WO1994012197A1 - Process for the manufacture of a preparation having immunomodulating activity and stimulating cytokine formation by extracting plants and plant residues - Google Patents

Process for the manufacture of a preparation having immunomodulating activity and stimulating cytokine formation by extracting plants and plant residues Download PDF

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Publication number
WO1994012197A1
WO1994012197A1 PCT/EP1993/003362 EP9303362W WO9412197A1 WO 1994012197 A1 WO1994012197 A1 WO 1994012197A1 EP 9303362 W EP9303362 W EP 9303362W WO 9412197 A1 WO9412197 A1 WO 9412197A1
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WO
WIPO (PCT)
Prior art keywords
concentrated
peat
extract
reaction
solution
Prior art date
Application number
PCT/EP1993/003362
Other languages
English (en)
French (fr)
Inventor
Jan Zbigniew Mioduszewski
Krystyna Witkiewicz
Magdalena Kowalska
Joanna Goral
Magdalena Klimecka
Original Assignee
Torf Establishment
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Torf Establishment filed Critical Torf Establishment
Priority to AU56295/94A priority Critical patent/AU5629594A/en
Publication of WO1994012197A1 publication Critical patent/WO1994012197A1/en

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/02—Medicinal preparations containing materials or reaction products thereof with undetermined constitution from inanimate materials
    • A61K35/10—Peat; Amber; Turf; Humus

Definitions

  • the present invention relates to a process for obtaining out of plants and plant residuesa non-toxic preparations having immunomodulating activity and stimulating the formation of cytokines (interferons, tumor necrosis factor, etc.) when administered to humans and animals.
  • cytokines interferons, tumor necrosis factor, etc.
  • Hyoshita Takuya et al (Chem. Abstr. 1974, 00, 47021j) teach a process in which peat is extracted several times with an 8% sodium hydroxide solution; the obtained extract is acid ⁇ ified, centrifuged to separate the solid particles, then dialyzed and purified on chromatographic columns.
  • the fraction is mainly (95 %) composed of polysaccharides and has antineoplastic activity.
  • the Polish patent application 279.475 covers a process of peat extraction with an acidified aqueous extracting medium.
  • the acidic ex ⁇ tract is neutralized and concentrated by means of ultra- filtration or reverse osmosis employing membranes having a retention limit - cut off - from 500 to 10.000 daltons.
  • the product is separated by means of column gel filtration or on anion exchange resins. Fractions differing in salt concentrations, and in the ratio of polysaccharides to gly- coproteides, may also be obtained. It is, however, indicated that all mixtures obtained are non-toxic and show immuno- tropic activity.
  • some secondary reactions between the extracted compounds may intentionally occur in a controlled direction and to a controlled extent so that new compounds are formed that in known - e.g. peat - extracts would either be absent, or be present in rather low concentrations only;
  • the processing of extracts thus obtained is modified so that valuable and beneficial components of the extract are not eliminated when the ballast substances - both extracted from natural peat and/or introduced with the extracting medium - are separated.
  • the organic substances related to the biological and thera ⁇ Treatmentic activity of peat derived preparations - including TTP (TOEPA TORF PREPARATION, Trademark protected in the name of TORF CORPORATION, Poland) - are Amadori rearrangement com- pounds and products obtained in the course of such rear ⁇ rangement of aldoses and aminoacid reaction products, i.e.
  • R1-CO-CH2-NH-R2 wherein R1 is a residue of 1-desoxy-2-keto-sugar, oligo- or polysac- charide in which such 1-desoxy-2-keto group is an end group of a carbon atom chain, and wherein R2 is a aminoacid re- sidue or peptide residue in which the end group is a free NH2- group not involved in the peptide bond.
  • the instant process allows to manufacture non-toxic prepa- rations, e.g. peat derived ones, having immunomodulating activity and stimulating cytokine (interferons, tumor necrosis factor and others) formation in the living organ ⁇ isms, suitable for therapeutic and preventive use in humans and animals.
  • non-toxic prepa- rations e.g. peat derived ones
  • immunomodulating activity and stimulating cytokine interferons, tumor necrosis factor and others
  • Raw peat is extracted; humic substances are separated from the obtained extract by means of precipita ⁇ tion in acidic environment; acidification is carried out with hydrochloric acid to pH 1,5 - 3,0; the precipitate is separated and the extract concentrated by evaporation under reduced pressure and/or by means of nano-filtration, simul- taneously eliminating inorganic salts; the pH of the concen ⁇ trated solution is brought to 6,0 - 7,1; the resulting mix ⁇ ture is concentrated to a thick syrup and heated up to 70- 90°C, preferably to 80°C until completion of the Amadori rearrangement of the aminoacid and/or peptide products sub- stituted at the terminal free NH2- group (not involved in any peptide bond) with a suitable 1-desoxy-2-ketose C1- radical, resulting from the reaction of simple sugars, oligo- and polysaccharides with aminoacids and/or peptides extracted from peat; the reaction is then stopped by cooling down the reaction
  • the peat extract to start with is a mixture of alkaline and water extracts obtained by means of extracting natural peat - optionally first washed with an acidic solu ⁇ tion - first with an alkaline aqueous solution and then with water.
  • Preferred raw peat is low in humification degree H6 to H10.
  • the Amadori rearrangement reaction is preferably carried out in a concentrated aqueous solution or concentrated water- alcohol solution.
  • the adsorption of substances to be eliminated from the final product is carried out from the neutral or slightly acidic solution; the columns are eluated with dis- tilled water and diluted alkaline aqueous solution. From the final solution, biologically and therapeutically active sub ⁇ stances are isolated in a solid form by spray-drying.
  • the Amadori rearrangement reaction can be monitored by means of a test reaction, i.e. reduction of potassium ferricyanide with the rearrangement products in alkaline solution.
  • a test reaction i.e. reduction of potassium ferricyanide with the rearrangement products in alkaline solution.
  • the potassium ferricyanide is reduced within the first minute by reducers such as ascorbic acid, within the first five minutes by the desired rearrangement products and within 15-30 minutes by reducing sugars.
  • the reduction product potassium ferrocyanide with ferrous sul- fate forms prussian blue.
  • Such reaction can be used for photometric quantitative monitoring of the rearrangement reaction. Samples of the reaction mixture are taken in pre ⁇ determined time intervals and subjected to the test reac- tion. The rearrangement reaction is stopped when the in ⁇ tensity of the prussian blue in a reduced sample is not increasing any longer.
  • the initial peat extract used in the process according to the invention being rich in sugars and amino-groups, is ob ⁇ tained in a way described in literature.
  • Preferred starting material is a low peat showing a high degree of humification (for example H6 to H10 in a scale known from Moor- und Torf-rete, ed. Karl-Hans Gottlich, Stuttgart, 3rd edition, 1990) and a carbon to nitrogen ratio of from 15 to 35.
  • Aqueous extracts may be obtained by any - preferably by a combination - of the above described methods, i.e. with aci ⁇ dic, neutral and/or alkaline aqueous extracting media.
  • the raw peat is first shortly (1 to 2 hours) ex ⁇ tracted with an acidic aqueous extraction medium in order to leak out inorganic substances, such as carbonates, metal oxides and the like; then for a prolonged time - of several to more than twenty hours - with aqueous alkaline extraction medium (for example a 0,2% to 0,6% NaOH-solution) at 20-50°C in order to dissolve humic and fulvo acids; and finally with water, preferably distilled water at 30-60°C for another 12 or more hours; the acidic extract is then discarded; the al ⁇ kaline and neutral extract are combined and further process ⁇ ed according to the present invention.
  • aqueous alkaline extraction medium for example a 0,2% to 0,6% NaOH-solution
  • the alkaline aqueous ex ⁇ tract is acidified to pH 1,5-3,0 - preferably 2,3-2,5 - in order to precipitate humic acids.
  • 6N hydrochloric acid is a suitable acidification medium.
  • the precipitates sediment within several to more than 20 hours, and the clear extract can be decanted. Centrifugation or filtration may be used to collect the remaining portion of the extract.
  • the clear solution is then concentrated in a rotary evapor ⁇ ator down to 1/10 of the initial volume.
  • concentration and elimination of inorganic salts may be a- chieved by means of nano-filtration while adapting the pH- value of the solution.
  • nano-filtration is used to define a modified reverse osmosis process in which membranes permeable only for small size molecules, such as simple in ⁇ organic salts, are used. Such membranes are not character ⁇ ised by a retention time but - more frequently - by per- meability for NaCl.
  • concentration may also be em ⁇ ployed in sequence in the same process.
  • the solvent is evaporated in a rotary vacuum evaporator; then, the solution is neutralized and subjected to nano- filtration until a concentration of 8-12 g dry solids in 100 ml of solution is reached.
  • the solution may be diluted in the course of nano-filtration with distilled water (dia- filtration) either periodically or in a continuous manner.
  • Cations of a bigger size such as Ca, Fe, Al, which migrate through the membranes much slower, may be substituted with sodium cation before the nano-filtration process is started by means of passing the solution to be concentrated through a column with a weak ion exchanger (for example AMBERLITE E IRC50) in sodium form.
  • a weak ion exchanger for example AMBERLITE E IRC50
  • the pH of the remaining solution is controlled and if necessary adjusted to 6,0-7,2, preferably to 6,5-6,8.
  • the solution is then placed in a ro ⁇ tary vacuum evaporator and is concentrated at temperature of 35-40°C down to approximately half of its initial volume (until a syrup-like, thick solution is obtained).
  • the temperature of the solution is raised to 70-90°C, preferably to 80°C, and heating is continued.
  • Test samples are taken at the beginning every 30 min, after 1,5 hours every 15 min. Monitoring of the reaction progress is accomplished by meas ⁇ uring the intensity of colour in the test reaction described above. If the intensity of the colour in the test reaction performed for a given sample is the same or lower than for the preceding sample, the reaction is stopped by rapidly cooling down the heating bath.
  • the reaction may be carried out in the fol ⁇ lowing manner:
  • the reaction mixture is subjected to the same concentration process as previously described. Then it is diluted with a 50% aqueous solution of ethanol in an amount sufficient for dissolving the product; then, the diluted solution is heated under reflux, and the progress of the re ⁇ action is monitored and controlled as described above, ex ⁇ cept that the solvent is evaporated from each sample prior to subjecting it to the test reaction.
  • alcohol is distilled off under reduced pressure.
  • the liquid that initially passed through the column is then com ⁇ bined with chosen eluate fractions selected for their desir- ed activity in order to obtain the product reaching a prede ⁇ termined ratio of activities measured in different tests and biological assays.
  • the biological assays are described in details below.
  • the most strongly adsorbed substances are eluated with a diluted alkali solution until the eluate is still neutral. Further fractions are discarded since they contain substances strongly inhibiting biological activity of the final preparation.
  • the solution obtained in the previous step is sterilized by means of membrane filtration and dried in a spray dryer under sterile conditions with an air inlet temperature of 180-185°C and an air outlet temperature of approx. 80°C.
  • the obtained powder is beige in colour and when stored - at room temperature, in an air-tight container in dark - maintains its biological activity for more than a year.
  • the present process for obtaining a bioactive product out of peat results in a 10 times higher yield than traditional methods according to Polish patent No. 124 110. This means that less of the valuable peat is used as a starting raw material. Furthermore the post-extraction peat is actually washed with water after the alkaline extraction process and therefore finylly is neutral (not contaminated with alkali or acids) and environmentally safe; it is not ecologically dangerous and may be used directly in agriculture.
  • any use of flammable organic solvents such as ethyl ether is eliminated. Due to the higher concentration of active sub ⁇ stances - also those of low molecular weight - the prepara ⁇ tion is resorbed more easily when administered orally, than a TTP obtained in a traditional way.
  • Example 1 The present invention is illustrated in more detail in the following examples: Example 1 :
  • the resulting solution was nearly neutral and diluted with distilled water to a total volume of 50 1 and subjected to nano-filtration in Lab Unit M20 (product of Dow Separation System, Denmark) equipped with flat membranes of a HC50PP type.
  • the total filtration area of such membranes was 0,36 m2 at a pressure of 20 atm and at a temperature of 21 °C.
  • the final volume of the filtrate was 7 liters.
  • the concentrated extract had a pH-value of 6,6 and therefore did not require any adjusting.
  • the concentrated extract as obtained above was charged into a vacuum rotary evaporator (BUCHI, Switzerland) and further concentrated under reduced pressure to a volume of 1,3 1.
  • the solution was brought to atmospheric pressure and to a temperature of 80°C; heating was continued under con ⁇ tinuous stirring by means of rotation.
  • the progress of the desired reaction was monitored by means of determining the intensity of colour in the samples taken after the test re- duction of potassium ferricyanide. After 135 min the colour intensity was stable or even slightly decreased.
  • the reac ⁇ tion was stopped by cooling it down to a temperature of 20° C.
  • the post-reaction mixture was now placed in a smaller .

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines Containing Plant Substances (AREA)
  • Compounds Of Unknown Constitution (AREA)
PCT/EP1993/003362 1992-12-02 1993-12-01 Process for the manufacture of a preparation having immunomodulating activity and stimulating cytokine formation by extracting plants and plant residues WO1994012197A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU56295/94A AU5629594A (en) 1992-12-02 1993-12-01 Process for the manufacture of a preparation having immunomodulating activity and stimulating cytokine formation by extracting plants and plant residues

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
PLP.296811 1992-12-02
PL92296811A PL170294B1 (pl) 1992-12-02 1992-12-02 Sposób otrzymywania z torfu aktywnego, nietoksycznego preparatuo dzialaniu immunomodulacyjnym i stymulujacym wytwarzanie w organizmie cytokin PL

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WO1994012197A1 true WO1994012197A1 (en) 1994-06-09

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PCT/EP1993/003362 WO1994012197A1 (en) 1992-12-02 1993-12-01 Process for the manufacture of a preparation having immunomodulating activity and stimulating cytokine formation by extracting plants and plant residues

Country Status (9)

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AU (1) AU5629594A (enrdf_load_stackoverflow)
CO (1) CO4180517A1 (enrdf_load_stackoverflow)
EE (1) EE03136B1 (enrdf_load_stackoverflow)
LT (1) LT3241B (enrdf_load_stackoverflow)
PE (1) PE55894A1 (enrdf_load_stackoverflow)
PL (1) PL170294B1 (enrdf_load_stackoverflow)
TW (1) TW272129B (enrdf_load_stackoverflow)
WO (1) WO1994012197A1 (enrdf_load_stackoverflow)
ZA (1) ZA938950B (enrdf_load_stackoverflow)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2818967A1 (fr) * 2001-01-04 2002-07-05 Evatex Dispositif de recyclage des eaux chargees en saumure et en polluants organiques
RU2219935C1 (ru) * 2002-07-09 2003-12-27 НИИ онкологии им. проф. Н.Н. Петрова Средство для профилактики рака

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE2846482A1 (de) * 1977-10-25 1979-04-26 Akad Wroclawiu Rolnicza Ein verfahren zur gewinnung eines neoplasmen gegenwirkenden praeparates aus angesaeuertem, alkalischen hydrolysat von torf
WO1992016600A1 (en) * 1991-03-16 1992-10-01 Torf Establishment Process for the extraction of peat and apparatus for carrying out the process
WO1992016216A1 (en) * 1991-03-16 1992-10-01 Torf Establishment Peat-derived bioactive products and pharmaceutical and cosmetic compositions containing them

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE2846482A1 (de) * 1977-10-25 1979-04-26 Akad Wroclawiu Rolnicza Ein verfahren zur gewinnung eines neoplasmen gegenwirkenden praeparates aus angesaeuertem, alkalischen hydrolysat von torf
WO1992016600A1 (en) * 1991-03-16 1992-10-01 Torf Establishment Process for the extraction of peat and apparatus for carrying out the process
WO1992016216A1 (en) * 1991-03-16 1992-10-01 Torf Establishment Peat-derived bioactive products and pharmaceutical and cosmetic compositions containing them

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
A.D. INGLOT ET AL.: "NEW NATURAL CLINICALLY USEFUL ORAL IMMUNOMODULATOR TOLPA R EXTRACTED FROM PEAT IS INTERFERON INDUCER IN HUMAN LEUKOCYTES", JOURNAL OF INTERFERON RESEARCH, vol. 11, no. S. 1, November 1991 (1991-11-01), pages S264 *
ANNA D. INGLOT ET AL.: "TOLPA* TORF PREPARATION (TTP*) INDUCES INTERFERON AND TUMOR NECROSIS FACTOR PRODUCTION IN HUMAN PERIPHERAL BLOOD LEUKOCYTES", ARCHIVUM IMMUNOLOGIAE ET THERAPIAE EXPERIMENTALIS, vol. 41, no. 1, 1993, pages 73 - 80 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2818967A1 (fr) * 2001-01-04 2002-07-05 Evatex Dispositif de recyclage des eaux chargees en saumure et en polluants organiques
RU2219935C1 (ru) * 2002-07-09 2003-12-27 НИИ онкологии им. проф. Н.Н. Петрова Средство для профилактики рака

Also Published As

Publication number Publication date
PE55894A1 (es) 1995-02-17
PL296811A1 (en) 1994-06-13
LTIP1509A (en) 1994-12-27
CO4180517A1 (es) 1995-06-07
PL170294B1 (pl) 1996-11-29
ZA938950B (en) 1994-08-02
EE03136B1 (et) 1998-12-15
TW272129B (enrdf_load_stackoverflow) 1996-03-11
LT3241B (en) 1995-04-25
AU5629594A (en) 1994-06-22

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