Herbal medicines for the prevention and treatment of Helicobacter pylori infection

Abstract

The present invention relates to the use of a herbal preparation characterized by the presence of four major compounds (expressed as a percentage of the total peak area in a GC / MS chromatogram, approximately 35% to approximately 50% carvacrol, approximately 10% to approximately 30% gamma-terpinene, approximately 10% to approximately 25% thymol, and approximately 8% to approximately 20% p-cymene) obtained by mixing at least two essential oils from species selected from the group consisting of Satureja L., Origanum L., and Thymus L., to prepare a dietary supplement or pharmaceutical product for the treatment and prevention of gastric Helicobacter pylori infection in humans. The mixture can be placed on a liquid or solid carrier and encapsulated in a non-gastro-resistant capsule. The present invention also relates to a dosing regimen of the herbal preparation for the treatment and prevention of Helicobacter pylori infection in patients, which successfully eradicates the bacteria without side effects.

Description

[Technical field]

[0001] The present invention relates to the therapeutic application of a herbal preparation with a specific chemical composition obtained by mixing two or more essential oils from the genera Satureja L., Origanum L. and Thymus L. to prepare a dietary supplement or medicine that is successful in eradicating pathogenic bacteria. More specifically, the present invention relates to the use of this herbal preparation to prepare a dietary supplement or medicine that is successful in eradicating Helicobacter pylori (H. pylori) in humans. [Background technology]

[0002] H. pylori inhabits the human stomach and can induce many serious gastrointestinal disorders, such as gastritis, peptic and duodenal ulcers, and gastric cancer. The World Health Organization (WHO) points out that approximately 50% of the world's population is infected with H. pylori, with different prevalence rates in developed (25-50%) and developing countries (70-90%). Although most patients are asymptomatic, a significant proportion (approximately 10-20%) of infected individuals develop peptic ulcers, and one-quarter of these patients (approximately 4.25%) develop severe ulcer complications, and 1-2% progress to gastric cancer. Gastric cancer is the second leading cause of cancer-related deaths worldwide (Kusters et al. (2006) Pathogenesis of H. pylori infection. Clin Microbiol Rev 19:449-490). Accordingly, in 1994, H. pylori was announced by the WHO as a human carcinogen. Furthermore, approximately 500 million people worldwide will sooner or later develop serious stomach lesions such as peptic ulcers due to Helicobacter pylori infection, and approximately 30 million people may develop gastric cancer.

[0003] There is a high need to achieve eradication of H. pylori from the gastric mucosa. To achieve this goal, a number of antibiotic regimens are used: amoxicillin, tetracycline, metronidazole, furazolidone, levofloxacin, and clarithromycin (Wannmacher (2011) Review of the evidence for H. pylori treatment regimens, 18 th Expert Committee on the Selection and Use of Essential Medicines). Currently, therapy for H. pylori infection consists of high doses of three to four different antibiotics plus adjuvant agents. However, this therapy exhibits very limited effectiveness, mainly due to the highly frequent occurrence of antibiotic-resistant strains of H. pylori. As an example: triple therapy with clarithromycin and amoxicillin / metronidazole plus a proton pump inhibitor (PPI) showed success rates of 84% and 76%, while quadruple therapy consisting of a PPI, bismuth, metronidazole and tetracycline showed a success rate of 87%. In fact, 20% of patients with H. pylori infection are not cured after the first course of antibiotic treatment and therefore require additional treatment cycles. Therefore, in 2017, the WHO included H. pylori in the "Global priority list of antibiotic-resistant bacteria to guide research, discovery and development of new antibiotics", developed by a group of 70 world-leading experts. Furthermore, all the mentioned treatments are often accompanied by severe gastrointestinal side effects such as diarrhea, nausea, vomiting, abdominal bloating and abdominal pain that patients find intolerable.

[0004] Furthermore, several alternative therapies based on microorganisms, peptides, polysaccharides and intragastric ultraviolet light irradiation have been proposed for eradicating H. pylori infection in humans, but were not effective in eradicating the bacteria (Ayala et al. (2014) Exploring alternative treatment for Helicobacter pylori infection, Word J Gastroenterol 20(6):1450-1469). Furthermore, recent studies on vaccines confirmed that no promising vaccine candidates have yet been found (Sutton & Boag (2019) Status of vaccine research and development for Helicobacter pylori, Vaccine 37:7295-7299).

[0005] Probiotics and some plant-based dietary supplements are used to prevent or reduce some of the side effects of antibiotic therapy, but no commercially available dietary supplements or other herbal products have been tested in humans to have anti-Helicobacter activity, improve the therapeutic response to antibiotics, or prevent recurrence of H. pylori infection in humans.

[0006] Essential oils obtained from aromatic plants are well-known antibacterial agents against many bacterial strains. Essential oils are complex mixtures of volatile organic compounds (mainly terpenes and phenylpropanes) obtained by hydrodistillation or steam distillation from aromatic plants. An essential oil from one aromatic plant can contain hundreds of different components. The chemical composition of the essential oil of a particular plant species is highly variable, both qualitatively and quantitatively. This variation is related to different factors, for example: intrinsic factors related to the plant, the interaction of the plant with its environment (such as soil type and climate), extrinsic factors related to the maturity of the plant, the plant organ selected for essential oil isolation, the time of harvest during the day, and the extraction method. The antibacterial power of an essential oil depends to a great extent on its chemical composition. Therefore, the strength of the antibacterial activity of an essential oil from a particular plant cannot be asserted without determining its chemical profile. Therefore, when investigating the possible applications of a particular essential oil, the chemical composition of each sample of the essential oil should be defined.

[0007] Some essential oils have shown antibacterial activity in in vitro bioassays, even against multidrug-resistant strains of H. pylori (Martin & Ernst (2003) Herbal medicines for treatment of bacterial infections: a review of controlled clinical trials. J Antimicrob Chemother 51:241-246; Ohno et al. (2003) Antimicrobial activity of essential oils against Helicobacter pylori. Helicobacter 8:207-215; Takeuchi et al. (2014) Natural products and food components with anti-Helicobacter pylori activities, World J Gastroenterol 20:8971-8978).

[0008] There is much in vitro evidence for the anti-H. pylori activity of essential oils obtained from species of the genera Satureja L., Origanum L. and Thymus L.

[0009] For example, the essential oils of Satureja bachtiarica, winter savory (Satureja montana) and summer savory (Satureja hortensis) show high anti-Helicobacter pylori activity in vitro (Bergonzelli et al. (2003) Essential oils as components of a diet-based approach to management of Helicobacter infection. Antimicrob Agents Chemother 47:3240-324; Falsafi et al. (2015) Chemical composition and anti-Helicobacter pylori effect of Satureja bachtiarica Bunge essential oil. Phytomed 22:173-177; Lesjak et al. (2016): Binary and tertiary mixtures of Satureja hortensis and Origanum vulgare essential oils as potent antimicrobial agents against Helicobacter pylori. Phytother Res 30:476-484).

[0010] Essential oils from oregano (Origanum vulgare) have shown anti-H. pylori activity in in vitro bioassays (Bergonzelli et al. (2003) Essential oils as components of a diet-based approach to management of Helicobacter infection. Antimicrob Agents Chemother 47:3240-324). In addition, carvacrol, the main component of oregano essential oil, has been shown to exhibit bactericidal activity against H. pylori strains in vitro (Marinelli et al. (2018) Carvacrol and its derivatives as antibacterial agents. Phytochem Rev 17:903-921; Ruiz-Rico et al. (2020) In vitro antimicrobial activity of immobilised essential oil components against Helicobacter pylori. World J Microbiol Biotechnol 36:3-9).

[0011] It has been shown that the essential oil of thyme (Thymus vulgaris) has an antibacterial effect against Helicobacter pylori (Esmaeili et al. (2012): Anti-Helicobacter pylori activities of soya powder and essential oils of Thymus vulgaris and Eucalyptus globulus. Open Microbiol J 6:65-69). Some other Thymus species also express anti-Helicobacter pylori activity (Dandlen et al. (2011): Antimicrobial activity, cytotoxicity and intracellular growth inhibition of Portuguese Thymus essential oils. Rev. Bras. Farmacogn 21:1012-1024). Furthermore, thymol, the main component of thyme essential oil, showed high bactericidal activity against Helicobacter pylori strains in vitro (Korona-Glowniak et al. (2020) The in vitro activity of essential oils against Helicobacter pylori growth and urease activity. Molecules 25:1-15).

[0012] In addition, the cyclic hydrocarbon p-cymene, present in high amounts in thyme essential oil, and gamma-terpinene, present in abundance in summer savory (Satureja hortensis) essential oil, have been shown to have no antibacterial activity against gram-negative bacteria (Korona-Glowniak et al. (2020) The in vitro activity of essential oils against Helicobacter pylori growth and urease activity. Molecules 25:1-15). However, it has been reported that some compounds that do not show antibacterial activity in the presence of other antibacterial agents show synergistic effects. For example, the presence of p-cymene together with carvacrol in essential oils can enhance the antibacterial activity of the essential oils (Ultee et al. (2002) The phenolic hydroxyl group of carvacrol is essential for action against the food-borne pathogen Bacillus cereus. Appl Environ Microbiol 68:1561-1568).

[0013] Furthermore, with regard to the activity of each single oil from the mixture, mixtures of essential oils were more effective, especially binary and ternary mixtures of summer savory (Satureja hortensis), oregano subsp. vulgare (Origanum vulgare subsp. vulgare) and Greek oregano (Origanum vulgare subsp. hirtum) essential oils (Lesjak et al. (2016): Binary and tertiary mixtures of Satureja hortensis and Origanum vulgare essential oils as potent antimicrobial agents against Helicobacter pylori. Phytother Res 30:476-484).

[0014] Regarding in vivo evidence in experimental animals, it was demonstrated that a mixture of summer savory (Satureja hortensis) and Greek oregano (Origanum vulgare subsp. hirtum) essential oils successfully eradicated 70% of H. pylori in mice without the use of antibiotics, while not eliciting any toxicity or changes in cytokine and chemokine balance (Harmati et al. (2017): Binary mixture of Satureja hortensis and Origanum vulgare subsp. hirtum essential oils: in vivo therapeutic efficiency against Helicobacter pylori infection. Helicobacter e12350).

[0015] However, there are no studies in the prior art conducted on humans to evaluate the therapeutic efficacy of essential oils obtained from Satureja sp., Origanum sp. and Thymus sp., their mixtures, or pure compounds isolated from these essential oils in eradicating H. pylori.

[0016] The above brief review clearly shows the great need to find new therapies for eradicating H. pylori infection in humans as well as the various approaches proposed to combat this bacterium. Summary of the Invention

[0017] Technical issues The object of the present invention is to provide a herbal formulation of defined chemical profile comprising a mixture of essential oils that is highly effective in eradicating H. pylori infection in humans.

[0018] A second object of the present invention is to provide a method for preparing the specified herbal formulation.

[0019] The third objective of the present invention is to establish a dosing regimen for treatment with herbal preparations to lead to successful eradication of H. pylori from the gastric mucosa in patients with confirmed H. pylori infection.

[0020] The fourth object of the present invention is to establish a treatment with a herbal preparation for eradicating H. pylori from the gastric mucosa without using any of the known drugs or antibiotics.

[0021] The fifth objective of the present invention is to establish a herbal medicine treatment for eradicating H. pylori from the gastric mucosa without causing unwanted side effects such as diarrhea, nausea, vomiting, abdominal distension and abdominal pain, which are very common in standard antibiotic therapy.

[0022] Disclosure of the Invention The present invention is the application of a herbal preparation - a mixture of certain essential oils - as a dietary supplement or medicine for the prevention and treatment of Helicobacter pylori infection in humans. The mixture is characterized by the presence of four main compounds in the following amounts, expressed as a percentage of the total area of ​​the peaks in the GC / MS chromatogram: about 35% to about 50% carvacrol, about 10% to about 30% gamma-terpinene, about 10% to about 25% thymol, and about 8% to about 20% p-cymene. The mixture can be obtained by mixing two or more essential oils from the species of Satureja L., Origanum L. and Thymus L. The mixture may be placed on a liquid or solid carrier and encapsulated in a non-gastro-resistant capsule. As disclosed herein, the preparation is suitable for use as a dietary supplement or herbal medicine for the treatment of H. pylori infection. Also disclosed herein is a successful regimen for eradicating H. pylori from the human gastric mucosa.

[0023] How to obtain the preparation (mixture of essential oils): The herbal preparation of the present invention must have a specific chemical profile to obtain high anti-H. pylori activity. It is characterized by the presence of four major compounds: about 35% to about 50% carvacrol, about 10% to about 30% gamma-terpinene, about 10% to about 25% thymol, and about 8% to about 20% p-cymene. The amounts are expressed as % of the total area of ​​all peaks in a chromatogram obtained by gas chromatography with mass spectrometry detection (GC-MS). In addition, the preparation may contain small amounts of compounds such as α-terpinene, trans-β-caryophyllene, α-pinene, β-pinene, β-myrcene, linalool, 1,8-cineole, and limonene, each present at 0.1 to 3% (expressed as % of the total area of ​​all peaks in a GC-MS chromatogram). Any other components of the preparation may be present up to 0.4% each. Preferably, the composition of the formulation (expressed as a % of the total area of ​​all peaks in the chromatogram) should be: about 38% to about 48% carvacrol, about 18% to about 28% gamma-terpinene, about 10% to about 12% thymol, about 9% to about 14% p-cymene, and alpha-terpinene, trans-beta-caryophyllene, alpha-pinene, beta-pinene, beta-myrcene, linalool, 1,8-cineole, and limonene, each present at 0.4 to 2.9%. The highest efficacy against H. pylori was obtained with a formulation with the following exact composition (expressed as % of the total area of ​​all peaks in the chromatogram): carvacrol 47.5%, γ-terpinene 18.5%, thymol 11.9%, p-cymene 13.6%, α-terpinene 1.55%, trans-β-caryophyllene 1.49%, linalool 1.24%, α-pinene 0.99%, β-myrcene 0.97%, 1,8-cineole 0.55%, limonene 0.46% and β-pinene 0.40%.

[0024] The preparation according to the present invention can be obtained by mixing two or more essential oils from the following plants, preferably by steam distillation: Satureja sp., Origanum sp. and Thymus sp. These plants are commonly used as spices or teas. More importantly, these essential oils are generally recognized as safe (GRAS) substances by the US Food and Drug Administration (FDA) and approved by the European Commission for use as flavorings in food.

[0025] Preferably, the herbal preparation of the present invention can be obtained by mixing the essential oils of Summer Savory (Satureja hortensis L.), Greek Oregano (Origanum vulgare subsp. hirtum L.) and Thymus vulgaris L. in the following ratios: 2:1:1, respectively. Since the chemical profile of essential oils varies greatly, mainly due to different environmental factors, cultivation methods of aromatic plants and isolation methods, the preferred chemical composition of each essential oil used to obtain the herbal preparation of the present invention is shown in the table below:

[0026] [Table 1]

[0027] The essential oils disclosed in this invention are obtained from the plant species Summer savory (Satureja hortensis L.), Greek oregano (Origanum vulgare subsp. hirtum L.) and Thymus vulgaris L. The plant material was collected at the flowering stage, air dried at 25-30°C and then ground in a blender. The essential oils were isolated by hydrodistillation in a Clavenger apparatus according to the procedure of Ph. EUR. IV (European Pharmacopeia, 2002). Briefly, 100 g of plant material was tapped with water and distillation continued for 3 hours. After cooling, the pure essential oil was removed from the Clavenger apparatus and dried over anhydrous sodium sulfate. After filtering through a Büchner funnel to remove sulfates, the essential oils were collected and stored at -20°C until use. The obtained essential oils of Summer Savory (Satureja hortensis L.), Greek Oregano (Origanum vulgare subsp. hirtum L.) and Thymus vulgaris L. were mixed in the following ratio: 2:1:1 respectively to obtain the herbal preparation of the present invention, which was further used for the treatment of human volunteers with Helicobacter pylori infection.

[0028] Possible applications of the present invention: The herbal preparation according to the present invention may be used as a dietary supplement or herbal medicine for the treatment or prevention of H. pylori infection. When used as a medicine, it can be applied alone or in combination with standard antibiotic therapy. The preparation (a specific mixture of essential oils) can also be placed in a liquid or solid carrier or encapsulated in a gelatin capsule. Each capsule should contain at least 50 mg of the preparation, preferably 65 mg of the preparation.

[0029] The efficacy of the herbal preparation of the present invention against H. pylori is demonstrated by both in vitro and in vivo studies.

[0030] The herbal formulation of the present invention was tested against 20 clinical strains of H. pylori classified into four different groups, including HpEurope, HpEAsia, HpAsia2, and HpAfrical. The majority of the strains were resistant to multiple antibiotics. The tests were performed using the agar dilution method. The minimum inhibitory concentration (MIC) was determined as the lowest concentration of the composition required to completely inhibit the growth of H. pylori. The minimum bactericidal concentration (MBC) was determined by the number of viable colonies, and the bactericidal effect of the product was expressed as the percentage reduction in the number of bacterial colonies compared to the untreated control. The results confirmed that the formulation was active against different antibiotic-resistant H. pylori strains with the same activity level, which means that the formulation of the present invention does not have selectivity for a specific type of H. pylori and the formulation does not have a specific cellular target. In other words, the essential oil is a complex mixture of numerous lipophilic compounds that penetrate the cell wall, disrupt the bacterial membrane, and interfere with integral membrane proteins. The complexity of this mixture completely prevents the microorganism from acquiring resistance. This implies that the formulation is suitable for eradicating all types of H. pylori, i.e. has the potential to be applied in treating patients worldwide, whatever the infection-causing strain of H. pylori (antibiotic-resistant or non-antibiotic-resistant).

[0031] The results of treatment of 28 human volunteers (divided into three groups) with the herbal preparation of the present invention are disclosed within this patent application.

[0032] The first group included 10 human volunteers with confirmed acute H. pylori infection who had mild symptoms of infection (abdominal bloating, stomach pain, high levels of stomach acid, cough, loss of appetite). The volunteers received gelatin capsules containing 65 mg of the formulation dissolved in 435 mg of sunflower oil (five volunteers) or placed on a solid carrier (five volunteers). Each volunteer received two capsules per day (65 mg of formulation per capsule), one in the morning and one in the evening, taken on an empty stomach 30 minutes before a meal, for 30 consecutive days, i.e. 130 mg of the herbal formulation daily and 65 mg twice daily for 30 consecutive days.

[0033] During these 30 days, they did not receive any other treatment for H. pylori infection and followed a normal diet plan, without alcohol only. Four and eight weeks after the end of treatment, the volunteers were tested for H. pylori stool antigens. Eradication of infection was achieved in 50% of cases. Therefore, we conclude that at least at the beginning of treatment, higher doses and longer durations of treatment should be used.

[0034] The second group included 15 human volunteers with confirmed acute H. pylori infection, with mild symptoms of infection (abdominal bloating, stomach pain, high levels of stomach acid, cough, loss of appetite). To compare with the dosage regimen applied to the first group, the dosage regimen applied in the second group was higher and longer at the beginning of the treatment. The volunteers received gelatin capsules containing 65 mg of the formulation dissolved in 435 mg of sunflower oil (7 volunteers) or placed on a solid carrier (8 volunteers). Each volunteer took 4 capsules per day, 2 in the morning and 2 in the evening, on an empty stomach 30 minutes before meals, for 15 consecutive days, then 2 capsules per day, 1 in the morning and 1 in the evening, on an empty stomach 30 minutes before meals, for another 30 consecutive days, i.e., 260 mg of the herbal preparation and 130 mg twice a day for 15 consecutive days, followed by 130 mg of the herbal preparation and 65 mg twice a day for another 30 consecutive days.

[0035] During these 45 days, they did not receive any other therapy for H. pylori infection and followed a normal diet plan, without alcohol only. Four and eight weeks after the end of the treatment, the volunteers were tested for H. pylori stool antigens. Complete eradication of H. pylori infection was confirmed in 14 out of 15 volunteers (eradication of infection was achieved in 93% of cases). Ten volunteers reported complete disappearance of symptoms of the disease (stomach pain, increased levels of gastric acid, abdominal bloating) 10 days after the start of the treatment, while the other four reported disappearance of symptoms by the end of the therapy. This suggests that the herbal preparation described in the present invention exerts an anti-inflammatory effect in addition to the antibacterial effect against H. pylori. During this treatment, none of the volunteers reported any gastrointestinal side effects that commonly occur during antibiotic therapy, such as diarrhea, nausea, vomiting or abdominal pain. These results strongly support that the preparation of the present invention is potent in eradicating H. pylori infection from the gastric mucosa, preferably without the use of antibiotics, and does not cause any unwanted side effects.

[0036] In a third group, three other volunteers with chronic infection (symptoms present for more than one year) received standard therapy (PPI twice a day + clarithromycin (2 x 500 mg) + amoxicillin (2 x 1000 mg)) together with a gelatin capsule containing 65 mg of the formulation of the invention dissolved in 435 mg of sunflower oil for two weeks. Four and eight weeks after the end of treatment, the volunteers were tested for H. pylori stool antigen and were found to be free of infection. This confirms that the formulation of the invention does not reduce the effectiveness of standard therapy, but rather enhances it.

[0037] Moreover, unlike all currently known and used therapeutic agents in the treatment of infections caused by H. pylori, the formulations disclosed herein are completely natural compositions and do not contain any synthetic compounds, making them preferable for individuals with allergies or hypersensitivities to antibiotics.

[0038] This preparation can also be used to prevent H. pylori infection in people susceptible to gastritis and H. pylori infection. For preventive purposes, the preparation can be taken once or twice a day for 60 consecutive days in a year, one capsule per day on an empty stomach 30 minutes before a meal, i.e., 65 mg of herbal preparation per day for 60 consecutive days. Preferably, this treatment should be performed at the end of summer or winter period.

[0039] The formulations of the present invention are particularly suitable for patients with proven H. pylori infection, especially those who prefer herbal therapy instead of using antibiotics, and even more preferably for those who have been previously treated with antibiotics but have not been cured after completing the antibiotic treatment.

Claims

1. A herbal preparation comprising essential oils from summer savory (Satureja hortensis L.), Greek oregano (Origanum vulgare subsp. hirtum L.) and thyme (Thymus vulgaris L.) in a ratio of 2:1:1 respectively for use in the treatment of gastric Helicobacter pylori infection in humans, wherein the herbal preparation is for oral administration; The herbal medicines are expressed as the percentage of the total peak area of ​​the GC / MS chromatogram. about 35% to about 50% carvacrol, about 10% to about 30% gamma-terpinene; about 10% to about 25% thymol, About 8% to about 20% p-cymene 23. A herbal medicine preparation comprising:

2. Expressed as a percentage of the total area of ​​the peaks in the GC / MS chromatogram, α-terpinene, trans-β-caryophyllene, α-pinene, β-pinene, β-myrcene, linalool, 1,8-cineole, and limonene were present in the herbal preparations at 0.4% to 2.9%, respectively. The herbal formulation of claim 1, further comprising:

3. The herbal preparations, expressed as a percentage of the total peak area of ​​the GC / MS chromatogram, are: about 38% to about 48% carvacrol; about 15% to about 25% gamma-terpinene; about 10% to about 12% thymol; about 10% to about 14% p-cymene, and α-terpinene, trans-β-caryophyllene, α-pinene, β-pinene, β-myrcene, linalool, 1,8-cineole and limonene, present at 0.4% to 2.9% in herbal preparations, respectively; The herbal preparation according to claim 1 or 2, comprising:

4. The herbal preparations, expressed as a percentage of the total peak area of ​​the GC / MS chromatogram, are: Carvacrol 47.5%, γ-Terpinene 18.5%, thymol 11.9%, p-cymene 13.6%, α-Terpinene 1.55%, Trans-β-caryophyllene 1.49%, Linalool 1.24%, α-pinene 0.99%, β-myrcene 0.97%, 1,8-Cineole 0.55%, Limonene 0.46% and β-pinene 0.40% The herbal preparation according to claims 1 to 3, comprising:

5. 5. The herbal preparation according to claims 1 to 4, formulated for twice daily administration in a 65 mg dosage form.

6. 5. The herbal preparation according to claims 1 to 4, formulated for twice daily administration in a 130 mg dosage form.

7. The herbal formulation according to claims 1 to 6, further comprising a liquid or solid carrier.

8. 8. The galenical formulation according to claim 7, wherein the liquid carrier is sunflower oil.

9. The galenical formulation according to claims 1 to 8, wherein the formulation is administered in the form of a capsule.

10. The herbal preparation according to claims 1 to 9, wherein the herbal preparation is administered in combination with one or more antibiotics.

11. The herbal preparation according to claims 1 to 10, wherein the herbal preparation is formulated for administration twice daily for 30 consecutive days in a 65 mg dosage form.

12. 11. The herbal preparation according to claims 1 to 10, wherein the herbal preparation is formulated for administration in a 130 mg dosage form twice daily for 15 consecutive days followed by administration in a 65 mg dosage form twice daily for 30 consecutive days.

13. A method for preparing the herbal preparation of claim 1, comprising the steps of: - collecting plant material of summer savory (Satureja hortensis L.), Greek oregano (Origanum vulgare subsp. hirtum L.) and thyme (Thymus vulgaris L.) at the flowering stage; - naturally drying the harvested plant material at 25-30°C to form a dried plant material; - grinding the dried plant material to form a ground material; - Extracting essential oils from the ground material; - drying the essential oil obtained using anhydrous sodium sulfate; - removing sulfates from the essential oil by filtration, and - mixing essential oils to form the herbal preparation of claim 1, The obtained essential oils of Summer Savory (Satureja hortensis L.), Greek Oregano (Origanum vulgare subsp. hirtum L.) and Thymus vulgaris L. are mixed in a ratio of 2:1:1 respectively.

14. 14. The method of claim 13, wherein extracting the essential oil comprises steam distillation of the ground material.

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