RU2793251C2 - Method for production of co2 ajan spruce pisea ajanensis extract - Google Patents

Abstract

FIELD: extraction.

SUBSTANCE: invention relates to a technology of extraction of natural raw materials, which can be used in food, perfumery and cosmetics, and pharmaceutical industry. A method for the production of CO₂ Ajan spruce Pisea ajanensis extract is proposed, characterized in that plant raw materials in the form of needles of Ajan spruce Pisea ajanensis, ground to a particle size of 5-6 mm, are subjected to extraction with a solvent, with a flow rate of 5-20 kg/kg of plant raw materials, as which supercritical fluid CO₂ is used with the mass fraction of ethanol of 5%, at a temperature of 31-55°C and pressure of 50-200 bar, for 60 min.

EFFECT: invention allows for provision of high efficiency of extraction of Ajan spruce Pisea ajanensis due to an increase in the mass fraction of a cosolvent, as which ethanol is used; increase in extraction of biologically active substances (BAS) with the yield of up to 5% of the weight of plant raw materials, in particular dihydroquercetin; reduction in temperature and pressure of extraction; as well as preservation of a composition of biologically active substances in the resulting product.

1 cl, 1 tbl, 1 dwg

Description

The invention relates to a technology for the extraction of natural raw materials, and in particular to methods for producing a CO ₂ extract, which can be used in the food, perfume, cosmetic and pharmaceutical industries.

A known method of obtaining CO ₂ extract, including the preparation of plant materials, followed by CO ₂ -extraction at a pressure of 20-40 MPa for 120 min [see. RF patent No. 2070053, IPC A61K 35/78, publication date 10.12.1996].

The disadvantage of analog is the fact that pure CO ₂ is an inefficient solvent for chemical compounds with a higher polarity.

As the closest analogue, a method for obtaining CO ₂ of ginseng extract was adopted, including its preparation, followed by extraction with supercritical fluid CO ₂ with a mass fraction of ethanol of 1.7-3.4% at a liquid flow rate of 250 g/min at a temperature of 31-70 ° C and a pressure of 20-40 MPa for 120 minutes [see. patent of the Russian Federation No. 2679634, IPC A61K 36/258, A61P 37/00, publication date 02/12/2019].

The disadvantages of the closest analogue are:

- extracting in effective amounts only six heat-labile ginsenosides from Far Eastern ginseng;

- long time, as well as high temperatures and pressures of extraction;

- a small yield of biologically active substances associated with the consumption of a small amount of co-solvent;

- destruction of some thermolabile biologically active components.

The task to be solved by the claimed invention is the development of a technology for obtaining CO ₂ extract of Ajan spruce Pisea ajanensis containing dihydroquercetin in effective amounts.

The technical result, which manifests itself in solving the problem, is expressed as follows:

- ensuring high efficiency of extraction of Ayan spruce Pisea ajanensis by increasing the mass fraction of the co-solvent, which is used as ethanol;

- the possibility of extracting biologically active substances (BAS) in effective quantities (with a yield of up to 5% by weight of plant materials), in particular dihydroquercetin;

- use of available, safe and easily separated ethanol co-solvent;

- reduction of time, reduction of temperature and pressure of extraction;

- preservation of the composition of biologically active substances in the resulting product;

- no hard-to-recycle waste and by-products.

The problem is solved by the fact that in the method of obtaining CO ₂ extract of Ayan spruce Pisea ajanensis , plant material in the form of needles of Ayan spruce Pisea ajanensis , crushed to a particle size of 5-6 mm, is subjected to solvent extraction with a consumption of 5-20 kg/kg of plant material, in which is used as supercritical fluid CO ₂ with a mass fraction of ethanol 5%, at a temperature of 31-55°C and a pressure of 50-200 bar for 60 minutes.

A comparative analysis of the set of essential features of the proposed technical solution and the set of essential features of the prototype and analogues indicates its compliance with the criterion of "novelty".

At the same time, the features of the claims solve the following functional tasks.

The feature “Ayan spruce needles are used as plant raw material” describes the type of plant material used.

Ayan spruce Pisea ajanensis (Pall.) Regel, Pine family - a plant growing in the central regions of the Khabarovsk Territory, in the southeast of Yakutia and the Amur Region, in the Primorsky Territory. Trees up to 35, rarely up to 40 m in height and up to 120 cm or more in trunk diameter. The natural range of Ayan spruce is located in the zone of active influence of the Pacific Ocean. The main function of the forest is raw material. It is necessary to find ways to increase the productivity of forests, one of the directions is to improve the nutrition of the forest stand. The level of content of the main elements N, P, K in the assimilation organs is used to diagnose the forest's need for nutrition and fertilizer. These elements determine the processes important for the life of plants: nitrogen - the production of organic matter, phosphorus - maintaining the energy balance, potassium - to regulate metabolic processes [1-3].

Ayan spruce needles contain a large amount of biologically active substances [4], including a complex of polyphenolic compounds, including bioflavonoids.

An important function of flavonoids in the cardiovascular system has been identified, namely their ability to lower blood pressure and improve endothelial function. Dihydroquercetin is a flavonoid with significant antihypertensive effects and many beneficial properties for the normal functioning of the cardiovascular system, it also has antiviral effects, which may be of particular importance in the current situation of the pandemic. In the course of numerous scientific studies, it has been proven that dihydroquercetin (taxifolin) has a beneficial effect on the state of the vascular wall of capillaries, reduces blood viscosity and prevents thrombosis. In people with bronchopulmonary diseases with pneumonia, chronic bronchitis, asthma, COPD (chronic obstructive pulmonary disease), it helps to reduce the frequency of attacks, the faster disappearance of cough, shortness of breath, swelling of the bronchi, improve bronchial patency, restore lung tissue and normal breathing.

The article [5] shows the impact of molecular mechanisms under the influence of taxifolin, identified in preclinical studies, which are relevant not only for the treatment of hypertension per se, but may also provide additional antiviral benefits that may be relevant for the treatment of patients with hypertension and SARS-CoV infection. -2.

Following the recent emergence of SARS-CoV-2 or coronavirus disease (COVID-19), drug discovery and vaccine development to combat this deadly infection is critical. In a study [6], the main enzyme in the SARS-CoV-2 replication apparatus, RNA-dependent RNA polymerase (RDRP), is subjected to a virtual screening analysis using a set of 1664 FDA-approved drugs, including sets of plant and synthetic derivatives. A set of 22 preparations showed a high docking score >-7. Notably, roughly one-third of the hits came from either natural products or biological molecules. Phytochemicals approved by the FDA included sennosides, digoxin, asiaticoside, glycyrrhizin, neohesperidin, dihydroquercetin, quercetin, and aloin. These approved natural products and phytochemicals are used as general tonics, antioxidants, cellular protectors and immune stimulants, and in various other systemic or topical applications.

The sign “Ayan spruce needles Pisea ajanensis , crushed to a particle size of 5-6 mm are used as plant raw materials” describes the optimal particle sizes of plant materials, which ensure more efficient extraction of biologically active substances.

Signs "vegetable raw materials are subjected to extraction with a solvent with a consumption of 5-20 kg/kg of vegetable raw materials, which is used as supercritical fluid CO ₂ with a mass fraction of ethanol 5%, at a temperature of 31-55 ° C and

pressure of 50-200 bar for 60 minutes” describe the optimal operating characteristics.

Changing the regime characteristics, both in the direction of decrease and increase, leads to a decrease in the efficiency of extraction of biologically active substances.

The drawing shows the yield of dihydroquercetin depending on temperature and pressure during solvent extraction with a consumption of 20 kg/kg of plant material for 60 minutes.

The inventive method is carried out as follows.

Pre-prepared vegetable raw materials.

The needles of the Ayan spruce Pisea ajanensis are crushed to a particle size of 5-6 mm and loaded into the vessel of the extraction apparatus of supercritical pressure Thar SFC, SN 3526551, USA, already heated with a hot casing, the temperature is controlled by a thermostat (±1°C). Carbon dioxide (CO ₂ ) was compressed using a compressor, the pressure is controlled by a metering valve.

Next, extraction is carried out with a solvent at a flow rate of 5-20 kg/kg of plant material, which is supercritical fluid CO ₂ with a mass fraction of ethanol 5%, at a temperature of 31-55°C and a pressure of 50-200 bar for 60 minutes.

The solution of biologically active substances in CO ₂ is subjected to decompression in the separator.

The yield of biologically active substances under various operating conditions is shown in Table 1.

Table 1

BAS output at different regime characteristics

Example No. Temperature, °С Pressure, bar Solvent consumption, kg/kg of vegetable raw materials Yield of biologically active substances, % by weight Example 1 31 50 5 0.28 Example 2 40 100 8 1.9 Example 3 50 150 12 4.3 Example 4 55 200 20 4.7

Based on Table 1, it can be concluded that a joint increase in temperature and extraction pressure contributes to a more efficient extraction of biologically active substances from plant materials.

Multicomponent mixtures were separated by HPLC using a Shimadzu LC-20 Prominence HPLC high-pressure liquid chromatograph (Shimadzu, Japan) equipped with an SPD-20A spectrophotometric detector and a Shodex ODP-40 4E reverse phase column.

The elution gradient program (water - acetonitrile) is as follows: 0-4 min, 100% H ₂ O, 0% acetonitrile (CH ₃ CN); 4-60 min, 100-25% H ₂ O, 0-75% CH ₃ CN; 60-75 min, 25-0% H ₂ O, 75-100% CH ₃ CN; control washing 75-120 min 0% H ₂ O, 100% CH ₃ CN. All HPLC analysis was done with an ESI detector at 230 nm and 330 nm; temperature 17°C. The injection volume was 1 ml.

BAS were identified by tandem mass spectrometry using an amaZon SL mass spectrometer (manufactured by BRUKER DALTONIKS, Germany) equipped with an ESI electrospray ionization source in negative and positive ion modes.

Optimized parameters are obtained as follows: ionization source temperature 70°C; gas flow 4 l/min; gas nebilizer (nebulizer) 7.3 psi; capillary voltage 4500 V; end plate bending voltage 1500 V; fragmentator voltage 280 V; collision energy 60 eV. The mass spectrometer was used in the scan range m/z 100-1.700 for MS and MS/MS. The capture rate was 1 spectrum/s for MS and 2 spectrum/s for MS/MS. Data collection was controlled by the Windows software for BRUKER DALTONIKS.

In FIG. It can be seen that at a temperature of 31°C and a pressure of 50 bar, the yield of taxifolin is minimal and amounts to 0.28 mg/g of plant material, and at a temperature of 55°C and a pressure of 200 bar it is maximum and amounts to 4.7 mg/g of plant material.

Thus, the claimed method allows to obtain CO ₂ extract of Ayan spruce Pisea ajanensis with a high content of dihydroquercetin at lower pressure and temperature in a shorter period.

Information sources

1. Voroshilova G.I., Koroleva R.A., Opritova Z.V. Diagnostic value of the leaf epidermis of conifers of the Soviet Far East // Tez. XIV Pacific Scientific Congress. Committee N. M.: Nauka, 1979. S. 64-65.

2. Grozdova N.B., Nekrasov V.I., Globa-Mikhailenko D.A. Trees, Shrubs and Lianas: A Reference Guide. M.: Lesnaya promyshlennost', 1986. 349 p.

3. Gubanov I.A., Ignatov M.S., Novikov V.S. Petelin D.A. Vascular plants // Flora and vegetation of the Tukuringa Ridge (Amur Region). M.: Publishing House of Moscow State University, 1981. S. 86-166.

4. Ilyina T.M. Chemical composition of Ajan spruce needles (Picea ajanensis) within its range // Vestnik KrasGAU. 2009. No. 10.

5. Bernatova, I.; Liskova, S. Mechanisms Modified by (−)–Epicatechin and Taxifolin Relevant for the Treatment of Hypertension and Viral Infection: Knowledge from Preclinical Studies. Antioxidants. 2021, 10, 467.

6 Kandeel, M.; Kitade, Y.; Almubarak, A. Repurposing FDA-approved phytomedicines, natural products, antivirals and cell protectives against SARS-CoV-2 (COVID-19) RNA-dependent RNA polymerase. PeerJ. 2020. 8:e10480. DOI 10.7717/peerj.10480.

Claims (1)

A method for producing CO ₂ extract of the Ayan spruce Pisea ajanensis , in which the plant material in the form of needles of the Ayan spruce Pisea ajanensis , crushed to a particle size of 5-6 mm, is subjected to solvent extraction with a consumption of 5-20 kg/kg of plant material, which is used as a supercritical fluid CO ₂ with a mass fraction of ethanol 5%, at a temperature of 31-55°C and a pressure of 50-200 bar for 60 min.

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