FORMULATIONS

TECHNICAL FIELD

The invention is related to obtaining a pharmaceutical or nutraceutical agent containing the essential oil of Rosmarinus officinalis with antimicrobial and antifungal properties.

PRIOR ART Today, natural sources such as herbal products have become very popular in the treatment of diseases.

Products obtained from plants and phytochemicals are included in the preparations used in the pharmaceutical field from past to present. There are many studies in the field of obtaining active ingredients against diseases from herbal extracts and natural substances, and it is known that Rosmarinus officinalis herbal products are conventionally preferred for their antibacterial, antiviral, and antifungal properties. However, new preparations/agents which have increased antimicrobial effects against diseases and are more effective need to be developed. Candidiasis is an infection caused by a yeast-like fungus. Its prevalence is increasing all over the world. Candida species cause a wide spectrum of infections, ranging from mucocutaneous involvement to invasive diseases that can affect any organ.

These infections range from mucosal involvement such as oropharyngeal and esophageal to more common and deeply located internal organ involvement, which is often observed in immunosuppressive patients.

Broad treatment methods are required for such a wide spectrum of diseases. There are topical and systemic treatment methods and prophylactic treatment regimens for these infections. Candida albicans is an important human pathogenic fungus. However, plant diseases are also an extremely important factor in the agricultural field, and phytopathogenic fungi belonging to different genus infect numerous crops and cause economic losses in agriculture.

In recent years, farmers all over the world have reported a decrease in the effectiveness of fungicides traditionally used to control plant diseases. At the same time, improper use of fungicides has resulted in increased fungal resistance and resistance to conventional antifungals such as currently used fungicides, benzimidazole and dicarbomosides have developed. To overcome this resistance, it is extremely important to discover new antifungal agents that can replace current control strategies. For these reasons, it is necessary to develop new active agents against microbial pathogens, especially Candida albicans and fungi such as Fusarium monili forme, Fusarium culmorum, Botrytis cinerea, which are important plant pathogens.

As a result, all the problems mentioned above made it necessary to make an innovation in the related technical field. BRIEF DESCRIPTION OF THE INVENTION

The present invention is related to the production of a pharmaceutical or nutraceutical preparation/agent to eliminate the above-mentioned disadvantages and to bring new advantages to the related technical field.

An object of the invention is to provide a pharmaceutical or nutraceutical preparation/agent with antimicrobial and antifungal properties.

An object of the invention is to provide the production of a pharmaceutical or nutraceutical preparation/agent in the form of a microemulsion with antimicrobial and antifungal properties.

To fulfill all the objects mentioned above and that will emerge from the detailed description below, the present invention is related to the production of a pharmaceutical or nutraceutical preparation/agent. Accordingly, said pharmaceutical or nutraceutical preparation/agent contains Rosmarinus officinalis essential oil. Thus, a pharmaceutical or nutraceutical agent with antimicrobial and antifungal properties can be obtained.

In the possible embodiment of the invention, the pharmaceutical or nutraceutical agent with the essential oil of Rosmarinus officinalis is in the form of a microemulsion. In this way, it is ensured that the microbial inhibition of the essential oil is increased.

The invention also is related to the production of a pharmaceutical or nutraceutical preparation/agent in the form of a microemulsion, which includes the process steps of

• Drying,

• Powdering

• Obtaining essential oil by distillation of plants containing the essential oil of Rosmarinus officinalis.

Said production of pharmaceutical or nutraceutical preparation/agent in the form of the microemulsion is characterized by obtaining the desired microemulsion formulation with the following process steps;

_® Mixing the essential oil substance in the range of 0.1-5% by weight with isopropyl myristate, which is oil in the range of 25-45% by weight,

® Adding thereto a substance selected from Span 60 and Chromophore EL substances or a combination of these as a surfactant in the range of 5-30% by weight,

_® Adding ethanol in a proportion of 35-50% by weight as a co-surfactant and one or a combination of distilled water substances as water ® mixing the prepared microemulsion formulation.

Thus, the production method of the pharmaceutical or nutraceutical agent in the form of microemulsion with antimicrobial and antifungal properties is provided.

In the possible embodiment of the invention, the microemulsion formulation of the essential oil obtained from the Rosmarinus officinalis plant is used as an antimicrobial and antifungal preparation/agent.

BRIEF DESCRIPTION OF THE FIGURE

Figure 1 shows the graph of the test results performed with the Poisoned PDA technique on the comparative % mycelial growth rates of the essential oil and microemulsion formulation. DETAILED DESCRIPTION OF THE INVENTION

In this detailed description, the subject of the invention is related to a pharmaceutical or nutraceutical preparation/agent with antimicrobial and antifungal properties; and it is explained with examples that do not have any limiting effect only for a better understanding of the subject.

Said pharmaceutical or nutraceutical preparation/agent contains essential oil obtained from the Rosmarinus officinalis plant. The essential oil of the invention is preferably obtained from the leaves of Rosmarinus officinalis.

The pharmaceutical or nutraceutical preparation/agent obtained in the invention is in the form of a microemulsion. In this way, the agent obtained in the form of the microemulsion is absorbed in the body quickly and with high efficiency. Microemulsion formulation is developed due to its various advantages such as providing controlled drug release and increasing the systemic and topical absorption of drugs. Microemulsions are highly advantageous systems compared to other drug forms. With the microemulsion formulation, the absorption of the drug is much higher and faster than other forms of drugs such as tablets and capsules. Also, due to the surfactant in its structure, it increases membrane fluidity, thus permeability, thus increasing drug absorption.

The pharmaceutical or nutraceutical preparation/agent of the invention is transformed into a microemulsion formulation of the essential oil after drying and powdering the leaves obtained from Rosmarinus officinalis plants. Said microemulsion form comprises an oily phase containing component, an aqueous phase containing component, one or two surfactants, and a co-surfactant component. The production of pharmaceutical or nutraceutical preparation/agent in microemulsion form is characterized by comprising the following process steps; i. Obtaining essential oil from the powdered plant, ii. Mixing the essential oil obtained in step (i) with another oil, iii. Adding co-surfactant, surfactant, and water. As the oil mentioned in step (ii), there may be at least one or mixtures of certain proportions of vegetable oils, synthetic oils, triglycerides, esters of fatty acids in certain proportions by weight. In a possible embodiment of the invention, isopropyl myristate is preferred as the oil phase. Thus, a non-toxic, biologically suitable oil was used.

Water, sodium chloride solution, buffers, propylene glycol, polyethylene glycols, or combinations thereof can be used in certain proportions by weight as said aqueous phase in step (iii). In a possible embodiment of the invention, Rosmarinus officinalis hydrosol is used as the aqueous phase.

At least one of alcohols, derivatives of glycols, polyglycerols, and propylene glycols in certain proportions by weight is used as the co-surfactant mentioned in step (iii). In a possible embodiment of the invention, ethanol is used as a co-surfactant. Thus, both solvent and protective properties were provided in the formulation.

As the surfactant mentioned in step (iii), non-ionic and ionic surfactants, lecithin, polyglycerol, fatty acid esters can be used in certain proportions by weight. In a possible embodiment of the invention, span 60 and chromophore EL were used as surfactants. Span 60 and Chromophore-EL are non-toxic materials and were preferred in the formulation.

In a possible embodiment of the invention, the production of pharmaceutical or nutraceutical preparation/agent in the form of microemulsion containing Rosmarinus officinalis essential oil is provided by the process of obtaining the desired microemulsion formulation with the following process steps;

_® Mixing the essential oil substance in the range of 0.1-5% by weight with isopropyl myristate, which is an oil in the range of 5-45% by weight, ® adding thereto a substance selected from Span 60 and Chromophore EL substances or a combination of these as a surfactant in the range of 5-50% by weight,

_® adding ethanol in a proportion of 25-50% by weight as a co-surfactant and one or a combination of distilled water substances as water ® mixing the prepared microemulsion formulation.

In a possible embodiment of the invention, in the Rosmarinus officinalis microemulsion formulation; the microemulsion was prepared by using Rosmarinus officinalis essential oil in the range of 0.1% -5% by weight, isopropyl myristate in the range of 5 g - 45 g, Span 60 in the range of 5 g - 25 g, Chromophore EL in the range of 1 g - 15 g, ethanol in the range of 25 g-50 g, distilled water in the range of 0.5 g - 20 g in 100 g of microemulsion formulation.

Example

Aboveground parts of R. officinalis were collected from Beykoz/lstanbul in 2018 and dried under suitable conditions in the shade. Herbarium samples are stored in Istanbul Medipol University Herbarium. The dried samples were powdered and R. officinalis essential oil was obtained in a Clevenger device.

Microemulsion formulation containing Rosmarinus officinalis essential oil was obtained by weighing another oil and Rosmarinus officinalis essential oil, adding surfactant and co-surfactant, and mixing with the last addition of water and its preparation. The titration method was used to create the microemulsion formulation. The mixtures of oil, surfactant, and co-surfactant were titrated at 25°C at 750 rpm using hydrosol. Triangle Phase Diagram Analysis Software was used to create a phase diagram. The microemulsion was formulated using isopropyl myristate as the oil phase and span 60 and chromophore EL was used as the surfactant, while ethanol was preferred as the co-surfactant and distilled water was preferred as the water. R. officinalis essential oil was dissolved by mixing with the oil in the formulation and added with stirring.

In the study, the antifungal activity of microemulsion formulations of essential oils obtained from Rosmarinus officinalis leaves against C. albicans, B. Cinerea, and different Fusarium species were determined.

The mycelial growth inhibitory activity of R. officinalis essential oil and R. officinalis microemulsion formulation against the important plant pathogens Fusarium moniliforme NRRL 2374, Fusarium culmorum NRRL 3288, and Botrytis cinerea AHU 9424 was examined comparatively.

Candida albicans ATCC 64548 strain was grown in RPMI broth and antifungal activity experiments were performed by microdilution method. The antifungal activity of plant pathogenic fungi was carried out by measuring their mycelial growth. A poisoned PDA technique has been used for this.

Table 1 shows the results of the analysis performed by Gas Chromatography-Mass Spectrometry (GC-MS), which is an analytical chemistry method, to determine the different compounds in the substance of R. officinalis essential oil. The main components of R. officinalis essential oil were identified as camphor (19.5%), verbenone (11.1%), borneol (8.3%), a-pinene (6.5%), and linalool (5.5%).

RRI Compound %

1014 Tricyclene 0.1

1032 a-Pinene 6.5

1072 a-Fenchene tr

1076 Camphene 1.5

1118 b-Pinene 0.4

1135 Thuja-2, 4(10)-diene 0.6

1159 d-3-Carene 1.1

1174 Myrcene 0.9

1176 a-Phellandrene 0.1

1183 p-Mentha-1,7(8)-diene (=Pseudolimonene) tr 1187 o-Cymene 0.1

1188 a-Terpinene 0.2

1203 Limonene 2.7

1213 1 ,8-Cineole 12.0

1225 (Z)-3-Hexenal tr

1244 2-Pentyl furan tr

1246 (Z)-p-Ocimene tr

1255 y-Terpinene 0.1

1265 3-Octanone 0.1

1266 (E)-p-Ocimene tr

1280 p-Cymene 1.5

1290 Terpinolene 0.2

1391 (Z)-3-Hexanol 0.1

1400 Nonanal 0.1

1445 Filifolone 0.2

1450 frans-Linalool oxide (Furanoid) 0.1

1452 a,r-Dimethylstyrene 0.1

1452 1-Octen-3-ol 0.4

1478 c/s-Linalool oxide (Furanoid) tr

1493 a-Ylangene 0.1

1497 a-Copaene 0.1

1499 a-Campholene aldehyde 0.1

1532 Camphor 19.5

1536 Pinocamphone 3.7

1541 Benzaldehyde tr

1553 Linalool 5.5

1571 frans-p-Menth-2-en-1 -ol 0.1

1586 Pinocarvone 0.4

1591 Bornyl acetate 0.4

1611 Terpinen-4-ol 1.3

1612 b-Caryophyllene 0.7

1638 c/s-p-Menth-2-en-1 -ol 0.1

1663 c/s-Verbenol 0.2 1670 trans- Pinocarveol 0.2

1683 frans-Ve rbenol 0.8

1687 a-Humulene 0.2

1700 p-Mentha-1,8-dien-4-ol (=Limonen-4-ol) 0.1

1704 g-Muurolene 0.1

1706 a-Terpineol 4.5

1719 Borneol 8.3

1725 Verbenone 11.1

1751 Carvone 0.2

1773 d-Cadinene 0.1

1805 a-Campholene alcohol 2.2

1823 p-Mentha-1(7),5-dien-2-ol tr

1838 (E)-p-Damascenone tr

1845 trans- Carveol 0.2

1864 p-Cymen-8-ol 0.4

1865 Isopiperitenone 0.1

1868 (E)-Geranyl acetone 0.3

1872 cis-Myrtanol 0.1

1879 trans- Myrtanol 0.1

1941 a-Calacorene tr

1949 Piperitenone 0.2

2008 Caryophyllene oxide 0.6

2029 Perilla alcohol 0.1

2030 Methyl eugenol 0.2

2045 Humulene epoxide-l tr

2071 Humulene epoxide-ll 0.2

2073 b-Caryophyllene alcohol tr

2088 1 -ep/-Cubenol 0.1

2161 Muurola-4,10(14)-dien-1-ol tr

2186 Eugenol 0.1

2187 T-Cadinol 0.1

2209 T-Muurolol tr

2211 Clovenol 0.1 2239 Carvacrol 0.1

2232 a-Bisabolol 0.1

2255 a-Cadinol 0.1

2324 Caryophylla-2(12),6(13)-dien-50-ol 0.2

(=Caryophylladienol II)

2365 (Z)-Methyl jasmonate 0.1

2384 Farnesyl acetone tr

2389 Caryophylla-2(12),6-dien-5a-ol ( =Caryophyllenol 0.50.2 /)

2392 Caryophylla-2(12),6-dien-5p-ol ( =Caryophyllenol

II)

2931 Hexadecanoic acid tr

Monoterpene Hydrocarbons 28.0

Oxygenated Monoterpenes 49.2

Sesquiterpene Hydrocarbons 12.4

Oxygenated Sesquiterpenes 2.3

Fatty acids tr

Others 1.4

Total 93 ^~ 3

RRI Relative retention indices calculated against n-alkanes % calculated from FID data tr Trace (< 0.1 %)

Table 1. GC-MS analysis results of R. Officinalis essential oil

In Table 2 below, the antimicrobial properties of R. officinalis essential oil and microemulsion formulation on C. albicans fungus, which is a sexually reproducing, diploid, yeast type fungus and which is a cause of oral and vaginal opportunistic infections in humans, were indicated.

Table 2. Antimicrobial activity (MIC values) of R. officinalis essential oil and its microemulsion formulation

When the results were examined comparatively, the antimicrobial effectiveness of the microemulsion formulation against C. albicans was given as the MIC value. The MIC value is the minimum inhibitory concentration and is considered to be the lowest concentration of antimicrobial agent that prevents the increase in cell number. When the antimicrobial activity results of R. officinalis essential oil and its microemulsion formulation are given in Table 2 are examined, it is seen that the microemulsion formulation as MIC has significantly increased against C. albicans compared to pure essential oil. While the MIC value of pure essential oil against C. albicans is 62.5 pg/mL, it is seen to be found as 15.6 pg/mL when it is made into microemulsion formulation. Therefore, Rosmarinus officinalis substance, which has been formed into microemulsion formulation, provides a much more effective antimicrobial antifungal agent compared to essential oil. Although the anti-candidal activity of pure R. officinalis essential oil was studied, as seen in this example, the MIC value of the essential oil was increased by 4 times by making a microemulsion formulation with the present invention, and it was made into a more effective formulation for use as an anticandidal preparation/agent.

In a possible embodiment of the invention, the mycelial growth inhibition of R. officinalis essential oil and its microemulsion formulation against plant pathogens was also determined comparatively. It is seen that a selective inhibition of approximately 50% is achieved as given in Figure 1. The micellar growth of F. culmorum, B. cinerea, and F. muniliforme pathogens was 50%, 33%, and 42%, respectively, when pure essential oil was used, while this ratio decreased to 12%, 15%, and 33%, respectively, when using essential oil formulated in the form of a microemulsion. As determined in the results, the microemulsion formulation of R. officinalis essential oil can be used as a pharmaceutical, nutraceutical preparation/agent, or pesticide with much more effective antimicrobial and antifungal properties than the essential oil. The antifungal activity of microemulsion formulation and pure essential oil against plant pathogenic fungi was investigated by measuring the mycelial growth of fungi with the poisoned PDA technique. For this, the medium and the substance were mixed and poured into Petri dishes. Then, the frozen and drug-treated medium was taken from the freshly developed fungus medium at a diameter of 4 mm and placed in the center of the Petri dish. After a week of incubation, the mycelial growth diameters of fungi were measured at the end of one week. It is thought that the substance that inhibits mycelial growth most can be used as an antifungal agent against plant pathogens. Mycelial growth inhibition means preventing the spread of the said fungus. Ketoconazole was used as a positive control and DMSO (Dimethyl sulfoxide) was used as a negative control. While there was no micellar growth in ketoconazole, mycelial growth was determined as 100% in DMSO.

The scope of protection of the invention is specified in the attached claims and it cannot be limited to what is explained in this detailed description for the sake of the example. A person skilled in the art can provide similar embodiments in the light of the above, without departing from the main theme of the invention.