PL248921B1 - Composition of hemp extract with cyclodextrin, method of obtaining it, use of the composition of hemp extract with cyclodextrin and cosmetic formulation - Google Patents

Abstract

The subject of the application is a composition of hemp extract with cyclodextrin and a method for its preparation, which is applicable in the pharmaceutical and cosmetic industries, and in the production of dietary supplements. The application also concerns the use of the composition of hemp extract with cyclodextrin as a cosmetic agent and cosmetic formulation. The composition of hemp extract with cyclodextrin is characterized in that the mass ratio of hemp extract to cyclodextrin is from 2:1 to 1:2, respectively. The cyclodextrin is selected from the group: alpha-cyclodextrin, beta-cyclodextrin, gamma-cyclodextrin, and hydroxypropyl-beta-cyclodextrin. The hemp extract is obtained by extraction of hemp raw material with supercritical CO2.

Description

Description of the invention

The invention concerns a composition of hemp extract with cyclodextrin and a method for its preparation, which finds application in the pharmaceutical and cosmetic industries, as well as in the production of dietary supplements. The invention also relates to the use of the composition of hemp extract with cyclodextrin as a cosmetic agent and cosmetic formulation.

International patent application number WO20028897A1 discloses water-soluble cannabinoid compositions in which the cannabinoid is complexed (inclusion complexes) with a cyclodextrin (the possibility of using alpha-, beta-, gamma-cyclodextrin is mentioned) and methods for preparing water-soluble cannabinoid complexes in which a reaction mixture comprising a cannabinoid component and a cyclodextrin component, uniformly dispersed in a polar solvent (e.g., water), is heated to a temperature just below the melting point of the cannabinoid component. A composition is disclosed in which the cyclodextrin component and the cannabinoid component may be in a molar ratio of from about 1:1 to about 10:1, from about 1:1 to about 5:1, from about 1:1 to about 4:1, from about 1:1 to about 3:1, or from about 1.5:1 to about 3:1. The document does not, however, disclose a method for obtaining the cannabinoid composition.

International patent application number WO20128767A1 discloses water-soluble compositions containing complexes of alpha-cyclodextrin and cannabinoids and/or complexes of alpha-cyclodextrin and terpenes, as well as methods for producing these compositions and their use in food products and as therapeutics. However, the document exclusively uses alpha-cyclodextrin and a composition using an alpha-cyclodextrin solution containing from about 15% w/v to about 40% w/v alpha-cyclodextrin. The patent document describes, among other things, a beverage containing a composition of an alpha-cyclodextrin complex with a cannabinoid. This beverage was tested on a group of eight volunteers, and none of them detected the taste or smell of cannabis and/or terpenes. However, patent document number WO20128767A1 does not mention a method for obtaining the cannabinoid composition. It is mentioned, however, that cannabinoids derived from plant extracts or obtained synthetically may be used.

A composition based on cannabinoids, hyaluronic acid, and certain herbal extracts for use in improving skin health is known from international patent application number WO22266767A1. The disclosed composition also contains gamma-cyclodextrin and derivatives thereof as an additive to improve the solubility of cannabinoids. In the disclosed composition, the solubility additive is used in a range of between 5 x 10 ^-3 and 7% by weight or 5 x 10 ^-3 and 15% by weight. The document does not provide information on the origin of the cannabinoid extract or its components.

U.S. Patent Application No. US2021401736A discloses a chewable gel composition comprising a gelling agent in an amount sufficient to provide a coherent gel product, a cannabinoid agent, and a complexing agent, wherein the complexing agent is selected to reduce the bitterness of the cannabinoid agent by forming a complex with the agent. Cyclodextrin is used as the complexing agent in an amount of at least 0.1%. The document provides no information on the origin of the cannabinoid extract or its components.

International patent application number WO17161387A1 discloses compositions and methods for treating and/or preventing diseases using natural extracts, their derivatives, or their components. Among the extracts are cannabinoid extracts. WO17161387A1 contains a recipe for extracting cannabinoid components with supercritical CO2 and using cyclodextrins to increase the solubility of the cannabinoid component and improve its activity. A composition is also described, which may be in the form of a cosmetic cream. However, the parameters of the supercritical CO2 extraction process described in WO17161387A1 are different from those described in this application. Furthermore, the document uses hydroxypropyl-alpha-cyclodextrin or hydroxypropyl-beta-cyclodextrin, and the process parameters for obtaining combinations with cyclodextrin are different from those described in this application.

Chinese patent document No. CN110787070A describes a method for preparing cannabis leaf extract, a method for preparing microcapsules containing the extract, and their use in cosmetics. The method for preparing cannabis leaf extract includes the following steps: mixing powdered and undried cannabis flowers and leaves with an alcohol solution and performing ultrasonic extraction at a temperature no higher than 60°C to obtain the extract.

The microcapsule preparation method involves the following steps: mixing beta-cyclodextrin, gum arabic monoglyceride, and hemp flower leaf extract to obtain an aqueous solution, homogenization, and spray drying. Furthermore, document CN110787070A discloses a cosmetic containing prepared ingredients in the form of microcapsules, which, when applied to the skin, has a restorative effect. The document uses a classic extraction method using an organic solvent, which is undoubtedly a disadvantage of this solution.

A method for obtaining hemp extract based on supercritical CO2 extraction is also known from patent document number WO21183467A1. However, the document does not mention the use of cyclodextrins.

The aim of the invention was to develop a hemp extract composition with cyclodextrin and a method for obtaining it. This would effectively extract and retain active ingredients from the hemp raw material within the cyclodextrin structure, creating a cyclodextrin-complete hemp extract system. Additionally, it was decided to ensure that active ingredients, such as cannabinoids and terpenes, were absorbed and stored within the cyclodextrin, improving their stability and bioavailability.

In another aspect, the aim of the invention was to develop an effective method for obtaining a hemp extract composition with cyclodextrin, which utilized a supercritical extraction technique based on the use of supercritical carbon dioxide (CO2), which enabled efficient and ecological extraction of active substances from the hemp raw material. A simple process was also developed for preparing the hemp extract-cyclodextrin combination, which does not require advanced equipment and is based on grinding hemp extract prepared using supercritical CO2 extraction with cyclodextrin without a solvent, and then drying the resulting mixture.

Another aspect of the invention was the use of a hemp extract-cyclodextrin composition as a cosmetic agent. Experimental studies have shown that the composition according to the invention is fully stable, exhibits higher antioxidant activity, and demonstrates significantly higher inhibition of selected pro-inflammatory enzymes, as well as higher permeability through a skin-imitating membrane compared to the pure extract. This means that the cyclodextrin-complete hemp extract system can be effectively used in the pharmaceutical and cosmetic industries, as well as in the production of dietary supplements. Furthermore, the developed combination guarantees complete physicochemical stability of the resulting cyclodextrin-complete hemp extract system. This means that the active ingredients remain intact during the extraction and storage processes, ensuring long-lasting and consistent product quality.

The essence of the invention is a composition of hemp extract with cyclodextrin characterized in that the mass ratio of hemp extract to cyclodextrin is from 2:1 to 1:2, respectively, the cyclodextrin is selected from the group: alpha-cyclodextrin, beta-cyclodextrin, gamma-cyclodextrin, hydroxypropyl-beta-cyclodextrin, and the hemp extract is obtained by extraction of hemp raw material with supercritical CO2.

Preferably, the composition is characterized in that the hemp extract has a cannabinoid and terpene profile that contains, expressed as % by weight relative to the total weight of the extract, the following components:

- CBD in amounts from 3.185 to 6.682,

- CBG in an amount from 0.001 to 0.008,

- CBDA in amounts from 0.121 to 3.340,

- CBN in the amount from 0.001 to 0.007,

- THC in the amount of 0.004 to 0.020,

- CBC in the amount from 0.001 to 0.038,

- THCA in the amount of 0.001 to 0.064,

It is particularly preferred that the mass ratio of hemp extract to cyclodextrin is 1:1.

Preferably, the hemp raw material is a hemp panicle.

Hemp panicles contain a higher concentration of valuable phytocannabinoids, such as CBD (cannabidiol) and CBG (cannabigerol), compared to leaves or stems, meaning that panicle extracts may contain more compounds with potential medicinal properties. Supercritical CO2 extraction allows for the controlled separation of desirable substances from unwanted contaminants. Using panicles in the extraction process can help minimize raw material waste because the entire plant is used, which is more ecologically sustainable. Furthermore, panicles are less contaminated than leaves or stems, resulting in a higher-quality extract. Furthermore, hemp panicles contain a higher concentration of cannabinoids, meaning the extract is more concentrated. This can lead to greater efficiency and cost savings in long-term use compared to the content of these active compounds in the leaves alone. Substances present in parts of the hemp panicle, such as terpenes and cannabinoids, can work together in what is known as a "co-extraction" (co-extraction). entourage effect, which means that they are more effective in treating certain diseases compared to isolated cannabinoids.

It is advantageous if the hemp raw material is previously crushed into fractions with a size ranging from 180 to 500 gm.

It is particularly advantageous when the hemp raw material is previously crushed to a fraction with a size in the range of 250-355 gm.

The fragmentation of the raw material affects the efficiency of the hemp extraction process. An increase in the content of active compounds was observed in the analyzed fractions with increasing fragmentation, up to fractions of 250-355 gm. Further fragmentation of the raw material negatively affected the content of active compounds in the analyzed fractions.

Preferably, the cyclodextrin is alpha-cyclodextrin.

Preferably, the extraction of hemp raw material with supercritical CO2 is carried out at a temperature of 30-70°C and at a pressure in the range of 6894757.29-41368543.76 Pa (1000-6000 psi).

It is particularly advantageous when the extraction of hemp raw material with supercritical CO2 is carried out at a temperature of 30-50°C and at a pressure in the range of 20684271.88-24131650.53 Pa (from 3000 to 3500 psi).

Most preferably, the extraction of hemp raw material with supercritical CO2 is carried out at a temperature of 50°C and a pressure of 22339013.63 Pa (3240 psi).

In another aspect, the invention also relates to a method for obtaining a composition of hemp extract with cyclodextrin, characterized in that it is carried out in the following steps: a) the hemp raw material is crushed to a particle size ranging from 180 to 500 gm; b) the crushed hemp raw material is subjected to extraction with supercritical CO2 at a temperature ranging from 30 to 70°C and at a pressure ranging from 6894757.29-41368543.76 Pa (1000-6000 psi); c) the obtained extract from the hemp raw material is combined with cyclodextrin in a mass ratio of 2:1-1:2 for a time ranging from 5-115 min.

Preferably, the hemp raw material is a hemp panicle.

Preferably, the hemp raw material is crushed to a particle size ranging from 250 to 355 gm.

Preferably, the supercritical CO2 extraction process is carried out in the temperature range of 30 to 50°C and at a pressure in the range of 20684271.88-24131650.53 Pa (3000 to 3500 psi).

It is particularly advantageous when the supercritical CO2 extraction process is carried out at a temperature of 50°C and a pressure of 22339013.63 Pa (3240 psi).

Preferably, the obtained hemp extract is combined with cyclodextrin in a 1:1 mass ratio for 60 minutes. It has been shown that as the preparation time of the system increases, the antioxidant activity of the system decreases.

It is particularly preferred when alpha-cyclodextrin is used as the cyclodextrin.

Preferably, the extract obtained from the hemp raw material is combined with cyclodextrin by grinding the cyclodextrin with the hemp extract for 60 minutes.

In yet another aspect, the invention also relates to the use of the composition as a cosmetic agent.

Preferably, the cosmetic formulation is characterized in that it contains a composition of hemp extract with cyclodextrin described above in an amount of 1 to 4% by weight and an oil in an amount of 5 to 60% by weight selected from the group: argan, jojoba or macadamia, ceramides in an amount of 5% by weight, a complex of three EAC vitamins in an amount of 3% by weight and a mixture of glyceryl caprylate/caprate in pentylene glycol (E-Leen Green C) in an amount of 2% by weight.

The primary advantage of the invention is the use of a supercritical CO2 extraction process, which enables the efficient and ecological extraction of active substances from hemp raw material without the need for solvents, particularly organic solvents. A key advantage of the invention is the use of hemp panicles as the raw material, which contain a higher amount of valuable phytocannabinoids, such as CBD (cannabidiol) and CBG (cannabigerol), compared to leaves or stems. Another advantage of the invention is that the preparation of the hemp extract-cyclodextrin system according to the invention does not require advanced equipment. This system is fully stable and characterized by improved activity thanks to the use of extract grinding.

PL 248921 Β1 hemp extract with cyclodextrin. These beneficial properties translate into potentially broad applications for the extract according to the invention. Additionally, the effective extraction and retention of active ingredients from the hemp raw material within the cyclodextrin structure is ensured, creating a cyclodextrin-complete hemp extract system. This means that active substances, such as cannabinoids and terpenes, are absorbed and stored within the cyclodextrin, improving their stability and bioavailability. It is also noteworthy that the biological activity of the resulting cyclodextrin-complete hemp extract system is improved. Experimental studies have shown that this system exhibits higher antioxidant activity and a significantly higher ability to inhibit selected pro-inflammatory enzymes compared to the pure extract. This means that the cyclodextrin-complete hemp extract system according to the invention can be effectively used in the pharmaceutical and cosmetic industries, as well as in the production of dietary supplements. The fundamental advantage of this invention is the complete physicochemical stability of the resulting cyclodextrin-whole hemp extract system. This means that the active ingredients remain intact throughout the extraction and storage processes, ensuring long-lasting and consistent product quality.

The subject of the invention is presented in more detail in the form of non-limiting embodiment examples.

Example 1. Analysis of the cannabinoid profile in the obtained hemp extracts

To determine the cannabinoid profile in the hemp extract and the content of active compounds, the HPLC method with isocratic elution was used; the parameters are presented in Table 1.

Table 1. Chromatographic separation parameters

Chromatographic separation parameter Value Stationary phase (column) CORTECS ShieldRP18 2.7 μιπ, 4.6 mm χ 150 nim Mobile phase A: 0.1% TF A (41%) B: Acetonitrile (59%) Flow rate 2.0 ml/min Column temperature 30°C Wavelength 228 nm Injection volume 10 μA

Using this method, the following compounds were determined:

- Delta-9-tetrahydrocannabinol (Delta-9-tetrahydrocannabinol) = THC,

- Delta-9-tetrahydrocannabinolic acid (Delta-9-tetrahydrocannabinolic acid) = THCA,

- Cannabidiol (Cannabidiol) = CBD,

- Cannabidiolic acid (Cannabidiolic acid) = CBDA,

- Cannabinol (Cannabinol) = CBN,

- Cannabigerol (Cannabigerol) = CBG,

- Cannabichromene (Cannabichromene) = CBC,

The cannabinoid profile in the hemp extract and the content of individual active compounds depending on the extraction conditions with supercritical CO2 and the fragmentation of the hemp raw material are presented in Table 2.

Table 2

Conditions Pressure Temperature Raw material CBD CBG CBD A CBN THC CBC THC A Sum a Wl 20684271.88 Pa (3000 psi) 30"C Not crushed 4.30 2 0.00 1 2,296 0.00 3 0.01 4 0.02 5 0.003 6,644 W2 41368543.76 Pa (6000 psi) 30°C Not crushed 3.18 5 0.00 2 1,700 0.00 2 0.01 1 0.02 0 0.001 4,921 W3 20684271.88 Pa (3000 psi) 50"C Not crushed 5.32 9 0.00 1 1,879 0.00 3 0.01 6 0.03 0 0.001 7,258 W4 41368543.76 Pa (6000 psi) 50°C Not crushed .3.91 0 0.00 3 2,436 0.00 2 0.01 3 0.02 5 0.001 6,389

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In 5 20684271.88 Pa (3000 psi) 30"C Grind. 5.64 1 0.00 1 2,578 0.00 3 0.01 9 0.03 6 0.001 8,279 W6 41368543.76 Pa (6000 psi) 30°C Grind. 5.38 6 0.00 2 2,433 0.00 2 0.01 7 0.03 1 0.001 7,873 W7 20684271.88 Pa (3000 psi) 50°C Grind. 6.23 7 0.00 1 2,179 0.00 3 0.02 0 0.03 8 0.789 9,265 W8 41368543.76 Pa (6000 psi) 50%: Grind. 4.97 2 0.00 3 3,340 0.00 3 0.01 8 0.03 2 0.001 8,369 W9 6894757.29 Pa (1000 psi) 30C Grind. 4.31 1 0.00 5 1,340 0.00 0.01 3 0.02 3 0.005 5,698 WIO 34473786.47 Pa (5000 psi) 30"C Grind. 4.42 4 0.00 5 1,375 0.00 2 0.01 3 0.02 4 0.005 5,847 Will 6894757.29 Pa (1000 psi) 70"C Grind. 4.51 9 0.00 5 1,405 0 _T 00 2 0.01 3 0.02 4 0.005 5,972 W12 34473786.47 Pa (5 (MOpsi) 7IJC Grind. 4.98 6 0.00 6 1,550 0.00 0.01 5 0.02 7 0.006 6,590 W13 20684271.88 Pa (3000 psi) 30°C Grind. 5.94 5 0.00 7 1,848 0.00 2 0.01 7 0.03 2 0.007 7,857 W14 20684271.88 Pa (3000 psi) 7O'C Grind. 6.57 0 0.00 7 2,042 0.00 2 0.01 9 0.03 5 0.007 8,684 W15 6894757.29 Pa (1000 psi) 5O'C Grind. 4.88 7 0.00 5 1,519 0.00 2 0.01 4 0.02 6 0.006 6,460 W16 34473786.47 Pa (5(X)0 psi) 5O'C Grind. 6.58 1 0.00 7 2,046 0.00 2 0.01 9 0.03 5 0.007 8,699 W17 20684271.88 Pa (3000 psi) 5U°C Grind. 7.08 6 0.00 8 2,203 0.00 3 0.02 1 0.03 8 0.008 9,366 WIS 20684271.88 Pa (3000 psi) 50°C Grind. 7.03 4 0.00 8 2,186 0.00 3 0.02 0 0.03 8 0.008 9,297 W19 22339013.63 Pa (3240 psi) 50"C Faction 1 3.82 4 0.00 2 0.431 0.00 1 0.01 1 0.00 1 0.001 4,097 W20 22339013.63 Pa (3240 psi) 50°C Faction 2 5.02 2 0.00 4 0.553 0.00 1 0.01 6 0.00 2 0.046 5,487 W21 22339013.63 Pa (3240 psi) 5()°C Faction 3 5.30 6 0.00 4 0.696 0.00 1. 0.01 7 0.00 2 0.051 5,844 W22 22339013.63 Pa (3240 psi) 50°C Faction 4 5.65 5 0.00 4 0.668 0.00 1 0.01 9 0.00 2 0.054 6,198 W23 22339013.63 Pa (3240 psi) 5O'C Faction 5 6.68 2 0.00 4 0.121 0.00 7 0.00 4 0.00 3 0.064 7,221 W24 22339013.63 Pa (3240 psi) 50"C Faction 6 5.98 6 0.00 3 0.551 0.00 2 0.00 6 0.00 3 0.056 6,421 W25 22339013.63 Pa (3240 psi) 50°C Faction 7 6.04 0 0.00 3 0.736 0.00 2 0.01 0 0.00 8 0.055 6,482 W26 22339013.63 Pa (3240 psi) 50°C Test 1 3.82 4 0.00 2 0.431 0.00 1 0.01 1 0.00 1 0.001 4,097 W27 22339013.63 Pa (3240 psi) 50°C Test 2 5.02 2 0.00 4 0.553 0.00 l 0.01 6 0.00 2 0.046 5,487 W28 22339013.63 Pa (3240 psi) 50°C Test 3 5.30 6 0.00 4 0.696 0.00 1 0.01 7 0.00 2 0.051 5,844 W29 22339013.63 Pa (3240 psi) 5fi°C Test 4 5.65 5 0.00 4 0.668 0.00 1 0.01 9 0.00 2 0.054 6,198 W30 22339013.63 Pa (3240 psi) 50"C Scaling 5.97 9 0.00 4 0.692 0.00 2 0.02 0 0.00 2 0.065 6.764

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Example 2. Supercritical CO2 extraction procedure

The supercritical extraction process is carried out using SFT-120 extractors. Approximately 400 g of hemp material from the panicle is placed in a 500 ml stainless steel container, thoroughly tamped to reduce dead space, and the container is sealed. After installing the container in the extractor before supplying CO2 to the system, an operating temperature of 50°C is achieved in the container and 75°C at the outlet nozzle. After the temperature stabilizes, Class 2 liquid CO2 is supplied to the system from an external cylinder, and the system pressure is then increased to 22339013.63 Pa (3240 psi), achieving supercritical CO2. The extraction is conducted dynamically, supplying fresh CO2 throughout the process to achieve a more efficient process while simultaneously collecting the resulting extract. The process continues for approximately 5 hours until the extract ceases to flow from the outlet nozzle. The system pressure is then reduced, allowing the CO2 to escape. Once the CO2 has completely escaped the system, the entire process is terminated. No other solvents, such as water or organic solvents, are used in the process.

Example 3. Selecting the method of preparing the raw material

The choice of the method of preparing the raw material, i.e. crushing it or leaving it uncrushed, was made based on the analysis of the cannabinoid content after extraction of the raw material in four randomly selected conditions.

Table 3. Extraction process conditions - comparison of the influence of the raw material fineness

Conditions Pan temperature Extraction vessel pressure W1 30°C 20684271.88 Pa (3000 PSI) W2 30°C 41368543.76 Pa (6000 PSI) W3 50°C 20684271.88 Pa (3000 PSI) W4 50°C 41368543.76 Pa (6000 PSI)

Table 4. Content of active compounds in unground raw material.

The obtained results were compared with the certificate provided by the supplier.

Relationship Conditions 1 Conditions 2 Conditions 3 Conditions 4 Certificate CBD 4.3024 3.1853 5.3292 3.9095 0.18 CBG 0.0011 0.0018 0.0012 0.0032 0.04 CBDA 2.2956 1.7002 1.8787 2.4359 1.18 CBN 0.0029 0.0022 0.0026 0.0021 <0.01 THC 0.0144 0.0108 0.0159 0.0129 <0.01 CBC 0.0247 0.0200 0.0302 0.0247 0.02 THCA 0.0032 0.0007 0.0007 0.0012 (1.03 Sum 6.6444 4.9210 7.2583 63893 1.74

Table 5. Content of active compounds in the ground raw material. The obtained results were compared against the certificate provided by the supplier.

Relationship Conditions 1 Conditions 2 Conditions 3 Conditions 4 Certificate CBD 5.6411 5.3863 6.2367 4.9723 0.18 CBG 0.0010 0.0016 0.0006 0.0028 0.04 CBDA 2.5782 2.4334 2.1791 3.3397 1.18 CBN 0.0028 0.0023 0.0025 0.0025 <0.01 THC 0.0193 0.0173 0.0203 0.0179 <0.01 CBC 0.0362 0.0313 0.0377 0.0323 0.02 THCA 0.0007 0.0006 0.7886 0.0012 0.03 Sum 8.2793 7.8727 9.2654 8.3687 1.74

The conducted research shows that the extraction of crushed raw material under each of the conditions proved to be more effective than the extraction of uncrushed raw material.

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Example 4. Procedure for grinding hemp material

The crushing process of the hemp material from the panicle was carried out using a millicontrol tube grinder, which placed loosely packed material into the mill. The grinding process was then carried out for 2 minutes at a blade speed of 5000 RPM. After grinding, the crushed material was subjected to sieve analysis to separate it into the desired fractions. The sieve analysis was performed using a sieve analyzer with an automatic vibration amplitude for 10 minutes, equipped with a set of seven sieves, which allowed for the separation of fractions.

Example 5. Determination of the content of active compounds depending on the size of the fragmentation of the plant raw material.

Due to the demonstrated impact of hemp material fragmentation on the efficiency of the extraction process, the raw material was again fractionated using sieve analysis to obtain fractions with a narrow range of fragmentation sizes and to identify the most optimal fractions in terms of active compound content. For this purpose, the raw material was divided into seven fractions with the following fragmentation size range:

Table 6. Range of raw material fragmentation size

Faction Raw material fineness range 1 > Imm 2 1 mm - 710 um 3 500 - 710 um 4 355 - 500 um 5 250 - 355 um 6 180 - 250 um 7 <180 μm

Next, the extraction process was carried out for each of the indicated fractions under previously optimized process conditions and the content of active compounds was determined depending on the fragmentation of the raw material.

Table 7. Cannabinoid content in individual fractions (%)

Relationship Faction 1 Faction 2 Faction 3 Faction 4 Faction 5 Faction 6 Faction 7 Certificate CBD 3.8239 5.0215 5.3060 5.6548 6.6820 5.9857 6.0403 0.18 CBG 0.0022 0.0035 0.0040 0.0040 0.0044 0.0032 0.0029 0.04 CBDA 0.4305 0.5526 0.6956 0.6675 0.1207 0.5509 0.7359 1.18 CBN 0.0008 0.0009 0.0013 0.0013 0.0067 0.0018 0.0018 <0.01 TIIC 0.0107 0.0162 0.0173 0.0189 0.0040 0.0056 0.0095 <0.01 CBC 0.0009 0.0020 0.0021 0.0023 0.0029 0.0028 0.0079 0.02 THCA 0.0013 0.0460 0.0507 0.0539 0.0638 0.0556 0.0553 0.03 Sum 4.0965 5.4872 5.8442 6,1978 7.2208 6.4214 6.4821 1.74

Based on the extraction performed on the hemp raw material divided into fractions of different fragmentation, the following conclusions were drawn:

- The fragmentation of the raw material affects the efficiency of the hemp raw material extraction process.

- An increase in the content of active compounds was observed in the analyzed fractions with increasing fineness up to fraction 5 with a size of 250-355 μΠΊ. Further fineness of the raw material negatively affected the content of active compounds in the analyzed fractions.

- Fractions 4-6 with sizes in the range of 180-500 μπι were selected as the optimal size fractions for further process optimization.

Example 6. Procedure for preparing a combination of hemp extract and cyclodextrin

A 1:1 mass ratio of hemp extract to cyclodextrin is prepared using a ball mill. Alpha-cyclodextrin is placed in a mixing vessel and mixed for 1 minute at 20 Hz. The hemp extract is then added and mixing continues for 60 minutes, depending on the experimental design, to achieve the desired combination of extract and cyclodextrin.

Example 7. Analysis of antioxidant activity

Hemp extract with alpha-cyclodextrin was tested for antioxidant activity using four different methods:

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- DPPH method

The DPPH (2,2-diphenyl-1-picrylhydrazyl) radical method is a spectrophotometric method that determines the degree of free radical neutralization by a given antioxidant. Under the influence of the antioxidant, the purple DPPH solution discolors to yellow as a result of electron transfer from the test solution to the DPPH radical.

- ABTS method

The ABTS radical-based antioxidant activity assay utilizes spectrophotometric measurement of the color change in the radical solution as a result of its neutralization reaction. The solution changes color from blue-green to colorless.

- CUPRAC method

The principle of the CUPRAC (CUPric Reducing Antioxidant Capacity) method is based on the spectrophotometric measurement of the absorbance of the neocuproine-copper Cu ⁺ complex formed. Under the influence of the tested antioxidant, the copper Cu2 ⁺ ions present in the solution are reduced and an orange-colored neocuproine complex is formed, exhibiting an absorbance maximum at a wavelength of 450 nm.

- FRAP method

The FRAP (ferric ion reducing antioxidant parameter) method for testing antioxidant capacity involves determining the ability of a test substance to reduce Fe3 ⁺ ions. The resulting Fe2 ⁺ ions are complexed by TPTZ (2,4,6-tripyridyl-S-thiazine), simultaneously producing an intense blue color with a maximum absorbance at 593 nm.

Table 8 summarizes the IC50 values for each method, i.e., the extract concentration required to inhibit 50% of the activity of a given radical. The lower the IC50 value, the more active the extract.

Table 8

Conditions DPPH ICso mg/ml ABTS ICfil mg/ml FRAP ŁCso ing/inl Cuprac ICso mg/ml W1 188,145 7,157 66,617 50,159 W2 199,249 10,224 71.243 61,364 W3 184,165 6,987 65,014 48,229 W4 190,127 7,054 67,015 50,339 W5 140,545 5,141 50,008 29,971 W6 154,328 5,873 58,144 38,334 W7 133,111 3,784 45,221 20,009 W8 138,327 4,664 48,987 22,574 W9 199,057 8,048 70,627 54,554 WIO 195,014 7,544 66,532 50,114 wu 196,127 7,884 69,098 53,114 W12 135,324 4,018 46,217 22.31 Wl3 141,322 4,557 50,331 29,951 W14 155,367 6,047 52,201 32,789 W15 177,987 6,224 54,329 35,245 W16 139,349 3,974 49,897 27,276 W17 133,331 3,807 46,667 24,713 WIS 129,367 3,718 45,834 23,134 W19 195,214 9,664 64,217 47,681 W20 190,554 7,609 61,024 42,555 W21 178,321 6,981 58,331 38,999 W22 155,654 6,129 51,384 32,555 W23 • 122,243- 3.314 40,091-' 18.145 W24 141,214 4,031 49,342 26,801 W25 142,339 4.122 49,877 27,999 W26 194,665 10.022 64,371 45,823 W27 189,314 9,873 63,673 44,781 W28 165,117 6,033 54,373 34,227 W29 126,334 3,401 42,149 21,834 W30 125,314 3,338 41,679 21,001

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Conclusions:

Analyzing the results presented in Table 8, the extract obtained under W23 conditions exhibits the most favorable antioxidant properties compared to the other samples. The IC50 values for this sample are significantly lower in all tested methods (DPPH, ABTS, FRAP, Cuprac), confirming its greatest ability to inhibit free radical activity.

In the DPPH, ABTS, FRAP, and Cuprac methods, W23 extract achieved the lowest IC50 values, indicating its effectiveness in both neutralizing free radicals and reducing iron and copper. This consistent and potent antioxidant activity makes W23 stand out as the most effective extract among all conditions tested.

Protocol for analysis of the ability to inhibit the hyaluronidase enzyme

Description: The Hyaluronidase Inhibitor Screening Assay utilizes a two-step turbidimetric reaction. The reagent stops the enzymatic reaction and creates turbidity with any residual hyaluronic acid in the well. The decrease in turbidity at 600 nm is directly proportional to the hyaluronidase activity in the sample.

Protocol for analysis of the ability to inhibit the elastase enzyme

Description: This procedure can be applied to elastase products using SucAla3-pNA as a substrate. Continuous spectrophotometric velocity determination (A410, light path = 1 cm) is based on the following reaction:

Elastase

SucAla -pNA + H*O —---SucAtaj + pNA

Table 9 presents the % inhibition for the tested extracts.

Table 9

Conditions Hyaluronidase % inhibition Elastase % inhibition W1 0.54 3,348 W2 0.333 2,174 W3 0.77 3,988 W4 0.561. 3,321 W5 2,548 14.455 W6 2,211 13,874 W7 3,318 18,664 W8 3,147 16,902 W9 0.333 2,541 WIO 0.478 2,907 Will 0.512 2,806 W12 3.098 16,032 W13 2,432 14,981 Wl4 2,052 13,325 W15 1,854 12,128 W16 2,655 14,202 W17 2,955 14,624 W18 3,153 15,016 W19 0.35 2,276 W20 0.417 2,392 W21 0.425 2,514 W22 1,715 11,927

PL 248921 Β1

gSgiiigl^ W24 2,752 15,482 W25 2,514 15,109 W26 0.408 2,292 W27 0.323 2,152 W28 1,652 11,671 W29 3,097 16,331 W30 3,111 16,703

Conclusions:

The extract obtained under W23 conditions demonstrated the highest hyaluronidase inhibition capacity, reaching a value of 3.514%. This confirms that the extract subjected to these conditions can be an effective inhibitor of hyaluronidase activity, which is significant because hyaluronidase is an enzyme involved in the degradation of hyaluronic acid. Similar to hyaluronidase, the W23 extract demonstrated the highest elastase inhibition capacity, reaching an impressive 20.174%. This indicates that this extract can be an effective inhibitor of elastase, an enzyme involved in the degradation of elastin, which is important for skin elasticity.

The extract obtained under the W23 condition appears particularly promising due to its high inhibition of both enzymes. This may indicate its therapeutic potential in protecting against processes related to the degradation of structural tissue components, which is important in delaying skin aging and maintaining skin elasticity.

Example 9. Correlation of odor masking effects with antioxidant activity values.

Table 10

Input data Results Trial number Mass ratio of extract to cyclodextrin expressed as the extract content in the system System preparation time [min] Specific density Odor masking Antioxidant activity (determined by the DPPH method) ICso [mg/mL] 1 0.33 5 1.6232 ± 0.0045 1 152.45 ±4.23 2 0.33 115 1.6242 ±0.0042 2 148.23 ±3.21 3 0.66 5 1.6126 ±0.0035 2 151.72 ±1.38 4 0.66 115 1.6284 ± 0.0023 3 149.72 ±2.35 5 0.33 60 L6L97 ± 0.0024 1 148.82 ±2.23 6 0.66 60 1.6264 ±0.0016 2 147.34 ±2.42 7 0.5 5 1.6312 ±0.0027 2 153.72 ± 1.24 8 0.5 115 1.6183 ± 0.0042 3 149.72 ± 2.9 L 9(C) 0.5 60 1.6273 ±0.0072 2 150.28 ±3.02 io(o 0.5 60 1.6183 ±0.0032 2 151.93 ±4.02

where the ability to mask the odor is marked on a scale of 0-1-2-3, where 0 is no masking of the odor and 3 is complete masking of the odor.

A weak negative correlation was observed between odor masking and antioxidant activity expressed as IC50, i.e., with increasing taste masking, the IC50 value decreases, i.e., antioxidant activity increases. Based on the obtained results, it seems that sample no. 8 shows

PL 248921 Β1 showed the best properties in terms of both odor masking and antioxidant activity, but its preparation required the longest time, which seems economically unjustified, especially since the differences in antioxidant activity between samples 8 and 9/10 were not statistically significant. Based on organoleptic observations of the preparation process, as well as the behavior of the system during further processing, the best processing properties were demonstrated by systems containing extract and cyclodextrin in a 1:1 ratio.

Example 10. Cosmetic formulations

As a result of the conducted research, the compositions of individual cosmetic formulations were developed; the full compositions, both qualitative and quantitative, are provided in Table 11.

Table 11

ointment cream serum balm oil Argan oil 5% Glyceryl Stearate Citrate 7% Hemp oil 25% 01ivem 100 6% Jojoba oil 60% Eucerin 45% Shea butter 8% 25% jojoba oil Hemp oil 16% Hemp oil 10% Lanolin 2% Organic wax 2% Glycerin 30% Nicociaca Oil 5% Polysorbate 8% Shea Butter 11% Jojoba oil 11% Vecticel AEC 5% 55% distilled water Glyceryl cocoate 8% Hemp system 4% Hemp system 1% Ceramides 5% Vecticell AEC 5% Vecticel AEC 5% Ceramides 5% 5% cosmetic glycerin Lialuronic acid 5% Ceramides 5% Panthenol 5% Distilled water 23% Ceramides 5% E-leen Green C 2% Lialuronic acid 5% Hemp system 2% Yecticell AEC 3% Distilled water 49% Hemp System oo/ J /0 E-Leen Green C 2% E-leen Green C 2% E-Leen Green C 2% Vecticell AEC 5% Hemp system 1% Lialuronic acid 5% E-Leen Green C 2%

Claims (20)

Patent claims 1. A composition of hemp extract with cyclodextrin characterized in that the mass ratio of hemp extract to cyclodextrin is from 2:1 to 1:2, respectively, the cyclodextrin is selected from the group: alpha-cyclodextrin, beta-cyclodextrin, gamma-cyclodextrin, hydroxypropyl-beta-cyclodextrin, and the hemp extract is obtained by extraction of hemp raw material with supercritical CO2. 2. A composition according to claim 1, characterized in that the hemp extract has a cannabinoid and terpene profile that contains, expressed as % by weight relative to the total weight of the extract, the following components: - CBD in amounts from 3.185 to 6.682, - CBG in an amount from 0.001 to 0.008, - CBDA in amounts from 0.121 to 3.340, - CBN in the amount from 0.001 to 0.007, - THC in the amount of 0.004 to 0.020, - CBC in the amount from 0.001 to 0.038, - THCA in the amount of 0.001 to 0.064, 3. The composition according to claim 1, characterized in that the mass ratio of hemp extract to cyclodextrin is 1:1. 4. The composition according to claim 1, characterized in that the hemp raw material is a hemp panicle. 5. Composition according to claim 4, characterized in that the hemp raw material is previously crushed to a fraction with a size in the range of 180-500 gm. 6. Composition according to claim 5, characterized in that the hemp raw material is previously crushed to a fraction with a size in the range of 250-355 gm. 7. The composition of claim 1, wherein the cyclodextrin is alpha-cyclodextrin. 8. The composition according to claim 1, characterized in that the extraction of the hemp raw material with supercritical CO2 is carried out at a temperature of 30-70°C and at a pressure in the range of 6894757.29-41368543.76 Pa (1000-6000 psi). 9. The composition according to claim 8, characterized in that the extraction of the hemp raw material with supercritical CO2 is carried out at a temperature of 30-50°C and at a pressure in the range of 20684271.88-24131650.53 Pa (3000 to 3500 psi). 10. The composition according to claim 9, characterized in that the extraction of the hemp raw material with supercritical CO2 is carried out at a temperature of 50°C and a pressure of 22339013.63 Pa (3240 psi). 11. A method for obtaining a composition of hemp extract with cyclodextrin, characterized in that it is carried out in the following stages: a) the hemp raw material is crushed to a particle size ranging from 180 to 500 gm, b) the crushed hemp raw material is extracted with supercritical CO2 at a temperature range of 30 to 70°C and a pressure in the range of 6894757.29-41368543.76 Pa (1000-6000 psi), c) the obtained extract from the hemp raw material is combined with cyclodextrin in a mass ratio of 2:1-1:2 for 5-115 minutes. 12. The method according to claim 11, characterized in that the hemp raw material is a hemp panicle. 13. The method according to claim 11, characterized in that the hemp raw material is crushed to a particle size in the range of 250 to 355 gm. 14. The method according to claim 9, characterized in that the supercritical CO2 extraction process is carried out in the temperature range of 30 to 50°C and under pressure in the range of 20684271.88-24131650.53 Pa (3000 to 3500 psi). 15. The method according to claim 10, characterized in that the supercritical CO2 extraction process is carried out at a temperature of 50°C and a pressure of 22339013.63 Pa (3240 psi). 16. The method according to claim 11, characterized in that the obtained extract from the hemp raw material is combined with cyclodextrin in a mass ratio of 1:1 for 60 minutes. 17. The method according to claim 11, characterized in that the cyclodextrin used is alpha-cyclodextrin. 18. The method according to claim 11, characterized in that the obtained extract from the hemp raw material is combined with cyclodextrin by grinding the cyclodextrin with the hemp extract for 60 minutes. 19. Use of the composition defined in claims 1 to 10 as a cosmetic agent. 20. Cosmetic formulation characterized in that it contains a composition of hemp extract with cyclodextrin described in claims 1-10 in an amount of 1 to 4% by weight and an oil in an amount of 5 to 60% by weight selected from the group: argan, jojoba or macadamia, ceramides in an amount of 5% by weight, a complex of three EAC vitamins in an amount of 3% by weight and a mixture of glyceryl caprylate/caprate in pentylene glycol (E-Leen Green C) in an amount of 2% by weight.