Technological Area:

This invention is about the ferula elaeochytris root extract solution developed to be used particularly in pharmaceutical industry and for use in diabetic erectile disfunction treatment.

State of Art:

Today, diabetes which causes harm in heart and veins, can also affect sex life. There are various antioxidants (such as vitamin E, selenium, alpha-lipoic acid, gamma-linoic acid) and substances and medicines such as phosphodiesterase enzyme inhibitors [Sildenafil (®Viagra), vardenafil (®Levitra), tadalafil (®Cialis)] released to the market for the treatment of diabetic impotence caused by diabetes. Today, it’s key important to use herbal products instead of synthetic ones by means of reducing possible adverse effects and cost (supported by scientific studies). But today, production of these herbal drugs developed as alternatives for already sold products, is quite low.

Among the existing researches there are studies made on various kinds of ferula, an endemic plant that’s used as aphrodisiac and grows in our country and it’s determined that some kinds have infertility treating characteristic. For this, the studies considered in the literature is mentioned below.

In the patent application numbered TR200707254, “Natural infertility treatment supplement for males and females (ferula root juice)” is described. The invention; is about an aromatic juice obtained by distilling the fresh root of ferula kinds growing in our country such as Ferula communis, Ferula orientelis, Ferula rigidula with styrax gum and currant in distillation boiler, that enables an increase in ovarian follicles and ovulation rate in females and sperm reproduction and quality in males.

In the patent application numbered TR201007344, “The powder obtained from the natural infertility treatment supplement aromatic juice for males and females” is described. The invention is about the aromatic juice obtained by distilling the fresh root of ferula kinds growing in our country such as Ferula communis, Ferula orientalis, Ferula rigidula with styrax gum and currant in distillation boiler, that enables an increase in ovarian follicles and ovulation rate in females and sperm reproduction and quality in males and further turning it into capsule and syrup.

In the patent application numbered TR201806575, “The feed additive triggering the stimulation of hormonal system” is described. The invention is about an economic and easy to use feed additive with a high success rate that triggers hormonal system in cattle and ovines, makes them to reach sexual maturity and estrous cycle. The invention is about the feed additive that contains ferula root, carob tree, ginseng, vitamins and minerals, affective aromatics and clinoptilolite which prevents the feed additive to get moldy and can be produced in powder, drop, solution, tablet, pellet, putty and injectable forms.

In the applications mentioned above what’s purposed is treating sexual diseases with various kinds of Ferula which’ s an endemic plant that grows in our country and used as an aphrodisiac. But in these studies, ferula communis, ferula orientelis and ferula rigidula kinds are used and they do not contain any information on diabetic impotency treatment. Besides the active substance of the same kinds of this plant growing in different locations, shows difference. For this reason, concerned inventions do not bear a scientific background so unconscious consumption of this plant poses an important problem.

In conclusion, there’s a need for a low cost, easy to produce new technology with antidiabetic effects, which can overcome the disadvantages mentioned above and can be used in diabetic impotency treatment, substantially increase spermatogenesis activity and has a decreasing effect on blood glucose.

Invention Definition:

This invention is the ferula elaeochytris root extract solution for use in erectile disfunction treatment and its’ characteristic is; a low cost, easy to produce new technology with antidiabetic effects, which can be used in diabetic impotency treatment, substantially increase spermatogenesis activity and has a decreasing effect on blood glucose.

[Eser, N., Buyuknacar, H.S., Cimentepe, O.O. et al. The effect of Ferula elaeochytris root extract on erectile dysfunction in streptozotocin-induced diabetic rat. Int J Impot Res 32, 186-194 (2020). http s - 81 in the article published in 1st April 2019 given at the link, there are the studies of the invention owner on this subject and in order to realize the purposes mentioned above and offered by the detailed description below the invention; meets the raw material needs of food market, pharmaceutical industry, cosmetics industry, feed industry and food supplement industries and ensures functional foods with high added value to be produced and high purity and quality products with no chemical substance contamination to be obtained.

With the invention, this endemic plant which’ s highly and unconsciously used by people, is supported by scientific and pharmacological data and ensured to be used in appropriate doses. It grants a great advantage to conduct the study with important information such as the kind of the ferula plant (Ferula elaeochytris) in the solution subject to invention, the region it was collected from (Kahramanmaras, Engizek plateau; 37° 33'39 "N, 41° 45'53" E ), and plant registration number (HURUB 4588-4589). In addition, with the study conducted, the appropriate amount of use of the ferula elaeochytris plant for diabetic impotency was determined and the mechanism behind its’ healing effect was proven. With the solution subject to invention, both the unconscious use will be prevented, and it will pose importance by means of shedding a light on further clinic trials. All the advantages of the method subject to invention will be understood more clearly with the help of the scheme given below and the detailed description written by referring to this scheme, for this reason assessment should be made considering this scheme and the detailed description.

Description of Figures:

The invention will be described by referring the figures enclosed, this way the characteristics of the invention will be understood and assessed more clearly, but this is not intended to limit the invention with these specific arrangements. Vice versa it’s intended to cover all alternatives, changes and equivalents which can be involved under the area the invention is defined in by the claims enclosed. It must be understood that the shown details are given only for the description of the preferred arrangements of the current invention and presented to provide the most utilizable and clear definition of both the formation of the methods and the rules and conceptual characteristics of the invention. In these figures;

Figure 1 Schematical view of the production method of the product subject to invention.

Figure 2 Graphical view of the trace samples showing the intracavernosal pressure measurement responses of the control, diabetes and diabetes+femla elaeochytris group rats.

Figure 3 Graphical view of the intracavernosal pressure increase responses (cmH20) of the control, diabetes, ferula, diabetes+ferula and ethyl alcohol group rats, *relevance according to the control responses (p<0.05, n= 8-25). + relevance according to the + “diabetes” group (p<0.05, n= 6-25).

Figure 4 Graphical view of the metabolic blood glucose decreasing effect of the ferula elaeochytris extract subject to invention. Description of References:

10. Drying

20. Making into powder

30. Extracting

40. Dissolving

50. Obtaining the oral solution

Description of Invention:

The invention consists of the steps of drying the ferula elaeochytris plant roots after purging it of the soil and impurities on the outer surface (10), grinding and making the dried roots into powder (20), extracting the powdered ferula elaeochytris plant in a solvent containing diethyl ether (30), solving the pure extract obtained by extraction process in its’ own solvent containing diethyl ether with at least 0.1 ml ethanol and 0.9 ml distilled water (40) and obtaining the oral solution with the root extract (50).

The invention is characterized with the step of obtaining the oral solution with the root extract (50) which has a positive effect on erectile disfunction disease.

The invention is characterized with the step of obtaining the oral solution (40) through using at least 40 mg/kg/mL pure extract.

Detailed Description of the Invention:

Extracting the ferula elaeochytris root used in the solution is as follows: First, the ferula elaeochytris root is purged of the impurities on its’ outer surface, cleaned in general and dried on a clean and dry ground under the sun without decaying (10), then they’re sifted through a 1 mm screen, grinded and turned into powder (20), after then they’re dried again (10). Dried sample is placed inside celluloid cartridges and these cartridges are closed with cotton. The ferula elaeochytris plant root (at least 20g) is added inside it and the cartridge is placed under a Soxhlet extraction tube. Fixed weighed extraction balloons are placed under the extraction tube. The solvent (diethyl ether about 320ml) is siphoned through and then filled to the half of the Soxhlet extraction tube. It’s extracted for about 4 hours (30). At the end of the extraction the balloon with the solvent in it is taken and tied to the evaporator and removed of the solvent. Then the balloon is hold on a stove set to 60 °C for 1 hour, cooled down to room temperature in desiccator and weighed.

The administration form of ferula elaeochytris root extract is by solving the pure extract obtained at the end of the extraction process in its’ solvent weighed at least 40 mg/kg/mL with at least 0.1 ml ethanol and 0.9 ml distilled water (40) and obtaining the oral solution (50).

The test group of the solution subject to invention: In this study, Wistar albino male rats (n=72) were divided into five groups; control group, diabetic group (60mg/kg; streptozotocin; i.p; single dose), Ferula elaeochytris group (40 mg/kg/day; intragastric; 8 weeks), diabetes+Ferula elaeochytris group (40 mg/kg/day) and ethanol group (%95, 1 ml/day). Ferula elaeochytris was dissolved in ethanol. Ferula elaeochytris and ethanol were given intragastrically for 8 weeks.

In vivo Intracavemosal Pressure Trials: Subject animals were held under proper conditions for 8 weeks, lied on operation table in supine position under sevoflurane anesthesia, Cavemosal root zone was penetrated into with a needle (23G) with one end mounted a polyethylene cannula (PE50- Clay Adams). This needle and cannula were tightly fixed at penis root zone and the other end of it was connected to a pressure transducer with a stopcock. The cavernosal nerve of the Major Pelvic Ganglion (MPG) localized at the lateral zone of the prostate was isolated and one end was placed on a platin tip electrode connected to the “Grass S88 stimulator”. Electrical stimulation was applied to cavernosal nerve for 1 minute (10V, 16Hz, 5ms duration) and intrapenis pressure was increased. Changes in intrapenis cavernosal pressure obtained with pressure transducers were transferred to a Data Collection System (BIOPAC-MP36). Responses were saved into the computer. Obtained intrapenis pressure increases were loaded into the ®GraphPad Prism software (San Diego, CA, USA), “mean ± S.E” of each subject group were found and analyzed using “Benferroni corrected t test” “analysis of variance (®ANOVA)” statistical software. As a result of this analysis, trace samples showing the intracavemosal pressure measurement responses of diabetes and diabetes+ferula elaeochytris group rats were given in Figure 2. They were given as a different graphical view in Figure 3.

In vitro Isolated Organ Bath Trials: Animals were decapitated with cervical dislocation and both two penile corpus cavemosal tissues of them were excised as a whole and put in a petri dish containing Krebs solution (mM: NaCl 119, KC1 4.6, CaCl2 1.5, MgCl2 1.2, NaHCCF 15, NaHPd* 1.2, glucose 11) and oxygenated with %95 O2 with the help of a cannula and cleaned of from environmental connective and fatty tissues. Two separate corpus cavernosum (crura) preparates were obtained from each rat as right and left. Cavemosal tissues were placed in 5 ml, externally heated organ baths containing 37 °C Krebs solution and ventilated with %95 O2 and % 5 CO2 with the help of a thin cannula (pH:7.4), under 700 mg tension. Top end was connected to an isometric transducer (MAY COM, FDT 10-A) with a rope. Tonus changes in the preparate were saved into computer through (®BIOPAC MP36 Systems, Inc.). Tissues that were applied 700 mg tonus at the beginning, were washed with fresh Krebs solution in 15 minutes intervals, put to 1 hour incubation and tissues were given 5 mM phenylephrine (alpha- 1 adrenergic receptor agonist), electrical field stimulation for neurogenic responses (ESS; 0.5 - 16 Hz, 30 V, 0.5 -1 ms duration); muscarinic receptor agonist for endothelial derived relaxing responses (Acetylcholine; cumulative; 0.05mM-100mM); nitric oxide donor to assess NO- guanite cyclase-cGMP pathway (sodium nitroprusside; cumulative; 0.05mM-100mM); and 10 mΐ Ferula elaeochytris extract and relaxing responses were monitored. Tissues were contracted with 100 mM papaverine for the percentage of relaxing responses and with KC1 (100 mM) for the percentage of contraction responses and the responses were loaded in ®GraphPad Prism software (San Diego, CA, USA) and “mean ± S.E” of each subject group were found and analyzed using “Benferroni corrected t test” “analysis of variance (®ANOVA)” statistical software. “P” values lower than 0.05 were taken statistically significant. Spermatogenesis activity: Testicle and cavemosal tissue samples taken from the groups were processed with %10 formalin fixation, after that micron thick sections were taken for histopathologic examination from the paraffin blocks prepared after tissue monitoring process in ®Leica brand tissue monitoring device. They were stained with H&E in ®Leica automatic stain device. They were evaluated under ®Nicon Eclipse E600 optical microscope. In order to assess spermatogenesis in testicle tissue, Johnsen tubular biopsy score method (JTBS) was used. Germinal epithelium maturation in seminiferous tubule was graded in 25 tubules using JTBS under 25X objective field. According to JTBS;

1. No sertoli cell nor germ cell in seminiferous tubule

2. No germ cell, only sertoli cell

3. Only spermatogonium as germ cell

4. Only a few spermatocyte cells (5/tubule)

5. No spermatozoa nor spermatid but a vast number of spermatocytes

6. Only a few spermatid (5/tubule)

7. No spermatozoa but a vast number of spermatids

8. Only a few spermatozoa

9. Germinal epithelia disorganized and agglomeration towards lumen, a vast number of spermatozoa

10. Germinal epithelium multi-rowed, accepted complete spermatogenesis in existence.

Blood Glucose Level: It was proven that there has been a significant decrease in the glucose levels of the animals given Ferula elaeochytris extract for 8 weeks.

Findings obtained in this study are as follows: It has proven that the oral chronic (8 weeks) use of Ferula elaeochytris root extract in some way, significantly prevented and/or recovered the neurogenic and/or endothelial disfunction derived in rats induced with diabetes. Significantly inhibiting the increased glucose levels in diabetes is the indication of the antidiabetic effect of this plant. The most important finding of our study is the chronic use of ferula elaeochytris root extract significantly increasing the spermatogenesis activity that was dramatically decreased due to the diabetes in testicles.

In the study subject to invention, the effect of ferula elaeochytris root extract on glucose was electrochemically examined with biosensor device. The measurements were taken with golden electrolyte with glucose oxidase enzyme bound with gelatin BSA, through Linear Pulse Voltmeter between 0,7 - 0,9 V range. In order to provide a physiological media, pH 7,5 phosphate buffer was used. At first, glucose was added into the media free of compounds, the measurement was taken due to its’ interaction with glucose oxidase enzyme. Then, (100 mM) b-lonone compound was added into the media, successive measurements were taken and obtained responses were recorded. At the end of the study, after adding b-lonone into the media, it was observed that there was a linear decrease in measurements due to the decrease in glucose and glucose oxidase enzyme interaction. It was seen that Ferula elaeochytris root extract has a metabolically blood glucose decreasing effect. And this was given as a graphic in Figure 4.