RU2740997C1 - Method of extracting polyphenols from ginger rootstocks - Google Patents

Abstract

FIELD: medicine; pharmaceuticals; food industry.

SUBSTANCE: invention refers to pharmaceutical and food industry and concerns extraction of biologically active substances from vegetal raw materials, and also relates to biological chemistry and molecular biology. Said technical result is achieved by adding to ginned rhizomes, 40 % aqueous solution of ethanol in ratio of 1:7 by weight, thoroughly stirring, boiling in a water bath for 15 minutes, then 10 days are infused at room temperature, centrifuged at 10,000 rpm for 30 minutes and produced in a supernatant extract of polyphenols; then centrifuge is passed through microcrystalline cellulose powder of same mass, as milled ginger rootstocks, and the polyphenol-enriched paste is dried in air for three days; obtained concentrate contains 2.50 ± 0.098 mg of polyphenolic compounds per 1 g of dried mass.

EFFECT: proposed invention is aimed at extraction of polyphenolic compounds from ginger roots of Zingiber officinale with subsequent concentration of these compounds on microcrystalline cellulose as an adsorbent.

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Description

The invention relates to the pharmaceutical and food industry and relates to the isolation of biologically active substances from plant materials, and also relates to biological chemistry and molecular biology.

There is a known method for isolating extractives from tea and coffee samples by boiling an aqueous solution (Tatarchenko I.A. Development of new types of tea and coffee products and improving the assessment of their quality: dis ... Candidate of technical sciences. - Krasnodar, 2015. - 181 s.).

The method has the following disadvantages:

- a multicomponent mixture is obtained, in which, in addition to polyphenolic compounds, other substances are present;

- in the extract after boiling of aqueous solutions, mainly polar compounds are present.

Known patented methods for the isolation of polyphenols from various herbal preparations using various organic solvents, followed by concentration of extracts (Rubchevskaya L.P. et al. Method of obtaining polyphenols: Pat. 2174011 from 12.07.1999, publ. 27.09.2001; Bradbury A., Kopp G. Polyphenol-enriched composition extracted from cocoa bean husks: Pat. 2392954 dated May 30, 2006, published on June 27, 2010; Yashunsky DV et al. Method of isolation of polyphenolic compounds of the stilbenes class of pinosylvin and methylpinosylvin from pine processing waste: Pat . 2536241 from 05.07.2011, publ. 20.12.2014). However, the known methods have the following disadvantages:

- the used solvents in such significant concentrations do not allow the use of the resulting extracts for therapeutic purposes;

- removal of solvents also entails noticeable losses of extractable substances;

- the degree of removal of side components in extracts is low.

There are also known methods for extracting polyphenolic compounds from ginger rhizomes using solvents of methanol, acetone and ethanol in various modifications: using outflow methods, soaking, using ultrasound (Sharif MF, Bennett MT The effect of different methods and solvents on the extraction of polyphenols in ginger (Zingiber officinale) // Jurnal Teknologi (Sciences and Engineering). - 2016. - Vol. 78, No. 11-2. - P. 49–54; Sasikala P., Chandralekha A., Chaurasiya RS et al. Ultrasound-assisted extraction and adsorption of polyphenols from ginger rhizome (Zingiber officinale) // Separation Science and Technology. - 2018. - Vol. 53, No. 3. - P.439–448. https://doi.org/10.1080/01496395.2017.1391290 ).

The known methods have disadvantages:

- the solvents used are toxic in the case of using the obtained extracts for medical purposes, as well as for their further use in experimental work with the involvement of animals;

- the proposed methods are rather complicated to implement;

- the concentration of the obtained extracts is achieved by evaporation of solutions, or by transferring the extract to another solvent.

There is a known prototype method for optimal isolation of polyphenolic compounds from ginger rhizomes, which consists in using ethanol as an extractant at moderate temperatures and extraction times (Mukherjee S., Mandal N., Dey A., Mondal B. An approach towards optimization of the extraction of polyphenolic antioxidants from ginger (Zingiber officinale) J Food Sci Technol . 2014; 51 (11): 3301-3308. https://doi.org/10/1007/s13197-012-0848-z). The prototype has the following disadvantages:

- the ethanol used is taken in a high concentration, which makes the final extract toxic for its further use for medical purposes;

- exposure time and temperature conditions do not allow to fully carry out the extraction of components;

- the method for concentrating the obtained extract is not given;

- the method for removing the extractant (ethanol) is not specified, as well as the method for concentrating polyphenols on any solid adsorbent.

The proposed invention is directed to the isolation of polyphenolic compounds from the rhizomes of ginger Zingiber officinale, followed by the concentration of these compounds on microcrystalline cellulose as an adsorbent. The proposed method also allows you to purify the isolated polyphenolic compounds from low molecular weight impurities, including ethanol.

The specified technical result is achieved by the fact that a 40% aqueous solution of ethanol is added to the crushed ginger rhizomes in a ratio of 1: 7 by weight, mixed thoroughly, boiled in a water bath for 15 minutes, then infused for 10 days at room temperature, centrifuged at 10,000 turns for 30 minutes and get the extract of polyphenols in the supernatant; then the centrifugate is passed through microcrystalline cellulose powder of the same mass as crushed ginger rhizomes, and the paste enriched with polyphenols is dried in air for three days; the resulting concentrate contains 2.50 ± 0.098 mg of polyphenolic compounds per 1 g of dried mass.

The structural features of the polyphenolic components of ginger make it possible to classify them as potential antioxidant compounds. The main components of the ginger rhizome are essential oil and phenolic compounds - gingerols and shogaols. Other components are zingerons and paradols:

As can be seen from the above structures, all these compounds are substances of oxyphenyl nature (phenolic base) with an optimal wick chain length. The presence of an oxyphenyl fragment is responsible for the antiradical properties of polyphenolic compounds (PFCs). The optimal length of the wick chain of polyphenolic compounds of ginger allows them not only to be fixed in the lipid bilayer of biological membranes, but also to circulate freely in the body fluids and thus provide a generalized antiradical and antioxidant effect. Gingerols are the most abundant compounds found in fresh roots. Shogaols, dehydrated derivatives of gingerols, are found only in small amounts in fresh root; mainly found in dried and heat-treated roots. The separation of compounds close in chemical structure, including PPS, into separate fractions is a very laborious task, since they are chemically very close, therefore it is preferable to separate them with a total mass (Nikolaev A.A., Vetoshkin R.V., Loginov P.V. Changes in rat testes proteoglycans under conditions of experimental chronic intoxication with sulfur-containing gas // Astrakhan Medical Journal. - 2012. - T. 7, No. 2. P.75–79).

Another important task is the concentration and purification of the isolated polyphenolic compounds from impurities. It was shown that polyphenols are well adsorbed by the surface and volume of polysaccharide fibers of cellulose and xylan due to intermolecular interactions (Costa TS, Rogez H., Pena RS Adsorption capacity of phenolic compounds onto cellulose and xylan. Food Science and Technology . 2015; 35 (2) : 314-320). In this regard, microcrystalline cellulose (MCC) was chosen as an adsorbent, which will efficiently absorb polyphenolic compounds due to hydrophobic interactions and pass through low molecular weight impurities, including ethanol.

The creation of a method for the isolation of polyphenolic compounds from ginger rhizomes was carried out by grinding and grinding dried rhizomes of Zingiber officinale, followed by extraction with a 40% ethanol solution on the basis of the Department of Chemistry of the Astrakhan State Medical University of the Ministry of Health of Russia. For grinding and grinding the dried rhizomes of ginger, 10-15 g of a sample were taken. The resulting powdery mass was poured with 40% aqueous ethanol solution, thoroughly mixed and the resulting mixture was placed in a water bath for 15 minutes. Next, they insisted for 10 days at room temperature, after which they were centrifuged at 10,000 revolutions in a CN-10001 centrifuge (Japan) for 30 minutes. The centrifugate was carefully passed through a previously prepared microcrystalline cellulose (MCC) powder of the same mass as the mass of crushed ginger rhizomes. The powder was prepared by grinding solid MCC briquettes in a mortar, after which this powder was placed on a paper filter installed in a glass funnel. Then, the mass impregnated with the polyphenol extract was removed from the funnel together with the filter, the filter itself with the resulting mass was straightened on a horizontal surface and dried in air for three days.

To determine the content of polyphenolic compounds in the dried mass, the polyphenols were measured in a centrifugate before passing the latter through the MCC powder, and then the content of polyphenolic compounds in the filtrate was measured. The difference between the content of polyphenols in the centrifugate and filtrate was used to judge the value of adsorption and, accordingly, the content of polyphenols in the resulting mass based on MCC.

To determine the content of polyphenolic compounds, we used the Folin-Chocalteu reagent, which is a mixture of phosphomolybdate and sodium phosphotungstate and obtained by a known method (Studopedia: [site]. URL: https://studopedia.ru/12_86442_laboratornaya-rabota--.html). The method for the quantitative determination of polyphenolic compounds (PPS) is based on the interaction of these compounds with the Folin-Ciocalteu reagent, giving a color reaction (Mukherjee S., Mandal N., Dey A., Mondal B. An approach towards optimization of the extraction of polyphenolic antioxidants from ginger (Zingiber officinale) J Food Sci Technol 2014; 51 (11): 3301-3308. Https://doi.org/10/1007/s13197-012-0848-z). Gallic acid is taken as a standard solution, which also gives a color reaction with the Folin-Chocalteu reagent. 1 ml of a solution of an extract of polyphenolic compounds or a solution of gallic acid (0-2 g / l) is mixed with 10 ml of distilled water and 1 ml of Folin-Chocalteu reagent is added. After 5 minutes, add 2 ml of a 20% Na ₂ CO ₃ solution, then incubate in the dark for 1 hour and measure the optical density on a spectrophotocolorimeter at a wavelength of 750 nm in a cuvette with a photometric layer thickness of 1 cm.The obtained optical density values are correlated with those in the calibration graph obtained using gallic acid as a standard solution. Next, the PFC is calculated in a given volume of extract. These manipulations are also performed with the extract passed through the MCC powder. Then the amount of absorption (adsorption) of PPS on the MCC is calculated. The content of PPS in the resulting mass based on MCC is expressed in mg / g of MCC powder by dividing the absorption of PPS (mg) by the mass of the powder (g).

The proposed method achieved the following results. Based on measurements of optical densities of standard solutions of gallic acid, a calibration graph was constructed (Fig. 1). The following acid concentrations were taken: 0.25; 0.50; 0.75; 1.00; 1.25; 1.50; 1.75 and 2.00 g / l. Next, the optical densities of 9 polyphenolic extracts from ginger rhizomes were measured: first, the optical densities of the extracts were measured before passing through the MCC powder (primary centrifugate), and then the optical densities of the extracts were measured after passing through the MCC powder (Table 1). The obtained values of optical densities were correlated with those in the calibration graph and the concentration of PPS (mg / l) in the centrifugate and filtrate was determined (table 2). Then the obtained concentration values were used to calculate the PPS content in extracts (in mg) by multiplying the concentration values by the solution volume. The difference between the content of polyphenols in the centrifugate and the filtrate was used to calculate the absorption of PPS by the particles of the MCC powder. The resulting value was converted into relative units by dividing the PFC absorption by the mass of the MCC powder applied to a paper filter. Below are examples of calculations (for the first three experiments) of the content of PPS concentrated on MCC as a solid carrier.

Example 1. For the experiment, we took 10 g of crushed ginger rhizomes. For this, 75 ml of a 40% ethanol solution (density 0.935 g / ml) was taken, which corresponds to 70 g of the solution. Thus, the ratio of crushed ginger rhizomes to ethanol solution 1: 7 by weight is achieved. The measured optical densities for the centrifugate and filtrate are 1.034 and 0.789, respectively. According to the calibration graph, we find that these values of optical densities correspond to PPS concentrations equivalent to the gallic acid content of 1.60 and 1.20 g / l. Received 50 ml of centrifugate and 48 ml of filtrate. Thus, from 75 ml of added 40% aqueous ethanol solution, 25 ml was the loss during extraction. We calculate the PFC content in the centrifugate and filtrate:

Centrifugate: 1.60 g / L × 0.05 L = 0.080 g = 80 mg

Filtrate: 1.20 g / l × 0.048 l = 0.0576 g = 57.6 mg ≈ 58 mg.

Absorption rate: 80 - 58 = 22 mg. Then the relative content in MCC (per 10 g of powder) is: 22 ÷ 10 = 2.2 mg / g.

Example 2. We also took 10 crushed ginger rhizomes. The measured optical densities for the centrifugate and filtrate are 1.105 and 0.790, respectively. According to the calibration graph, we find that these optical densities correspond to PPS concentrations of 1.72 and 1.20 g / l. 50 ml of centrifugate and 48 ml of filtrate were also obtained. We calculate the PFC content in the centrifugate and filtrate:

Centrifugate: 1.72 g / L × 0.05 L = 0.086 g = 86 mg

Filtrate: 1.20 g / l × 0.048 l = 0.0576 g = 57.6 mg ≈ 58 mg.

Absorption rate: 86 - 58 = 28 mg. Then the relative content in MCC (per 10 g of powder) is: 28 ÷ 10 = 2.8 mg / g.

Example 3. Took 12 g of ginger rhizomes. 84 g of a 40% ethanol solution was added, which corresponds to a volume V = m / ρ = 84: 0.935 = 90 ml. 62 ml of centrifugate and 60 ml of filtrate were obtained. The measured optical densities for the centrifugate and filtrate are 0.980 and 0.721, respectively. According to the calibration graph, we find that these optical densities correspond to PPS concentrations of 1.50 and 1.12 g / l. We calculate the PFC content in the centrifugate and filtrate:

Centrifugate: 1.50 g / L × 0.062 L = 0.093 g = 93 mg

Filtrate: 1.12 g / L × 0.060 L = 0.0672 g = 67.2 ≈ 67 mg.

Absorption rate: 93 - 67 = 26 mg. Then the relative content in MCC (per 12 g of powder) is: 26 ÷ 12 = 2.2 mg / g.

Example 4. Took 10 g of ginger rhizomes. 75 ml of a 40% ethanol solution was added. Received 54 ml of centrifugate and 52 ml of filtrate. Optical densities for the centrifugate and filtrate are 1.092 and 0.801, respectively. According to the calibration graph, we find that these optical densities correspond to PPS concentrations of 1.65 and 1.25 g / l. We calculate the PFC content in the centrifugate and filtrate:

Centrifugate: 1.65 g / L × 0.054 L = 0.089 g = 89 mg

Filtrate: 1.25 g / L × 0.052 L = 0.065 g = 65 mg.

Absorption rate: 89 - 65 = 24 mg. Then the relative content in MCC (per 10 g of powder) is: 24 ÷ 10 = 2.4 mg / g.

Calculations for experiments 4-9 are made in a similar way. Then the obtained data were statistically processed. Calculations have shown that the average PPS content in the resulting mass based on MCC is 2.50 ± 0.098 mg / g. The data obtained are summarized in Table 2.

The results obtained have been tested. The identification of polyphenolic compounds was carried out using IR spectroscopy (Fig. 2). The IR spectra of the isolated polyphenols contain bands due to bond vibrations characteristic of the main structural elements of phenolic compounds: aromatic rings, carbonyl, alcohol, and phenolic hydroxyl groups. The presence of phenolic hydroxyl is confirmed by absorption bands (pp) at 1230 and 1410 cm ^-1 . The presence of a methoxy group at the aromatic ring is confirmed by pp. at 2850 cm ^-1 . The presence of an aromatic ring is proved by pp. at 1600 cm ^-1 . The keto group surrounded by aliphatic radicals gives pp. at ≈ 1700 cm ^-1 . The presence of a side wick chain is confirmed by paragraphs. at 1440-1480 cm ^-1 , and the presence of the –CH ₂ CO– group is confirmed by pp. at 1400-1440 cm ^-1 . Thus, in accordance with FIG. 2, the following elements of PFC are proved: the presence of an aromatic ring connected to a hydroxyl group (phenolic part), the presence of a second phenolic hydroxyl, in which a hydrogen atom is replaced by a methyl group (methoxy group), and a wick chain, in which there is a keto group. All these elements are present in gingerols and shogaols, as well as zingerols and paradols.

Contained in 1 g of PPS powder (2.5 mg) make up the daily dose for rats, calculated in accordance with the characteristics of metabolism (Zakharov A.A. Morphogenesis of the internal organs of the male reproductive system of experimental animals during immunosuppression and immunostimulation in postnatal ontogenesis: dis. .. Doctor of Medical Sciences. - Lugansk, 2019. - 356 p.). Thus, the resulting powder containing PFC can be further used as a dosed dietary supplement in experimental studies.

Thus, the proposed method makes it possible to effectively extract polyphenolic compounds from ginger rhizomes with subsequent concentration of these compounds on microcrystalline cellulose. The method also makes it possible to purify the isolated polyphenolic compounds from low molecular weight impurities, which makes it possible to further use them in experimental work and clinical studies.

Claims (1)

A method for isolating polyphenols from ginger rhizomes, which consists in extracting polyphenolic compounds from Zingiber officinale ginger rhizomes using an ethanol solution, characterized in that a 40% aqueous solution of ethanol is added to the crushed ginger rhizomes in a ratio of 1: 7 by weight, mixed thoroughly, boiled in a water bath for 15 minutes, then infused for 10 days at room temperature, centrifuged at 10,000 rpm for 30 minutes and a polyphenol extract is obtained in the supernatant; then the centrifugate is passed through microcrystalline cellulose powder of the same mass as the crushed ginger rhizomes, and the paste enriched with polyphenols is dried in air for three days; the resulting concentrate contains 2.50 ± 0.098 mg of polyphenolic compounds per 1 g of dried mass.

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