PL246730B1 - Antibacterial preparation from the biomass of fallen tree leaves and method of its production - Google Patents

Abstract

The subject of the application is an antibacterial preparation from the biomass of fallen leaves of selected tree species (of natural origin) intended for use in the treatment of bacterial infections, especially bacterial infections caused by bacteria of the Staphylococcus genus, in humans and animals, and a method of producing it. The subject of the invention is also a method of obtaining an antibacterial preparation.

Description

Description of the invention

The subject of the invention is an antibacterial preparation of natural origin from the biomass of fallen leaves of selected tree species (of natural origin) intended for use in the treatment of bacterial infections, especially bacterial infections caused by bacteria of the Staphylococcus genus, in humans and animals, and a method for its production.

For thousands of years, medicinal plants have played a significant role in the treatment of many diseases, including infectious diseases. Some naturally occurring chemical compounds have served as the basis for the development of a large percentage of clinically proven drugs. Modern medicine uses plants that are rich in a wide variety of secondary metabolites, such as tannins, terpenoids, alkaloids and flavonoids, with research-proven antimicrobial properties.

The use and discovery of drugs and dietary supplements derived from plants for the treatment of infectious diseases or infections is also currently a point of interest of the medical community. Infectious diseases remain a significant cause of morbidity and mortality worldwide. Despite significant advances in microbiology and virology and the possibility of controlling microorganisms, epidemics caused by drug-resistant pathogens and previously unknown pathogens pose a huge threat to global public health. These negative health trends require a global initiative to develop new strategies for the prevention and treatment of infectious diseases, including the use of natural products.

Since the advent of synthetic antibiotics in the early 20th century, the use of plant derivatives as antimicrobial agents has become less popular. However, there are reasons why interest in the antimicrobial activity of plant extracts is growing. An important issue is the phenomenon of bacterial resistance to the antibiotics used and the growing public awareness of the overuse of traditional antibiotics. In addition, many people are interested in greater autonomy in their medical care. Plant compounds can be purchased without a prescription from herbal suppliers in natural food stores.

To date, thousands of plant species have been studied for their activity against various strains of bacteria in vitro, and many medicinal plants have been shown to be active against a wide range of Gram-positive and Gram-negative bacteria. Medicinal plants offer significant potential for the development of new antibacterial therapies and adjunctive therapies to antibiotic therapy.

The use of so-called "natural antibiotics" of herbs or plants is also known, among which we find everyday food products to which folk tradition attributes antibacterial or antioxidant properties. Before antibiotics appeared on the market, it was precisely this type of product that was used to combat diseases. We can include: garlic, honey, oregano oil, ginger, cranberries, turmeric, or horseradish. Currently, the products mentioned are used as supporting agents, accompanying other medications in order to increase immunity, mainly during seasonal ailments such as colds, runny nose, or sore throat.

Bacterial infections are one of the most serious problems of humanity, which particularly affects underdeveloped regions and is a significant factor in reducing the quality of life and increasing mortality. The widespread, often unjustified use of antibiotics deepens this problem. New strains of bacteria resistant to the drugs used are appearing, which additionally complicates the fight against infections.

The problem of antibiotic-resistant bacterial strains is currently very widely discussed in the scientific community, medical institutions and public media. There is a well-founded opinion that at the current stage of medical development, a serious problem may arise in the short term with the invention of an effective synthetic bactericidal agent that would allow for safe treatment of patients, disinfection of hospitals and treatment of animals. Hence, there is a constant demand for alternative products, especially of natural origin.

Staphylococcus bacteria are considered to be among the most common and dangerous bacteria. These microorganisms can cause dozens of different diseases, from skin and respiratory diseases to damage to the nervous system and sepsis.

The practical use of plants or their parts for medical or technical purposes is widely known. It has also become the subject of intellectual property protection. The use of plant leaves as a natural raw material in various applications is known, and so from the description CN 105668684 A the use of phoenix leaves as an adsorbent of antibiotics and/or organic pollutants in sewage is known. These leaves do not require special treatment, which means that they are easily separated from the water body, and no secondary pollutants are created in the adsorption process. From another description of the invention DE 10345343 an extract of ivy leaves with anti-inflammatory action is known.

From the publication of Amina Cherif Haouat et al. "Antimycobacterial activity of Populus alba leaf extracts", Journal of Medicinal Plant Research, Vol. 7(16), p. 1015-1021, 25 April, 2013, there is known plant material in the form of branches with leaves, which was collected in Morocco in the month of March. From the presented material it is clear that the tested extract was prepared from leaves collected directly from the tree.

Similarly, in the article by Sabina Dahija et al. "Antimicrobal and cytotoxic activity of Alnus glutinosa (L.) Gaertn, A incana (L.) Moench, and A.virdis (Chaix) DC. Extracts", Journal of Health Science 2016;6(2): 1-5, it was revealed that the leaves were also obtained directly from the tree in the month of June.

Both of the above publications disclose biological material in the form of leaves, which were collected directly from trees, i.e. green leaves. On the other hand, the subject of our invention is material from fallen leaves collected in autumn in Poland.

In the indicated publications there is no mention or information about preparations prepared from fallen leaves. This is a key difference, because you cannot compare leaves (as such) just because they come from the same trees. They are biological material, their structure and chemical content change, which is why it is so important when and how they are collected. Our material is naturally fallen leaves.

Depending on the season, and therefore the environmental conditions in which the plant lives, its metabolic processes are variable, because the plant adapts to the living conditions. Therefore, if the metabolic processes undergo dynamic changes, then also the content of metabolites (chemical compounds) in plant tissues varies depending on, among others, the season. Therefore, a leaf picked directly from the tree is characterized by a different content of metabolites compared to a naturally fallen leaf. Therefore, in these two cases of leaves, a different set of chemical compounds is responsible for antibacterial activity. They are not the same, and extracts obtained from green/fallen leaves and collected in spring/autumn do not have analogous properties.

It will be similar with the concentration, where the antibacterial concentration of 160 milligrams/millilitre contained in the above article is more than a thousand times higher than the MIC values obtained by the inventors of the present invention, which are 62.5-125 micrograms/millilitre, and such a difference cannot be described as analogous properties. Therefore, the preparation of the applicant of the subject invention is much more active.

The product that is the subject of this invention, as a preparation from the biomass of fallen leaves, fits perfectly into this current trend, constituting an effective tool in the fight against bacterial infections.

Due to the unique, unusual source of raw material, which is the waste biomass of fallen leaves, the product in question has an advantage over other natural antibacterial preparations.

The research results obtained in the subject invention indicate the possibility of using a preparation obtained from the biomass of fallen leaves of selected tree species as an antibacterial agent. The proposed bactericidal agent of natural origin is characterized by high antibacterial activity. It is important to use for its production easily available in large quantities of natural raw materials, which have not found wide practical application so far and were customarily treated as waste.

The research conducted by the team of creators focused on obtaining new substances of natural origin with antibacterial activity. One source of such substances turned out to be collected fallen leaves from two species of trees, White Poplar (Populus alba) and Black Alder (Alnus glutinosa).

The aim of the present invention is to create a preparation from the biomass of fallen leaves of White Poplar and Black Alder for use against bacterial infections.

The essence of the invention is an antibacterial preparation of natural origin, which contains:

- as an active ingredient it contains an extract from the biomass of fallen leaves of white poplar (Populus alba) and/or black alder (Alnus glutinosa),

- is in the form of a dry residue obtained after extraction of white poplar and/or black alder leaves and evaporation of the extract,

- contains dry residue obtained from the extract of White Poplar leaves in the MIC concentration range of 15-62.5 pg/ml,

- contains dry residue obtained from the extract of Black Alder leaves in the MIC concentration range of 31-62.5 pg/ml.

The subject of the invention is also an antibacterial preparation of natural origin as defined above for use in bacterial infections, especially bacterial infections caused by bacteria of the Staphylococcus genus, in humans and animals.

Preferably, the preparation is used to treat bacterial infections caused by Staphylococcus aureus bacteria.

Preferably the preparation is intended for oral administration.

Preferably, the preparation is intended for topical use in the form of an ointment or solution, including micellar solutions, emulsions and suspensions.

The preparation is preferably used as a dietary supplement.

The preparation is preferably used as a cosmeceutical.

Preferably the preparation is in liquid form.

The subject of the invention is also a method for obtaining the preparation defined above, which comprises:

a) drying the biomass of collected white poplar and/or black alder leaves at 37°C for three days,

b) grinding the dried material to obtain fine dust,

c) subjecting the dried material to a continuous extraction process using a series of organic solvents to obtain extracts selected from the group consisting of chloroform and methanol,

d) subjecting the individual extracts to a distillation process under reduced pressure at a temperature not exceeding 50°C until the solvent is completely removed,

(e) isolation of the solid residue which constitutes the actual preparation suitable for use.

Advantages of the present invention:

- has specific antibacterial activity against Staphylococcus bacteria, including clinical strains of this microorganism,

- easily available raw material in large quantities,

- use of leaves, which are waste biomass,

- high effectiveness, low toxicity,

- compliance with the principles of sustainable development,

- natural origin.

The terms used above and in the description and claims have the following meanings:

MIC - minimum concentration that inhibits bacterial growth.

Golden staphylococcus - also known as Staphylococcus aureus.

Natural origin - means from the biomass of fallen leaves of White Poplar (Populus alba) and/or Black Alder (Alnus glutinosa) trees.

Pathogen - interchangeably microorganism

Preparation - means fine dust, otherwise known as fines, or dry extract.

Table description:

Table 1 - presents the MIC values (pg/ml) of the tested preparations against selected bacterial species.

Table 2 - presents the MIC values (pg/ml) of the tested preparations against selected species of Staphylococcus bacteria.

Table 3 - shows the study of the activity of leaf preparations on clinical isolates of Staphylococcus aureus bacteria.

The invention is illustrated by the following non-limiting embodiments.

Example 1

The research was carried out on leaves collected from various sites in November 2014 and 2015 (two seasons).

The collected leaves were dried at 37°C for three days. Then the dried material was ground to obtain fine dust (fines), from which extracts were obtained in the process of continuous extraction with a series (sequence) of organic solvents selected from the group consisting of hydrocarbons, chlorinated derivatives, alcohols, ethers, esters, volatile organic acids. Preferably, the organic solvent is hexane (or a fraction of petroleum with a boiling point in the range not exceeding from 30 to 150°C), chloroform, methanol.

The individual extracts were subjected to a distillation process under reduced pressure at a temperature not exceeding 50°C until the solvent was completely removed. The obtained residues (preparations) were used in further biological studies. In order to determine the antibacterial potential, the MIC values were determined for each of the two preparations using the standard microdilution method recommended by the Clinical and Laboratory Standards Institute (CLSI). The obtained results clearly indicate that the preparations obtained by the authors most strongly inhibit the growth of one of the tested bacterial species, Staphylococcus aureus (Table 1).

Due to such strong selectivity of the preparations, further studies were conducted, but they were limited to bacteria of the staphylococcus genus. The two species most susceptible to the preparations were Staphylococcus aureus and Staphylococcus auricularis (Table 2).

Example 2

Toxicological analysis of leaf preparations

The analysis was performed on three human cell lines (HaCaT, BJ and HepG2) and on Danio rerio fish used as a model.

Cell studies have shown that the tested preparations from poplar leaves in the concentration range of 1-200 μg/ml did not show any toxic effects. Preparations from black alder did not show any significant toxic effects on cells in the concentration range of 1 to 30 μg/ml.

Studies on fish have shown that poplar preparations do not affect fish development in concentrations of up to 500 mg/ml. Alder preparations are safe for fish in concentrations of up to 100 μg/ml.

Example 3

Study of the activity of leaf preparations on clinical isolates of Staphylococcus aureus bacteria

The results are presented in Table 3

MIC values are expressed in μg/ml, - means no activity, leaf preparations were tested in the concentration range of 3.9-1000 μg/ml, antibiotics were used in concentrations of 1.9-500 μg/ml, 0.19-50 μg/ml, 0.01-5 μg/ml. Antibiotics were used as positive controls.

The obtained results indicate that the tested preparations inhibit the growth of the clinical isolates tested.

The preparation obtained:

- is used to treat bacterial infections in humans and animals. Due to the lack of toxic effects on living organisms, in a preferred embodiment of the invention the preparation is intended for oral administration.

- can also be used for top application in the form of an ointment or solution, including micellar solutions, emulsions and suspensions.

- can be used as a dietary supplement or cosmeceutical.

- the dry residue preparation may be administered per se, for example in a gelatin capsule or wafer capsule.

- the preparation can also be administered in tablet form, in combination with excipients approved for use in humans, such as fillers, binders, disintegrants, preservatives, glidants, anti-adhesive substances and others used in the art of drug preparation and known to those skilled in the art.

- the preparation may also be in the form of a liquid, for example an elixir, syrup or suspension, containing, in addition to the dry residue, a liquid carrier such as water, alcohol or another liquid and auxiliary substances such as dispersing agents, emulsifiers, preservatives, flavourings and fragrances and others necessary to obtain a stable preparation.

Claims (9)

Patent Claims 1. An antibacterial preparation of natural origin, characterized in that - as an active ingredient it contains an extract from the biomass of fallen leaves of white poplar (Populus alba) and/or black alder (Alnus glutinosa), - is in the form of a dry residue obtained after extraction of white poplar and/or black alder leaves and evaporation of the extract, - contains dry residue obtained from the extract of White Poplar leaves in the MIC concentration range of 15-62.5 pg/ml, - contains dry residue obtained from the extract of Black Alder leaves in the MIC concentration range of 31-62.5 pg/ml. 2. An antibacterial preparation of natural origin as defined in claim 1 for use in the treatment of bacterial infections, especially bacterial infections caused by bacteria of the Staphylococcus genus, in humans and animals. 3. The preparation according to claim 2, characterized in that it is for the treatment of bacterial infections caused by Staphylococcus aureus bacteria. 4. The preparation according to claim 2, characterized in that it is intended for oral administration. 5. The preparation according to claim 2, characterized in that it is intended for topical use in the form of an ointment or solution, including micellar solutions, emulsions and suspensions. 6. The preparation according to claim 2, characterized in that it is for use as a dietary supplement. 7. The preparation according to claim 2, characterized in that it is for use as a cosmeceutical. 8. The preparation according to claim 1, characterized in that it is in liquid form. 9. A method for obtaining a preparation as defined in claim 1, characterized in that it comprises: a) drying the biomass of collected white poplar and/or black alder leaves at 37°C for three days, b) grinding the dried material to obtain fine dust, c) subjecting the dried material to a continuous extraction process using a series of organic solvents to obtain extracts selected from the group consisting of chloroform and methanol, d) subjecting the individual extracts to a distillation process under reduced pressure at a temperature not exceeding 50°C until the solvent is completely removed, e) isolating the solid residue, which constitutes the actual preparation suitable for use.