The object of the invention is a composition having an adaptogenic activity, based on an extract of Eleutherococcus senticosus and a method of preparation of a composition having an adaptogenic activity, based on an extract of Eleutherococcus senticosus.

From US20110014305 a method is known for inhibiting acetyl CoA carboxylase activity in a subject in need of such treatment, comprising administering a composition containing a therapeutically effective amount of Eleutherococcus. The Eleutherococcus extract is prepared in a process comprising the steps of: contacting Eleutherococcus with an alcohol-containing solvent in order to extract soluble materials from plant material and to remove solids, thereby obtaining the Eleutherococcus extract, indirectly concentrating the Eleutherococcus extract and isolating or separating the concentrated Eleutherococcus extract with a solvent of medium to high polarity in order to obtain the final Eleutherococcus extract. The solvent of medium to high polarity is selected from the group consisting of isobutyl alcohol, n-butanol, n-butyl acetate, chloroform, ethyl acetate or a mixture thereof.

In turn, W02017030167 discloses a memory-improving drug composition for use in neurological disorders, containing Eleutherococcus and naringenins. The composition may also contain excipients, binders, disintegrants, coating agents, glidants, dyes, flavours and stabilizers, emulsifiers, absorption enhancers, surfactants, pH regulators, preservatives, antioxidants and the like.

CN104288245 discloses a method for ultrasonic alcohol extraction of Eleutherococcus. The document CN 101357203 describes a method for preparing an anti-cancer composition, based on ultrasonic chloroform extraction of e.g. Eleutherococcus, followed by alcohol extraction of the obtained extract.

Known compositions do not always meet pharmacopoeial requirements. Research has shown that 26% of the compositions available on the market, containing E. senticosus and P. ginseng did not contain the minimum acceptable amount of active compounds. Therefore, it is the object of the invention to develop a method for preparing a composition containing the biggest possible spectrum of active substances obtained from Eleutherococcus senticosus.

A composition having an adaptogenic activity, based on an extract of Eleutherococcus senticosus contains chlorogenic acid in an amount of 2-3% by weight, protocatechuic acid in an amount of 0.01 - 0.02% by weight, eleutheroside B + E in an amount of 1-2% by weight, polyphenols in an amount of 30 - 40% by weight, flavonoids in an amount of 20 - 30% by weight, naringenin in an amount of 1 - 2% by weight. A method of preparation of a composition having an adaptogenic activity, based on an extract of Eleutherococcus senticosus according to the invention comprises preparation of ground roots of Eleutherococcus senticosus, which are then subjected to ultrasound assisted extraction with chloroform in order to isolate a lipophilic fraction. Preferably, the extraction with chloroform is carried out three times, and also preferably, each extraction is followed by decanting and filtering the extract, immersing the raw material in chloroform again and extraction.

The raw material after extraction is dried, preferably under a stream of liquid nitrogen in order to remove chloroform. The dried raw material is extracted with methanol in order to isolate the hydrophilic fraction. Preferably, the extraction is carried out for 10-30 minutes. Also preferably, the methanol extraction is repeated three times, and also preferably, each extraction is followed by decanting and filtering the extract, immersing the raw material with methanol again and extraction. The extracts obtained are concentrated, preferably in a vacuum evaporator, and lyophilised, and then combined, preferably in a ratio of 3:7 - chloroform extract : methanol extract.

Naringenin is added to the combined extracts, preferably in a ratio of 2: 1 - the combined extracts : naringenin. The combined ingredients are homogenised by known methods.

The invention is shown in an embodiment which does not limit the scope of protection.

Embodiment

10 g of ground roots of E. senticosus were subjected to ultrasound assisted extraction (UAE) three times with chloroform, 3x50 ml. After each extraction cycle (15 min), the extract was decanted and filtered, the raw material was immersed again in chloroform and extraction was carried out. After the third cycle, the raw material was dried under a stream of liquid nitrogen in order to remove chloroform, and then extracted three times with 75% methanol, 3x50 ml. After each extraction cycle (15 min), the extract was decanted and filtered, the raw material was immersed again in 75% methanol and extraction was carried out. 150 ml of chloroform extract and 150 ml of 75% methanol extract were obtained. The extracts were concentrated using a vacuum evaporator and then lyophilised. The extracts were combined and naringenin was added in a ratio (mg) of 3:7:5 (chloroform extract : 75% methanol extract : naringenin) and homogenised.

The resulting composition, containing total extract from Eleutherococcus senticosus roots with the addition of naringenin, was used to determine its effect on the induction of morphological changes in A549 cells (lung squamous cell tumour line).

In the first stage, a non-toxic dose of the composition was determined on cell line A549, for concentrations of 300, 150, 75, 37.5, 18.8, 9.4, 4.7, 2.35 pg/mL. Microscopic readings of changes in cell morphology after 72 hours of stimulation of the cells with the preparation, indicating cytotoxicity (CTE, cytotoxic effect), were made on a scale of: 0 - no cytotoxicity; 1 - up to 25%; 2 - 50%; 3 - 75%; 4 - 100% cytotoxicity. No cytotoxicity has been demonstrated for the 37.5 pg/mL dose. The value obtained was used for further research.

The proliferative activity of the composition was demonstrated by an increase in the number of human peripheral blood leukocytes, PBMCs. The resulting composition, containing total extract from Eleutherococcus senticosus roots with the addition of naringenin, was used to determine the effect on the proliferation of human peripheral blood leukocytes.

Antiviral activity of VSV (Vesicular stomatitis Virus, Indiana strain; ATTC VR-1238 ™, Rhabdoviridae)

HAdV-5 - human type 5 adenovirus (strain Adenoid 75; ATTC VR-5 ™, Adenoviridae)

Chemical analysis of the composition

Content of eleutherosides was determined by HPLC-DAD, and phenolic acids by LC-ESI-MS/MS.

Residual solvents used for extraction were determined by HPLC-DAD and GC:

- chloroform: content below detection level

- methanol: content below detection level

The method of preparation of the composition used is safe - it can be concluded that there is no binding of methanol and chloroform in other chemical structures. This is important for the safe use of the composition by humans. At the same time, the composition provides the body with a full set of extracted metabolites from the same raw material (polar and non-polar) which are responsible for the indicated adaptogenic effect.

Biological analysis of the composition, taking into account various recipe compositions.

The effect on the synthesis of pro- and anti-inflammatory cytokines (interleukin 2 and 10; IL-2 and IL-10) after stimulation of human leukocytes with the composition, at a concentration of 37.5 pg/ml, ELISA assay.

- IL-2: Any significant changes in IL-2 level were not observed (value below detection)

- IL-10: the composition did not increase the level of secreted interleukin

Antiviral and/or virucidal activity against HAdV-5 (human adenovirus type 5 (strain Adenoid 75; Adenoviridae ATTC VR-5™). AVA assay

- There was no virucidal and antiviral activity of the tested composition, at a concentration of 37.5 pg/ml, relative to HAdV-5.

Adaptogenic activity - effect on the proliferation of human peripheral blood leukocytes and determination of the degree of leukocyte proliferation after stimulation with the composition during 96h.

- The composition, at a concentration of 37.5 pg/ml, statistically significantly increased the proliferation of human leukocytes (150% of the control value).

Effect of the composition on the level of natural resistance of human peripheral blood leukocytes (cell resistance assay against infection with the VSV indicator virus). VSV (Vesicular stomatitis Virus, strain Indiana; ATTC VR-1238 ™, Rhabdoviridae)

- A statistically significant decrease in VSV replication after treatment with the composition (by 1.5 log) at a concentration of 37.5 pg/ml was found.