LT3241B - Process for the manufacture of a preparation having immunomodulating activity and stimulating cytokine formation by extracting plants and plant residues - Google Patents

Abstract

A non-toxic preparation having immunomodulating activity and stimulating cytokine (interferons, tumor necrosis factor and others) formation is manufactured by extracting plants and plant residues and is suitable for therapeutic and preventive use in humans and animals. From the extract, preferably from peat extract, humic substances are eliminated by means of acidification with hydrochloric acid to pH 1,5 - 3,0 and separation. The extract is then concentrated by evaporation under reduced pressure and/or by means of nano-filtration with simultaneous elimination of inorganic salts. Next, the pH of the concentrated solution is brought to 6,0 - 7,1. The resulting mixture is then concentrated to a thick syrup and heated up to 70-90 DEG C until completion of Amadori rearrangement reaction of the products resulting from the reaction of simple sugars, oligo- and polysaccharides with amino acids and/or peptides extracted from peat. The reaction is then stopped by cooling and freed from hydrophobic substances - which would reduce the immunotropic activity of the product - by selective adsorption on a specific resin. The product thus obtained is converted into a sterile solid form by spray-drying.

Description

The present invention relates to the recovery of non-toxic preparations of plants and plant residues having immunomodulatory activity and promoting the formation of cytokines (interferons, tumor necrosis factors, etc.) when administered to humans and animals.

Numerous disclosures have been made regarding the production of various extracts, especially from peat with immunotropic or antitumor activity.

Hyoshita Takuya et al. (Chem. Abstr. 1974, 00, 47021j) discloses a process in which peat is extracted several times with 8% sodium hydroxide solution; the resulting extract is acidified, centrifuged to remove solids, then dialyzed and purified by chromatography on a column. The fraction that has antineoplastic activity is predominantly (95%) polysaccharides.

St. Tolpa et al. (Polish Patent No. 124110) describes the same peat extract as far as its further treatment is concerned. After acidification, the extract is basified, then acidified again, concentrated by evaporation, neutralized, concentrated again, extracted with alcohol / water, and further partially concentrated and extracted with ether or another water immiscible organic solvent. The aqueous layer is then separated and purified on a chromatographic column with a filtering gel. However, it is not specified which of the fractions could be considered to constitute the desired end product.

Instead of the alkaline extraction medium already known from the two documents mentioned above, Polish patent application no. 279475 (Tarchominskie Zakiady Farmaceutyczne POLFA, published BUP 24/90) protects the peat extraction process, where the extraction solvent is an acidified aqueous medium.

The acidic extract is neutralized and concentrated by ultrafiltration or reversible osmosis using membranes with a retention range of 500 to 10,000 Daltons. The product is then separated by filtration through a column of fresh or anionic resin. Fractions varying in salt concentration and in the ratio of polysaccharides to glycoproteins can also be obtained. However, it is noted that all mixtures obtained are non-toxic and show immunotropin activity.

The processes described above have a number of common features; It has long been possible to isolate active substances from natural raw materials while preserving the active substances, thus avoiding side reactions that may lead to degradation or inactivation of the active substances. Unfortunately, none of the prior art known solutions provide information on the exact structure or chemical nature of compounds which, by their presence, result in a biologically active product (being a mixture of various substances). There is very little information on polysaccharides and their use in separation techniques for low molecular weight fractions (dialysis and ultrafiltration); this indicates that the products obtained are attempting to combine biological activity with high molecular weight fractions.

In these studies of peat extracts, it has been found that the extracts obtained, even under preservation conditions, exhibit relatively low immunotropic activity or have poorly separable ballasts, mainly inorganic salts, the presence of which limits the use of the product in the desired pharmaceutical formulations; they also increase the labor and energy costs of downstream processing, and even render the product unusable for a number of therapeutic applications.

The main object of the present invention is to provide a novel method of extraction of plants and their residues and treatment of extracts, which enables to obtain preparations with higher yields which have immunotropic properties that can promote cytokine formation and which have no known process deficiencies.

It has been unexpectedly found that each batch of product retains its immunotropic activity within the desired range and the yield of organic matter in the final product is significantly increased when the extraction conditions are modified according to one or more of the following:

- some of the side effects may specifically occur in a controlled direction and to a controlled degree, resulting in the formation of new compounds known in the art, e.g. peat extracts, if any, or only in small concentrations;

modifies the treatment of the extracts thus obtained in such a way that valuable and therapeutic components of the extract are not removed when segregated by ballast and extracted from natural peat and / or derived from the extraction medium.

Organic substances related to the biological and therapeutic activity of peat-derived preparations, including TTP (TOLPA TORF PREPARATION, trademark issued by TORF CORPORATION, Poland), are Amadori rearrangement compounds and products derived from such aldoses and amino acid reaction products, that is, compounds of the general formula R ₁ -CO-CH ₂ -NH-R ₂ , wherein R _x is a residue of 1-deoxy-2-keto sugar, an oligo- or polysaccharide wherein such 1-deoxy-2-keto group is a terminal carbon an atom chain group, and wherein R ₂ is an amino acid residue or a peptide residue in which the terminal group is a free, non-peptide bond, NH _{2 group} . The process for preparing such compounds, as well as their pharmaceutical applications, is disclosed in PCT / EP93 / 00327.

The present invention provides that extracts of plants and plant residues that are rich in both substrates required for the synthesis of such active compounds can be obtained, i. y. sugars, oligo- and polysaccharides on the one hand, just like amino acids and peptides on the other, and finally having organic acids and / or compounds with at least one active methylene group as a catalyst for the reaction described above.

This process allows the production of non-toxic preparations, e.g. derived from peat, which has immunomodulatory activity and promotes the production of cytokines (interferons, tumor necrosis factors, etc.) in living organisms, suitable for therapeutic and prophylactic use in humans and animals. Untreated peat is extracted and the humic substances are separated from the extract obtained by precipitation in acidic medium; acidification with hydrochloric acid to pH 1.5-3.0; separating the precipitate and concentrating the extract by evaporation under reduced pressure and / or nanofiltration, whereupon removing inorganic salts; raising the pH of the concentrated solution to 6.0-7.1; the resulting mixture is concentrated to a thick syrup and heated to 70-90 ° C, preferably to 80 ° C, until completion of Amadori regrouping of the products formed by reaction of simple sugars, oligo- and / or polysaccharides extracted from peat with amino acids and / or peptides; non-peptide bond formation) in the NH ₂ group is substituted by a suitable C _x radical of 1-deoxy-2-ketose; then quenching by cooling the reaction mixture; hydrophobic substances which reduce the immunotropic activity of the product are removed from the reaction mixture by selective adsorption onto a specific adsorbing resin such as AMBERLITE XAD type, preferably XAD-2 or XAD-4, or any other resin having similar properties.

which is derived from the water

Ideally, when the extract is treated, a mixture of the alkaline and aqueous extracts, trapping the natural peat, first with an alkali solution, and then with water, may be washed with an acid solution before extraction. Natural marsh peat with a degree of humification of H 6 to Hl0 is best.

Amadori rearrangement reaction is best performed in concentrated aqueous solution or concentrated aqueous-alcoholic solution.

Ideally, adsorption of the substances to be removed from the final product is carried out from a neutral or weakly acidic solution; wash the columns with distilled water and dilute aqueous alkaline solution. From the final solution, biologically and therapeutically active substances are isolated by spray drying in solid form, the Amadori rearrangement reaction can be controlled by a test reaction, i. y. reaction of the reduction of potassium hexacyanoferrate (III) with alkaline media by products of rearrangement. At room temperature, potassium hexacyanoferrate (III) is reduced per minute by reducing agents such as ascorbic acid, within five minutes by redeployment products and by 15-30 minutes by reducing sugars. The reduction product potassium hexacyanoferrate (II) with iron (III) sulfate forms Berlin Blue. Such a reaction can be used for photometric quantitative monitoring of the rearrangement reaction. Take samples of the reaction mixture at appropriate intervals and perform a test reaction. The rearrangement reaction stops when the intensity of the Berlin blue color in the reduced sample no longer increases.

Unexpectedly, it has been found that in a mixture of treated substrates, such as peat extract,

The formation of Amadori rearrangement products is easier than in a laboratory mixed formulation of the same sugars and amino acids.

It has further been found that some of the substances contained in the peat extract increase the biological effectiveness of the final formulations obtained by the process of the present invention compared to analogous synthetic products, while other substances decrease such efficacy.

The initial peat extract used in the process of the present invention, which is rich in sugars and compounds with amino groups, is obtained as described in the literature. The best starting material is peat marsh with a high degree of humification (for example, H6 to H10 on a scale known from the book: Moor und Torf-Kunde, Karl-Hans Gotlich, Stutgart, 3rd ed. 1990), and when carbon to nitrogen is between 15 and 35.

Aqueous extracts can be obtained by any of the methods described above, preferably in combination, i. y. using acidic, neutral and / or alkaline extraction media. Ideally, natural peat is initially extracted briefly (1 to 2 hours) in an acidic aqueous extraction medium to leach inorganic materials such as carbonates, metal oxides and the like; thereafter, over a period of several hours to more than twenty hours, extraction in an alkaline aqueous medium (for example, 0.2% to 0.6% sodium hydroxide solution) at 20-50 ° C to dissolve the humic and fulvic acids; and finally extracting with water, preferably distilled water, at 30-60 ° C for a further 12 hours or more; the acidic extract is then removed; the alkaline and neutral extracts are mixed and further processed according to the present invention.

It is known that peat after extraction is difficult to separate from the extract and that expensive extracts such as centrifugal separators are required to obtain clear extracts with good yield. Such difficulties can be eliminated by using special extractors in which the solid part of the peat is in a stationary bearing and the extracting medium circulates through it. Details of the extraction process will be provided elsewhere in the description.

According to the present invention, the alkaline aqueous extract is acidified to a pH of 1.5-3.0, preferably 2.3-2.5, which causes the humic acids to fall into the precipitate. 6 mol / L hydrochloric acid is suitable for acidification. Precipitate formation occurs over a period of several to more than 20 hours, and the resulting clear excipient can be decanted. Centrifugation or filtration could be used to collect the remainder of the extract.

The clear solution is then concentrated in the rotary evaporator to a tenth of the initial volume. Otherwise, concentration and removal of inorganic salts can be accomplished by adjusting the pH of the solution by nanofiltration. The term nanofiltration is used to define an altered reversible osmosis process where the membranes used are permeable only to small size molecules such as simple inorganic salt molecules. They do not use retention time to characterize such membranes, but more often divide them by sodium chloride conductivity. Satisfactory results were found to be obtained with HC5OPP-type membranes having 40-60% conductivity on sodium chloride or CA865PP-type membranes having 66-74% conductivity on sodium chloride (Dow product, Denmark). In the process with these or similar membranes, it is possible to carry out electrolyte removal along with solution concentration with practically no loss of low molecular weight organic matter, which is considered to be the most important process factor in the present invention (whereas other higher molecular weight components are , contributing to the final activity of the desired product that is ultimately obtained.

The two methods of concentration just described can be used one after the other in the same process. Preferably, the solvent is initially evaporated in a vacuum rotary evaporator; then the solution is neutralized and subjected to nanofiltration until it reaches a concentration of 8 to 12 g of solid dry residue in 100 ml of solution. If any particular application requires a higher degree of desalination, i. y. less inorganic salts must be present, the solution can be either periodically or continuously diluted with distilled water during nanofiltration (diafiltration). Larger cations, such as Ca, Fe, Al, which migrate much more slowly across membranes, may be replaced by sodium ions prior to nanofiltration by passing the solution to be concentrated through a column of weak cation exchanger (such as AMBERLITE E IRC50) with sodium-substituted ions.

At the end of the nanofiltration, the pH of the residual solution is followed and adjusted to 6.0-7.2, preferably 6.5-6.8, if necessary. The solution is then transferred to a rotary evaporator and concentrated at about 35-40 ° C to about half its initial volume (until a thick, syrupy solution is formed). The temperature is now raised to 7090 ° C, preferably up to 80 ° C, at atmospheric pressure with constant stirring, and heating is continued. Test samples are taken every 30 minutes and after 1.5 hours every 15 minutes. The progress of the reaction is monitored by measuring the intensity of the color produced in the test reaction described above. If the color intensity after the described test reaction with a given sample is equal to or less than that of the previous sample, the reaction is terminated by sudden cooling of the heating bath.

Alternatively, the reaction may be carried out in the following manner: the reaction mixture is concentrated in the same manner as described above. It is then diluted with 50% aqueous ethanol sufficient to dissolve the product; the further diluted solution is heated to reflux and the reaction is monitored and controlled as described above, except that the solvent is evaporated from each sample prior to the test reaction. When the reaction is complete, the alcohol is distilled off under reduced pressure. After determining the dry solids content of the reaction mixture, dilute the latter with distilled water to form a 10-12% solution, which is then slowly passed through a column packed with a nonionic XAD-type adsorbent (Rohm & Haas product), preferably XAD2 or XAD-4. type. When in contact with this adsorbent, hydrophobic substances are the most strongly adsorbed, whereas hydrophilic substances such as sugars are the weakest adsorbent. Collect the liquid leaving the column. The column is then washed with distilled water and the eluate collected in separate fractions. Each fraction is analyzed chromatographically and / or tested for activity in biological samples. The column then passes initially through the liquid with fractions of eluate selected according to their required activity to give a product with a predetermined ratio of activity measured in different assays and biological assays. Biological tests are described in detail below. Subsequently, the strongly adsorbed substances are washed with dilute alkali solution while the eluate is still neutral. Subsequent fractions are rejected because they contain substances that strongly inhibit the biological activity of the final formulation.

The solution obtained in the previous step is sterilized by membrane filtration and dried under sterile conditions in a spray dryer at an inlet air temperature of 180-185 ° C and an outlet air temperature of about 80 ° C.

The resulting powder is beige and remains biologically active for more than a year when stored at room temperature in air-tight containers and in the dark.

The activity of the final product described above is assessed by the following tests showing the effect of the preparation on the individual immune system responses:

1. Determination of the percentage of splenocytes producing E-rosettes according to Bach and Dardenn (Cell. Immunol. 3, 116, 1972)

2. Determination of splenocytes producing IgM-type hemolytic antibodies by the method of Jerne modified by Mishell and Dutton (J. Exp. Med. 126, 423-442, 1967).

3. Determination of Haemagglutination of 19S + 7S and 7S Antibodies in Active Haemagglutination Assay by Adler (J. Immunol. 95, 26-38, 39-47, 1965)

4. Mouse thymocyte 20-well culture with hydrocortisone survival test (cytotoxic test)

5. Interferon and TNF Induction Assay (PCT / EP93 / 00327)

In all of the above tests, the product of the present invention exhibits stimulatory activity at concentrations ranging from 0.05 to 1 mg / kg (tests 1 to 3), 0.05 to 10 µg / ml (test 4) and 10 when compared to control blank controls. up to 100 µg / ml (Test 5).

As regards the other properties of the product obtained according to the invention, it should be noted that the respective properties fall within the limits defined by the TTP (Tolpa Torf Preparation) issued by the Polish Ministry of Health, the data of which are given in the standard of analysis of this preparation.

The provided process for extracting the bioactive product from peat yields a yield 10 times higher than traditional methods according to Polish patent no. 124110. This means that less valuable peat is used, which is the raw material. Moreover, peat is actually washed with water after extraction with an alkaline solution, so that after the process they are neutral (not contaminated with alkali or acids) and environmentally friendly; they are not environmentally hazardous and can be used directly in agriculture.

In addition, the process of the present invention does not use any flammable organic solvent such as diethyl ether. Due to the higher concentration of active substances, including low molecular weight, the preparation is more easily absorbed by the oral route than by the conventional method of TTP.

The following examples further illustrate the present invention:

Example 1: 45 kg of peat with a moisture content of 64-65% is crushed, sieved through a 14 mm sieve and poured into a semi-industrial sized extractor specially designed for peat extraction. Through the peat layer from the 180 L feed tank, very slowly, allowing the liquid level in the extractor to rise to about 1 cm / min, allows the extraction of 0.4% sodium hydroxide solution. When the entire peat layer is submerged under the liquid, the flow rate is increased to 500 rpm. Recycle the extract for 27 hours at 40 ± 2 ° C. The power pump is then switched off; switches the supply valves to a 120 l container with distilled water and continues the circulation of the washer fluid at the same temperature for another 16 hours.

The alkaline solution and aqueous extracts combine; the extract is decanted to give 240 l of a liquid having a pH of 12.1. The collected liquid is then treated separately by dividing it into two halves.

Example 2: 120 l of the extract obtained according to the procedure described in Example 1 are acidified to pH 2.1 using 1.61 6 mol / L hydrochloric acid. Leaves the acidified mixture for sedimentation for 24 hours. After this time, 75 L of the clear solution is decanted and the remaining 25 L is recovered by filtration. Both clear solutions are combined and concentrated under reduced pressure in a rotary glass evaporator (SIMAX) until the volume drops to 19 1. The pH of the concentrated solution is increased to 6.8 by the addition of caustic soda. The resulting solution is nearly neutral, diluted to 50 L with distilled water, and then subjected to nanofiltration in a Lab Unit M20 (Dow Separation System, Denmark, manufacturing) equipped with HC5OPP membranes. Such membranes have a total filtration area of 0.36 m, a pressure of 20 atm and a temperature of 21 ° C. The final volume is 7 liters.

The pH of the concentrated extract is 6.6 and therefore does not require any adjustment.

The resulting concentrated extract is transferred to a vacuum rotary evaporator (BUCHI, Switzerland) and further concentrated under reduced pressure to 1.3 1. Further, the solution is heated to 80 ° C under atmospheric pressure; heating continues with continuous stirring. The course of the desired reaction is followed by determining the color intensity of the samples taken after the potassium hexacyanoferrate (III) reduction test. After 135 min the color intensity stabilizes or even slightly decreases. The reaction is stopped by cooling the reaction mixture to 20 ° C. After the reaction, the available mixture is transferred to a smaller (laboratory) evaporator and further concentrated under reduced pressure to 800 ml (now containing 13.25 g of dry solid per 100 ml).

The concentrated solution is filtered and passed through a column of 26 mm diameter filled with 270 ml of XAD-2 adsorbent at a rate of 0.8 ml / min. Collect the liquid and wash the column with distilled water. Collect three successive fractions (1 to 3) of 160, 240 and 400 ml respectively. Continue to use 0.1% sodium hydroxide in the eluent and collect the liquid until the eluate has a pH above 7.0; this fourth 220 ml fraction has a pH of 7.0. The composition of the fractions is determined chromatographically (horizontal liquid chromatography on paper). Fraction # 1, 2, 300 ml of fraction no. 3 and 150 ml fraction no. After mixing, the solution is concentrated to 100 ml and then mixed with a portion of the liquid initially passing through the column. The final solution is sterilized by filtration under sterile conditions through a membrane with 0.22 µm pores, then dried under sterile conditions in a spray dryer at an inlet air temperature of 185 ° C and an outlet air temperature of 81 ° C. The total yield of the powder obtained is 96 g.

Example 3: The remainder of the 120 L extract is acidified (as in Example 2 above) to pH 2.3, separating the humic acid precipitate as described in Example 2 to give 98 L of acidified clear solution. The solution is neutralized to pH 6.7 with 4 M NaOH and left for 12 hours. It is then filtered and subjected to nanofiltration under the same conditions as described in Example 2. The final volume of the filtrate is 101. Further, 20 L of distilled water are added; the resulting solution is finally concentrated to 7 1. Further treatment is carried out in the same manner as in Example 2 except that Adamori proceeds with the formation of the rearrangement products for 110 min. The final powder yield is 84 g.

Claims (5)

DEFINITION OF INVENTION 1. A process for the preparation of a preparation having an immunomodulatory activity and promoting the formation of cytokines in living organisms, suitable for the treatment and prophylaxis of animals, which comprises extracting the natural plants and their residues by separating the humic substances from the obtained extract by acid precipitation. -the humic substances are removed from the extract by acidifying it to pH 1.5-3.0 and separating the precipitate, further concentrating the extract by evaporation under reduced pressure and / or nanofiltration, whereupon the inorganic salts are removed, then the pH of the concentrated solution is raised to 6.0 -7.1, -concentrates the resulting mixture to a thick syrup and warms it to 70-90 ° C, preferably to 80 ° C, until Amadori's rearrangement of the products formed by reaction with simple sugars extracted from peat, oligo- and polysaccharides with amino acids and / or some free peptides, peptide bond formation) in the -NH ₂ group is substituted by a suitable C-radical of 1-deoxy-2-ketose, The reaction is stopped by cooling the reaction mixture by removing the hydrophobic substances (which would reduce the immunotropic activity of the product) by selective adsorption using a specific absorbent resin of type AMBERLITE XAD, preferably XAD-2 or XAD-4 or another resin having similar properties. 2. A process according to claim 1, characterized in that the initial extract is a mixture of alkaline and aqueous extracts obtained from natural untreated, preferably low-peat peat with a degree of humidity of H6-H10, which can then be washed with acid before being extracted with water. solution. 3. A process according to claim 1 or 2, wherein the Amadori pre-regrouping reaction is carried out in a concentrated aqueous solution or a concentrated aqueous alcoholic solution. 4. A process according to claims 1-3, wherein the substances which are not present in the final product are adsorbed from the acidic or neutral solution on the columns, which are then washed with distilled water and dilute alkali solutions. 5. A process according to claims 1-4, characterized in that the final solution is spray-dried while sterilizing.