP HPLC
17 DM water Demineralized Water

According to data from the Massachusetts Male Aging Study, up to 52% of men between the ages of 40 and 70 are affected by ED. The risk factors for erectile dysfunction include neurologic problems (multiple sclerosis, stroke), depression and medications for treating diabetes, hypertension, heart disease and depression.

Approved medication in the US is the PDE 5 enzyme inhibitor Sildenafil (Viagra). Unapproved therapies, some of which are available in the US and some elsewhere, include Tadalafil (Cialis), vardenafil (Levitra), yohimbine, phentolamine, trazodone, apomorphine and nutraceuticals.

The present invention discloses a pharmaceutical composition for improving male sexual dysfunction. The said composition is significantly efficient and effective for improving erectile dysfunction in males.

The present invention provides an herbal pharmaceutical composition comprising of an active compound Teuhetenone which is effective in treating the disorder of erectile dysfunction and exhibits minimum or no side effects.

Experiments have been conducted on the isolated compound Teuhetenone and its derivatives for its potential ability to be used for male sexual dysfunction. Since anti-diabetic property of Teuhetenone is known, experiments have been conducted to examine the effect of Teuhetenone on an Erectile Dysfunction animal model.

All the experimental work has been done following the protocols approved by Institutional Animal Ethics Committee (IAEC) constituted under guidelines of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA).

Extraction of Active Compound:
Extraction is the process of separating the pharmaceutically active mixture of naturally active compounds contained inside plant materials or tissues using selective solvents using the standard procedure. It can be also defined as the treatment of the plant material with solvent, whereby the medicinally active components are dissolved and most of the inactive matter remains undissolved. Therefore, the objective of all extraction is to separate the soluble plant metabolites, leaving behind the insoluble cellular residue.

Damiana leaves were the source of raw material for extraction of Teuhetenone A. The extraction of active compounds from Damiana were performed by trials in batches with water, 50% ethanol and ethyl acetate wash followed by water extraction. It was observed that extraction process with water yields the best quantity of active compounds.
Isolation of Teuhetenone
Purification of the compound of interest using HPLC is the process of separating or extracting the target compound from other (possibly structurally related) compounds or contaminants. Each compound should have a characteristic peak under certain chromatographic conditions. Depending on what needs to be separated and how closely related the samples are, the chromatographer chooses the conditions, such as the proper mobile phase, flow rate, suitable detectors and columns to get an optimum separation.

Identification of compounds by HPLC is a critical part of any HPLC assay. Thus, the combination of HPLC and MS enables rapid and accurate identification of compounds in medicinal herbs. In order to distinguish any compound by HPLC, a detector must first be chosen. Once the detector is chosen and is set to optimal detection settings, a separation assay must be developed. The parameters of this assay be such that a clean peak of the known sample is observed from the chromatograph. The identifying peak is desired to have a reasonable retention time and is to be well separated from irrelevant peaks at the detection levels which the assay will be performed.

Accordingly, in some embodiments, the disclosure provides an efficient method for isolation of the active compound of interest, Teuhetenone. Isolation of active compound Teuhetenone A was first done with traditional manual column chromatography method and then using flash chromatography method. Both methods give a good yield. However, the flash chromatography method has clear advantages over the traditional method. It was seen that in a single column a higher yield as well as greater degree of purity is achieved in flash chromatography as compared to manual chromatography.

Therefore, the present invention provides a method of isolation of active compound in an efficient manner and hence is scalable and cost effective.

Separation of Teuhetenone A using traditional manual column chromatography
The isolation and identification of the active compound Teuhetenone A by traditional manual column chromatography was conducted in the following steps.

Column 1:
In the first column, specific quantity of water extract was introduced in the column which got adsorbed on silica. Further the glass column was packed with suitable quantity of silica. The column was then eluted with ethyl acetate, then with 10% methanol in ethyl acetate and finally with 15% methanol in ethyl acetate. This 15% methanol fraction contained the targeted compound.

Column 2:
In the second column, the 15% methanol fraction from column 1 was introduced and adsorbed on silica. The glass column was again packed with silica. Column was eluted with chloroform, then with 10% methanol in chloroform, followed with 15% methanol and then with 20% methanol in chloroform. This 20% methanol fraction contained the targeted compound.

Column 3:
The 20% methanol fraction from column 2 was adsorbed on silica in column 3. The glass column was packed with silica. The column was then eluted with chloroform, further with 12% methanol in chloroform and then with 16% methanol in chloroform.

In total 9 fractions were collected and the pure compound of Teuhetenone was isolated.

The HPLC column parameters of this manual isolation method is as mentioned below:
Mobile Phase: The solvents used in the mobile phase of the column are as below:
A. 0.1% Formic acid
B. Acetonitrile

Column parameters: The various parameters used in the column are given below:
Column : Inertsil ODS 250×4.6×5 µ
Column Temp : 30°C
Sample temperature : 25°C
Wavelength: : 280nm
Flow rate : 0.8 ml/min
Injection volume : 20 µL
Run time : 50 min
Elution : Gradient

The Damiana Extract Chromatogram and Isolated compound chromatogram are as illustrated in Figures 1 and 2 respectively.

The Mass Spectroscopy performed with direct tunning to mass method is illustrated in figure 3 and NMR Spectroscopy of 1H NMR AND 13C NMR are illustrated in Figures 4 (a) and 4 (b) respectively.

Separation of Teuhetenone A using Flash chromatography
The separation of the active compound Teuhetenone was also conducted using the Flash chromatography method. The results from this method are more encouraging. The particulars of the trial are as presented below.
Mobile Phase:
A. Chloroform
B. Methanol

Column parameters-
Column: Silica 40 g
Column Temp: 30°C
Sample temperature: 25°C
Wavelength: 280nm
Flow rate: 40 ml/min.
Run time: 30 min.
Elution: Gradient

Sample preparation and loading:
Crude water extract of a specific quantity was dissolved in DM water and then liquid extract injected in silica column. About 8-10 injections per column are carried put.

It was found that using 3 g of extract and 6 hours’ time, 200 mg separation of 90% pure compound was achieved, showing a recovery of 7%. The result is as illustrated in Figure 5.

ADMET Analysis Test Report:
In another embodiment of the invention, ADMET analysis tests were carried out for analysis and evaluation of the active compound Teuhetenone A of the proposed pharmaceutical composition, for its efficiency and toxicity. The ADMET studies were first carried out on the active compounds available in the market for treating male sexual dysfunction. These compounds are Sildenafil and Tadalafil which are primarily and popularly used for mitigating conditions of erectile disorders.

Chemical absorption, distribution, metabolism, excretion, and toxicity (ADMET), play key roles in drug discovery and development. A high-quality drug candidate should not only have sufficient efficacy against the therapeutic target, but also show appropriate ADMET properties at a therapeutic dose. A successful drug should achieve a finely tuned combination of biochemical behavior, pharmacokinetics and safety. In addition to high potency and selectivity, desirable absorption, distribution, metabolism, excretion and toxicity (ADMET) profile is equally critical to the success of a drug candidate.

Software used:
There are various software available to check the prediction of ADMET properties. Here the software used were the ADMETlab2.0 https://admetmesh.scbdd.com/ to check the prediction of ADMET properties. ADMETlab 2.0 was built using the Python web framework of Django and deployed on an elastic compute service from Aliyun running an Ubuntu Linux system. The web access was enabled via the Nginx web server and the interactions between Django and proxy server were supported by Uwsgi. This application was developed based on the Model-View-Template (MVT) framework. The model layer maps the business objects to the database objects. The view layer is a business logic layer, responsible for performing the access to the deep learning models, delivering the data to be shown on the template layer, and handling the upload and download of files. The template layer provides the visualization of results, page rendering, integration of documentation, etc. The uploaded and downloaded files, pre-trained models and model predictions were stored in the server.

ADMET studies:
The computational ADMET predication (absorption, distribution, metabolism, excretion, and toxicity) are constitutive methods used in modern drug discovery to predict the drug pharmacokinetics and toxicity. ADMET properties are necessary for the selection and development of drug candidates. The ADMET properties for the compounds Sildenafil and Teuhetenone A were estimated using ADMETlab. The ADMET properties as derived from ADMETlab server reveal that the Sildenafil and Teuhetenone A had better Human Intestinal Absorption (HIA) score. Greater HIA denotes that the compound could be better absorbed from the intestinal tract upon oral administration. The penetration through the Blood Brain Barrier (BBB Penetration) came out to be a best drug candidate. However, determining the toxicity of chemical compounds is necessary to identify their harmful effects on humans, animals, plants or the environment.

The chemical structure of Sildenafil and its ADMETlab 2.0 evaluation result is illustrated in Figure 6. Similarly, the chemical structure of Teuhetenone and its ADMETlab 2.0 evaluation result is illustrated in Figure 7. There are various other properties which are listed below in Tables 2 and 3 in respect of Sildenafil and Teuhetenone A respectively.

Table:2 Performance of the classification models incorporated into the ADMETlab 2.0 platform for Sildenafil
Category Model Points
Absorption Caco-2 Permeability -4.887
MDCK Permeability 1.9e-05
Pgp-inhibitor ++
Pgp-substrate +++
HIA ---
Distribution PPB 95.990%
VD 2.313
BBB Penetration --
Fu 4.578%
Metabolism CYP1A2 inhibitor --
CYP1A2 substrate +++
CYP2C19 inhibitor --
CYP2C19 substrate +++
CYP2C9 inhibitor --
CYP2C9 substrate +
CYP2D6 inhibitor ---
CYP2D6 substrate ++
CYP3A4 inhibitor +
CYP3A4 substrate +++
Excretion CL 9.920
T1/2 0.279
Toxicity hERG Blockers +
H-HT ++
DILI +++
AMES Toxicity --
Rat Oral Acute Toxicity ---
FDAMDD ++
Skin Sensitization ---
Carcinogenicity ++
Respiratory Toxicity ++
Bioconcentration Factors 0.576
IGC50 2.385
LC50FM 3.202
LC50DM 4.401
Physicochemical Property Molecular Weight (MW) 474.200
Volume 456.038
Density 1.040
nHA 10
nHD 1
nRot 7
nRing 4
MaxRing 9
nHet 11
fChar 0
nRig 25
Flexibility 0.280
Stereo Centers 0
TPSA 113.420
logS -3.952
logP 2.168
logD 2.593

Table:3 Performance of the classification models incorporated into the ADMETlab 2.0 platform for Teuhetenone A
Category Model Points
Absorption Caco-2 Permeability -4.652
MDCK Permeability 2.2e-05
Pgp-inhibitor -
Pgp-substrate ---
HIA ---
Distribution PPB 77.265%
VD 0.792
BBB Penetration +++
Fu 31.045%
Metabolism CYP1A2 inhibitor ---
CYP1A2 substrate +++
CYP2C19 inhibitor ---
CYP2C19 substrate ++
CYP2C9 inhibitor ---
CYP2C9 substrate --
CYP2D6 inhibitor ---
CYP2D6 substrate ---
CYP3A4 inhibitor ---
CYP3A4 substrate -
Excretion CL 6.966
T1/2 0.809
Toxicity hERG Blockers ---
H-HT -
DILI ---
AMES Toxicity --
Rat Oral Acute Toxicity -
FDAMDD --
Skin Sensitization --
Carcinogenicity ++
Respiratory Toxicity ++
Bioconcentration Factors 0.483
IGC50 2.011
LC50FM 2.582
LC50DM 3.836
Physicochemical Property Molecular Weight (MW) 194.130
Volume 211.303
Density 0.919
nHA 2
nHD 1
nRot 0
nRing 2
MaxRing 10
nHet 2
fChar 0
nRig 12
Flexibility 0.000
Stereo Centers 2
TPSA 37.300
logS -2.378
logP 1.589
logD 1.448

Note-the prediction probability values are transformed into six symbols: 0-0.1(---), 0.1-0.3(--), 0.3-0.5(-), 0.5-0.7(+), 0.7-0.9(++), and 0.9-1.0(+++).

The ADMET properties for the compounds Tadalafil were estimated using ADMETlab. Greater HIA denotes that the compound could be better absorbed from the intestinal tract upon oral administration. The penetration through the Blood Brain Barrier (BBB Penetration) came out to be a best drug candidate. However, determining the toxicity of chemical compounds is necessary to identify their harmful effects on humans, animals, plants or the environment. The chemical structure of Tadalafil and its ADMETlab 2.0 evaluation result is illustrated in Figure 8. There are various other properties which are listed below in Table 4.

Table 4: Performance of the classification models incorporated into the ADMETlab 2.0 platform for Tadalafil
Category Model Points
Absorption Caco-2 Permeability -5.127

MDCK Permeability TheMadin-Darby canine kidney: 2.4e-05
Pgp-inhibitor (Permeability glycoprotein) --
Pgpsubstrate (permeability glycoprotein) ++
HIA (Human Intestinal Absorption) ---
Distribution PPB (Plasma protein binding) 95.927%
VD (Volume Distribution) 0.651
BBB Penetration (Blood Brain Barrier) -
Fu 2.175%
Metabolism CYP1A2inhibitor (Cytochrome P4501) --
CYP1A2 substrate +++
CYP2C19 inhibitor (Cytochrome P4502) +++
CYP2C19 substrate ++
CYP2C9 inhibitor (Cytochrome P450 family 2 subfamily C member 9) ++
CYP2C9 substrate ++
CYP2D6 inhibitor (Cytochrome P450 2D6) ---
CYP2D6 substrate --
CYP3A4 inhibitor (Cytochrome P450 3A4) +++
CYP3A4 substrate +++
Excretion CL 10.530
T1/2(half-life of the reaction) 0.191
Toxicity hERG Blockers (Human ether à-go-go related gene) ---
H-HT -
DILI +++
AMES Toxicity ---
Rat Oral Acute Toxicity ---
FDAMDD +++
Skin Sensitization --
Carcinogenicity ++
Respiratory Toxicity ++
Bioconcentration Factors 0.595
IGC50(Inhibition Growth concentration) 2.072
LC50FM (Lethal Dose) 3.946
LC50DM 5.008
Physicochemical Property Molecular Weight (MW) 389.140
Volume 382.152
Density 1.018
nHA (No of hydrogen acceptor) 7
nHD (No of hydrogen donor) 1
nRot (No of rotatable bonds) 1
nRing (No of rings) 6
MaxRing (Maximum no of rings) 17
nHet (No of heteroatoms) 7
fChar (Formal charge) 0
nRig (No of rigid bonds) 32
Flexibility 0.031
Stereo Centers 2
TPSA (Topological polar surface area) 74.870
logS (Measuring solubility) -4.850
logP (Measuring lipophilicity) 2.911
logD (Distribution Coefficient) 2.452
Note-the prediction probability values are transformed into six symbols: 0-0.1(---), 0.1-0.3(--), 0.3-0.5(-), 0.5-0.7(+), 0.7-0.9(++), and 0.9-1.0(+++)

Docking Studies
Cyclic guanosine monophosphate-specific phosphodiesterase type 5 is an enzyme from the phosphodiesterase class. It is found in various tissues, most prominently the corpus cavernosum and the retina. It has also been recently discovered to play a vital role in the cardiovascular system. The phosphodiesterase (PDE) isozymes, found in several tissues including the rod and cone photoreceptor cells of the retina, belong to a large family of cyclic nucleotide PDEs that catalyze cAMP and cGMP hydrolysis.

The interest in PDEs as molecular targets of drug action has grown with the development of isozyme selective PDE inhibitors that offer potent inhibition of selected isozymes without the side-effects attributed to nonselective inhibitors such as theophylline. Sildenafil, vardenafil, tadalafil, and avanafil are PDE5 inhibitors that are significantly more potent and selective than Zaprinast and other early PDE5 inhibitors. PDE5 is an enzyme that accepts cGMP and breaks it down. Sildenafil, vardenafil and tadalafil are inhibitors of this enzyme, which bind to the catalytic site of PDE5. Both inhibitors bind with high affinity and specificity, and cGMP-binding to the allosteric sites stimulates binding of PDE5 inhibitors at the catalytic site.

Molecular Docking
Molecular Docking is an important component of computer-assisted drug discovery. It helps in predicting the intermolecular framework formed between a protein and ligand and outputs the appropriate binding between the molecules. Docking was performed by AutoDock 4.2.6 program, using the implemented empirical free energy function and the Lamarckian Genetic Algorithm (LGA). The grid maps were calculated using AutoGrid. In all dockings, a grid map with 58 x 52 x 48 points and a grid-point spacing of 1.000Å was applied.

The best conformation with the lowest docked energy was chosen from the docking search. The interactions of complex protein-ligand conformations including hydrogen bonds and bond lengths were analyzed using Pymol software, UCSF Chimera and Accelrys Discovery Studio Visualizer software.

Comparative Docking Analysis Report
Yet another embodiment of the disclosure examines the docking aspects of the active compound Teuhetenone A with three proteins namely, Estradiol 17 beta-dehydrogenase1, 17-Beta-Hydroxysteroid-Dehydrogenase, and cGMP-specific 3',5'-Cyclic Phosphodiesterase. The study also reports the docking analysis of the said three proteins with a popular treatment option available in the market viz. Tadalafil. The details of the study and the results along with interpretation are presented in the following paragraphs.

Docking analysis
PDB ID: 1EQU
Protein name: Protein (estradiol 17 beta-dehydrogenase1) Organism(s): Homo Sapiens, Resolution: 3.00 Å, Sequence Length= 327, Uniport ID - P14061, Gene Names: HSD17B1, E17KSR, EDH17B1, EDH17B2, EDHB17, SDR28C1.

PDB ID: 1FDT
Protein name: 17-Beta-Hydroxysteroid-Dehydrogenase, Organisms(s): Homo sapiens, Sequence Length= 327, Resolution: 2.20 Å Uniprot ID: P14061, Gene Names: HSD17B1, E17KSR, EDH17B1, EDH17B2, EDHB17, SDR28C1

PDB ID: 1EQU
Protein name: PROTEIN (ESTRADIOL 17 BETA-DEHYDROGENASE 1), Organisms(s): Homo sapiens, Sequence Length= 327, Uniprot ID -P14061, Gene Names: HSD17B1, E17KSR, EDH17B1, EDH17B2, EDHB17, SDR28C1

PDB ID: 1FDT
Protein name: 17-BETA-HYDROXYSTEROID-DEHYDROGENASE, Organisms(s): Homo sapiens, Sequence Length= 327, Uniprot ID -P14061, Gene Names: HSD17B1, E17KSR, EDH17B1, EDH17B2, EDHB17, SDR28C1

PDB ID: 1XOZ
Protein name: cGMP-specific 3',5'-cyclic phosphodiesterase, Organisms(s): Homo sapiens, Sequence Length= 364, Uniprot ID -O76074, Gene Names: PDE5A, PDE5

The Grid Data information is as given in Table 5 below.
Table 5: Grid data info:
Grid Point Spacing Even Number of User-specified Grid Points
1.000 Angstroms 40x-points,40y-points,48z-points
1.000 Angstroms 40x-points,40y-points,50z-points
1.000 Angstroms 40x-points,40y-points,40z-points
1.000 Angstroms 40x-points,40y-points,40z-points
1.000 Angstroms 40x-points,40y-points,40z-points

Ligands used:
1)Ligand name: Teuhetenone A, Molecular Formula: C12H18O2, Molecular Weight: 194.27 PubChem ID: 15153216
2)Ligand name: Tadalafil, Molecular Formula: C22H19N3O4, Molecular Weight: 389.4 PubChem CID: 110635
The details of Ligands used are mentioned in Table 6 as below.

Table 6: Details of Ligands used:
Proteins Name
Ligand Name Binding Energy (kcal/mol) No. of H Bonds Interacting residue Final Intermolecular Energy (kcal/mol) vdW + Hbond + desolv Energy (kcal/mol) Electrostatic
Energy (kcal/mol) Torsional Free Energy (kcal/mol)
1EQU TEUHETENONE A. -5.55 01
H1:3.78Å THR:190(H1),
PHE:192,
ILE:14,
VAL:188. -5.89 -5.85 -0.04 +0.30
1FDT TEUHETENONE A -6.70 02
H1:3.41Å
H2:3.39Å GLU:202(H1)
ARG:206(H2)
LEU:95
LEU:102 -7.03 -6.93 -0.10 +0.30
1EQU TADALAFIL -7.11 02
H1:3.19Å
H2:2.02Å ARG:37(H1),
SER:12(H2),
THR:41,
SER:11,
VAL:196,
LEU:93,
ALA:191.
-7.43 -7.33 -0.11 +0.30

1FDT TADALAFIL -7.54 01
H1:2.74Å GLU:202(H1),
LEU:197,
GLY:198,
ALA:101,
VAL:196,
MET:193.
-7.83 -7.82 -0.01 +0.30
1XOZ TADALAFIL -6.65 01
H1:2.50Å MET:816(H1),
LEU:804,
PHE:820 -6.95 -6.95 -0.00 +0.30

Selection of Ligands:
A few of the ligands were got from the PubChem drug databases and docked against Protein estradiol 17 beta-dehydrogenase 1 (PDB ID: 1EQU) 17-Beta-Hydroxysteroid-Dehydrogenase (1FDT), Protein estradiol 17 beta-dehydrogenase 1 (PDB ID: 1EQU) 17-Beta-Hydroxysteroid-Dehydrogenase (1FDT) and cGMP-specific 3', 5'-cyclic phosphodiesterase (1XOZ) with high AutoDock V4.2 software. The ligand names are Teuhetenone A (-5.55 kcal/mol), Teuhetenone A (-6.70kcal/mol), Tadalafil (-7.11kcal/mol), (-7.54 kcal/mol) and (-6.65kcal/mol). For the selection of ligands in the current examination, the existing ligands for the mentioned proteins were chosen from a series of literature review. The existing quantities of ligands are 02 which are chosen to promote perceptions.

Protein preparation-
The crystal structure of Target proteins was retrieved from Protein Data Bank (PDB) with PDB ID 1EQU, 1FDT, 1EQU, 1FDT and 1XOZ and was carried further for more studies of docking process.

Discussion:
The best conformation with the lowest docked energy was chosen from the docking search. On performing the docking of Protein (ESTRADIOL 17 BETA-DEHYDROGENASE 1 (1EQU), and 17-BETA-HYDROXYSTEROID-DEHYDROGENASE (1FDT) with Teuhetenone A (-5.55 kcal/mol), Teuhetenone A (-6.70kcal/mol) and Protein (ESTRADIOL 17 BETA-DEHYDROGENASE 1 (1EQU), 17-BETA-HYDROXYSTEROID-DEHYDROGENASE (1FDT) and cGMP-specific 3',5'-cyclic phosphodiesterase (1XOZ) with Tadalafil (-7.11kcal/mol), Tadalafil (-7.54 kcal/mol) and Tadalafil (-6.65kcal/mol) it was observed that the binding energy shown by the protein and ligand is good at Tadalafil (-7.54kcal/mol). Number of torsions are chosen from 0-6, and if any ligand shows more than 6 it is adjusted to 6. Hydrogen bond interactions are also calculated and mentioned, presence of H-bonds depicts stable interaction between ligand and protein. Discovery studio 2020 Client and Chimera software are used to depict Hydrogen bonds, 2-D images and protein-ligand interactions images for a good visualization of the docking. According to the docking studies, the constituents (Tadalafil) showed highest binding affinity towards the protein 17-BETA-HYDROXYSTEROID-DEHYDROGENASE (1FDT) as compared to the Teuhetenone A. Thus, it is needed to be tested and evaluated in the laboratory for further analysis.

Docking Analysis Report-PDE5 (Human Phosphodiesterase-5)
In another embodiment, the docking analysis was carried out of the compounds Sildenafil and Teuhetenone A with the protein cGMP-specific 3', 5’-cyclic phosphodiesterase. The details and discussion aspects are presented here.
PDB ID: 1XOZ
Protein name: cGMP-specific 3’, 5’-cyclic phosphodiesterase, Organisms(s): Homo sapiens, Sequence Length= 364, Resolution: 1.37 Å Uniprot ID: O76074, Gene Names: PDE5A, PDE5

Grid data info:
Grid Point Spacing =1.000Angstroms,
Even Number of User-specified Grid Points = 58-points
52-points
48z-points
Coordinates of Central Grid Point of Maps= (37.245, 39.890, 20.428)
Minimum coordinates in grid = (8.245, 13.890, -3.572)
Maximum coordinates in grid = (66.245, 65.890, 44.428)

Ligands used:
1) Ligand name: Sildenafil, Molecular Formula: C22H30N6O4S, Molecular Weight: 474.6, PubChem CID: 135398744
2) Ligand name: Teuhetenone A, Molecular Formula: C12H18O2, Molecular Weight: 194.27, PubChem CID: 15153216

Information on Docking binding energy and protein-ligands interaction and Lipinski’s Rule are illustrated in Table 7 & 8 respectively.

Table 7: Docking binding energy and protein-ligands interaction information

Proteins Name Ligand Name Binding Energy (kcal/mol) No. of H Bonds Interacting residue Final Intermolecular Energy (kcal/mol) vdW + Hbond + desolv Energy (kcal/mol) Electrostatic
Energy (kcal/mol) Torsional Free Energy (kcal/mol)

1XOZ Sildenafil -7.95 01
H1:2.89Å GLN:817(H1)
VAL:782
PHE:786
PHE:820
ASP:764
LEU:804
LEU:725 -9.80 -8.53 -1.27 +2.09
Teuhetenone A -6.14 01
H1:2.01Å TYR:612(H1)
ILE:768
LEU:765
ALA:779
VAL:782
ALA:767
PHE:820 -6.44 -6.44 -0.00 +0.30

Table 8: Lipinski’s Rule information:

Sr. No. Name of Co-former Mol
Weight
(g/mol) XLogP3 Hydrogen Bond Donor Hydrogen Bond Acceptor Rotatable Bond
1 Sildenafil 474.6 1.5 1 8 7
2 Teuhetenone A 194.27 1.2 1 2 0

Table 9: Ligands Mol formula and 2D structure information
Sr. No. Ligand Name Mol Formula 2D Structure

1. Sildenafil C22H30N6O4S

2. Teuhetenone A C12H18O2

Selection of Ligands:
A few of the ligands were got from the PubChem drug databases and docked with cGMP-specific 3', 5'-cyclic phosphodiesterase (1XOZ) protein in AutoDock V4.2 software. The ligand names are Sildenafil (-7.95kcal/mol), Teuhetenone A (-6.14kcal/mol). The chemical name and structure as shown in Table 9 above. For the selection of ligands in the current examination, the existing ligands for the mentioned proteins were chosen from a series of literature review. The existing quantities of ligands are two which are chosen to promote perceptions.

Ligand Preparation-
The 3-D structure of inhibitors with their respective PubChem CID were redeemed and saved in SDF format. Furthermore, ligand preparations were continued by taking the 3-D structure of all the ligands and were introduced in Pymol software for conversion of 3-D structure from SDF to PDB format. Using Pymol software metals were also removed from the ligands structure for an appropriate docking study. The prepared ligands were saved in PDB format for further docking studies.

Protein preparation-
The crystal structure of Target proteins was retrieved from Protein Data Bank (PDB) with PDB ID 1XOZ and was carried further for more studies of docking process.

The best conformation with the lowest docked energy was chosen from the docking search. The interactions of complex protein-ligand conformations including hydrogen bonds and bond lengths were analyzed using Pymol software, UCSF Chimera and Accelrys Discovery Studio Visualizer software.

Discussion:
The best conformation with the lowest docked energy was chosen from the docking search. On performing the docking of the cGMP-specific 3', 5’-cyclic phosphodiesterase (1XOZ) with Sildenafil, Teuhetenone A it was observed that the binding energy shown by the protein and ligand is good at Sildenafil (-7.95 kcal/mol). Number of torsions are chosen from 0-6, and if any ligand shows more than 6 it is adjusted to 6. Hydrogen bond interactions are also calculated and mentioned, presence of H-bonds depicts stable interaction between ligand and protein. Discovery studio 2020 Client and Chimera software are used to depict Hydrogen bonds, 2-D images and protein-ligand interactions images for a good visualization of the docking. On performing the docking of the Protein name cGMP-specific 3', 5'-cyclic phosphodiesterase (1XOZ) is Sildenafil (-7.95kcal/mol), Teuhetenone A (-6.14kcal/mol). It was observed that the binding energy shown by the protein and ligand is good. The Molecular docking of cGMP-specific 3',5'-cyclic phosphodiesterase(1XOZ) Binding Domain Complexed with Sildenafil shows 3D model of the interactions and the 2D interaction patterns and H-bond interaction as illustrated in Figure 22. The Molecular docking of cGMP-specific 3',5'-cyclic phosphodiesterase(1XOZ) Binding Domain Complexed with Teuhetenone A shows 3D model of the interactions and the 2D interaction patterns and H-bond interaction as illustrated in Figure 23.

Extra Docking Information:
Essentially, the aim of molecular docking is to give a prediction of the ligand-receptor complex structure using computation methods. Docking can be achieved through two interrelated steps: first by sampling conformations of the ligand in the active site of the protein; then ranking these conformations via a scoring function. Ideally, sampling algorithms should be able to reproduce the experimental binding mode and the scoring function should also rank it highest among all generated conformations. From these two perspectives, we give a brief overview of basic docking theory.

AutoDock uses a computationally (relatively) inexpensive “hybrid” force field that contains terms based on molecular mechanics as well as empirical terms. The prediction of absolute binding energies may be less accurate compared to more computationally expensive, purely force field-based methods, but this semi-empirical approach is considered as well-suited for the relative rankings.

The AutoDock semi-empirical force field includes intramolecular terms, a “full” desolvation model, and also considers directionality in hydrogen bonds. The conformational entropy is calculated from the sum of the torsional degrees of freedom. Water molecules are not modelled explicitly though, but pair-wise atomic terms are used to estimate the water contribution (dispersion/repulsion, hydrogen bonding, electrostatics, and desolvation), where weights are added for calibration (based on experimental data). The evaluation step in a nutshell: firstly, calculate the energy of ligand and protein in the unbound state. Secondly, calculate the energy of the protein-ligand complex. Then take the difference between 1 and 2.

Where P refers to the protein, L refers to the ligand, V are the pair-wise evaluations mentioned above, and ?S~conf~ denotes the loss of conformational entropy upon binding (R Huey et al., 2006).

Binding energy is a measure of the affinity of ligand-protein complex or is the difference between the energy of complex and the sum of energies of each molecule separately. Ligand efficiency is binding energy per atom of ligand to protein. Intermolecular energy is the energy between non-bounded atoms that is the energy between atoms separated by 3-4 bonds or between atoms in different molecules. Desolvation energy is the electrostatic and/or van der Waals energy loss of the interaction between ligand or protein and solvent upon binding. Electrostatic energy is the non-bounded energy, which is different from electrostatic desolvation energy. It is the change on the electrostatic non bounded energy of ligand or protein upon binding. Total energy is the sum of changes of all energetic terms included in scoring function of ligand or protein upon binding, plus the changes upon binding of the entropic terms. Torsion energy is related to dihedral term of internal energy.
Inhibition constant is an indication of how potent an inhibitor is, it is concentration required to produce half maximum inhibition. The electrostatic potential around each of the docked molecules is calculated by solving the linearized Poisson-Boltzman equation, using the finite-differences method as implemented in the program Delphi (Klapper et al. 1986; Honig and Nicholls 1995). The calculations are performed on a fine grid, 0.5 å, producing accurate estimates of the potential. The calculation of the potential is separate from our docking procedure (implemented in a computer program named MolFit), which reads the potential files for the two molecules, together with the necessary data regarding the grid interval and the origin of the potential grid. Thus, in principle, potentials calculated with other programs or with different force field parameters can be read in and used for describing the electrostatic character of the docked molecules.

Docking analysis report-Interpretation report all the three proteins along with Tadalafil
• PDB ID: 1EQU
Protein name: PROTEIN (ESTRADIOL 17 BETA-DEHYDROGENASE 1), Organisms(s): Homo sapiens, Sequence Length= 327, Uniprot ID -P14061, Gene Names: HSD17B1, E17KSR, EDH17B1, EDH17B2, EDHB17, SDR28C1

• PDB ID: 1FDT
Protein name: 17-BETA-HYDROXYSTEROID-DEHYDROGENASE, Organisms(s): Homo sapiens, Sequence Length= 327, Uniprot ID -P14061, Gene Names: HSD17B1, E17KSR, EDH17B1, EDH17B2, EDHB17, SDR28C1

• PDB ID: 1XOZ
Protein name: CGMP-SPECIFIC 3',5'-CYCLIC PHOSPHODIESTERASE, Organisms(s): Homo sapiens, Sequence Length= 364, Uniprot ID -O76074, Gene Names: PDE5A, PDE5

Grid data info:
The details of Grid data information are given below and in Table 10.
Grid Point Spacing =1.000Angstroms,
Even Number of User-specified Grid Points = 40x-points
40y-points
40 z-points
Table 10: Grid data info:
Coordinates of Central Grid Point of Maps Minimum coordinates in grid Maximum coordinates in grid
(17.595, 20.696, 42.058)
(-2.405, 0.696, 22.058) (37.595, 40.696, 62.058)
(42.599, -0.813, 30.861) (22.599, -20.813, 10.861) (62.599, 19.187, 50.861)
(37.245, 39.890, 20.428) (17.245, 19.890, 0.428) (57.245, 59.890, 40.428)

Ligands used:
1) Ligand name: Tadalafil, Molecular Formula: C22H19N3O4, Molecular Weight: 389.4 PubChem CID: 110635. The Ligand information and the Lipinski’s Rule information are presented in Tables 11 and 12 respectively.

Table 11: Ligand information:
Proteins Name
Ligand Name Binding Energy (kcal/mol) No. of H Bonds Interacting residue Final Intermolecular Energy (kcal/mol) vdW + Hbond + desolv Energy (kcal/mol) Electrostatic
Energy (kcal/mol) Torsional Free Energy (kcal/mol)
1EQU

TADALAFIL -7.11 02
H1:3.19Å
H2:2.02Å ARG:37(H1),
SER:12(H2),
THR:41,
SER:11,
VAL:196,
LEU:93,
ALA:191. -7.43 -7.33 -0.11 +0.30
1FDT
TADALAFIL -7.54 01
H1:2.74Å GLU:202(H1),
LEU:197,
GLY:198,
ALA:101,
VAL:196,
MET:193.
-7.83 -7.82 -0.01 +0.30
1XOZ
TADALAFIL -6.65 01
H1:2.50Å MET:816(H1),
LEU:804,
PHE:820 -6.95 -6.95 -0.00 +0.30

Table 12: Lipinski’s Rule information:
Sr. no. Name of Co-former Mol
Weight
(g/mol)
XLogP3 Hydrogen Bond Donor Hydrogen Bond Acceptor Rotatable Bond
1 Tadalafil 389.4 2.3 1 4 1

Table 13: Lipinski’s rule of five and Information of Ligands:
Sr. no. Ligand Name Mol Formula 2D Structure
1 Tadalafil C22H19N3O4

The Lipinski’s rule of five and Information of Ligands are presented in Table 13 as above.
The molecular docking of Protein (Estradiol 17 Beta-Dehydrogenase 1 (1EQU) binding domain complexed with Tadalafil is illustrated in Figure 24.

Interpretation:
Human17beta-hydroxysteroid dehydrogenase type 1 (17ß-HSD1) is a steroid-converting enzyme that has long been known to play critical roles in estradiol synthesis and more recently in dihydrotestosterone (DHT) inactivation, showing a dual function that promotes breast cancer cell proliferation. This enzyme belongs to the family of oxidoreductases, specifically those acting on the CH-OH group of donors with NAD+ or NADP+ as acceptor. The systematic name of this enzyme class is estradiol-17beta: NAD(P)+ 17-oxidoreductase. Other names in common use include 20alpha-hydroxysteroid dehydrogenase, 17beta,20alpha-hydroxysteroid dehydrogenase, 17beta-estradiol dehydrogenase, estradiol dehydrogenase, estrogen 17-oxidoreductase, and 17beta-HSD. This enzyme participates in androgen and estrogen metabolism.

17ß-Hydroxysteroid dehydrogenases (17ß-HSD, HSD17B), also 17-ketosteroid reductases (17-KSR), are a group of alcohol oxidoreductases which catalyze the reduction of 17-ketosteroids and the dehydrogenation of 17ß-hydroxysteroids in steroidogenesis and steroid metabolism. This includes interconversion of DHEA and androstenediol, androstenedione and testosterone, and estrone and estradiol.

Selection of Ligands:
A few of the ligands were got from the PubChem drug databases and docked against Protein (ESTRADIOL 17 BETA-DEHYDROGENASE 1 (1EQU), 17-BETA-HYDROXYSTEROID-DEHYDROGENASE (1FDT) and cGMP-specific 3',5'-cyclic phosphodiesterase (1XOZ) with high AutoDock V4.2 software. The ligand names is Tadalafil (-7.11kcal/mol) (-7.54 kcal/mol) and (-6.65kcal/mol). For the selection of ligands in the current examination, the existing ligands for the mentioned proteins were chosen from a series of literature review. The existing quantities of ligands are 01 which are chosen to promote perceptions.

Ligand Preparation-
The 3-D structure of inhibitors with their respective PubChem CID were redeemed and saved in SDF format. Furthermore, ligand preparations were continued by taking the 3-D structure of all the ligands and were introduced in Pymol software for conversion of 3-D structure from SDF to PDB format. Using Pymol software metals were also removed from the ligands structure for an appropriate docking study. The prepared ligands were saved in PDB format for further docking studies.

Protein preparation-
The crystal structure of Target proteins was retrieved from Protein Data Bank (PDB) with PDB ID 1EQU, 1FDT and 1XOZ and was carried further for more studies of docking process.

Discussion:
The best conformation with the lowest docked energy was chosen from the docking search. On performing the docking of Protein (ESTRADIOL 17 BETA-DEHYDROGENASE 1 (1EQU), 17-BETA-HYDROXYSTEROID-DEHYDROGENASE (1FDT) and cGMP-specific 3',5'-cyclic phosphodiesterase (1XOZ) with Tadalafil(-7.11kcal/mol), (-7.54 kcal/mol) and (-6.65kcal/mol) it was observed that the binding energy shown by the protein and ligand is good at Tadalafil(-7.54kcal/mol). Number of torsions are chosen from 0-6, and if any ligand shows more than 6 it is adjusted to 6. Hydrogen bond interactions are also calculated and mentioned, presence of H-bonds depicts stable interaction between ligand and protein. Discovery studio 2020 Client and Chimera software are used to depict Hydrogen bonds, 2-D images and protein-ligand interactions images for a good visualization of the docking. Molecular docking of 17-BETA-HYDROXYSTEROID-DEHYDROGENASE (1FDT) Binding Domain Complexed with TADALAFIL shows 3D model of the interactions and the 2D interaction patterns and H-bond interaction as illustrated in Figure 25. Figure 26 illustrates Molecular docking of cGMP-specific 3',5'-cyclic phosphodiesterase (1XOZ) Binding Domain Complexed with TADALAFIL showing 3D model of the interactions and the 2D interaction patterns and H-bond interaction.

Animal studies
In an embodiment of the invention, study of the effect of the new pharmaceutical formulation on Paroxetine induced Erectile dysfunction in rats has been presented.

After one weeks of acclimatization, the animals were randomly divided into eight groups with 6 animals in each group. A positive control group was included and treated with the formulation of the present invention, with active compound from Damiana to compare the ED stimulating activity. The other groups were treated with Sildernafil and Tadalafil from a class of medications called Phosphodiesterase (PDE) inhibitors, used to treat ED. Phosphodiesterase-5 (PDE5) inhibitors are known as erectile dysfunction drugs [EDDs]. Sildenafil, vardenafil, tadalafil, and avanafil are classified as PDE5 inhibitors and are indicated for the treatment of men with ED.

Objectives of the study
1. To induce erectile dysfunction by oral administration of paroxetine.
2. To study the effect of SAVA 22 on sexual behavior in paroxetine induced erectile dysfunction in rats.
3. To study the effect of SAVA 22 on penile tissue nitric oxide level in paroxetine induced erectile dysfunction in rats.
4. To study the effect of SAVA 22 on blood glucose, Phospho diesterase 5, testosterone,17ß-hydroxysteroid dehydrogenase and 17ß- ketosteroid reductase levels in paroxetine induced erectile dysfunction in rats.
5. To study the effect of SAVA 22 on heart rate, systolic and diastolic blood pressure in paroxetine induced erectile dysfunction in rats.

To ascertain the effect of SAVA 22 on sexual behavior, estrous female rats were paired with treated male rats. Female rats were induced to estrous by administration of estradiol benzoate and progesterone, prior to the commencement of sexual behavioral studies. The male rat sexual behavioral study was conducted after the training of the male Wistar rats with the sexually receptive females for three consecutive days. Behavioral observations were conducted 4 h after progesterone administration.

The sexual behavior parameters examined are as follows.
1. Mounting number (the number of mounts without intromission from the time of introduction of the female to the male)
2. Mount latency (time from introduction of the female until the first mount with pelvic thrusting)
3. Intromission number (the number of intromissions from the time of introduction of the female until the end of the experiment)
4. Intromission latency (time from introduction of the female until the first mount with pelvic thrusting and vaginal penetration)

The sexual behavior parameters are further explained below.
During sexual arousal, nitric oxide (NO) is released from nerve terminals and endothelial cells in the corpus cavernosum. NO activates guanylate cyclase to convert guanosine triphosphate (GTP) into cyclic guanosine monophosphate (cGMP), triggering a cGMP-dependent cascade of events. NO increases cGMP, which results in relaxation of the smooth muscle, creating an increased blood flow. Sildenafil therapy restored nitric oxide synthase activity and decreased reactive oxygen species signaling, resulting in improved erectile function.

The blood glucose level was measured using the commercially available glucometer strips.
The heart rate and systolic and diastolic blood pressure of the male animals were measured. The levels of serum testosterone, 17ß–hydroxysteroid dehydrogenase and 17–ketosteroidreductase were determined in serum samples. The specific activity of PDE-5 was calculated and expressed as nmol/min/mg protein and Nitric oxide content in penile tissue homogenate was also estimated.

Summary of the animal studies-
The animals were divided into 8 groups of 6 animals each as Vehicle control, Paroxetine ED control, Standard Sildenafil citrate, new pharmaceutical formulation (low, medium and high dose groups), Tadalafil and SAVA 22 oral groups. Paroxetine was administered at a suitable the dose to all the groups except for vehicle control group, for 21 days. The new pharmaceutical formulation was administered at three doses of low, medium and high subcutaneously to the groups 4, 5 and 6 for 21 days. Sildenafil citrate of standard oral dosage was administered to the standard group for 21 days. Tadalafil was administered in the 7th group orally and SAVA 22 was administered orally in the 8th group.

Summary of Results -
Effect of SAVA 22 on Mounting Latency (s) in Paroxetine induced Erectile Dysfunction
In this study, administration with Paroxetine significantly decreased the mounting latencies of the male rats when compared with vehicle control rats. However, treatment of rats with SAVA 22 (high dosage) and Sildenafil the alterations caused by paroxetine in male rats through a significant decrease in mounting latencies as compared with Paroxetine treated rats. There were no significant alterations in the mounting latencies upon treatment of rats with SAVA 22 of lower dosages. Also, the mounting latencies was found to be significantly decreased in rats treated with tadalafil and oral SAVA 22. (Figure 9 and Table 30).
Effect of SAVA 22 on Mounting Frequency in Paroxetine induced Erectile Dysfunction
Treatment of rats with Paroxetine significantly altered the mounting frequencies of the male rats when compared with vehicle control rats. However, treatment of rats with SAVA 22 (high dosage) and Sildenafil significantly increased the mounting frequencies as compared to paroxetine treated rats. Whereas in rats treated with SAVA 22 (lower two dosages), there was no significant change in mounting frequency. It was found to be significantly increased in rats treated with tadalafil and oral SAVA 22 as compared to Paroxetine treated rats. (Figure 10 and Table 30).

Effect of SAVA 22 on Intromission Latency (s) in Paroxetine induced Erectile Dysfunction
Administration with Paroxetine significantly increased the intromission latencies of the male rats when compared with vehicle control rats. However, treatment of rats with SAVA 22 (high dosage) and Sildenafil reversed the alterations caused by Paroxetine in male rats through a significant decrease in intromission latencies as compared with Paroxetine treated rats. There were no significant alterations upon treatment of rats with SAVA 22 (lesser two dosages) when compared with Paroxetine treated rats. The intromission latencies were found to be significantly decreased in rats treated with tadalafil and oral SAVA 22 when compared to Paroxetine treated rats (Figure 11 and Table 30).

Effect of SAVA 22 on Intromission Number in Paroxetine induced Erectile Dysfunction
Administration of rats with paroxetine significantly decreased the intromission numbers in the male rats showing decrease in libido and erection. However, treatment of rats with SAVA 22 (high dosage) and Sildenafil significantly increased the mounting frequencies as compared to paroxetine treated rats. Whereas in rats treated with SAVA 22 of lower two dosages, there was no significant change in mounting frequency seen. The intromission numbers were found to be significantly increased in rats treated with tadalafil and SAVA 22 (oral) when compared to Paroxetine treated rats (Figure 12 and Table 30).

Effect of SAVA 22 on penile tissue Nitric Oxide (mmol/mg of tissue) in Paroxetine induced Erectile Dysfunction
The result of the effect of paroxetine, SAVA 22 on penile tissue nitric oxide level in rats is presented in Figure 13. Administration of Paroxetine significantly reduced the NO level when compared with the vehicle control group. However, treatment of rats with SAVA 22 (high dosage) significantly elevated the NO level. In rats treated with SAVA 22 of lower two dosages no significant change in NO level was seen. The penile tissue nitric oxide level was found to be significantly increased in rats treated with tadalafil and SAVA 22(oral) when compared to Paroxetine treated rats (Figure 13 and Table 30).

Effect of SAVA 22 on penile tissue Phosphodiesterase 5 activity (nmol/min/mg of protein) in Paroxetine induced Erectile Dysfunction
The result of the effect of paroxetine, SAVA 22 and Sildenafil on penile tissue nitric oxide level in rats is presented in Figure 14. Administration of Paroxetine significantly reduced the phosphodiesterase 5 level when compared with the vehicle control group. However, treatment of rats with SAVA 22 (high dosage) and Sildenafil significantly elevated the phosphodiesterase 5 level as compared to Paroxetine treated rats. Treatment with SAVA 22 of lower two dosage caused no significant change in phosphodiesterase 5 level but was found to be significantly decreased in rats treated with tadalafil and SAVA 22 (oral) when compared to Paroxetine treated rats (Figure 14 and Table 31).

Effect of SAVA 22 on Blood Glucose Level (mg/dl) in Paroxetine induced Erectile Dysfunction
The result of the effect of Paroxetine, Tadalafil, SAVA 22 and Sildenafil on blood glucose level in rats is presented in Figure 15. No significant difference was observed on blood glucose level on Paroxetine administration in rats when compared with vehicle treated rats. There was no significant change in blood glucose in any of the treatment groups (Figure 15 and Table 30).

Effect of SAVA 22 on serum Testosterone (ng/dL) in Paroxetine induced Erectile Dysfunction
The result of the effect of paroxetine, SAVA 22 and Sildenafil on testes tissue testosterone level in rats is presented in Figure 16. Administration of Paroxetine significantly reduced the testosterone level when compared with the vehicle control group. Treatment of rats with Sildenafil significantly reduced the testosterone level as compared to Paroxetine treated rats. However, on treatment of rats with SAVA 22 (all dosages), there was no significant change in testosterone level was observed as compared to Paroxetine treated rats. The serum testosterone level was found to be significantly decreased in rats treated with tadalafil when compared to Paroxetine treated rats (Figure 16 and Table 31).

Effect of SAVA 22 on serum17ß-Hydroxysteroid dehydrogenase (Unit/min/mg protein) in Paroxetine induced Erectile Dysfunction
The result of the effect of Paroxetine, SAVA 22 and Sildenafil on serum 17ß-Hydroxysteroid dehydrogenase level in rats is presented in Figure 17. Administration of Paroxetine significantly reduced the 17ß-Hydroxysteroid dehydrogenase level when compared with the vehicle control group. Treatment of rats with Sildenafil significantly reduced these levels as compared to Paroxetine treated rats. However, treatment of rats with SAVA 22 (all dosages) no significant change in 17ß-Hydroxysteroid dehydrogenase level was observed. The said level was found to be significantly decreased in rats treated with tadalafil when compared to Paroxetine treated rats (Figure 17 and Table 31).

Effect of SAVA 22 on testes 17ß-Ketosteroid reductase (Unit/min/mg protein) in Paroxetine induced Erectile Dysfunction
The result of the effect of Paroxetine, SAVA 22 and Sildenafil on testes 17ß-Ketosteroid reductase level in rats is presented in Figure 18. Administration of Paroxetine significantly reduced the 17ß-Ketosteroid reductase level when compared with the vehicle control group. Treatment of rats with Sildenafil significantly reduced the said level as compared to Paroxetine treated rats. However, treatment of rats with SAVA 22 (all dosages) caused no significant change in the said level. The 17ß-Ketosteroid reductase level was found to be significantly decreased in rats treated with tadalafil when compared to Paroxetine treated rats (Figure 18 and Table 31).

Effect of SAVA 22 on Heart rate (Beats per minute (bpm)) in Paroxetine induced Erectile Dysfunction
The result of the effect of paroxetine, SAVA 22 and Sildenafil on heart rate in rats is presented in Figure19. Treatment of rats with Sildenafil significantly increased the heart rate when compared with the Paroxetine treated rats. Whereas treatment of rats with SAVA 22 (all dosages) did not cause significant change in heart rate when compared to vehicle control group. Further, the heart rate was significantly decreased in SAVA 22 (all dosages) treated groups when compared to Sildenafil treated rats. The heart rate was found to be significantly increased in rats treated with tadalafil and SAVA 22 oral when compared to Sildenafil treated rats (Figure 19 and Table 31).

Effect of SAVA 22 on Systolic BP (mmHg) in Paroxetine induced Erectile Dysfunction
The result of the effect of paroxetine, SAVA 22 and Sildenafil on systolic BP in rats is presented in Figure 20. The treatment of rats with Sildenafil significantly increased the systolic BP when compared with the Paroxetine treated rats. Whereas treatment of rats with SAVA 22 (all dosages) did not cause significant change in systolic BP when compared to vehicle control group. Further treatment of rats with SAVA 22 (all dosages) significantly decreased the systolic BP when compared to Sildenafil treated rats. The systolic BP was found to be significantly decreased in rats treated with tadalafil and SAVA 22 (oral) when compared to Sildenafil treated rats (Figure 20 and Table 31).

Effect of SAVA 22 on Diastolic BP (mmHg) in Paroxetine induced Erectile Dysfunction
The result of the effect of paroxetine, SAVA 22 and Sildenafil on diastolic BP in rats is presented in Figure 21. The treatment of rats with Sildenafil significantly increased the diastolic BP when compared with the Paroxetine treated rats. Whereas treatment of rats with SAVA 22 (all dosages) did not cause significant change in heart rate when compared to vehicle control group. Further, treatment of rats with SAVA 22 (all dosages) caused a significant decrease in diastolic BP when compared to Sildenafil treated rats. The diastolic BP was found to be significantly decreased in rats treated with tadalafil and SAVA 22 (oral) when compared to Sildenafil treated rats (Figure 21 and Table 31).

Conclusion of animal studies
In conclusion it can be stated that the new pharmaceutical formulation (at the particular high dosage) was found to cause significant improvement in libido. The observed effect is mediated through increase in nitric oxide by inhibiting the phosphodiesterase. The observed effect is devoid of cardiac adverse effects. However, the said formulation at other tested low and medium doses did not cause any improvement in libido.
Preparation of oral pharmaceutical formulation

Process of preparation of pharmaceutical composition
In an embodiment, the present invention relates to the process of preparation of a pharmaceutical composition with active compound Teuhetenone and its derivatives for improving male sexual dysfunction.

In a preferred embodiment the formulation comprises of Teuhetenone A, (4aS,8S)-8-hydroxy-4a,8-dimethyl-4,5,6,7-tetrahydro-3H-naphthalen-2-one, a natural product found in a wild shrub Damiana, organisms like Teucrium heterophyllum, Alpinia oxyphylla, and other organisms with data available.

In an embodiment, the oral dosage form of formulation also comprises of premix of excipients such as F melt type C, further also comprises ethanol as solvent and magnesium stearate as an additive acting as the lubricant.

In another embodiment, the present invention relates to a process of preparing a pharmaceutical composition for oral delivery of Teuhetenone A which is prepared using following steps:

Preparation of Tablet-
It must be ensured that the temperature of manufacturing area be NMT 25°C and humidity be NMT 50.0% RH.
Step 1: Drug solution preparation:
a. Dissolving specific quantity of Teuhetenone in a suitable solvent to yield a premix solution.

Step 2: Wet granulation:
a. Taking F Melt (Type C) as per dispensing qty. & sifting through 40# sieve.
b. Co-sifting F Melt (Type C) and color through 80# Sieve and mixing in polybag for a specific time.
c. Transferring sifted blend of F Melt (Type C) and color in SS bowl and granulating using drug solution of Step 1.

STEP 3: Drying:
a. Drying the wet granules of Step 2 c in oven at a particular temperature for a specific time.

STEP 4: Sizing and lubrication:
a. Passing the dried granules through 40# sieve and charging into blender.
b. Adding sifted lubricating agent to blender and mixing for a specific time.
c. Transferring the material to polybag and using for compression.

STEP 5: Compression
Compressing the blend using suitable tools with specified parameters.
The lubricating agent used is selected from a group comprising of magnesium stearate, sodium stearyl fumarate, stearic acid, calcium stearate, hydrous magnesium silicate or talc.

HPLC Analysis
The HPLC analysis of the pharmaceutical composition comprising of Teuhetenone and its derivatives is as provided in the following paragraphs.

Standard solution Preparation:
For the preparation of standard solution ,2.5 mg of standard transfer was weighed accurately into a 25 ml volumetric flask and to that about 20 mL of diluent were added, sonicated to dissolve, then cooled and the volume was made up with diluents and mixed well.

Sample Preparation:
For the preparation of sample tablet blend was weighed and transferred into a 25 ml volumetric flask. To this diluent is added, sonicated to dissolve, cooled and volume made up with diluents and mixed well.

Chromatograms:
The formulation chromatogram showed a prominent peak of Teuhetenone which was analyzed and confirmed. The standard chromatogram is illustrated in Figure 27 and the tablet chromatogram is illustrated in Figure 28.

Analytical method validation report for assay of Teuhetenone: By HPLC
Experimental studies were carried out to develop a simple, precise, specific, accurate, cost-effective, and validated RP HPLC method according ICH guidelines for the estimation and routine analysis of assay of Teuhetenone in pure and pharmaceutical formulations. The properties of the active compound Teuhetenone are as given below in Table 14.

Table 14: Compound properties:
Name Molecular Structure Chemical Name Molecular Formula Molecular
weight
Teuhetenone Teuhetenone C12H18O2
194.2

Scope:
In-house reverse phase HPLC method for the assay of Teuhetenone was considered for analytical method validation.

Objective:
The objective of the work was to validate the HPLC method for the assay of Teuhetenone as per the ICH guidelines Q2(R1).

Equipment / instrument / material needed:
The equipment and instruments used in this validation were calibrated, maintained, and used within their calibrated period. The instruments used for this validation work are tabulated as below-
Table 15: Instrument information:
Sr. No. Name of Instrument/Equipment Make Model Instrument/ Equipment ID
1. HPLC system equipped with quaternary gradient pump Waters Allience-e-2695 SM/RND/AD/INST/005
2. UV/Photo Diode Array detector water LC-2010CHT SM/RND/AD/INST/005

3. Analytical Balance Sansui Vibra HTR-220E SM/RND/AD/INST/009
Mettler XP26 SM/RND/AD/INST/021
4. Sonicator PCI Analytics 15L300 SM/RND/AD/INST/028

Column used:
The details of the columns used are illustrated in the table below.
Table 16: Column parameters:
Sr. No. Name of Column Make Part No. & Sr. No. Column ID
1. Inertsil ODS C-18,
(4.6 x 250 mm), 5µm. GL Sciences 20H0143918 ADL-381

Working Standard used for the validation study is as given below:
Table 17: Working standard:
Sr. No. Name of Standard B. No./ Lot No. Potency Validity
1. Teuhetenone TD-01 96.00%
Table 18: Materials used during the validation study:
Sr. no. Name Grade Make Batch /Lot No.
1 Acetonitrile Analytical Reagent Rankem T045C21
2 Methanol Analytical Reagent Rankem R215B21
3 Purified water Mili Q Mili Q NA
4 Sodium Hydroxide Analytical Reagent Rankem 849230306JT
5 Hydrochloric acid Analytical Reagent Rankem K009D21
6 Hydrogen Peroxide Rankem Rankem G102E19
7 0.45µm Nylon syringe filter NA MDI SN5189D

Analytical method:
Chromatographic conditions:
Mobile Phase A:
1 ml of Orthophosphoric acid was mixed in 1000ml of water, filtered through 0.45 µm filter and sonicated to degas and then used.
Mobile Phase B:
Acetonitrile was used in mobile phase B.

Diluent:
Methanol was used as a diluent.

Chromatographic parameters:
Column : Inertsil ODS C18 250 x 4.6 mm and 5µm.
Column Temperature : 35°C
Wavelength : 278 nm
Flow rate : 1 mL/min
Injection Volume : 20 ?L
Run time : 20 minutes
Retention Time : About 13 minutes.
Elution : Gradient. ( as shown in Table 19)

Table 19: Column parameters: Gradient Program:
Time (minutes) % Mobile phase: A % Mobile phase: B
0.1 100 0.0
8.0 90 10
10 70 30
12 40 60
15 40 60
17 100 0.0
20 100 0.0

Procedure:
Teuhetenone Standard stock solution preparation:
About 2.5 mg of Teuhetenone standard was weighed accurately in to a 25 mL volumetric flask. About 20 mL of diluent was added to the flask and was sonicated to dissolve the standard. The solution was cooled, and volume made up with diluent. 1 ml of the solution was diluted in to a 10 ml volumetric flask, diluent added to make up the volume and mixed well.

Analytical Method Validation Parameters
System Suitability:
To assess system suitability of the method, the repeatability, theoretical plates, tailing factor
and retention time of five replicate injections of standard Teuhetenone were used and the
%RSD values were calculated. The five replicates of standard solution were chromatographed and the peak response recorded.

Procedure:
The diluent, standard preparation and sample preparations were separately injected into the chromatograph. The chromatograms were recorded, and the responses measured for the analyte peaks.

Validation results: System Suitability
System suitability was demonstrated by making five replicate injections of standard solution as per the analytical method. The peak area sums of Teuhetenone peaks were recorded. Theoretical plates and tailing factor in each replicate injection were recorded and results are reported in Table 20.

Table 20: System Suitability
Inj. No. Area Tailing factor Theoretical plates
1 235564 1.4 132227
2 235372 1.4 131562
3 234733 1.4 133670
4 235332 1.4 134134
5 235564 1.4 135762
Average 235313 NMT 2.0 NLT 2000
Standard deviation 341.4103
% RSD 0.15

Acceptance criteria for the validation
• The %RSD of area of sum of Teuhetenone peaks for five replicate standard injections should not be more than 2.0.
• The numbers of Theoretical plates should not be less than 2000. Tailing factors should not be more than 2.0 for Teuhetenone peak.

Conclusion:
As % RSD of the peak area, Theoretical plates and Tailing factors were found within acceptance criteria as defined in system suitability criteria, it indicates an acceptable level of precision for repeatability studies.

Linearity & Range:
The Linearity of method was demonstrated by preparing solutions over the concentration levels ranging from 1% to 150% of working concentration. These solutions injected in triplicate into the system and the peak area of analyte peak recorded.
Linearity graph of concentration Vs average peak area of analyte was plotted separately.
The correlation co-efficient between concentration (ppm) & peak area slope and intercept evaluated. The results are given in Table 21 and graph of concentration Vs peak area of analyte.

Table 21: Linearity Study
Linearity Level Conc. (%) Conc. (ppm) Area
1 1 0.101 2168
2 5 0.506 10347
3 10 1.012 20218
4 20 2.024 42610
5 40 4.048 86162
6 80 8.096 180053
7 100 10.120 223364
8 120 12.144 257828
9 150 15.180 306261
Slope 20929.51
Intercept 1807.8111
Correlation Coefficient 0.998
bias

The findings with respect to the linearity and range are illustrated in Figure 29.
Conclusion:
Based on the above observations the method is found to be Linear at given range.

Forced degradation:
The forced degradation study was performed on drug product and placebo as per the validation protocol. The sample and placebo as per the validation protocol were degraded in the following manner (Refer to Table 22).

Table 22: Forced degradation study conditions
Sr. No. Condition Degrading agents /
condition Exposure period
1 Alkali degradation 1 N NaOH for 1 Hrs.
2 Acid degradation 1 N HCl for 1 Hrs.
3 Oxidative degradation 3% H2O2 for 1 Hrs.

The interference due to diluent and placebo, peak purity results and absolute % degradation in Teuhetenone in degraded samples are reported in Table 23.
Table 23: Peak purity data & absolute % degradation
Sr.
No. Stress type Diluent and degradation diluent Interference Placebo and degradation placebo Interference Teuhetenone Peak purity
Result Absolute % Degradation
of Teuhetenone
1 Alkali degradation No No Passes 8.6%
2 Acid degradation No No Passes 10.2%
3 Oxidative degradation No No Passes 9.8%

Acceptance criteria used for these studies are as enumerated below.
• Diluent and degradation blank solutions should not show any interference at the retention time corresponding to the peaks of Teuhetenone.
• Placebo and degradation placebo solutions should not show any interference at the retention time corresponding to the peaks of Teuhetenone.
• Peak purity of Teuhetenone should pass in control sample and all the degradation samples.
• All degradant peaks should be well separated from the Teuhetenone peaks.

Conclusion:
Based on the above observations the method was found to be specific to the degradants and stability indicating.

Precision:
The Precision of an analytical procedure expresses the closeness of agreement between a series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions.
• System precision:
Standard solution was prepared as described in analytical method and injected in six replicates. Result obtained is given in Table 24.

Table 24: System Precision
Inj. No Area
1 227214
2 228137
3 227643
4 227778
5 228694
6 230051
Average 228253
Standard deviation 1011.7299
% RSD 0.44

Acceptance criteria:
• % RSD of peak areas of analyte for six replicate injections of standard solution should not be more than 2.0.

Conclusion:
% RSD of the area found within acceptance criteria indicates an acceptable level of precision for repeatability studies.

The developed RP-HPLC method for the determination of Teuhetenone is simple, precise, accurate, reproducible, and highly sensitive. The developed method was validated based
on ICH guidelines Q2(R1). Hence, this method can be used for the routine determination of Teuhetenone in pure and pharmaceutical formulations.

EXAMPLES
For a better understanding of this invention, the following examples are presented. These examples are for the purpose of illustration only and are not to be construed as limiting the scope of the invention in any manner.

Damiana Extraction Trial:
The source of raw material for extraction of Teuhetenone A was Damiana leaves. The extraction of active compounds from Damiana were performed by trials in various batches with water, 50% ethanol and Ethyl acetate wash followed by water extraction. The results of the extraction trials are provided below in Table 25. It was observed that extraction process with water yields the best quantity of active compounds. The percentage yield of different batches is as given in Table 25.

Table 25: Damiana extraction trial
S. N. Batch no. Solvent
% TD-1
1 RDP/TD/011 Water 4.59
2 RDP/TD/012 50% Ethanol 3.46
3 RDP/TD/013 Water 4.49
4 RDP/TD/014 50% Ethanol 3.81
5 RDP/TD/015 Ethyl acetate wash followed by water extraction 3.06

Teuhetenone A Isolation Trial:
Isolation was done with traditional manual column chromatography method and using flash chromatography method. Both methods give a good yield. However, the flash chromatography method has clear advantages which are compared in Table 26 as below.

It can be seen that in a single column a higher yield as well as greater degree of purity can be achieved in flash chromatography as compared to manual chromatography.

Table 26: Comparison: Traditional method vs Advance method
Sr. No Parameter Column chromatography
(Traditional method) Flash chromatography
1 Weight of extract 20 gm 20 gm
2 Weight of silica gel 500 gm Pre-packed column
3 Solvent consumptions 7-8 Lit. 10 lit
4 Time required for final purification 30-40 days 5-6 days
5 Quantity received after purification 50-60 mg 180-200 mg
6 Capacity 50 gm extract 200-250 gm
7 purity For more than 90% purity need to do at least 3 columns In single column 85% purity can be achieved

The isolation and identification of the active compound Teuhetenone A using (manual column chromatography method) was carried out in the following steps.
Column 1:
15 gm of water extract adsorbed on 30 gm of silica (60-120#)
Glass column packed with 120 gm of silica (100-200#)
Column eluted with ethyl acetate then 10% methanol in ethyl acetate
and finally, 15% methanol in ethyl acetate

15% methanol fraction contains the targeted compound
Qty- 2.5 gm

Column 2: 2.5 gm of fraction from column 1 adsorbed on 5 gm of silica (60-120#)
Glass column packed with 60 gm of silica (100-200#)
Column eluted with Chloroform then 10% methanol in chloroform, 15% methanol,
20% methanol in chloroform

20% methanol fraction contains the targeted compound (Qty- 850 mg)

Column 3: 850 mg of fraction from column 2 adsorbed on 2 gm of silica (60-120#)
Glass column packed with 20 gm of silica (100-200#)
Column eluted with Chloroform then 12% methanol in chloroform and 16% methanol
in chloroform

Total 9 fractions were collected.

Pure compound Qty- 170 mg
Purity: 96% by HPLC
Recovery: 1.13%

HPLC Method:
Mobile Phase:
A) 0.1% Formic acid
B) Acetonitrile

Column: Inertsil ODS 250×4.6×5 µ
Column Temp: 30°C
Sample temp: 25°C
Wavelength: 280nm
Flow rate: 0.8 ml/min.
Injection volume: 20 µL
Run time: 50 min.
Elution: Gradient (Table 27)

Gradient Program:

Table 27: Gradient program
Time %A %B
0.010 100 00
10.0 90 10
35.0 60 40
40.0 40 60
44.0 40 60
45.0 100 00
50.0 100 00

The Damiana Extract Chromatogram and Isolated compound chromatogram are as illustrated in Figure 1 and 2 respectively.

The Mass Spectroscopy performed with direct tunning to mass method is illustrated in Figure 3 and NMR Spectroscopy of 1H NMR AND 13C NMR are illustrated in Figures 4 (a) and 4 (b) respectively.

Separation of Teuhetenone A using flash chromatography
Mobile Phase:
A) Chloroform
B) Methanol
Column: Silica 40 g
Column Temp: 30°C
Sample temperature: 25°C
Wavelength: 280nm
Flow rate: 40 ml/min.
Run time: 30 min.
Elution: Gradient (as shown in Table 28)

Sample preparation and loading:
Crude water extract of a specific quantity was dissolved in DM water and then liquid extract injected in silica column. About 8-10 injections per column can be done.

Gradient Program:

Table 28: Gradient Program:

Time Duration in minutes %A %B
0.010 0 100 0
1.2 1.2 100 0
8.4 7.2 87.4 12.6
10.7 2.3 87.4 12.6
12.2 1.5 84.8 15.2
14.9 2.7 84.8 15.2
27.4 12.5 65 35.0
27.7 0.3 62.7 37.3
28.0 0.3 60 40
30.0 2 100 0

The result is as illustrated in Figure 5. Using 3 g extract and 6 hours’ time, achieved 200 mg separation of 90% pure compound was achieved, showing a recovery of 7%.

Animal study to check the effect of SAVA 22 on erectile dysfunction.
Animal studies were conducted to study the effect of SAVA 22 on paroxetine induced erectile dysfunction in rats.
The animals were divided into 8 groups and administered or treated with the medical formulations as mentioned in Table 29 below.

Table 29: Grouping of animals

Sr. No. Group No. Name No. of animals
1 I Vehicle (water) control via s.c. injection 6
2 II Paroxetine (PAR) (10 mg/kg) via oral gavage 6
3 III Paroxetine (10 mg/kg) and Sildenafil citrate (SC) (5 mg/kg) via oral gavage 6
4 IV Paroxetine and SAVA 22 ED 0.1 mg/rat of 250 g in 100 µl of plain water s.c 6
5 V Paroxetine and SAVA 22 ED 0.2 mg/rat of 250 g in 100 µl of plain water s.c 6
6 VI Paroxetine and ED0.4 mg/rat of 250 g in 100 µl of plain water s.c 6
7 VII Paroxetine and Tadalafil 5 mg/kg, Oral 6
8 VIII Paroxetine and SAVA 22 Oral 6
Total Male animals 48
To study the ED function number of female animals required 36

The drugs were administered from the given sample at the volume of 0.1 ml, s.c. once daily for 21 days. On the last day, rats were subjected to sexual behavioral study. To ascertain the effect of SAVA 22 on sexual behavior, estrous female rats were paired with treated male rats. Female rats were induced to estrous by administration of estradiol benzoate at a dose of 2 µg/kg body weight and progesterone, 500 µg/kg body weight at 48 and 4 h, respectively prior to the commencement of sexual behavioral studies. The male rat sexual behavioral study was conducted after the training of the male Wistar rats with the sexually receptive females for three consecutive days. Behavioral observations were conducted 4 h after progesterone administration.

The summary of observation is presented as below:
The animals were divided into 8 groups of 6 animals each as Vehicle control, Paroxetine ED control, Standard sildenafil citrate, SAVA 22 (low, medium and high dose groups), Tadalafil and SAVA 22 oral groups. Paroxetine was administered at the dose of 10 mg/Kg to all the groups except for vehicle control group, for 21 days. The SAVA 22 was administered at the doses of (0.1 mg/rat), (0.2 mg/rat) and (0.4 mg/rat) subcutaneously to the groups 4, 5 and 6 for 21 days. Sildenafil citrate (5 mg/Kg) oral was administered to the standard group for 21 days. Tadalafil was administered at 5 mg/Kg orally and SAVA 22 was administered orally in the 8th group.

Summary of Results-
Effect of SAVA 22 on Mounting Latency (s) in Paroxetine induced Erectile Dysfunction
In this study, administration with Paroxetine (10 mg/kg) significantly (p<0.001) decreased the mounting latencies of the male rats when compared with vehicle control rats. The time taken to mount on the female animal was delayed upon administration of Paroxetine. However, treatment of rats with SAVA 22 (0.4 mg/rat) and Sildenafil reversed the alterations caused by paroxetine in male rats through a significant (p<0.001) decrease in mounting latencies as compared with Paroxetine treated rats. There were no significant alterations in the mounting latencies upon treatment of rats with SAVA 22 (0.1 mg/rat and 0.2 mg/rat) when compared with Paroxetine treated rats. The mounting latencies was found to be significantly (p<0.001) decreased in rats treated with tadalafil (5 mg/kg, oral) and SAVA 22 oral when compared to Paroxetine treated rats (Figure 9 and Table 30).
Table 30: Effect of SAVA 22 on Paroxetine-induced Erectile Dysfunction in rats
Parameters/
Groups Mounting Latency (sec) Mounting Frequency Intromission Latency (sec) Intromission Number NO (µmol/mg of tissue) Blood Glucose (mg/dL)
Vehicle Control 139.3±
4.998 14.33 ±
0.8433 187.00 ±
4.179 8.500 ±
0.995 66.67 ±
3.232 125.3 ±
4.794
Paroxteine ED Control (10 mg/kg) 238.3 ±
4.602c 4.833 ±
0.6009c 285.5 ±
4.217c 2.833 ±
0.3073c 34.5 ±
2.405c 113.00±
5.41
SC (5 mg/kg) oral 135.5 ±
2.045*** 11.67 ±
0.4944*** 186.00 ±
1.966*** 7.5 ±
0.6708*** 155.7 ±
5.044*** 119.8±
6.337
SAVA 22 ED
(0.1 mg/rat) oral 221.3 ±
2.996 5.333 ±
0.4216 269.5 ±
2.861 3.167 ±
0.3073 37.17 ±
1.956 135.8±
12.53
SAVA 22 ED
(0.2 mg/rat) oral 222.7 ±
6.07 6.333 ±
0.6667 270.5 ±
6.602 3.667 ±
0.333 41.67 ±
2.472 120.8±
4.636
SAVA 22 ED
(0.4 mg/rat oral 140.5 ±
2.837*** 12 ±
1.238*** 189.3 ±
2.848*** 7.667 ±
0.4216*** 136.7 ±
2.445*** 123±
3.821

SC – Sildenafil Citrate
Values are expressed as mean ± SEM, n = 6
One way ANOVA followed by Dunnet’s ‘t’ test
cp<0.001 when compared to vehicle control.
**p<0.01; ***p<0.001 when compared to Paroxetine ED control
$p<0.001 when compared to SC.

Effect of SAVA 22 on Mounting Frequency in Paroxetine induced Erectile Dysfunction
In this study, treatment of rats with Paroxetine (10 mg/kg) significantly (p<0.001) altered the mounting frequencies (number of mountings) of the male rats. Paroxetine (10 mg/kg) administration caused a significant (p<0.001) decrease in mounting frequencies when compared with vehicle control rats. However, treatment of rats with SAVA 22 (0.4 mg/rat) and Sildenafil significantly (p<0.001) increased the mounting frequencies as compared to paroxetine treated rats. Whereas in rats treated with SAVA 22 (0.1 mg/rat and 0.2 mg/rat), there was no significant change in mounting frequency seen when compared to paroxetine treated rats. The mounting frequencies was found to be significantly (p<0.001) increased in rats treated with tadalafil (5 mg/kg, oral) and SAVA 22 oral when compared to Paroxetine treated rats (Figure 10 and Table 30).

Effect of SAVA 22 on Intromission Latency (s) in Paroxetine induced Erectile Dysfunction
In this study, administration with Paroxetine (10 mg/kg) significantly (p<0.001) increased the intromission latencies of the male rats. The time taken for intromission in the female animal was delayed upon administration of Paroxetine. However, treatment of rats with SAVA 22 (0.4 mg/rat) and Sildenafil reversed the alterations caused by Paroxetine in male rats through a significant (p<0.001) decrease in intromission latencies as compared with Paroxetine treated rats. There were no significant alterations in the intromission latencies upon treatment of rats with SAVA 22 (0.1 mg/rat and 0.2 mg/rat) when compared with Paroxetine treated rats. The intromission latencies were found to be significantly (p<0.001) decreased in rats treated with tadalafil (5 mg/kg, oral) and SAVA 22 oral when compared to Paroxetine treated rats (Figure 11 and Table 30).

Effect of SAVA 22 on Intromission Number in Paroxetine induced Erectile Dysfunction
In this study, administration of rats with paroxetine (10 mg/kg) significantly (p<0.001) decreased the intromission numbers in the male rats. This confirms that administration of paroxetine decreases libido and erection. However, treatment of rats with SAVA 22 (0.4 mg/rat) and Sildenafil significantly (p<0.001) increased the mounting frequencies as compared to paroxetine treated rats. Whereas in rats treated with SAVA 22 (0.1 mg/rat and 0.2 mg/rat), there was no significant change in mounting frequency seen when compared to paroxetine treated rats. The intromission numbers were found to be significantly (p<0.001) increased in rats treated with tadalafil (5 mg/kg, oral) and SAVA 22 oral when compared to Paroxetine treated rats (Figure 12 and Table 30).

Effect of SAVA 22 on penile tissue Nitric Oxide (mmol/mg of tissue) in Paroxetine induced Erectile Dysfunction
The result of the effect of paroxetine, SAVA 22 on penile tissue nitric oxide level in rats is presented in Figure 13. Administration of Paroxetine significantly (p<0.001) reduced the NO level when compared with the vehicle control group. However, treatment of rats with SAVA 22 (0.4 mg/rat) significantly (p<0.001) elevated the NO level as compared to Paroxetine treated rats. In rats treated with SAVA 22 (0.1 mg/rat and 0.2 mg/rat) no significant change in NO level was seen as compared to Paroxetine treated rats. The penile tissue nitric oxide level was found to be significantly (p<0.001) increased in rats treated with tadalafil (5 mg/kg, oral) and SAVA 22 oral when compared to Paroxetine treated rats (Figure 13 and Table 30).

Effect of SAVA 22 on penile tissue Phosphodiesterase 5 activity (nmol/min/mg of protein) in Paroxetine induced Erectile Dysfunction
The result of the effect of paroxetine, SAVA 22 and Sildenafil on penile tissue nitric oxide level in rats is presented in Figure 14. Administration of Paroxetine significantly (p<0.001) reduced the phosphodiesterase 5level when compared with the vehicle control group. However, treatment of rats with SAVA 22 (0.4 mg) and Sildenafil significantly (p<0.001) elevated the phosphodiesterase 5 level as compared to Paroxetine treated rats. Treatment with SAVA 22 (0.1 mg/rat and 0.2 mg/rat) caused no significant change in phosphodiesterase 5 level as compared to Paroxetine treated rats. The penile tissue phosphodiesterase 5level was found to be significantly (p<0.001) decreased in rats treated with tadalafil (5 mg/kg, oral) and SAVA 22 oral when compared to Paroxetine treated rats (Figure 14 and Table 31).

Table 31: Effect of SAVA 22 on Paroxetine-induced Erectile Dysfunction in rats
Parameters/
Groups Phospho diesterase 5 activity (nmol/min/
mg of protein) Testosterone (ng/dL) 17ß- Hydroxysteroid dehydrogenase (Unit/min/mg protein) 17-ß- Ketosteroid reductase (Unit/min/
mg protein) Heart rate (beats per minute bpm) Systolic BP (mmHg) Diastolic BP (mmHg)
Vehicle Control 3.26 ±
0.1412 33.39 ±
2.89 0.155 ±
0.004 1.577 ±
0.029 376.8 ±
11.6 126.7 ±
2.128 87.83±
1.181
Paroxteine ED Control (10 mg/kg) 6.517±
0.1302c 18.24 ±
1.114 c 0.135 ±
0.005 c 1.35 ±
0.019c 399.3 ±
5.533 124.3 ±
2.817 82.45±
0.7686
SC (5 mg/kg), Oral 2.067 ±
0.2512*** 9.345 ±
0.071** 0.110 ±
0.003** 1.168 ±
0.037*** 526.6 ±
8.82*** 142.3 ±
2.817*** 107.5±
1.588***
SAVA 22 ED
(0.1 mg/rat) oral 6.5 ±
0.09661 18.77 ±
1.171 0.138 ±
0.005 1.33 ±
0.011 421.7 ±
8.64$ 126.1 ±
0.8188$ 89.11±
2.14$
SAVA 22 ED
(0.2 mg/rat) oral 6.383 ±
0.04773 18.42 ±
1.121 0.130 ±
0.0057 1.35 ±
0.019 417.6 ±
5.037$ 129.2 ±
1.396$ 97.83±
1.181$
SAVA 22 ED
(0.4 mg/rat) oral 3.167 ±
0.04944*** 18.04 ±
1.297 0.126 ±
0.0066 1.372 ±
0.016 389.1 ±
14.76$ 122.3 ±
2.104$ 90.22±
2.331$

SC – Sildenafil Citrate
Values are expressed as mean ± SEM, n = 6
One way ANOVA followed by Dunnet’s ‘t’ test
cp<0.001 when compared to vehicle control.
**p<0.01; ***p<0.001 when compared to Paroxetine ED control
$p<0.001 when compared to SC.

Effect of SAVA 22 on Blood Glucose Level (mg/dl) in Paroxetine induced Erectile Dysfunction
The result of the effect of Paroxetine, Tadalafil, SAVA 22 and Sildenafil on blood glucose level in rats is presented in Figure 15. No significant difference was observed on blood glucose level on Paroxetine administration in rats when compared with vehicle treated rats. There was no significant change in blood glucose in any of the treatment groups (Figure 15 and Table 30).

Effect of SAVA 22 on serum Testosterone (ng/dL) in Paroxetine induced Erectile Dysfunction
The result of the effect of paroxetine, SAVA 22 and Sildenafil on testes tissue testosterone level in rats is presented in Figure 16. Administration of Paroxetine significantly (p<0.001) reduced the testosterone level when compared with the vehicle control group. Treatment of rats with Sildenafil significantly (p<0.01) reduced the testosterone level as compared to Paroxetine treated rats. However, on treatment of rats with SAVA 22 (0.1 mg/rat, 0.2 mg/rat and 0.4 mg/rat), there was no significant change in testosterone level was observed as compared to Paroxetine treated rats. The serum testosterone level was found to be significantly (p<0.001) decreased in rats treated with tadalafil (5 mg/kg, oral) when compared to Paroxetine treated rats (Figure 16 and Table 31).

Effect of SAVA 22 on serum17ß-Hydroxysteroid dehydrogenase (Unit/min/mg protein) in Paroxetine induced Erectile Dysfunction
The result of the effect of Paroxetine, SAVA 22 and Sildenafil on serum 17ß-Hydroxysteroid dehydrogenase level in rats is presented in Figure 17. Administration of Paroxetine significantly (p<0.001) reduced the 17ß-Hydroxysteroid dehydrogenase level when compared with the vehicle control group. Treatment of rats with Sildenafil significantly (p<0.01) reduced the 17ß-Hydroxysteroid dehydrogenase level as compared to Paroxetine treated rats. However, treatment of rats with SAVA 22 (0.1 mg/rat, 0.2 mg/rat and 0.4 mg/rat) no significant change in 17ß-Hydroxysteroid dehydrogenase level was observed as compared to Paroxetine treated rats. The serum 17ß-Hydroxysteroid dehydrogenase level was found to be significantly (p<0.01) decreased in rats treated with tadalafil (5 mg/kg, oral) when compared to Paroxetine treated rats (Figure 17 and Table 31).

Effect of SAVA 22 on testes 17ß-Ketosteroid reductase (Unit/min/mg protein) in Paroxetine induced Erectile Dysfunction
The result of the effect of Paroxetine, SAVA 22 and Sildenafil on testes 17ß-Ketosteroid reductase level in rats is presented in Figure 18. Administration of Paroxetine significantly (p<0.001) reduced the 17ß-Ketosteroid reductase level when compared with the vehicle control group. Treatment of rats with Sildenafil significantly (p<0.001) reduced the 17ß-Ketosteroid reductase level as compared to Paroxetine treated rats. However, treatment of rats with SAVA 22 (0.1 mg, /rat 0.2 mg/rat and 0.4 mg/rat) caused no significant change in 17ß-Ketosteroid reductase level as compared to Paroxetine treated rats. The 17ß-Ketosteroid reductase level was found to be significantly (p<0.001) decreased in rats treated with tadalafil (5 mg/kg, oral) when compared to Paroxetine treated rats (Figure 18 and Table 31).

Effect of SAVA 22 on Heart rate (Beats per minute (bpm)) in Paroxetine induced Erectile Dysfunction
The result of the effect of paroxetine, SAVA 22 and Sildenafil on heart rate in rats is presented in Figure 19. Treatment of rats with Sildenafil significantly (p<0.001) increased the heart rate when compared with the Paroxetine treated rats. Whereas treatment of rats with SAVA 22 (0.1 mg/rat, 0.2/rat and 0.4 mg/rat) did not cause significant change in heart rate when compared to vehicle control group. Further, the heart rate was significantly (p<0.001) decreased in SAVA 22 (0.1 mg/rat, 0.2/rat and 0.4 mg/rat) treated groups when compared to Sildenafil treated rats. The heart rate was found to be significantly (p<0.001) increased in rats treated with tadalafil (5 mg/kg, oral) and SAVA 22 oral when compared to Sildenafil treated rats (Figure 19 and Table 31).

Effect of SAVA 22 on Systolic BP (mmHg) in Paroxetine induced Erectile Dysfunction
The result of the effect of paroxetine, SAVA 22 and Sildenafil on systolic BP in rats is presented in Figure 20. The treatment of rats with Sildenafil significantly (p<0.001) increased the systolic BP when compared with the Paroxetine treated rats. Whereas treatment of rats with SAVA 22 (0.1 mg/rat, 0.2/rat and 0.4 mg/rat) did not cause significant change in systolic BP when compared to vehicle control group. Further treatment of rats with SAVA 22 (0.1 mg/rat, 0.2 mg/rat and 0.4 mg/rat) significantly (p<0.001) decreased the systolic BP when compared to Sildenafil treated rats. The systolic BP was found to be significantly (p<0.001) decreased in rats treated with tadalafil (5 mg/kg, oral) and SAVA 22 oral when compared to Sildenafil treated rats (Figure 20 and Table 31).

Effect of SAVA 22 on Diastolic BP (mmHg) in Paroxetine induced Erectile Dysfunction
The result of the effect of paroxetine, SAVA 22 and Sildenafil on diastolic BP in rats is presented in Figure 21. The treatment of rats with Sildenafil significantly (p<0.001) increased the diastolic BP when compared with the Paroxetine treated rats. Whereas treatment of rats with SAVA 22 (0.1 mg/rat, 0.2/rat and 0.4 mg/rat) did not cause significant change in heart rate when compared to vehicle control group. Further, treatment of rats with SAVA 22 (0.1 mg/rat, 0.2 mg/rat and 0.4 mg/rat) caused a significant (p<0.001) decrease in diastolic BP when compared to Sildenafil treated rats. The diastolic BP was found to be significantly (p<0.001) decreased in rats treated with tadalafil (5 mg/kg, oral) and SAVA 22 oral when compared to Sildenafil treated rats (Figure 21 and Table 31).

Conclusion of the study:
The SAVA 22 (0.4 mg/rat) was found to cause significant improvement in libido. The observed effect is mediated through increase in nitric oxide by inhibiting the phosphodiesterase. The observed effect is devoid of cardiac adverse effects. However, SAVA 22 at the other tested doses did not cause any improvement in libido.

Teuhetenone Tablet Preparation:
The formulation of oral dosage in the form of tablet and its constituents are presented in Table 32 as below.
Table 32: Tablet information
Teuhetenone Tablet
Ingredient Mg/Tab %w/w For 300 Tablets (g)
SAVA 22 0.4 0.57 0.12
F melt type C 69.1 98.71 20.73
Ethanol q. s q. s q. s
Magnesium Stearate 0.5 0.71 0.15
Total 70.00 100.00 21.00

The stepwise procedure for the preparation of the oral dosage of the new pharmaceutical formulation is disclosed below.
It must be ensured that the temperature of manufacturing area be NMT 25°C and humidity NMT 50.0% RH
Step 1: Drug solution preparation:
Dissolving 120 mg of Teuhetenone in 12 ml of ethanol.

Step 2: Wet granulation:
a. Taking F Melt (Type C) as per dispensing qty. & sifting through 40# sieve.
b. Co-sifting F Melt (Type C) and color through 80# Sieve and mixing in polybag for 5 mins.
c. Transferring sifted blend of F Melt (Type C) and Color in SS bowl and granulating using drug solution of STEP 1.

STEP 3: Drying:
Drying the wet granules of STEP 2 c in oven at 60°C for 15 mins.

STEP 4: Sizing and lubrication:
a. Passing the dried granules through 40# sieve and charging into blender.
b. Adding sifted magnesium stearate to blender and mixing for 5 mins.
c. Transferring the material to polybag and using for compression.

STEP 5: Compression
Compressing the blend using the following in process parameters with the tooling as mentioned. (as shown in Table 31)

Table 31: Compression tools
Tools Description Embossing
Upper Punch 5.5 mm, Standard concave round, plain on both side Plain
Lower Punch 5.5 mm, Standard concave round, plain on both side Plain
Die 5.5 mm, round ---

The tablet parameters are as disclosed in Table 32 below:

Table 32: Tablet Parameters:
B. No Appearance Tablet weight Hardness DT
TD-01 White color uncoated tablet, round, plain on both sides. 69.0 mg – 72.0 mg 30 to 50 N 25 sec to 45 sec

The lubricating agent used is selected from a group comprising of magnesium stearate, sodium stearyl fumarate, stearic acid, calcium stearate, hydrous magnesium silicate or talc.

HPLC Analysis
The HPLC analysis of the pharmaceutical composition comprising Teuhetenone and its derivatives is as provided in the following paragraphs.

Standard solution Preparation:
For the preparation of standard solution ,2.5 mg of standard transfer was weighed accurately into a 25 ml volumetric flask and to that about 20 mL of diluent were added, sonicated to dissolve, then cooled and the volume was made up with diluents and mixed well.

Sample Preparation:
For the preparation of sample 70 mg of tablet blend is weighed accurately and transferred into a 25 ml volumetric flask. To this about 20 mL of diluent is added, sonicated to dissolve, cooled and volume made up with diluents and mixed well.

Chromatograms:
The formulation chromatogram showed a prominent peak of Teuhetenone which was analyzed and confirmed. The standard chromatogram is illustrated in Figure 27 and the tablet chromatogram is illustrated in Figure 28.
,CLAIMS:We claim:
1. A method of isolating of Teuhetenone or its derivatives from a mixture comprising one or more additional bioingredients or impurities, comprising the steps of:
a. Dissolving crude water extract containing Teuhetenone or its derivatives along with additional bioingredients or impurities, in demineralized water in the range of 25-75 mg/ ml to prepare a mixture;
b. Injecting one ml of the mixture as prepared in step (a) for 8-10 times to the stationary phase of a column comprising of silica;
c. eluting Teuhetenone or its derivatives in gradient manner with a mobile phase;
wherein the mobile phase comprises one or more solvents, such that the active compound flows through the column with a retention time that is different from one or more other ingredients of the mixture;
wherein the details of the phases and process parameters ranges are as shown below:
Stationary Phase silica Silica: in the range of 20-60 gm
Mobile Phase First Solvent (A): Chloroform
Second Solvent (B): Methanol
Temperature of Mixture in the range of 15-35 °C
Temperature of column in the range of 20-40 °C
Wavelength in column in the range of 200-350 nm
Flow rate in column in the range of 25-55 ml/min
Mobile Phase run time In the range of 20-40 min

d. detecting Teuhetenone or its derivatives by analyzing the chromatogram;

wherein better yield with high purity is achieved in lesser time by using single column.

2. A process of preparing a pharmaceutical composition of Teuhetenone or its derivatives along with excipients, comprising the steps of:
(i) dissolving the specific quantity of an active compound in a suitable solvent to yield a premix solution;
(ii) taking the premix of excipients as per dispensing quantity and sifting through 40# sieve;
(iii) co-sifting premix of excipients and color through 80# Sieve and mixing in polybag for a specific time;
(iv) transferring sifted blend of premix of excipients and color in a stainless-steel bowl and granulating the drug solution of Step (i);
(v) drying the wet granules of step (iv) in oven at 60°C for 15 mins;
(vi) passing the dried granules through 40# sieve and charging into a blender;
(vii) adding sifted lubricating agent to the blender and mixing for 5 mins;
(viii) transferring the blend material to a polybag for using for compression; and
(ix) compressing the blend using compression tools,
wherein, the manufacturing area temperature is maintained at NMT 25°C and the humidity is maintained at NMT 50.0% RH;
wherein the said active compound is Teuhetenone or its derivatives;
to obtain a composition for improving male sexual dysfunction.
3. The process as claimed in claim 2, wherein the quantity of active compound is in the range of 100mg to 140 mg.
4. The process as claimed in claim 2, wherein the solvent used is ethanol.
5. The process as claimed in claim 2 or claim 4, wherein the quantity of solvent is in the range of 10 ml to 14 ml.
6. The process as claimed in claim 2, wherein said premix of excipients comprises of five pharmaceutical excipients consisting of carbohydrates, inorganic ingredients and disintegrants.
7. The process as claimed in claim 2, wherein said lubricating agent is selected from a group comprising of magnesium stearate, sodium stearyl fumarate, stearic acid, calcium stearate, hydrous magnesium silicate or talc.
8. The process as claimed in claim 2 or claim 3, wherein the quantity of active compound is 120 mg.
9. The process as claimed in claim 2 or claim 4, wherein the quantity of solvent is 12 ml.
10. A pharmaceutical composition consisting of active compound along with excipients comprising of:
1) Teuhetenone or its derivatives : 0.1 to 10% by weight
2) Premix of excipients : 50 to 99.9 % by weight:
3) Ethanol : q.s.; and
4) Magnesium stearate : 0.1 to 10% by weight:
11. The pharmaceutical composition as claimed in claim 10, wherein the quantity of active compound is 0.57 % by weight.
12. The pharmaceutical composition as claimed in claim 10, wherein the quantity of premix of excipients is 98.71% by weight.
13. The pharmaceutical composition as claimed in claim 10, wherein the quantity of magnesium stearate is 0.71% by weight.
14. The pharmaceutical composition as claimed in claim 10, wherein the composition is formulated as oral dosage.
15. A pharmaceutical composition as claimed in claim 10 which is capable of improving or mitigating male sexual dysfunction
16. A process for extraction of active compounds from Damiana, comprising extraction with water, wherein the active compounds extracted are effective in improving male sexual dysfunction.