CA2083061C - Peat-derived bioactive products and pharmaceutical and cosmetic compositions containing them - Google Patents
Peat-derived bioactive products and pharmaceutical and cosmetic compositions containing them Download PDFInfo
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Abstract
The peat-derived bioactive product contains not more than 70%, preferably not more than 60% by weight of inorganic salts, especially of sodium chloride, based on dry solids.
It is obtainable by a process wherein a highly concentrated aqueous solution of inorganic salts, especially of sodium chloride containing peat-derived bioactive ingredients is diluted with demineralized water and subjected to reverse osmosis in order to desalinate the solution, inorganic salts being removed, and wherein the resulting solution is concentrated and clarified, and, optionally, in at least one further step, sterilized and/or spray-dried. A
pharmaceutical formulation containing a peat-derived bioactive product, in the form of a gel, is prepared by combining a sterile alcoholic herb extract with sterile glycerol, a sterile aqueous solution of previously powdered peat-derived bioactive product and a sterile menthol solution; the resulting mixture is gradually combined with colloidal silica to convert the liquid composition into gel form, the weight ratio of liquid mixture to silica preferably being from 90:10 to 94:6. A cosmetic preparation such as a gel, ointment, balm, shampoo, bath salt lotion etc. contains as active ingredient the instant peat-derived bioactive product in a quantity of 0.01.-10% by weight, preferably 0.05-1% by weight, more preferably 0.05-0.1% by weight.
It is obtainable by a process wherein a highly concentrated aqueous solution of inorganic salts, especially of sodium chloride containing peat-derived bioactive ingredients is diluted with demineralized water and subjected to reverse osmosis in order to desalinate the solution, inorganic salts being removed, and wherein the resulting solution is concentrated and clarified, and, optionally, in at least one further step, sterilized and/or spray-dried. A
pharmaceutical formulation containing a peat-derived bioactive product, in the form of a gel, is prepared by combining a sterile alcoholic herb extract with sterile glycerol, a sterile aqueous solution of previously powdered peat-derived bioactive product and a sterile menthol solution; the resulting mixture is gradually combined with colloidal silica to convert the liquid composition into gel form, the weight ratio of liquid mixture to silica preferably being from 90:10 to 94:6. A cosmetic preparation such as a gel, ointment, balm, shampoo, bath salt lotion etc. contains as active ingredient the instant peat-derived bioactive product in a quantity of 0.01.-10% by weight, preferably 0.05-1% by weight, more preferably 0.05-0.1% by weight.
Description
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_ 1 _ PEAT-DERIVED ~IOACTI~fE PRODUCTS AND PHARMACEUTICAL AND COSMETIC
CDt~4~OSITIONS CONTAINING T(~IEM
The present invention relates primarily to novel peat-derived bioactive products and to a process .for producing such products.
The invention also relates to pharmaceutical and cosmetic compositions containing these products and to processes =or preparing pharmaceutical and/or cosmetic formulations containing said peat-derived bioactive products.
It is known to extract peat by various methods using various extraction media and to use such extraets, containing peat-derived bioactive ingredients, for cosmetic and pharmaceutical purposes.
On o= these known processes is described in Polish patent specification No. 124110 (Chemical Abstracts 101(10), 78854e).
According to this prior art process peat-derived bioactive products are obtained by primary and secondary alkaline hydrolysis of an air-dried raw peat material, followed by acidification of the thus obtained hydrolysate and separation o~
insoluble solid parts with subseguent second alkali~atio.~., acidification of the c'_ear liguid phase and elim~.nar__on c~
ballast substances by means o~ alcohol and etner eat=act;on. ..
said process, the aqueous phase from the organic e_ct=act_on is a liauid neat-derived bioactive product.
The known liquid product, being a solution ov peat--dewived active ingredients in a highly concentrated, near-,~ saturatea acrueous solution of sodium chloride, obtained according to t:~.°
above cited Polish patent specification, is unstab'~e wPen stores for a long time and, moreover, contains - regardinc the b=o-logical activity of the composition - a large excess one~~tra_ inorganic substances. As a bulk product, it is ha=d to handl°, store and process.
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~'O 92/ 16216 f ~'~~~2~~~ 1 -It is the main object of the present invention to provide a product which is not afflicted with these disadvantages, i.e. a product which is stable and which can easily be formulated into pharmaceutical and veterinary products as well as be introduced, either in solid state or in any suitable solution, into cosmetic preparations.
In particular, with respect to the use of a peat-derived bio-active product for pharmaceutical purposes, i.e. for the production of pharmaceutical compositions, there was a strong need for providing a solid form which would be well suited for this purpose. As previaus attempts aiming at concentration o' the known aqueous solution of peat-derived bioactive substances and desalination of the same failed to give a positive result due to the occlusion of active ingredients in a crystallising solid phase, causing decrease of biological activity pa ~ze composition, it was very hard to find a suitable process °or converting the liquid composition into powder form.
Unexpectedly, it was found that a positive result is achievable if, prior to concentration, the liquid composition is L-rst diluted several times, i.e. by water volumes several times the volume of the composition.
Accordingly, the prey ent invention provides a process by whic~ a peat-derived bioactive product, featuring the above advantageous characteristics, is obtainable from a highly concentrated aqueous inorganic salt - especially sodium chloride - solutio:~
containing peat-derived bioactive ingredients, by diluting said solution with demineralised water, e.g. distilled water, ~ollow~-ed by reverse osmosis, concentration and clarification. A solut-ion so obtained can easily be converted into a sterile and solid product, well suited to the intended purposes, by stervlizatio.~.
and spray-drying. Dilution is preferably effected with quantit-ies of water several times, preferably 5 to 8 times, the quanti-ty of the concentrated solution to be diluted.
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Thus, in one aspect, the present invention provides a process for producing a peat-derived bioactive product. A
concentrated solution of a peat extract containing inorganic salts and peat-derived bioactive ingredients is subjected to processing steps comprising: (a) a dilution step wherein the solution is diluted with demineralized water, (b) a subsequent desalination step wherein the diluted solution is subjected to reverse osmosis in order to remove inorganic salts from the solution, such that the desalinated solution contains no more than 70% by weight, based on dry solids, of the inorganic salts being removed, (c) a concentrating and purifying step wherein the desalinated solution is concentrated and clarified, and (d) optionally, a sterilizing and/or drying step wherein the concentrated and clarified solution is sterilized and/or spray-dried, the steps yielding the peat-derived bioactive product.
In another aspect, the invention provides a process for producing a peat-derived bioactive products from a highly concentrated aqueous solution of inorganic salts containing peat-derived bioactive ingredients. The solution is diluted with demineralized water, the dilution being effected with water volumes several times the volume of the solution to be diluted. The diluted solution is subjected to reverse osmosis in order to desalinate the solution. Inorganic salts being removed, the desalinated solution is concentrated and clarified.
The instant process, applied to the processing of a product obtained according to Polish patent specification No.
124110, consists in expelling residual organic solvents from a post-extraction aqueous phase, separating insoluble parts by filtration under reduced pressure through a sintered ceramic material, diluting the permeate with several times the volume of distilled water and subjecting it to desalination by reverse osmosis to separate excessive mineral salts, mainly sodium chloride, as a permeate. Then, the desalinated solution is concentrated, clarified by centrifugation and sterilized by filtration through a membrane filter, e.g. a Millipore(R) filter. The resulting microbiologically clean solution may optionally be spray-dried. The sterilized product (liquid or solid) may be formulated into a cosmetic pharmaceutical or veterinary composition. Optionally, the concentrated and clarified solution may be used - without sterilisation and spray-drying - in any suitable dilution as a component in numerous cosmetic compositions.
Preferably, in a spray-drying step, the inlet temperature is set to about 180°C, while the outflow temperature is set to about 90°C.
While the process of the present invention is described above in combination with the process according to Polish patent specification 124110, its use is not restricted to such combination, but is applicable generally in the context of a process for obtaining a peat-derived bioactive product from a highly concentrated aqueous solution of inorganic salts, especially of sodium of sodium chloride, containing peat-derived bioactive ingredients.
The peat-derived bioactive products provided by the present invention do not contain more than 70s by weight, preferably not more than 60% by weight of inorganic salts, especially of sodium chloride. Since a sodium chloride 4a concentration as low as possible would be desired for an optimal product, especially for pharmaceutical applications and such cosmetic applications where higher concentrations are required, i.e. for face care, lower sodium chloride concentrations, such as 55% and even lower, are most preferred, especially when obtainable by the steps of dilution and reverse osmosis.
Thus, in one aspect, the invention provides a water soluble peat-derived bioactive product, containing not more than 70% by weight sodium chloride, based on dry mass. In a further aspect, the invention provides a peat-derived bioactive product containing not more than 70% by weight of inorganic salts, based on dry solids.
In another aspect, the invention provides a peat-derived bioactive product obtainable by desalination of a concentrated aqueous solution of peat extract, which solution contains inorganic salts and water-soluble peat-derived bioactive ingredients and originates from an extraction process comprising alkaline hydrolysis of raw peat material, the bioactive product containing not more than 70%, by weight of inorganic salts, based on dry solids.
Where the process is terminated with the concentration and clarification steps, the product is a concentrated (or thickened) solution. "Concentrated peat extract", referred to in this specification, is a dark-brown liquid of a density of 1.02 - 1.09 g/ml and has a content of dry solids of not less than 5% by weight. The chloride ion content in dry solids, calculated as NaCl, is not higher than 70%, preferably not higher than 60%, and the pH value of a 1%
aqueous solution is 5.0-6.5, generally about 6Ø The lack 4b of a further sterilization step may not be detrimental in certain cases, e.g. for certain cosmetic uses of said concentrated peat extract.
On the other hand, the sterilization step will be mandatory, particularly when the product is intended for the preparation of pharmaceutical compositions. Particularly in such a case, the further step of spray-drying is most preferred, if not mandatory. The product resulting after such a spray-drying step is in powder form and thus particularly suited to the preparation of certain pharmaceutical compositions. A most preferred product of this type is the product commercialized under the designation "TOLPA~R~ Torf Preparation", TOLPA~R~ being a registered trade mark of Torf Corporation, u1. Mydlana 2, Wroclaw, Poland. The abbreviation TTP will be used in the course of this specification to designate said product.
The present invention also relates to pharmaceutical compositions containing as active ingredient a peat-derived bioactive product as hereinbefore described, particularly a product which contains not more than 70% by weight, preferably not more than 60% by weight, of inorganic salts, especially sodium chloride, based on dry solids, together with a pharmaceutically acceptable r. S!
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W~ 92/I6216 PCT"/EP92/Otk191 _ 5 carrier. The peat-derived bioactive product contained in such pharmaceutical compositions is preferably TTP as defined above.
The pharmaceutical preparation contains the peat-derived bio-active product and the gharmaceutically acceptable carrier material, preferably in a weight ratio of between about 1:5 and 1:25, and most preferably between 1:9 and 1:19.
The present invention furthermore relates to a process for preparing a pharmaceutical formulation containing a peat-derived bioactive product, in the form of a gel. This process is cha-racterised in that a sterile alcoholic herb extract is combined with sterile glycerol, a sterile aqueous - preferably concen-trated - solution of previously powdered peat-derived bioactive product and a sterile menthol solution, and that the resulting mixture is gradually combined with colloidal silica 'to convert the liquid composition into gel form, the weight ratio of liauid mixture to silica being from 90:10 to 94:6. Preferably TTP is used as the powdered or concentrated peat-derived bioactive product.
The present invention also relates to a process for preparing a pharmaceutical formulation containing a peat-derv ved bioactive product, in the form of an ointment. Th9.s process is charact-erised in that a sterile herb extract is gradually combined with a sterile solution of powdered peat-derived bioactive product, that the resulting mixture is gelled with the addition of col-loidal silica and that the gel thus obtained is triturated with a previously sterilized mixture of fatty components, such as eucerine and petrolatum, preferably with a weight ratio of liquid components to silica of about 30:20 and of gel to fatty composition of between 32:68 and 34:66. Also here, preferably TTP is used as the powdered or concentrated peat-derived - bioactive product.
Cosmetic preparations, which may comprise herbal extracts as well as other auxiliary and enriching components, fragrant compositions and carrier materials allowed for cosmetic use, C?i IRr:~~~~ ~~~ J~"
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WO 92!16216 PCT/EP92/00491 contain the peat-derived bioactive product according to the present invention in an amount of 0.01-10~ by weight, preferably 0.05-1.00 by weight, and most preferably 0.05-0.10 by weight.
Carrier materials may be aqueous solutions of alcohols, all types of emulsions, gels, foaming compositions and fatty carriers. Use of one specific carrier selected from the group of the above mentioned substances allows formulation of various types of cosmetic preparations according to the invention, such as tonics, creams, balsams, cleaning milks etc. for daily body care as well as shampoos, hair balms, foaming bath compositions, al? with the addition of peat-derived bioactive produc:.s.
Peat-derived bioactive products (peat extracts in abbreviacec form) stemming from original yaw peat material, such as famonc others) therapeutic mud, contain well balanced quantities o~:
mineral and organic compounds, such as mineral salts of ~~e following -elements: B, Si, Ab, Fe, Mg, Mn, Cu, Sn, Ni, Ca, ~c and Na; organic compounds, such as aminoacids in free form ant as salts; polysaccharides, partially degraded/reaeted - _.. she course of hydrolysis - 'to desoxysaccharides and%or aminosacc'.~.-arides. Peat, in particular therapeutic muc, is known ant =°-cognised -as a material of biological plant and microorganism origin; due to its contents of nourishing and stimul ar_~.ng cor,.-aonents it has beneficial effects an humans and mammals; she==-fore, peat-derived bioactive compositions contain the above-mentioned substances in proportions characteristic for the '-=v-ing organisms; this is considered to be an explanation of the advantageous effects of cosmetic and pharmaceutical pr~parat~o:a containing peat-derived active products and compositions.
Particularly good effects of the new cosmetic compositions are observed when herb extracts are also present. in the =ormulation.
Selection of a suitable herb extract is based on a known typical use of such extracts in cosmetics, modifying the activity of preparations and thus enabling the content of cosmetic prepar-ations to be matched with demands and needs of indivic~aals to be treated.
~tJBSTiTIJTE SHEET
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'!W~ 92/16216 PCT/EP92/00491 The present in~rention is better characterised and explained in the following examples.
Exa~le 1 Starting with 1000 kg of air-dried raw peat material, following e.g. the known procedure according to Polish patent No. 124110, a solution of peat-derived bioactive ingredients in a saturated aqueous solution of sodium chloride was obtained in a quantity of about 10 liters. The solution was filtered through a sintered ceramic filter under reduced pressure in order to clarify the solution before desalination of the same, The clear solution thus obtained contains about 95~ of NaC'_ in a dry mass. The dr.:
mass constitutes about 32~ by weight of the solution. The volume of this clear solution is about 7 liters.
The clear solution is diluted with 5 to 8 M mes the quantity o.
distilled water and in diluted form is subjected to a desalin-ation step carried out by using a reverse osmosis technique using a DDS apparatus. Desalination was carried out for 3 ~o hours, whereby the excess of mineral salts - mainly NaC'_ - is separated in the form of the permeate. The desalinated compos-ition contained approx. 6~-70a.of sodium chloride in the soi'_ds.
The solution thus obtained, or 6-7 liters by volume, was con-centrated 4-5 times in a Buechi rotating evaporator, so tat a concentrated sol~ition containing approx. 20~ of dry mass was obtained. The resulting concentrated solution was clarified using a Biofuga-Heraeus centrifugal apparatus (flow separator) and then sterilised by filtering through a Millipore~R) ~-lte=.
The resulting microbiologically pure solution was spray-dried in an Anhydro dryer with the outflow temperature se't at 90°C and supply inlet temperature set at 180°C. The yield of dried oow~:e~
was approx. 200 g.
Example 2:
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W~ 92/1626 P(.'T/1EP92/fl(kl9l _ g ._ The product obtained in Example 1 was used for preparing gel and ointment pharmaceutical compositions, containing also hero ex-tracts synergistically improving the therapeutic effect with , respect to certain diseases. For example, a gel and ointment against varicose ulcer of the shank was prepared as follows: , 20 g of hippocastanaceous extract, 10 g of calendula extract, 60 g of glycerol, 0.1 g of TTP in the form of a powder obtained as described in Example 1 above, 0.1 g of salicylic acid, 1.0 g of distilled water and 8.8 g of Aerosil(R) (colloidal silica) were used in order to obtain a gel form of the preparation.
hiquid (non-volatile) ingredients were sterilised before use, by means of heating under reflux for two hours. Herb extracts were combined with glycerol and an aqueous solution of TTP and also with menthol, and silica was gradually added to the obtained mixture, under continuous stirring.
Similarly, in order to obtain an ointment composition, t:~.e following ingredients were used: 20 g of hippocastanaceous ex-tract, 10 g of calendula extract, 0.1 g of salicylic acid, 0.1 g of TTP in powdered form obtained as described in example 1 and 2.0 g o~ Aerosil(R) (colloidal silica).
As fatty components, a mixture of the following substances was used: 22 g of eucers.:ne and 45.8 g of petrolatum. :lerb e~:.rac~s were sterilised by heating under reflux for approx. ? hours.
Eucerine -and petrolatum were similarity sterilised. ~iauic in-gredients wer a carefully combined with silica to obtain a ge'_, which in turn was triturated with sterilised and 'arty com-ponents cooled down to room temperature. A stable aintmen~ was obtained which did not separate when stored.
The gel and ointment obtained above were simultaneously applied in. the treatment of varicose ulcer of the shank. Ulcers were treated with the gel preparation while the surrounding, non-affected skin was treated with ointment. Addition c~ colloidal silica is believed to be responsible for prompt dess,~cation ~~~~TITUT~ S~-l~~T
, ~~ ~) 4~ l~' ~7 ~7 d ?3 !J ?' ii a,3 iV0 9~/162i6 PCf/EF'92/~Od91 g _ while the herbal and peat-derived ingredients are believed to be responsible for 'the curing effect of the preparation. Fatty components helped to keep elastic the crust and the skin. The results obtained were compared with a control group of patients treated in a classic way. Those who received the new treatment were selected from a group of patients suffering from the disease for many months (sometimes years) without noticeable positive effects. Patients treated with compositions according to the invention showed better results - already within the fir st few weeks - than control patients, _Example 3:
Products obtained in Example 1 were used to pr°par~ pharmaceut-ical formulations in the form of tablets, or granules to be placed in capsules.
A sterile pe at-derived bioactive composition ;n pcwdered form was combined with a carrier in a weight ratio of 1:9. As a carrier, ~IYVATEX(P)TL (tradename of F_astman-:Codak), a .mixture o-_.
lactose and lubricating substances, was used in a weight =atio of 44:1. Lactose of 50 mesh particle size and ivIYVAT°X(~)TT~ were finely disintegrated so that approx. .70% o= its mass passed through a 100 mesh screen. A part of the resulting mixture of active composition and carrier was formulated into tablets con-taining ~ mg of aci~ive ingredients. The total. mass oz -ac'.~.
tablet was SO mg. 'The other part of the same rni:<tare of active camoosition -and a carrier was granulated using c.~. oz ec'nanol (40% by volume). Granules were sieved and ground ~._ necessary and then filled in capsules in such a quantity that each capsu~~.e contained 5 mg o.f active ingredients by using TTP in mixture with a carrier at a ratio of 19:1.
The tablets obtained as above were tested is order to measure the time of their disintegration in an ar'~i~icial gastric juice at 37°C +/- z°C using Erweka equipment. The artificial gastric juice was prepared as follows: 2.0 g of sodium chloride and 3.~g oz pepsin were dissolved in 7 ml of hydroc~loric acid and SUBST1TUT~ SNEET
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WO 92116216 P~T~1;P92/00491 distilled water was added up to a total volume of 1000 ml. The pH-value of the resulting solution was approx. 1.2.
l3esintegration time of a tablet, having a diameter of 5.1 mm and a total mass of 0.0498 g, was 5 minutes.
Further examples relate to numerous cosmetic preparations according to the present invention, having different forms composition and being designed for different applications, containing the beneficial addition of bioactive ingredients derived from peat. Among others, preparations such as tonics, balms, creams, milks, shampoos, foaming bath compositions etc.
are described.
Example 4:
A reaction vessel equipped with a stirrer was charged with 150 g of camomile extract obtained by the extraction of camomile in-florescence with a 1:1 ethanol:water solution, as we';l as i g o~
TTP as described above. SO g of glycerol were added :,o the mixture obtained. The three substances were stirred to obtain a uniform mixture. Subsequently, a second mixture as previously formulated, was introduced into the same vessel. It comprised 340 g or a 95:5 ethanol: water solution, 1 g or salicyl'_c acid and 0.5 g of menthol.. The two mixtures were combined b~ st;rring to form a uniform solution: Next, 3 g of a fragrant composition TILIANA H4308 were added. TILIANA H4308 is a product o~ ~abryka Syntetykow Zapachowych Pollena-Aroma (Synthetic Fragrance works Pollena-Aroma), of r,Varsaw, Poland. The solution was then brought to a total volume of 1000 ml by adding 454.5 g or distil'_ed water; stirring was continued until a homogeneous mixture was obtained.
In the above procedure, 86~ glycerol, menthol and water accord-ing to the requirements of Polish Pharmacopea FP T_V and ethanol in a concentration of 95~ according to the Polish industrial standard BN-75/6193-01 were used.
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~V~ 9211b216 PCT/EP92/(j0491 The concentrated peat extract used was a dark-brown liquid or a density 1.020-1.090 g/ml and a content of dry solids not less than 5~; the pH value of a l~ aqueous solution was 5.0-6.5.
Camomile extxact was a red-brown liquid of a density of 0.9160-0.9503 g/ml, and ethanol content of 52-56~ by volume.
The tonic preparation obtained above is suitable for all kinds of skin. It is a clear liquid without any solids. Its colour is yellow. The pH value is 4.28 and the ethanol content is 45.920 by volume. Total acidity calculated as salicylic acid content was not less than 0.1~ by weight, namely 0.23a by weight. The preparation being stored for 12 months did not lose any of the above characteristic features.
E_x_ample 5:
The procedure described in Example 4 above was repeated, the only difference being that instead of camomile extract and the TILIAIdA H4308 fragrant composition, a marigold flowers extract and a composition SINUS H4625 (also a product of Fabryka Syn-tetykow Zapachowych Pollena-Aroma) were used in the same way and the same molar and volume ratios. The resulting tonic prepar-ation is suitable for dry and fragile skin. Similar==y '-.- was a clear liquid without any solid particles. The pH value was 4.30, total ethanol content was 45.82 by volume and total acidity was 0.27$ by weight. When stored, the preparation was unchainged after 12 months like the preparation obtained acca-rding to Example 4.
Example 6:
The procedure of Example 4 was repeated, except that an ext=acs of sage leaves was chosen instead of camomile extract, and the fragrance LELIA 90368 (Pollena-Aroma, Warsaw) was chosen instead of TILIANA H4308. The extract of sage leaves was obtained by ex-tracting dried gage leaves with ethanol at 50°C and had a brown-ish colour, a characteristic sage odour and a density of C.916C
to 0.9503; it contained 52 to 56~ of ethanol. The resulting fac°
care agent is particularly su°itable for greasy skin. It is a SUBSTITUTE SH~~T
., ,.
~,i3(..~'le~~~~i9 WO 92/16216 ~CT/IEP92/(30491 - '12 -clear and homogeneous liquid having a dark yellow colour; the pH
value, the ethanol content and the acidity were comparable with the values of the care agent according to Example 4.
Example 7:
The following composition proved to be a particularly effective gel for avoiding or treating periodontosis:
24.0 g camomile extract 3.0 g sage leaf extract 0.3 g salicylic acid 0.2 g menthol v C.1 g TTP
to 100.0 g commercial gel base Example 8:
The following carefully weighed components were introduces =~ro a reaction vessel of a volume of 2000 m1, equipped with a mechanical stirrer:
270 g of camomile extract obtained by extraction of camomile inflorescence with 50o ethanol; the extract was a =ed-brow~
liquid, having a density of 0.9160-0.9503 g%ml and a~
ethanol content of approx. 55o by volume - 50 g of glycerol,'86~, according to the requirements o. °olis Pharmacopea E'P IV
- 30 g of a saponaria officinalis extract obtained by ext=act~:=g saponaria of.ficinalis roots with 70~ ethanol; the e:c~=acs was a red-brown liquid, the density was 0.9630-0.9810 glm~-, and the ethanol content approx. 75~ by volume - 1.0 g of the inventive concentrated peat ext=act, being a da='-:
brown liquid having a density of 1.02-1.09 g/mi; not more than 2~ chloride ions calculated as sodium chloride; the c~.~
solids content was no's less than 5~; the pH value oa 1~
water solution of the extract was 6Ø
The ingredients listed above were mixed thoroughly. ~ pr°-~=ous_y prepared solution of 1 g of salicylic acid in 260 g of ethanol was added thereto. To :he combined solution, 383 :~
SUSST'ITL~TE SHEET
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;.i J t 3 i,~ r W~ 92116216 PtJT/EI'~2/Od~l9l -. 13 -distilled water and 5 g of fragrant composition TILIANA H3408 were added and stirred until a uniform solution was obtained.
The preparation was analysed and stored in retail size bottles of 200 ml volume. The resulting preparation was suitable as a hair care preparation. It was a clear, slightly opalescent liquid, containing approx. 45~ by volume of ethanol; the pH
value was 4.5; the total acidity calculated as salicylic acid was not less than 0.1~ by weight. The preparation is suitable for blonde hair. During 12 months storing the preparation re-mained unchanged in i'ts features.
Example 9:
The procedure described in Example 8 was followed except that instead of camomile extract and TILIANA H4308 composition there were used in the same sequence and ratio: horsetail herb extaact and the fragrant composition FINUS H4625. Horsetail herb extract was a green-brown l,;quid of a density oz 0.9160-0.9503 g/ml and an ethanol ccntent or 55~ by volume.
The resulting preparation was suitable for a1'_ kinds of ;~a=. -..
was a clear and transparent liquid without any soled parts, yellow-brown in colour. pH value, ethanol content, total acidity as well as stability after a 12 months storing period were the same as those of a preparation described in Example 5.
Fxam 1e 10:
The procedure as described in Example 8 was repeated with com-parable results. The only difference was that, instead of camo-mile extract and TILIANA H4308 composition, stinging nettle leaves extract and fragrant composition LELIA 90368 (product o~
the same Fabryka Syntetykow Zapachowych Pollena-Aroma) in the same sequence and ratio were used. 'The herb extract used was olive-green in colour, had a density of 0.9160-0.9503 g/ml and an ethanol content of approx: ~5~ by volume. The preparation was suitable for all kinds of hair.
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'1~'~O 92116216 Pf'f/E)°92100491 Example 11:
rn general, cosmetic milks are dispersions of fatty substances acting in both chemical and mechanical ways on the skin. In .
fact, due to a convenient way of application and better interaction of the fluid and the skin, it is very appropriate to use liquid, more specifically emulsion creams. They can easily penetrate to deeper layers of the skin and thus prevent changes of the skin due to age. Cosmetic milks are used mainly to clean a dry and fragile skin. Accordingly, they must not contain any aggressive volatile oils, while frequently they contain suitable herb extracts like camomile extract or wheat germ extract. Addition of peat-derived bioactive products to such cosmetic milks further improves their positive effects. In particular, the new recipe is as follows:
0.05 g TTP
aloe extract 20.00 g glycerol 3.00 g eucerine 2.00 g white paraffin oil 1.00 g triethylamine 1.00 g Aerosil(R) (colloidal silica)4.00 g Example 12:
Improved regenerative results were observed when TT° and carefully selected fatty carriers were used in a c7.assic nourishing and regenerative cream formula. TTP is used in an amount of 0.01 -1.00 by weight in combination with a herb extract (selection depends on the type of skin for which the cream is intended) in an amount of at least 0.05 - 1.00 by weight, antibacterial preparation in an amount of 0.05 - l.OOj by weight, synthetic fragrant composition in an amount of 0.01 -0.05~ by weight and a fatty carrier in the form of a water emulsion, constituting 97.00 - 99.500 by weight of the whole composition. The fatty composition needs to be a good carrier for the active ingredients and to be well accepted by the skin.
Preferably, it is an emulsion of. (a1.1 amounts ir_ o by weight) 35-45 eucerine, 8-14 petrolatum, 2.5-4 olive oil, 6-10 glycerol ci iRRTfTi fTF SH~~T
-~ u, ,~- y:~ ~s r ~ ~
t .
-~ 3.~ J ~.# 'tJ t. .' wo 9zn~zr~ ~cri~~z~o~a91 and 35-40 water. Preferred are marigold flower herb extracts extract, camomile extract, and the like.
thyme extract Preferred recipes are lows:
as fol 39.00 partsby weight 1. Eucerine Petrolatum 11.50 partsby weight Olive oil 3.13 partsby weight Glycerol 7.80 partsby weight Water 38.00 partsby weight NIPAGINA A (anti-bacterial preparation) 0.40 partsby weight TTP
0.05 partsby weight Marigold extract 0.10 partsby weight rance 0 02 parts~weiaht fra ti th g c e Syn total 100.00 by weight parts 00 parts by weight 2. Eucerine .
Petrolatum 8.50 carts by weight O1'_ve oil 3.08 parts by weight Glycerol 7.90 parts by weight Water 38.00 parts by weight NIPAGINA A (anti-bacterial preparation) 0.40 partsby weight TTP 0.05 partsby weight Camomile extract 0.02 partsby weight Synthetic fragx'ance_ 0.05 parts5v weight total 100.00 parts by weicJnc Example 13:
An after-shave preparation contains TTP as a peat ext=act in an amount. of 0 . 01 -- 1 ~ by weight, herb e:ctracts in ar. amount of 1 -30~ by weight, glycerol in an amount of 1-8o by weight, salicylic acid and menthol in aqueous-alcohol solution.
Preferred herb extracts are: camomile, marigold, thyme, aloe extract and similar beneficial herb extracts. Addition of glycerol is also beneficial due to its influence on the elasticity of the skin. Tt speeds up the spreading of the preparation on the face as well as the penet~ation into t:ze . . ..., .~-....-r-a ,'~'-r= c~ ~-7 C ~T
~1 ~-. .;
~~ S_7 ! 7 rw.1 ~,~ L' .~'_ lV~. !16216 PC_'T/~Ptl2/U0491 16 _.
deeper layers o~ the skin, thus enhancing the beneficial effects of the active peat composition and herb extracts. .
A preferred recipe is as follows:
0.10 parts byweight Camomile extract 15.00 parts byweight Glycerol 5.00 pats byweight Menthol 0.10 parts byweight Salicylic acid 0.10 parts byweight Ethanol (conc.95~) 10.00 parts byweight Fragrant composition 0.30 parts byweight Distilled water ad 100.00 parts by weight Example 14:
A shampoo composition was prepared according to the ~oliowing recipe:
Fuller's herb e:ctract 15.60 g 7.50% byweignt Stinging nettle leaves extract20.00 g 10.00% byweight GAMAL SBS-11 (detergent) 30.00 g 15.00% byweight GAMAL NO-3 (detergent) 20.00 g 10.00% byweight ' 0.40 g 0.20 byweight tina Asep ethanol 1.60 g 0.8U% byweight BRONOPOL (preservative) 0.04 g 0.02% byweight Sodium chloride 6.00 g 3.00% byweight W 106.96 53.48% b~weight t a er a total 200.0 0 100.00 %
g by weight To 92 parts by weight of the above shampoo composition~8 parts by weight of T'rP were added to obtain 100 parts by weight oz a shampoo according to the invention. Other herb ertrac~s car, be used in place of stinging-nettle leaves e:ct-ract.
Example 15:
The following shampoo composition was prepared:
Horse chestnut extract 13.00 g Marigold extract 22.00 g GAMAL SBS-11 30.00 g GAMAL NO-3 20.00 g w ~zmsz~s ~~c:rm~zivog9i _ 17 Aseptina 0.40 g Ethanol 1.60 g BRONOPOL 0.04 g Sodium chloride 6.00 g Water 106.96 a total 200.00 g To 95 parts by weight of the above composition, 5 parts by weight of TTP were added to obtain 100 parts by weight of shampoo according to the present invention.
Example 16:
A tooth paste contains TTP as a concentrated peat extract, in an amount of 0.01-0.10% by weight, etheral oils or their compositions or else fruit essences in an amount of 1-10% by weight, glycerol in an amount of S-10g~ by weight, herb extwac~s in an amount of 0.10-10~ by weight and cleaning substances in a::
amount of 20-35% by weight dispersed in water in an amount of 45-60% by weight, and dyes and whitening components in an amount of 1-2~ by weight.
Titanium dioxide may be used as a whitening component; TTP
itself may be used as an anti-bacterial additive; sage leaves, camomile or marigold flowers extracts may be used as benef'_cial preferred herb extracts.
The preferred recipe is as follows:
Precipitated calcium carbonate 150.00 g Magnesium carbonate 60.00 g Glycerol 70.00 g Herb extract x.00 g TTP 0.50 g Titanium dioxide 10.00 g Etheral oils (or mint, lemon, etc essence) 5.00 g water 400.00g Dye trace S~BBTITL~'TE ~HBET
~ .~ J ~i K.1 ~ ~-a t ~~r ~ o i_l ~J 1~ ~i ..~..
WO 9. 0216 PC'f1E~92/1H~491 _ 1g _ E_xamnle 17:
Bath salt preparation: In the course of the process for obtaining a peat-derived bioactive product according to the present invention, in particular when oonverting a liquid form into a powdered one, there is the desalination step in which sodium chloride is separated as a by-product. In said by-product, 95g constitutes sodium chloride, other mineral salts separated are calcium salts, magnesium salts, mainly chlorides and sulfates; these salty products also contain some organic peat-derived low molecular compounds occluded within the crystal structure of these inorganic salts. These organic compounds are components of TTP and are - among others - polysaccharides, aminoacids, fulvic acids and the like. The presence of these components in the salty by-products is beneficial when sail is used as a bath salt, because they may add additional bene=icial effects to the standard activity of bath salt. Accordingly ~..is by-product was tested for its chemical and physical grope=~ies in the Balneologic Institute in Poznan, Poland, to find ouc whether it can be used in cosmetic baths. Since the T_nsti~u~°
has found no undesired entity in the salt, it was approved =o.
cosmetic use.
The preferred recipe is as follows:
Salt (NaCl) containing occluded TTP 97.00 g Pine etheral oil or etheral oils composition 3.00 g Example 18:
A new hair balm contains TTP in an amount of 0.01-1% by weigh herb extracts in an amount of 0.01-10% by weight, anti-elec~ro-static components in an amount of 3-4% by weight, components preventing excessive drying of hair and skin in an amount o= v,:p to 2~ by weight, glycerol in an amount or 1-5% by weight, pre-servative and stabilisers in an amount of 0.05-0.50% by weigi:~
and water to 100 by weight.
As an anti-electrostatic component, the present balm contains ar.
alcoholic solution of trimethylamine and ammonium chloride salt, obtained from fatty animal-derived amines; as 'thickening agent -~UBST1TUTB ~~E~~T
V1'~ ~2/1621G PC.T/EP92/00~91 acting also as stabilizing agent - as agent cosmetic alcohol;
preventing excessive dryness of hair skin - t oils, and plan acting simultaneously as co-emulsifying glycerol agents; and for easying spreading and penetration of balm, in the particular of its active ingredients TTP and herb acidic extracts, As an en-vironment stops multiplication of bacteriar the balm according to the invention contains citric acid fumaric or acid in an amount of 0.1~ as well as a preservativeknown as BRONOPOL
and fragrant compositions.
The preferred recipe is as follows:
Alcoholic solution of trimethylamine and ammonium Chloride salts 3-4~ by weight Cosmetic alcohols 3-4~ by weight T'rP 0.01-10$ weight by Thickened herb extracts 0.01-10~ weight by Glycerol 1.5a by weight Plant oils up to 2; by weight Citric or fumaric acid 0.1% by weight BRONOPOL 0.1$ by weight Fragrant composition 0.3~ by weight Distilled Water to 100 weight by Fxamole 19:
Cosmetic masks are well known cosmetic preparations serving many different purposes. As therapeutic mud has a known beneficial effect on the skin and body, it was believed that also post-extraction peat obtained in the process of separation of bio-active peat-derived compositions from peat may be used in cos-metic applications. Post-extraction peat contains a solution, o active bodies freed in the alkaline hydrolysis process due 'to extremely high sorptive properties of peat after neutralisation;
It was therefore found to be a valuable component of cosmetic masks. To enrich the post-extraction peat with more of the valuable components, natural therapeutic mud and humic acid fractions were added which are present in natural peat and separated in a process for obtaining peat-derived bioactive compositions .from the alkaline hydrolysate. Such a composition c.i ~czCTIT'i iTF fiHE~~
Zi 7~ ?j ~~ i~
we ; mz~6 ~criE~ziot~am was tested in the above-mentioned ~alneologic Institute and was found suitable for cosmetic use.
The preferred recipe is as follows:
post-extraction peat 100.0 g Natural therapeutic mud or peat 20.0 g Humic acid fraction 10.0 g Magnesium carbonate 10.0 g Zinc oxide 5.0 g Citric acid or the like 0.1 g Herb extract or powdered plant material 5.0 g Distilled water q~s' The following statements and explanations relate to the bio-logical aspect of the products of the present invention, _.e. to the bioactive characteristics and to the compatibility o~ these products, particularly with a view to their usefulness as phar-maceuticals. The following abbreviations will be used below:
TTP TOLPA(R) Torf Preparation (trademark of Torf Corporation, Wroclaw, u1. Mydlana 2, °oland) IFN Interferon exists as a ubiquitous cytokine (tissue hcrmone).
IFN genes are present in all cells. IFN is mainly i~.duced by proteins or glyco-proteins. Substances stimulating the IFN
genes for the production of IFN are called inducers. The process of the IFN induction is a highly regulates, soph-isticated biochemical process; negative and positive regul-atory genes controlling IFN production have been recognized.
Small amounts of IFN may be produced spontaneoulsy, without any detectable induces. Such IFNs are sometimes named "physiological IFNs". IFN exists in nature in three main molecular forms:
IFN-a (or leukocyte IFN), IFN-~i (fibroblast TFN), and IFN-Y (immune IFN).
IFN-a and IFN-(i are type I--IFNs, IFN-Y is the type II-IFN.
~U~STITLIT~ ~H~~T
GZ a ~,a ~; ~I r~ c.3 :J _~.
~~fO 92/16216 hCT/EF'92100~b~1 The major biological activities of IFNs are antiviral, anti-proliferative (anticancer), and immunomodulatory activities.
Various forms of IFN are produced commercially as the natural and recombinant preparations and are used as drugs for treatment of neoplastic, viral, and several other diseases.
CTL - cytotoxic T lymphocyte.
NK cells - natural killer cell.
IL-1, IL-2 well known interleukins stimulating the prolifer-ation of T cells and other lymphoid ce'_'_s including 3 cells;
RPMT_ 1640 tissue culture medium for the growt'n of human and other leukocytes (abbreviation of the Roswell ?ark Memor~.a'_ T_nstitute, Buffai0);
FCS = fetal calf serum (for assays with leukocytes it has to be pre-tested because it may contain mitogenic substances mimicking the action of interleukins);
EMCV -= encephalomyocarditis virus, mouse oicorna virus non-pathogenic fox humans, it is often used as a challenge virus in the TFN bioassays;
A-X49 human adenocarcinoma cell line - used in t:ze i=N bio-assays because of its high sensitivity fo.r. IFN-a,~, and Y.
The line is recommended for such use by the wii0 e~pe=~s on IFN standardization;
MTT 3-(4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide. Reagent used to measure the cell kill or cell growth in several bioassays, using the ELTSA scanners (kiansen et al., J. Immunol. Meth. 1989, 119, 203-210);
m tnhTlTi iT~ C,''Ld~~"T
.7 r~ ~ ~~ ~ .9 ~.$ U 9~ to ti lVO . 16Z 16 PCI"/EP921(10491 _ zz L929 the mouse cell line, commonly used for assaying mouse ZFN and human or mouse TNF;
TNF tumor necrosis factor, cytokine, (relatively small pro-tein, very susceptible to the proteolytic enzymes), produced by monocytes and macrophages (TNF-a known also as cachectin factor causing cachexia in humans and animals);
produced after stimulation with LPS (lipopolysaccharides), viruses, bacteria, and many other agents, very toxic for many virus-infected and neoplastic cells; may also act as a growth factor for fibroblasts. Connected with inflammaLOry reactions. The related form TNF-p (lymphotaxin) is produces mainly by T cells and some other tolls.
PBL peripheral blood leukocytes, normal human ieukocy=es from healthy blood donors, isolated from "buffy coats"
(interphase between red cells and plasma). The respons~ve-ness of PBL from individual donors to various cytok'_ne '_..
ducers appears to depend on a genetic set-up of the donors.
The high responders and non-responders have been identifies.
This refers also to the response of PBL _to TTO, The variation in the individual response to inducers c~ =FN
or TNF is more visible when Weak inducers a.r.~ useri than whe::
very strong induc ers, like viruses, are employed. This is due to the fact that the reaction to weak inducers is o_ the "all or none" type whereas viruses almost always induce de-tectable amounts of the cytokines.
Tolerance to inducer: Called also a hyporeactivity state. Occurs after administration of a single dose of an induces, e.g.
after 20 h of exposure of PBL to an induces (e. g. vir:a);
the cells stop further production of IFN. The hyporeactivity state lasts usually about seven days. It may be complete or partial. Such reactivity makes the therapeutic application of strong inducers difficult and/or ineffective. weak T_FN
~UBSTITUTc ~H~BT
92/1b216 pf~~~~i'/1~.
inducers either do not induce the hyporeactivity state or the tolerance is small.
Several natural drugs which are extracted from medicinal plants possess immunomodulating properties. TTP appears to be one of them. TTP alters many different immune functions in vitro and in viva. It initiates balanced immunostimulation, with the capabil-ity of non-specifically activating all effector pathways (CD4 helper, weakly CD8 suppressor, CTL, NK cell, and activated macrophage) without cytotoxicity for norrnal tissues.
TTP has a restorative effect on normal wound healing. Low doses of TTP weakly stimulate IL-1 and IL-2 production. High doses may inhibit the cytokine synthesis.
The respective tests with TT° were carried out by the following methods:
PBL from healthy blood donors were pur.='_ed 'oy amrnoniLm chlcrice treatment. "'he culture medium was RPMI 1640 plus 1,0~ PCS.
proximately'8 x 1C6 cells/ml were cultured far 20 h at 3?°C, S~
COZ. The antiviral activity of interferon was assayed by ~MC'J
cytopathic effect inhibition in human A549 cells. The MTT method to measure cell ki7.1 was also used.
TNF activity was measured in =929 cells. T_n order to de='_ne =='a type, the individual IFN samples were trAated with different anti-iFN -sera for 1 h. Their antiviral activities were compared with non-treated preparations.
The tests indicate TTP stimulates the production of endogenous interferons (IFNs) and tumor necrosis factor ('rNF). The response is dose-related.
Seven assayed cocnmercial batches of TTP had comparable biolog-ical activity as immunostimulant and the cytokine inducers.
3~~~~~~~TE ~~~CT
.. , y , ',i ;; 3 '.? ~ 7 7 j...
~:: ~ E ~-.l P.j i~ a~,1 ~1.
w zm6zy6 ~c°ri~~zioo~~~
- 24 _ Considerable variation in the response to TTP of leukocytes of the individual blood donors have been observed. PBL of several donors wrere found to be unresponsive. This may reflect a genetic background.
Potent polyclonal antisera were used, such as anti-IFN-a (Cantell), anti-IFN-a Ly (Namalwa) from K. Fantes, and anti-IFN-Y (Cantell) to neutralize antiviral activity in the supernatants of PBL treated for 20 h with TTP.
The results of th.e neutralization assays were found to resemble the finger prints of individual blood donors. In other words, proportions of IFN types produced by the individuals varied con-siderably. The separation of PHL into adherent and non-adherent fractions may potentiate the induced cytokine synthesis.
The hyporeactivity (tolerance) a the induction of ~FN by "dDV
observed 20 h after the initial stimulation of PBL with TTY was either minimal or absent.
All of the seven batches of TTP tested in human PBL as I=N and TNF inducers were found to be active in inducing _TFN and/or TNF.
The optimal concentration of TTP for IFN induction was 30-100 p.g/ml'and for TNF induction 100-200 ~.~g/ml. The dose of 200ug/ml may be subtoxic for PBL, but the synthesis of TNE" occurs much faster than that of IFN and faster than the devE:i.oprnenL oz moderate cytotoxici t:y .
The conclusion of the foregoing is the experience that the active principle in TTP pharmaceuticals is an immunoactive fraction of an extract from peat containing organic, primarily bound sugars, aminoacids, uronic acids, hurnic ,=acid substances and mineral salts including microelernents. Tire LD~O in animals is >2400 mg/kg per os.
~.. r hr ~ ~.t r.~ > > a 3 .~A.
V ~2/162i6 PCT/~pf2/0049i No mutagenic, genotoxic, embryotoxic, teratogenic as carcino-genic activity of TTP was found. TTP shows no allergenic pro-perties and had no topically irritating activity.
The therapeutic indications include chronic and recurring respiratory tract inflammations and lower leg varicose ulcers, supglementing the treatment of vaginal erosions, and periodontal diseases.
There are clinical observations suggesting that TTP may be use-ful as an immunomodulator in the supporting therapy of several neoplastic diseases. TTP is used r,_-ally (5 mg tablets) or topically.
Of particular relevance and importance appears to be the fact that PBL _treated with TTP for 20 h at 37°C do not develop the hyporeactivity state because they retain the capacity to produce IFN after induction with NDV (Newcastle Disease Virus), a very potent IFN induces.
The attached Tables 1-24 relate to various biological, e.g.
toxicological, hematological and immunological tests. 'these Tables appear to be self-explanatory and provide for the bio-logist pertinent information relating to compatibility and bio-logical activity of T'I'P.
It is worth noting that the concentration of active ingredients in pharmaceuticals, as indicated in claim 13, may be higher than in cosmetic compositions (as indicated in claim 14) due to the following reasons:
Pharmaceuticals are prepared in unit dosages, wherein the con-tent of active ingredients is under strict control; e.g. in tablets, the concentration is adapted to 'the size of the tablet containing the effective daily dose (or a part of it) of the active ingredient.
SI~~ST1~'UT~ ~~~~ l' ~, r i ;vi ~~ ;~ ,.~ .~.
fft /16226 P~T/Ek'92IU043~
_ 2 The concentration of the active ingredient, e.g. cf 'TTP, in granules with which capsules are filled, preferably is only 5 by weight in order to achieve a sufficient tablet size and to allow an appropriate operation of the capsulating machine.
Another reason is that most pharmaceuticals are administered orally, and the active ingredient is distributed within the whole body..~Even though it reaches the skin cells in a very low concentration, the therapeutic effect is remarkable.
Furthermore, .cosmetic compositions - contrary to pharmaceuticals - are used in rather uncontrolled quantities, applied locally, with a different penetration rate to different cells.
Some compositions, such as a shampoo which is nearly immediately washed out, have short contact times with the body or hair, and may contain relatively more of the beneficial component; others are applied several times a day and therefore should have a lower content of the active ingredient.
Finally, as in the case of tooth paste, being in contact with the mucous membrane i.n the mouth, active ingredient penetration is much easier than through the skin, and the concentration of the active ingredient may be lower.
SUBSTITUTE SH~~T
;'.q _::.g ': .~ ~ a n a hf ~ y v 1J ;"~' .,.
9xmszxs ~~'iE~zi~aa91 ;_ TAB. ACUTE TOxI CITY.EFFECT OEZALLY ADMINISTERED
ON BIOCHEMICAL F BLOODOF
THE PARAMETERS RABBITS
O
Examined Parameters Sex Dose Day of TT_? Test Creatinxne TotalProteinY-Globulin ;
~
g/kg ~mg%) lg/1) ~9~) 0.0 0 1.090.03 65.0 =3.49 0.53 Ø00 ~
7 I 1.03=0.02 62.0--_0.330.50 Ø01 _ ~ 2.0 0 1.06=0.01 58.0 ~1.9b I 0.50x0.02 ~
7 I 1.14=0.38 57.8 =1.54 i 0.54-_0.01 5.0 0 ~ 0.96-_0.12 70.3 .3.52 I 0.55-_0.00 ~ I
7 I 0.94Ø13 60.0 _2.34 j 0.57x0.00 i 0.0 0 0.80_-0.11 73.8 -_3.330.72 =0.06 I
~ 7 , 0.82X0.13 75.3 -_1.020.79 =0.03 ~
I 3.0 0 ~ 0.88-0.05 64.6 .2.36 ~, _0.'0 M . 0.52 j 7 I 1.24Ø34 64.5 =2.03 0.67 =~J.07 , .,.0 0 0.85_-0.03 58.5 -3.19 0.57 =0.03 i 7 i 1.04-0.12 57.0 =4.00 0.50 -~J.J1 .
'.'AB. ACJTE TOXIC:':"_'. ___"ECT IST:?ED
~ OF '"'"
ORtIL:.'! C~I
ADMI'I ='='-~--' .~C'_'IVT_'~'_ ':'P.ANSAMINAScS =N ScRUM ?.a.33I':'S
OF 'rT.:=OF .
'T':?E
I I i Exam=ned ~a~arneLers Se ~ Dose i ay r D o ; ~~mD ~ ~:esL Aianine ~spar ag~.~...
g%k~ j a11t1:1OL~3nSAe~a S2 SL_-35e llTt;nOL~atl t ~ ~ iU T_U
_ .-0.0 __ 0 ; 8.07 X1.20 33.33 ~3.:i0 j 7 I 9.07 Ø33 28.57 2.0 0 ~ 10.33 =1.76t 18.33 =..09 I , I 12 33 .3.52 0.00 x...03 I i i 5.0 ~ 0 ! 11.33 -'._56- - ''3,33~_.52 I 7 i 11.00 =3.05 20.07 =..06 0.0 0 10.33 :.-1.76 X3.00 =3.05 ~ 7 ~ 12.00 =2.04; 25.67 .4.'.'i M 2.0 I 0 9.00 -_1.00~ ;8.00 =..52 i ' 7 9.00 =0.57; 20.66 . .53 i 5.0 0 8.00 -_1.00I - 00 18.00 =1 7 11.33 -1.66; 23.00 :
=
~~ c~~~~~
s; . ~ CJ s;~ ry to 'a ., r.r L~ ~~i m.) s1 ~z«6z~6 ~~rr~z~~io~a~r TAB. 3 ACUTE TOXICITY. EFFECT OF ORALLY ADMINIST~t~
TTP ON THE I~riIATOLOGIC?.L PARAMETERS OF RABBTTS
Examined I
parameters Sex Dose Day of TTP Test Hemoglobin Hematooszt Leukocytes Erytzrocytesl I ~ ~ --glkg (mg%) (%) I x x 10 0.0 0 12.4=0.4438<3_1.857.32 =0.435.21=0.20 I
7 1 11.8=0.6337.7.1.206.30 x0.661 -_0.11 1 6.11 2.0 0 11.5._0.3136.7Ø885.69 0.531 _0.65 I I 5.d6;
r i 7 1 12.3_-0.4838.30.56 5.74 =0.76j --0.'.
i 5.69 j 0 112.3_0.1440.7--2.57.35 _0.991 Ø20 5.0 1 5.42 I ~ 111.6=0.2337.3Ø33, =1.791 -_0.19 ! 7 8.6a 5.08 0 ~ 0 X12.0=0.8637.3X1.20111.10_0.631 =0.26 0 -~~3 , I 7 111.9=0.6135.7.1.8510.80=0.941 =0.79 ' .~..i4 !
!9 0 112.3-_0.26X43.7-4.80~ =2.001 _0.65 ! 9.94 ~.tn 2.0 7 11.8=0.39134.%=2.02111.06_1.081 =0.50 ~.37 5.0 j 0 j12.7Ø6338.7_2.40j =1.361 :0.10 9.52 -.9 jl0.oX1.04,4Ø2.08I -_1..3! _0.6a 8.87 ..,_ '_~~B. -. C:IRONTC =~OXTCT'='<_'. ==~5C'.' OF ORALu..Y aDMTNIST°?c-0 T'.'? ON '.'HE MASd OF T4;; ORGANS OF ?ABBI'=S
Dose of TTP (mg/kg) I
i ' I ~ 0.0 ! 60 i 150 ~ 0.0 I 60 160 ;Sex ; .
(Organ ! Mass of Organ '_a ~ of Tocal Mass j t I I i jLung 0.72 X0.08 0.61 =0.1710.67 Ø0? 0.50 -0.05j0.54 _0.0910.1 =0.:.
I I
(Heart 0.23 Ø01 0.22 Ø02 0.25 _-0.03 0.24 -0.020.23 ~O.OtIO.?3 _'J.J2:
I I I
ISoleen 10.057=0.01 0.051=0.000.079=0.02 0.05 ~0.01~O.Oo =0.01i,0.J4 =O. J1 I i i ~ I, ;
jLiver 12.35 =0.1513.02 Ø08 3.22 -_0.37 2.32 =0.091'.77 _J.2813.12 -O.Jo.
1 ~ 1 !
:Cidney 0.59 =0.030.52 =0.0210.60 ~0.01I0.48 X0.0310.6_ _J.0110.49 -O. J3 ., Adrenal Gland 0.0160.01 0.015_0.01 0.020=0.01 0.013=0.01'0.013=0.02!0.012=0.0 Ovaries I I ~
with Uterus-0.58 _~0.04 0.39 =0.02 0.60 Ø04 - -TestiC
ales ~ - - - X0.30 -0.01 0.32 _0.0310.30 _0.02' ~U$s~s~U~~ ~~"~~~T
,~-.~;:
,a 3 S (.~ e. ~ ':9 ~,.t . 92/ 1 b21 b PCT/EP92/00491 Tpg, 5 CHRONIC TOX%CXTY. EFFECT OF Ok~ALLY ADMTPIISTERED
TTY ON THE H;rMATOLOGICAL FARAMETERS OF RABBITS
Examined parameters Sex Dose Week l TTP of Hemoglobin HematocritLeukoc yT~esrythsocytes E -' g/kg Test (mgt) (~ ) x , I 10- x 0 0 12 X0.05138.2.1.71i 9.33=0.7915.10Ø56 N! . 7 . =0.1540.7=0.331- 9.39=1.1816.34-_0.23 ~ 12.3 6 3.0 0.35141.7=1.30 9.38=1.1217.32=0.70 , i 12 13.6 =0.19'40.7~0.57j,11.23 _1.1015.30Ø46 SO ~ 0 12.9 ~O.d6135.7=1.821 7.62=0.7815.14;0.47 I
6 13.3 -u.35d1.0=:c.10~ 0.00=1. 0.3_".;.
";
12 13.7 =0.11j39.3=0.94 9.36=1.0?I6.19=0.19 i ' 0 X14.20 =0.2?36.7=1.811 8.19=0.1515.34.'J.;6 150 '.
I 3 11 25j 39.5=1.531 3.33=0.'815.6d=0.-10 05 =0 i ~ 6 . . =1.451 3.18=0.0610.34='J.10 (12 =0.53f37.3 .
I ~ 12 . Ø32!39.0 =0.63 8.24Ø0-110.~4=0.54 ~ 113.70 .
0 ~ 0 112.6 =0.=7160.2 :0.831 7.07=1.16i~.".,=0.17 j . ~ 3 ~i2.1 ~0.~3~39.3 -0.721 3. =1.011.,.'~=J.12 i6 j i ; 5 112.4 =0.86142_3 -1.20; 9.79=t.69~...36=0.?4 i ',~ j12.5 =0.87137.7 =2.181 10.?0=2.1614.00wJ.~9 i : 0 113.2=1.20135.0 =1.701 7.44=0.9615.47=~J.50 ; ;
t t I~ 6 113.0=0.55139.3 -1.201 7.25=0.96i7.08=0.?4 .
i 12 112.3=0.21/3'.. =4.331 9.31=0.35io.~'_'=0.32 y p '.'S.' _1.15138.3 =1.401 8.50=0.5416. =0.'J8 15p J9 i 3 '13.1 _1.10147.0 =2_901 3.10=0.76j5.35=~7 0 12 =0.92j41.0=1.15j 6.35-0.3115.61_~
~i I 12 . =0.701-'=1.'J=I.OOj 8.15=l.5di~.92='J.?3 13.1 ~IIBSTITUTE SH~~T
;~ ,:, ,r., d~ " t' 4 i a ,, r'-, i. C i c3 is ~. !.
1~(3 »/1~21G PC:l'/E~2/00~197 'TAB. 6 EFFECT OF TTP ON THE PHAGOCYTIC ACTIVITY OF
NEUTROPHTLS IId THE PERIPHERAL BLOOD OF RABBITS
Phagacytic Activity Neutrophil~s ~
Neutrophiles Group n (1000/1 NBT+ L.H L.W
mm ) U
Control 1 ml 6 1.9 2.3 2. 14.315.117.6154.560.659.81 7.:3'.511 ?3S 7.53 v .0 1 4 y4 ~4 O~t9.4 .4.3c6.8I~0.77 -_0.86=0.4 i 9 1 6 6 -3 . . __ _ . _ .
. . . _ .
TTD I, (Test *O *O *O * * * I
1) 5 mg/kg15 i.2 1.718.426.327.2 69.374.98.751U.39.38 2.0 59.2 _.v. I~0.9 _0.7.7.1.7.1Ø7_7.0~=0.7_-7.0=7.91-i.18-i.?S=1.0;
I
I TTP I ~ I
I ('='esta I xo *o *o *o ~c , _ z y .
15 mg/kgi 2.8 1.6 1.615.522.125.563.069.576.Si 3.5:3.:0 -.i6 ii. I =0.8_0.5'_1.0'_4.9_-7.0=9.11_7.0=7.1=3.91_0.33 -i.04.1.0 v.
.. Number of Test Animals ?9S ohosDhate bu=~er solution 0 3e.ore the administration o~ '~'='?
? 3 days a=ter the administration or TTP
Ld i.'I'tf :j #.~r ~
~s~ 9zr ~ sx a s pcri;v ~~i~a9 ~
_ 1 _ PEAT-DERIVED ~IOACTI~fE PRODUCTS AND PHARMACEUTICAL AND COSMETIC
CDt~4~OSITIONS CONTAINING T(~IEM
The present invention relates primarily to novel peat-derived bioactive products and to a process .for producing such products.
The invention also relates to pharmaceutical and cosmetic compositions containing these products and to processes =or preparing pharmaceutical and/or cosmetic formulations containing said peat-derived bioactive products.
It is known to extract peat by various methods using various extraction media and to use such extraets, containing peat-derived bioactive ingredients, for cosmetic and pharmaceutical purposes.
On o= these known processes is described in Polish patent specification No. 124110 (Chemical Abstracts 101(10), 78854e).
According to this prior art process peat-derived bioactive products are obtained by primary and secondary alkaline hydrolysis of an air-dried raw peat material, followed by acidification of the thus obtained hydrolysate and separation o~
insoluble solid parts with subseguent second alkali~atio.~., acidification of the c'_ear liguid phase and elim~.nar__on c~
ballast substances by means o~ alcohol and etner eat=act;on. ..
said process, the aqueous phase from the organic e_ct=act_on is a liauid neat-derived bioactive product.
The known liquid product, being a solution ov peat--dewived active ingredients in a highly concentrated, near-,~ saturatea acrueous solution of sodium chloride, obtained according to t:~.°
above cited Polish patent specification, is unstab'~e wPen stores for a long time and, moreover, contains - regardinc the b=o-logical activity of the composition - a large excess one~~tra_ inorganic substances. As a bulk product, it is ha=d to handl°, store and process.
C?i 1'i~C~l~l 1~~ wH~~
(l , C n ~ ~f a J ~a t.~ .~
~'O 92/ 16216 f ~'~~~2~~~ 1 -It is the main object of the present invention to provide a product which is not afflicted with these disadvantages, i.e. a product which is stable and which can easily be formulated into pharmaceutical and veterinary products as well as be introduced, either in solid state or in any suitable solution, into cosmetic preparations.
In particular, with respect to the use of a peat-derived bio-active product for pharmaceutical purposes, i.e. for the production of pharmaceutical compositions, there was a strong need for providing a solid form which would be well suited for this purpose. As previaus attempts aiming at concentration o' the known aqueous solution of peat-derived bioactive substances and desalination of the same failed to give a positive result due to the occlusion of active ingredients in a crystallising solid phase, causing decrease of biological activity pa ~ze composition, it was very hard to find a suitable process °or converting the liquid composition into powder form.
Unexpectedly, it was found that a positive result is achievable if, prior to concentration, the liquid composition is L-rst diluted several times, i.e. by water volumes several times the volume of the composition.
Accordingly, the prey ent invention provides a process by whic~ a peat-derived bioactive product, featuring the above advantageous characteristics, is obtainable from a highly concentrated aqueous inorganic salt - especially sodium chloride - solutio:~
containing peat-derived bioactive ingredients, by diluting said solution with demineralised water, e.g. distilled water, ~ollow~-ed by reverse osmosis, concentration and clarification. A solut-ion so obtained can easily be converted into a sterile and solid product, well suited to the intended purposes, by stervlizatio.~.
and spray-drying. Dilution is preferably effected with quantit-ies of water several times, preferably 5 to 8 times, the quanti-ty of the concentrated solution to be diluted.
~~J~~TITtJT~ ~H~~T
Thus, in one aspect, the present invention provides a process for producing a peat-derived bioactive product. A
concentrated solution of a peat extract containing inorganic salts and peat-derived bioactive ingredients is subjected to processing steps comprising: (a) a dilution step wherein the solution is diluted with demineralized water, (b) a subsequent desalination step wherein the diluted solution is subjected to reverse osmosis in order to remove inorganic salts from the solution, such that the desalinated solution contains no more than 70% by weight, based on dry solids, of the inorganic salts being removed, (c) a concentrating and purifying step wherein the desalinated solution is concentrated and clarified, and (d) optionally, a sterilizing and/or drying step wherein the concentrated and clarified solution is sterilized and/or spray-dried, the steps yielding the peat-derived bioactive product.
In another aspect, the invention provides a process for producing a peat-derived bioactive products from a highly concentrated aqueous solution of inorganic salts containing peat-derived bioactive ingredients. The solution is diluted with demineralized water, the dilution being effected with water volumes several times the volume of the solution to be diluted. The diluted solution is subjected to reverse osmosis in order to desalinate the solution. Inorganic salts being removed, the desalinated solution is concentrated and clarified.
The instant process, applied to the processing of a product obtained according to Polish patent specification No.
124110, consists in expelling residual organic solvents from a post-extraction aqueous phase, separating insoluble parts by filtration under reduced pressure through a sintered ceramic material, diluting the permeate with several times the volume of distilled water and subjecting it to desalination by reverse osmosis to separate excessive mineral salts, mainly sodium chloride, as a permeate. Then, the desalinated solution is concentrated, clarified by centrifugation and sterilized by filtration through a membrane filter, e.g. a Millipore(R) filter. The resulting microbiologically clean solution may optionally be spray-dried. The sterilized product (liquid or solid) may be formulated into a cosmetic pharmaceutical or veterinary composition. Optionally, the concentrated and clarified solution may be used - without sterilisation and spray-drying - in any suitable dilution as a component in numerous cosmetic compositions.
Preferably, in a spray-drying step, the inlet temperature is set to about 180°C, while the outflow temperature is set to about 90°C.
While the process of the present invention is described above in combination with the process according to Polish patent specification 124110, its use is not restricted to such combination, but is applicable generally in the context of a process for obtaining a peat-derived bioactive product from a highly concentrated aqueous solution of inorganic salts, especially of sodium of sodium chloride, containing peat-derived bioactive ingredients.
The peat-derived bioactive products provided by the present invention do not contain more than 70s by weight, preferably not more than 60% by weight of inorganic salts, especially of sodium chloride. Since a sodium chloride 4a concentration as low as possible would be desired for an optimal product, especially for pharmaceutical applications and such cosmetic applications where higher concentrations are required, i.e. for face care, lower sodium chloride concentrations, such as 55% and even lower, are most preferred, especially when obtainable by the steps of dilution and reverse osmosis.
Thus, in one aspect, the invention provides a water soluble peat-derived bioactive product, containing not more than 70% by weight sodium chloride, based on dry mass. In a further aspect, the invention provides a peat-derived bioactive product containing not more than 70% by weight of inorganic salts, based on dry solids.
In another aspect, the invention provides a peat-derived bioactive product obtainable by desalination of a concentrated aqueous solution of peat extract, which solution contains inorganic salts and water-soluble peat-derived bioactive ingredients and originates from an extraction process comprising alkaline hydrolysis of raw peat material, the bioactive product containing not more than 70%, by weight of inorganic salts, based on dry solids.
Where the process is terminated with the concentration and clarification steps, the product is a concentrated (or thickened) solution. "Concentrated peat extract", referred to in this specification, is a dark-brown liquid of a density of 1.02 - 1.09 g/ml and has a content of dry solids of not less than 5% by weight. The chloride ion content in dry solids, calculated as NaCl, is not higher than 70%, preferably not higher than 60%, and the pH value of a 1%
aqueous solution is 5.0-6.5, generally about 6Ø The lack 4b of a further sterilization step may not be detrimental in certain cases, e.g. for certain cosmetic uses of said concentrated peat extract.
On the other hand, the sterilization step will be mandatory, particularly when the product is intended for the preparation of pharmaceutical compositions. Particularly in such a case, the further step of spray-drying is most preferred, if not mandatory. The product resulting after such a spray-drying step is in powder form and thus particularly suited to the preparation of certain pharmaceutical compositions. A most preferred product of this type is the product commercialized under the designation "TOLPA~R~ Torf Preparation", TOLPA~R~ being a registered trade mark of Torf Corporation, u1. Mydlana 2, Wroclaw, Poland. The abbreviation TTP will be used in the course of this specification to designate said product.
The present invention also relates to pharmaceutical compositions containing as active ingredient a peat-derived bioactive product as hereinbefore described, particularly a product which contains not more than 70% by weight, preferably not more than 60% by weight, of inorganic salts, especially sodium chloride, based on dry solids, together with a pharmaceutically acceptable r. S!
',t fzt ~ 1 ~_) R/ l~ 1.f E.
W~ 92/I6216 PCT"/EP92/Otk191 _ 5 carrier. The peat-derived bioactive product contained in such pharmaceutical compositions is preferably TTP as defined above.
The pharmaceutical preparation contains the peat-derived bio-active product and the gharmaceutically acceptable carrier material, preferably in a weight ratio of between about 1:5 and 1:25, and most preferably between 1:9 and 1:19.
The present invention furthermore relates to a process for preparing a pharmaceutical formulation containing a peat-derived bioactive product, in the form of a gel. This process is cha-racterised in that a sterile alcoholic herb extract is combined with sterile glycerol, a sterile aqueous - preferably concen-trated - solution of previously powdered peat-derived bioactive product and a sterile menthol solution, and that the resulting mixture is gradually combined with colloidal silica 'to convert the liquid composition into gel form, the weight ratio of liauid mixture to silica being from 90:10 to 94:6. Preferably TTP is used as the powdered or concentrated peat-derived bioactive product.
The present invention also relates to a process for preparing a pharmaceutical formulation containing a peat-derv ved bioactive product, in the form of an ointment. Th9.s process is charact-erised in that a sterile herb extract is gradually combined with a sterile solution of powdered peat-derived bioactive product, that the resulting mixture is gelled with the addition of col-loidal silica and that the gel thus obtained is triturated with a previously sterilized mixture of fatty components, such as eucerine and petrolatum, preferably with a weight ratio of liquid components to silica of about 30:20 and of gel to fatty composition of between 32:68 and 34:66. Also here, preferably TTP is used as the powdered or concentrated peat-derived - bioactive product.
Cosmetic preparations, which may comprise herbal extracts as well as other auxiliary and enriching components, fragrant compositions and carrier materials allowed for cosmetic use, C?i IRr:~~~~ ~~~ J~"
r,~ ~ J: i~ s~ v ~" :.~ ~.3 ~> ~? :~.
WO 92!16216 PCT/EP92/00491 contain the peat-derived bioactive product according to the present invention in an amount of 0.01-10~ by weight, preferably 0.05-1.00 by weight, and most preferably 0.05-0.10 by weight.
Carrier materials may be aqueous solutions of alcohols, all types of emulsions, gels, foaming compositions and fatty carriers. Use of one specific carrier selected from the group of the above mentioned substances allows formulation of various types of cosmetic preparations according to the invention, such as tonics, creams, balsams, cleaning milks etc. for daily body care as well as shampoos, hair balms, foaming bath compositions, al? with the addition of peat-derived bioactive produc:.s.
Peat-derived bioactive products (peat extracts in abbreviacec form) stemming from original yaw peat material, such as famonc others) therapeutic mud, contain well balanced quantities o~:
mineral and organic compounds, such as mineral salts of ~~e following -elements: B, Si, Ab, Fe, Mg, Mn, Cu, Sn, Ni, Ca, ~c and Na; organic compounds, such as aminoacids in free form ant as salts; polysaccharides, partially degraded/reaeted - _.. she course of hydrolysis - 'to desoxysaccharides and%or aminosacc'.~.-arides. Peat, in particular therapeutic muc, is known ant =°-cognised -as a material of biological plant and microorganism origin; due to its contents of nourishing and stimul ar_~.ng cor,.-aonents it has beneficial effects an humans and mammals; she==-fore, peat-derived bioactive compositions contain the above-mentioned substances in proportions characteristic for the '-=v-ing organisms; this is considered to be an explanation of the advantageous effects of cosmetic and pharmaceutical pr~parat~o:a containing peat-derived active products and compositions.
Particularly good effects of the new cosmetic compositions are observed when herb extracts are also present. in the =ormulation.
Selection of a suitable herb extract is based on a known typical use of such extracts in cosmetics, modifying the activity of preparations and thus enabling the content of cosmetic prepar-ations to be matched with demands and needs of indivic~aals to be treated.
~tJBSTiTIJTE SHEET
~~.s~~~..
'!W~ 92/16216 PCT/EP92/00491 The present in~rention is better characterised and explained in the following examples.
Exa~le 1 Starting with 1000 kg of air-dried raw peat material, following e.g. the known procedure according to Polish patent No. 124110, a solution of peat-derived bioactive ingredients in a saturated aqueous solution of sodium chloride was obtained in a quantity of about 10 liters. The solution was filtered through a sintered ceramic filter under reduced pressure in order to clarify the solution before desalination of the same, The clear solution thus obtained contains about 95~ of NaC'_ in a dry mass. The dr.:
mass constitutes about 32~ by weight of the solution. The volume of this clear solution is about 7 liters.
The clear solution is diluted with 5 to 8 M mes the quantity o.
distilled water and in diluted form is subjected to a desalin-ation step carried out by using a reverse osmosis technique using a DDS apparatus. Desalination was carried out for 3 ~o hours, whereby the excess of mineral salts - mainly NaC'_ - is separated in the form of the permeate. The desalinated compos-ition contained approx. 6~-70a.of sodium chloride in the soi'_ds.
The solution thus obtained, or 6-7 liters by volume, was con-centrated 4-5 times in a Buechi rotating evaporator, so tat a concentrated sol~ition containing approx. 20~ of dry mass was obtained. The resulting concentrated solution was clarified using a Biofuga-Heraeus centrifugal apparatus (flow separator) and then sterilised by filtering through a Millipore~R) ~-lte=.
The resulting microbiologically pure solution was spray-dried in an Anhydro dryer with the outflow temperature se't at 90°C and supply inlet temperature set at 180°C. The yield of dried oow~:e~
was approx. 200 g.
Example 2:
~I Ii~STiTUTc S1-~E~T
~, -, c7 r -a .a .,~ r. a ~~~ a,~ ~ r ~ ~ ~.~ .!.
W~ 92/1626 P(.'T/1EP92/fl(kl9l _ g ._ The product obtained in Example 1 was used for preparing gel and ointment pharmaceutical compositions, containing also hero ex-tracts synergistically improving the therapeutic effect with , respect to certain diseases. For example, a gel and ointment against varicose ulcer of the shank was prepared as follows: , 20 g of hippocastanaceous extract, 10 g of calendula extract, 60 g of glycerol, 0.1 g of TTP in the form of a powder obtained as described in Example 1 above, 0.1 g of salicylic acid, 1.0 g of distilled water and 8.8 g of Aerosil(R) (colloidal silica) were used in order to obtain a gel form of the preparation.
hiquid (non-volatile) ingredients were sterilised before use, by means of heating under reflux for two hours. Herb extracts were combined with glycerol and an aqueous solution of TTP and also with menthol, and silica was gradually added to the obtained mixture, under continuous stirring.
Similarly, in order to obtain an ointment composition, t:~.e following ingredients were used: 20 g of hippocastanaceous ex-tract, 10 g of calendula extract, 0.1 g of salicylic acid, 0.1 g of TTP in powdered form obtained as described in example 1 and 2.0 g o~ Aerosil(R) (colloidal silica).
As fatty components, a mixture of the following substances was used: 22 g of eucers.:ne and 45.8 g of petrolatum. :lerb e~:.rac~s were sterilised by heating under reflux for approx. ? hours.
Eucerine -and petrolatum were similarity sterilised. ~iauic in-gredients wer a carefully combined with silica to obtain a ge'_, which in turn was triturated with sterilised and 'arty com-ponents cooled down to room temperature. A stable aintmen~ was obtained which did not separate when stored.
The gel and ointment obtained above were simultaneously applied in. the treatment of varicose ulcer of the shank. Ulcers were treated with the gel preparation while the surrounding, non-affected skin was treated with ointment. Addition c~ colloidal silica is believed to be responsible for prompt dess,~cation ~~~~TITUT~ S~-l~~T
, ~~ ~) 4~ l~' ~7 ~7 d ?3 !J ?' ii a,3 iV0 9~/162i6 PCf/EF'92/~Od91 g _ while the herbal and peat-derived ingredients are believed to be responsible for 'the curing effect of the preparation. Fatty components helped to keep elastic the crust and the skin. The results obtained were compared with a control group of patients treated in a classic way. Those who received the new treatment were selected from a group of patients suffering from the disease for many months (sometimes years) without noticeable positive effects. Patients treated with compositions according to the invention showed better results - already within the fir st few weeks - than control patients, _Example 3:
Products obtained in Example 1 were used to pr°par~ pharmaceut-ical formulations in the form of tablets, or granules to be placed in capsules.
A sterile pe at-derived bioactive composition ;n pcwdered form was combined with a carrier in a weight ratio of 1:9. As a carrier, ~IYVATEX(P)TL (tradename of F_astman-:Codak), a .mixture o-_.
lactose and lubricating substances, was used in a weight =atio of 44:1. Lactose of 50 mesh particle size and ivIYVAT°X(~)TT~ were finely disintegrated so that approx. .70% o= its mass passed through a 100 mesh screen. A part of the resulting mixture of active composition and carrier was formulated into tablets con-taining ~ mg of aci~ive ingredients. The total. mass oz -ac'.~.
tablet was SO mg. 'The other part of the same rni:<tare of active camoosition -and a carrier was granulated using c.~. oz ec'nanol (40% by volume). Granules were sieved and ground ~._ necessary and then filled in capsules in such a quantity that each capsu~~.e contained 5 mg o.f active ingredients by using TTP in mixture with a carrier at a ratio of 19:1.
The tablets obtained as above were tested is order to measure the time of their disintegration in an ar'~i~icial gastric juice at 37°C +/- z°C using Erweka equipment. The artificial gastric juice was prepared as follows: 2.0 g of sodium chloride and 3.~g oz pepsin were dissolved in 7 ml of hydroc~loric acid and SUBST1TUT~ SNEET
"~p yl o.? 6 .~ A'1 t ~~~~r_~~~~~.~_~.
WO 92116216 P~T~1;P92/00491 distilled water was added up to a total volume of 1000 ml. The pH-value of the resulting solution was approx. 1.2.
l3esintegration time of a tablet, having a diameter of 5.1 mm and a total mass of 0.0498 g, was 5 minutes.
Further examples relate to numerous cosmetic preparations according to the present invention, having different forms composition and being designed for different applications, containing the beneficial addition of bioactive ingredients derived from peat. Among others, preparations such as tonics, balms, creams, milks, shampoos, foaming bath compositions etc.
are described.
Example 4:
A reaction vessel equipped with a stirrer was charged with 150 g of camomile extract obtained by the extraction of camomile in-florescence with a 1:1 ethanol:water solution, as we';l as i g o~
TTP as described above. SO g of glycerol were added :,o the mixture obtained. The three substances were stirred to obtain a uniform mixture. Subsequently, a second mixture as previously formulated, was introduced into the same vessel. It comprised 340 g or a 95:5 ethanol: water solution, 1 g or salicyl'_c acid and 0.5 g of menthol.. The two mixtures were combined b~ st;rring to form a uniform solution: Next, 3 g of a fragrant composition TILIANA H4308 were added. TILIANA H4308 is a product o~ ~abryka Syntetykow Zapachowych Pollena-Aroma (Synthetic Fragrance works Pollena-Aroma), of r,Varsaw, Poland. The solution was then brought to a total volume of 1000 ml by adding 454.5 g or distil'_ed water; stirring was continued until a homogeneous mixture was obtained.
In the above procedure, 86~ glycerol, menthol and water accord-ing to the requirements of Polish Pharmacopea FP T_V and ethanol in a concentration of 95~ according to the Polish industrial standard BN-75/6193-01 were used.
SLJBBTITUTc SNE~T
~,e ~y~ !J 3 l ~ ;;l l s.
~V~ 9211b216 PCT/EP92/(j0491 The concentrated peat extract used was a dark-brown liquid or a density 1.020-1.090 g/ml and a content of dry solids not less than 5~; the pH value of a l~ aqueous solution was 5.0-6.5.
Camomile extxact was a red-brown liquid of a density of 0.9160-0.9503 g/ml, and ethanol content of 52-56~ by volume.
The tonic preparation obtained above is suitable for all kinds of skin. It is a clear liquid without any solids. Its colour is yellow. The pH value is 4.28 and the ethanol content is 45.920 by volume. Total acidity calculated as salicylic acid content was not less than 0.1~ by weight, namely 0.23a by weight. The preparation being stored for 12 months did not lose any of the above characteristic features.
E_x_ample 5:
The procedure described in Example 4 above was repeated, the only difference being that instead of camomile extract and the TILIAIdA H4308 fragrant composition, a marigold flowers extract and a composition SINUS H4625 (also a product of Fabryka Syn-tetykow Zapachowych Pollena-Aroma) were used in the same way and the same molar and volume ratios. The resulting tonic prepar-ation is suitable for dry and fragile skin. Similar==y '-.- was a clear liquid without any solid particles. The pH value was 4.30, total ethanol content was 45.82 by volume and total acidity was 0.27$ by weight. When stored, the preparation was unchainged after 12 months like the preparation obtained acca-rding to Example 4.
Example 6:
The procedure of Example 4 was repeated, except that an ext=acs of sage leaves was chosen instead of camomile extract, and the fragrance LELIA 90368 (Pollena-Aroma, Warsaw) was chosen instead of TILIANA H4308. The extract of sage leaves was obtained by ex-tracting dried gage leaves with ethanol at 50°C and had a brown-ish colour, a characteristic sage odour and a density of C.916C
to 0.9503; it contained 52 to 56~ of ethanol. The resulting fac°
care agent is particularly su°itable for greasy skin. It is a SUBSTITUTE SH~~T
., ,.
~,i3(..~'le~~~~i9 WO 92/16216 ~CT/IEP92/(30491 - '12 -clear and homogeneous liquid having a dark yellow colour; the pH
value, the ethanol content and the acidity were comparable with the values of the care agent according to Example 4.
Example 7:
The following composition proved to be a particularly effective gel for avoiding or treating periodontosis:
24.0 g camomile extract 3.0 g sage leaf extract 0.3 g salicylic acid 0.2 g menthol v C.1 g TTP
to 100.0 g commercial gel base Example 8:
The following carefully weighed components were introduces =~ro a reaction vessel of a volume of 2000 m1, equipped with a mechanical stirrer:
270 g of camomile extract obtained by extraction of camomile inflorescence with 50o ethanol; the extract was a =ed-brow~
liquid, having a density of 0.9160-0.9503 g%ml and a~
ethanol content of approx. 55o by volume - 50 g of glycerol,'86~, according to the requirements o. °olis Pharmacopea E'P IV
- 30 g of a saponaria officinalis extract obtained by ext=act~:=g saponaria of.ficinalis roots with 70~ ethanol; the e:c~=acs was a red-brown liquid, the density was 0.9630-0.9810 glm~-, and the ethanol content approx. 75~ by volume - 1.0 g of the inventive concentrated peat ext=act, being a da='-:
brown liquid having a density of 1.02-1.09 g/mi; not more than 2~ chloride ions calculated as sodium chloride; the c~.~
solids content was no's less than 5~; the pH value oa 1~
water solution of the extract was 6Ø
The ingredients listed above were mixed thoroughly. ~ pr°-~=ous_y prepared solution of 1 g of salicylic acid in 260 g of ethanol was added thereto. To :he combined solution, 383 :~
SUSST'ITL~TE SHEET
6~ ~i i:) 'r:' 1'e ,(~ .~
;.i J t 3 i,~ r W~ 92116216 PtJT/EI'~2/Od~l9l -. 13 -distilled water and 5 g of fragrant composition TILIANA H3408 were added and stirred until a uniform solution was obtained.
The preparation was analysed and stored in retail size bottles of 200 ml volume. The resulting preparation was suitable as a hair care preparation. It was a clear, slightly opalescent liquid, containing approx. 45~ by volume of ethanol; the pH
value was 4.5; the total acidity calculated as salicylic acid was not less than 0.1~ by weight. The preparation is suitable for blonde hair. During 12 months storing the preparation re-mained unchanged in i'ts features.
Example 9:
The procedure described in Example 8 was followed except that instead of camomile extract and TILIANA H4308 composition there were used in the same sequence and ratio: horsetail herb extaact and the fragrant composition FINUS H4625. Horsetail herb extract was a green-brown l,;quid of a density oz 0.9160-0.9503 g/ml and an ethanol ccntent or 55~ by volume.
The resulting preparation was suitable for a1'_ kinds of ;~a=. -..
was a clear and transparent liquid without any soled parts, yellow-brown in colour. pH value, ethanol content, total acidity as well as stability after a 12 months storing period were the same as those of a preparation described in Example 5.
Fxam 1e 10:
The procedure as described in Example 8 was repeated with com-parable results. The only difference was that, instead of camo-mile extract and TILIANA H4308 composition, stinging nettle leaves extract and fragrant composition LELIA 90368 (product o~
the same Fabryka Syntetykow Zapachowych Pollena-Aroma) in the same sequence and ratio were used. 'The herb extract used was olive-green in colour, had a density of 0.9160-0.9503 g/ml and an ethanol content of approx: ~5~ by volume. The preparation was suitable for all kinds of hair.
~I )RRT1T~ )T~ ~~~~T
~' S ,-~; 5~i ~ tr-p re i~,! ! j t) ~~ S ~ . .
'1~'~O 92116216 Pf'f/E)°92100491 Example 11:
rn general, cosmetic milks are dispersions of fatty substances acting in both chemical and mechanical ways on the skin. In .
fact, due to a convenient way of application and better interaction of the fluid and the skin, it is very appropriate to use liquid, more specifically emulsion creams. They can easily penetrate to deeper layers of the skin and thus prevent changes of the skin due to age. Cosmetic milks are used mainly to clean a dry and fragile skin. Accordingly, they must not contain any aggressive volatile oils, while frequently they contain suitable herb extracts like camomile extract or wheat germ extract. Addition of peat-derived bioactive products to such cosmetic milks further improves their positive effects. In particular, the new recipe is as follows:
0.05 g TTP
aloe extract 20.00 g glycerol 3.00 g eucerine 2.00 g white paraffin oil 1.00 g triethylamine 1.00 g Aerosil(R) (colloidal silica)4.00 g Example 12:
Improved regenerative results were observed when TT° and carefully selected fatty carriers were used in a c7.assic nourishing and regenerative cream formula. TTP is used in an amount of 0.01 -1.00 by weight in combination with a herb extract (selection depends on the type of skin for which the cream is intended) in an amount of at least 0.05 - 1.00 by weight, antibacterial preparation in an amount of 0.05 - l.OOj by weight, synthetic fragrant composition in an amount of 0.01 -0.05~ by weight and a fatty carrier in the form of a water emulsion, constituting 97.00 - 99.500 by weight of the whole composition. The fatty composition needs to be a good carrier for the active ingredients and to be well accepted by the skin.
Preferably, it is an emulsion of. (a1.1 amounts ir_ o by weight) 35-45 eucerine, 8-14 petrolatum, 2.5-4 olive oil, 6-10 glycerol ci iRRTfTi fTF SH~~T
-~ u, ,~- y:~ ~s r ~ ~
t .
-~ 3.~ J ~.# 'tJ t. .' wo 9zn~zr~ ~cri~~z~o~a91 and 35-40 water. Preferred are marigold flower herb extracts extract, camomile extract, and the like.
thyme extract Preferred recipes are lows:
as fol 39.00 partsby weight 1. Eucerine Petrolatum 11.50 partsby weight Olive oil 3.13 partsby weight Glycerol 7.80 partsby weight Water 38.00 partsby weight NIPAGINA A (anti-bacterial preparation) 0.40 partsby weight TTP
0.05 partsby weight Marigold extract 0.10 partsby weight rance 0 02 parts~weiaht fra ti th g c e Syn total 100.00 by weight parts 00 parts by weight 2. Eucerine .
Petrolatum 8.50 carts by weight O1'_ve oil 3.08 parts by weight Glycerol 7.90 parts by weight Water 38.00 parts by weight NIPAGINA A (anti-bacterial preparation) 0.40 partsby weight TTP 0.05 partsby weight Camomile extract 0.02 partsby weight Synthetic fragx'ance_ 0.05 parts5v weight total 100.00 parts by weicJnc Example 13:
An after-shave preparation contains TTP as a peat ext=act in an amount. of 0 . 01 -- 1 ~ by weight, herb e:ctracts in ar. amount of 1 -30~ by weight, glycerol in an amount of 1-8o by weight, salicylic acid and menthol in aqueous-alcohol solution.
Preferred herb extracts are: camomile, marigold, thyme, aloe extract and similar beneficial herb extracts. Addition of glycerol is also beneficial due to its influence on the elasticity of the skin. Tt speeds up the spreading of the preparation on the face as well as the penet~ation into t:ze . . ..., .~-....-r-a ,'~'-r= c~ ~-7 C ~T
~1 ~-. .;
~~ S_7 ! 7 rw.1 ~,~ L' .~'_ lV~. !16216 PC_'T/~Ptl2/U0491 16 _.
deeper layers o~ the skin, thus enhancing the beneficial effects of the active peat composition and herb extracts. .
A preferred recipe is as follows:
0.10 parts byweight Camomile extract 15.00 parts byweight Glycerol 5.00 pats byweight Menthol 0.10 parts byweight Salicylic acid 0.10 parts byweight Ethanol (conc.95~) 10.00 parts byweight Fragrant composition 0.30 parts byweight Distilled water ad 100.00 parts by weight Example 14:
A shampoo composition was prepared according to the ~oliowing recipe:
Fuller's herb e:ctract 15.60 g 7.50% byweignt Stinging nettle leaves extract20.00 g 10.00% byweight GAMAL SBS-11 (detergent) 30.00 g 15.00% byweight GAMAL NO-3 (detergent) 20.00 g 10.00% byweight ' 0.40 g 0.20 byweight tina Asep ethanol 1.60 g 0.8U% byweight BRONOPOL (preservative) 0.04 g 0.02% byweight Sodium chloride 6.00 g 3.00% byweight W 106.96 53.48% b~weight t a er a total 200.0 0 100.00 %
g by weight To 92 parts by weight of the above shampoo composition~8 parts by weight of T'rP were added to obtain 100 parts by weight oz a shampoo according to the invention. Other herb ertrac~s car, be used in place of stinging-nettle leaves e:ct-ract.
Example 15:
The following shampoo composition was prepared:
Horse chestnut extract 13.00 g Marigold extract 22.00 g GAMAL SBS-11 30.00 g GAMAL NO-3 20.00 g w ~zmsz~s ~~c:rm~zivog9i _ 17 Aseptina 0.40 g Ethanol 1.60 g BRONOPOL 0.04 g Sodium chloride 6.00 g Water 106.96 a total 200.00 g To 95 parts by weight of the above composition, 5 parts by weight of TTP were added to obtain 100 parts by weight of shampoo according to the present invention.
Example 16:
A tooth paste contains TTP as a concentrated peat extract, in an amount of 0.01-0.10% by weight, etheral oils or their compositions or else fruit essences in an amount of 1-10% by weight, glycerol in an amount of S-10g~ by weight, herb extwac~s in an amount of 0.10-10~ by weight and cleaning substances in a::
amount of 20-35% by weight dispersed in water in an amount of 45-60% by weight, and dyes and whitening components in an amount of 1-2~ by weight.
Titanium dioxide may be used as a whitening component; TTP
itself may be used as an anti-bacterial additive; sage leaves, camomile or marigold flowers extracts may be used as benef'_cial preferred herb extracts.
The preferred recipe is as follows:
Precipitated calcium carbonate 150.00 g Magnesium carbonate 60.00 g Glycerol 70.00 g Herb extract x.00 g TTP 0.50 g Titanium dioxide 10.00 g Etheral oils (or mint, lemon, etc essence) 5.00 g water 400.00g Dye trace S~BBTITL~'TE ~HBET
~ .~ J ~i K.1 ~ ~-a t ~~r ~ o i_l ~J 1~ ~i ..~..
WO 9. 0216 PC'f1E~92/1H~491 _ 1g _ E_xamnle 17:
Bath salt preparation: In the course of the process for obtaining a peat-derived bioactive product according to the present invention, in particular when oonverting a liquid form into a powdered one, there is the desalination step in which sodium chloride is separated as a by-product. In said by-product, 95g constitutes sodium chloride, other mineral salts separated are calcium salts, magnesium salts, mainly chlorides and sulfates; these salty products also contain some organic peat-derived low molecular compounds occluded within the crystal structure of these inorganic salts. These organic compounds are components of TTP and are - among others - polysaccharides, aminoacids, fulvic acids and the like. The presence of these components in the salty by-products is beneficial when sail is used as a bath salt, because they may add additional bene=icial effects to the standard activity of bath salt. Accordingly ~..is by-product was tested for its chemical and physical grope=~ies in the Balneologic Institute in Poznan, Poland, to find ouc whether it can be used in cosmetic baths. Since the T_nsti~u~°
has found no undesired entity in the salt, it was approved =o.
cosmetic use.
The preferred recipe is as follows:
Salt (NaCl) containing occluded TTP 97.00 g Pine etheral oil or etheral oils composition 3.00 g Example 18:
A new hair balm contains TTP in an amount of 0.01-1% by weigh herb extracts in an amount of 0.01-10% by weight, anti-elec~ro-static components in an amount of 3-4% by weight, components preventing excessive drying of hair and skin in an amount o= v,:p to 2~ by weight, glycerol in an amount or 1-5% by weight, pre-servative and stabilisers in an amount of 0.05-0.50% by weigi:~
and water to 100 by weight.
As an anti-electrostatic component, the present balm contains ar.
alcoholic solution of trimethylamine and ammonium chloride salt, obtained from fatty animal-derived amines; as 'thickening agent -~UBST1TUTB ~~E~~T
V1'~ ~2/1621G PC.T/EP92/00~91 acting also as stabilizing agent - as agent cosmetic alcohol;
preventing excessive dryness of hair skin - t oils, and plan acting simultaneously as co-emulsifying glycerol agents; and for easying spreading and penetration of balm, in the particular of its active ingredients TTP and herb acidic extracts, As an en-vironment stops multiplication of bacteriar the balm according to the invention contains citric acid fumaric or acid in an amount of 0.1~ as well as a preservativeknown as BRONOPOL
and fragrant compositions.
The preferred recipe is as follows:
Alcoholic solution of trimethylamine and ammonium Chloride salts 3-4~ by weight Cosmetic alcohols 3-4~ by weight T'rP 0.01-10$ weight by Thickened herb extracts 0.01-10~ weight by Glycerol 1.5a by weight Plant oils up to 2; by weight Citric or fumaric acid 0.1% by weight BRONOPOL 0.1$ by weight Fragrant composition 0.3~ by weight Distilled Water to 100 weight by Fxamole 19:
Cosmetic masks are well known cosmetic preparations serving many different purposes. As therapeutic mud has a known beneficial effect on the skin and body, it was believed that also post-extraction peat obtained in the process of separation of bio-active peat-derived compositions from peat may be used in cos-metic applications. Post-extraction peat contains a solution, o active bodies freed in the alkaline hydrolysis process due 'to extremely high sorptive properties of peat after neutralisation;
It was therefore found to be a valuable component of cosmetic masks. To enrich the post-extraction peat with more of the valuable components, natural therapeutic mud and humic acid fractions were added which are present in natural peat and separated in a process for obtaining peat-derived bioactive compositions .from the alkaline hydrolysate. Such a composition c.i ~czCTIT'i iTF fiHE~~
Zi 7~ ?j ~~ i~
we ; mz~6 ~criE~ziot~am was tested in the above-mentioned ~alneologic Institute and was found suitable for cosmetic use.
The preferred recipe is as follows:
post-extraction peat 100.0 g Natural therapeutic mud or peat 20.0 g Humic acid fraction 10.0 g Magnesium carbonate 10.0 g Zinc oxide 5.0 g Citric acid or the like 0.1 g Herb extract or powdered plant material 5.0 g Distilled water q~s' The following statements and explanations relate to the bio-logical aspect of the products of the present invention, _.e. to the bioactive characteristics and to the compatibility o~ these products, particularly with a view to their usefulness as phar-maceuticals. The following abbreviations will be used below:
TTP TOLPA(R) Torf Preparation (trademark of Torf Corporation, Wroclaw, u1. Mydlana 2, °oland) IFN Interferon exists as a ubiquitous cytokine (tissue hcrmone).
IFN genes are present in all cells. IFN is mainly i~.duced by proteins or glyco-proteins. Substances stimulating the IFN
genes for the production of IFN are called inducers. The process of the IFN induction is a highly regulates, soph-isticated biochemical process; negative and positive regul-atory genes controlling IFN production have been recognized.
Small amounts of IFN may be produced spontaneoulsy, without any detectable induces. Such IFNs are sometimes named "physiological IFNs". IFN exists in nature in three main molecular forms:
IFN-a (or leukocyte IFN), IFN-~i (fibroblast TFN), and IFN-Y (immune IFN).
IFN-a and IFN-(i are type I--IFNs, IFN-Y is the type II-IFN.
~U~STITLIT~ ~H~~T
GZ a ~,a ~; ~I r~ c.3 :J _~.
~~fO 92/16216 hCT/EF'92100~b~1 The major biological activities of IFNs are antiviral, anti-proliferative (anticancer), and immunomodulatory activities.
Various forms of IFN are produced commercially as the natural and recombinant preparations and are used as drugs for treatment of neoplastic, viral, and several other diseases.
CTL - cytotoxic T lymphocyte.
NK cells - natural killer cell.
IL-1, IL-2 well known interleukins stimulating the prolifer-ation of T cells and other lymphoid ce'_'_s including 3 cells;
RPMT_ 1640 tissue culture medium for the growt'n of human and other leukocytes (abbreviation of the Roswell ?ark Memor~.a'_ T_nstitute, Buffai0);
FCS = fetal calf serum (for assays with leukocytes it has to be pre-tested because it may contain mitogenic substances mimicking the action of interleukins);
EMCV -= encephalomyocarditis virus, mouse oicorna virus non-pathogenic fox humans, it is often used as a challenge virus in the TFN bioassays;
A-X49 human adenocarcinoma cell line - used in t:ze i=N bio-assays because of its high sensitivity fo.r. IFN-a,~, and Y.
The line is recommended for such use by the wii0 e~pe=~s on IFN standardization;
MTT 3-(4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide. Reagent used to measure the cell kill or cell growth in several bioassays, using the ELTSA scanners (kiansen et al., J. Immunol. Meth. 1989, 119, 203-210);
m tnhTlTi iT~ C,''Ld~~"T
.7 r~ ~ ~~ ~ .9 ~.$ U 9~ to ti lVO . 16Z 16 PCI"/EP921(10491 _ zz L929 the mouse cell line, commonly used for assaying mouse ZFN and human or mouse TNF;
TNF tumor necrosis factor, cytokine, (relatively small pro-tein, very susceptible to the proteolytic enzymes), produced by monocytes and macrophages (TNF-a known also as cachectin factor causing cachexia in humans and animals);
produced after stimulation with LPS (lipopolysaccharides), viruses, bacteria, and many other agents, very toxic for many virus-infected and neoplastic cells; may also act as a growth factor for fibroblasts. Connected with inflammaLOry reactions. The related form TNF-p (lymphotaxin) is produces mainly by T cells and some other tolls.
PBL peripheral blood leukocytes, normal human ieukocy=es from healthy blood donors, isolated from "buffy coats"
(interphase between red cells and plasma). The respons~ve-ness of PBL from individual donors to various cytok'_ne '_..
ducers appears to depend on a genetic set-up of the donors.
The high responders and non-responders have been identifies.
This refers also to the response of PBL _to TTO, The variation in the individual response to inducers c~ =FN
or TNF is more visible when Weak inducers a.r.~ useri than whe::
very strong induc ers, like viruses, are employed. This is due to the fact that the reaction to weak inducers is o_ the "all or none" type whereas viruses almost always induce de-tectable amounts of the cytokines.
Tolerance to inducer: Called also a hyporeactivity state. Occurs after administration of a single dose of an induces, e.g.
after 20 h of exposure of PBL to an induces (e. g. vir:a);
the cells stop further production of IFN. The hyporeactivity state lasts usually about seven days. It may be complete or partial. Such reactivity makes the therapeutic application of strong inducers difficult and/or ineffective. weak T_FN
~UBSTITUTc ~H~BT
92/1b216 pf~~~~i'/1~.
inducers either do not induce the hyporeactivity state or the tolerance is small.
Several natural drugs which are extracted from medicinal plants possess immunomodulating properties. TTP appears to be one of them. TTP alters many different immune functions in vitro and in viva. It initiates balanced immunostimulation, with the capabil-ity of non-specifically activating all effector pathways (CD4 helper, weakly CD8 suppressor, CTL, NK cell, and activated macrophage) without cytotoxicity for norrnal tissues.
TTP has a restorative effect on normal wound healing. Low doses of TTP weakly stimulate IL-1 and IL-2 production. High doses may inhibit the cytokine synthesis.
The respective tests with TT° were carried out by the following methods:
PBL from healthy blood donors were pur.='_ed 'oy amrnoniLm chlcrice treatment. "'he culture medium was RPMI 1640 plus 1,0~ PCS.
proximately'8 x 1C6 cells/ml were cultured far 20 h at 3?°C, S~
COZ. The antiviral activity of interferon was assayed by ~MC'J
cytopathic effect inhibition in human A549 cells. The MTT method to measure cell ki7.1 was also used.
TNF activity was measured in =929 cells. T_n order to de='_ne =='a type, the individual IFN samples were trAated with different anti-iFN -sera for 1 h. Their antiviral activities were compared with non-treated preparations.
The tests indicate TTP stimulates the production of endogenous interferons (IFNs) and tumor necrosis factor ('rNF). The response is dose-related.
Seven assayed cocnmercial batches of TTP had comparable biolog-ical activity as immunostimulant and the cytokine inducers.
3~~~~~~~TE ~~~CT
.. , y , ',i ;; 3 '.? ~ 7 7 j...
~:: ~ E ~-.l P.j i~ a~,1 ~1.
w zm6zy6 ~c°ri~~zioo~~~
- 24 _ Considerable variation in the response to TTP of leukocytes of the individual blood donors have been observed. PBL of several donors wrere found to be unresponsive. This may reflect a genetic background.
Potent polyclonal antisera were used, such as anti-IFN-a (Cantell), anti-IFN-a Ly (Namalwa) from K. Fantes, and anti-IFN-Y (Cantell) to neutralize antiviral activity in the supernatants of PBL treated for 20 h with TTP.
The results of th.e neutralization assays were found to resemble the finger prints of individual blood donors. In other words, proportions of IFN types produced by the individuals varied con-siderably. The separation of PHL into adherent and non-adherent fractions may potentiate the induced cytokine synthesis.
The hyporeactivity (tolerance) a the induction of ~FN by "dDV
observed 20 h after the initial stimulation of PBL with TTY was either minimal or absent.
All of the seven batches of TTP tested in human PBL as I=N and TNF inducers were found to be active in inducing _TFN and/or TNF.
The optimal concentration of TTP for IFN induction was 30-100 p.g/ml'and for TNF induction 100-200 ~.~g/ml. The dose of 200ug/ml may be subtoxic for PBL, but the synthesis of TNE" occurs much faster than that of IFN and faster than the devE:i.oprnenL oz moderate cytotoxici t:y .
The conclusion of the foregoing is the experience that the active principle in TTP pharmaceuticals is an immunoactive fraction of an extract from peat containing organic, primarily bound sugars, aminoacids, uronic acids, hurnic ,=acid substances and mineral salts including microelernents. Tire LD~O in animals is >2400 mg/kg per os.
~.. r hr ~ ~.t r.~ > > a 3 .~A.
V ~2/162i6 PCT/~pf2/0049i No mutagenic, genotoxic, embryotoxic, teratogenic as carcino-genic activity of TTP was found. TTP shows no allergenic pro-perties and had no topically irritating activity.
The therapeutic indications include chronic and recurring respiratory tract inflammations and lower leg varicose ulcers, supglementing the treatment of vaginal erosions, and periodontal diseases.
There are clinical observations suggesting that TTP may be use-ful as an immunomodulator in the supporting therapy of several neoplastic diseases. TTP is used r,_-ally (5 mg tablets) or topically.
Of particular relevance and importance appears to be the fact that PBL _treated with TTP for 20 h at 37°C do not develop the hyporeactivity state because they retain the capacity to produce IFN after induction with NDV (Newcastle Disease Virus), a very potent IFN induces.
The attached Tables 1-24 relate to various biological, e.g.
toxicological, hematological and immunological tests. 'these Tables appear to be self-explanatory and provide for the bio-logist pertinent information relating to compatibility and bio-logical activity of T'I'P.
It is worth noting that the concentration of active ingredients in pharmaceuticals, as indicated in claim 13, may be higher than in cosmetic compositions (as indicated in claim 14) due to the following reasons:
Pharmaceuticals are prepared in unit dosages, wherein the con-tent of active ingredients is under strict control; e.g. in tablets, the concentration is adapted to 'the size of the tablet containing the effective daily dose (or a part of it) of the active ingredient.
SI~~ST1~'UT~ ~~~~ l' ~, r i ;vi ~~ ;~ ,.~ .~.
fft /16226 P~T/Ek'92IU043~
_ 2 The concentration of the active ingredient, e.g. cf 'TTP, in granules with which capsules are filled, preferably is only 5 by weight in order to achieve a sufficient tablet size and to allow an appropriate operation of the capsulating machine.
Another reason is that most pharmaceuticals are administered orally, and the active ingredient is distributed within the whole body..~Even though it reaches the skin cells in a very low concentration, the therapeutic effect is remarkable.
Furthermore, .cosmetic compositions - contrary to pharmaceuticals - are used in rather uncontrolled quantities, applied locally, with a different penetration rate to different cells.
Some compositions, such as a shampoo which is nearly immediately washed out, have short contact times with the body or hair, and may contain relatively more of the beneficial component; others are applied several times a day and therefore should have a lower content of the active ingredient.
Finally, as in the case of tooth paste, being in contact with the mucous membrane i.n the mouth, active ingredient penetration is much easier than through the skin, and the concentration of the active ingredient may be lower.
SUBSTITUTE SH~~T
;'.q _::.g ': .~ ~ a n a hf ~ y v 1J ;"~' .,.
9xmszxs ~~'iE~zi~aa91 ;_ TAB. ACUTE TOxI CITY.EFFECT OEZALLY ADMINISTERED
ON BIOCHEMICAL F BLOODOF
THE PARAMETERS RABBITS
O
Examined Parameters Sex Dose Day of TT_? Test Creatinxne TotalProteinY-Globulin ;
~
g/kg ~mg%) lg/1) ~9~) 0.0 0 1.090.03 65.0 =3.49 0.53 Ø00 ~
7 I 1.03=0.02 62.0--_0.330.50 Ø01 _ ~ 2.0 0 1.06=0.01 58.0 ~1.9b I 0.50x0.02 ~
7 I 1.14=0.38 57.8 =1.54 i 0.54-_0.01 5.0 0 ~ 0.96-_0.12 70.3 .3.52 I 0.55-_0.00 ~ I
7 I 0.94Ø13 60.0 _2.34 j 0.57x0.00 i 0.0 0 0.80_-0.11 73.8 -_3.330.72 =0.06 I
~ 7 , 0.82X0.13 75.3 -_1.020.79 =0.03 ~
I 3.0 0 ~ 0.88-0.05 64.6 .2.36 ~, _0.'0 M . 0.52 j 7 I 1.24Ø34 64.5 =2.03 0.67 =~J.07 , .,.0 0 0.85_-0.03 58.5 -3.19 0.57 =0.03 i 7 i 1.04-0.12 57.0 =4.00 0.50 -~J.J1 .
'.'AB. ACJTE TOXIC:':"_'. ___"ECT IST:?ED
~ OF '"'"
ORtIL:.'! C~I
ADMI'I ='='-~--' .~C'_'IVT_'~'_ ':'P.ANSAMINAScS =N ScRUM ?.a.33I':'S
OF 'rT.:=OF .
'T':?E
I I i Exam=ned ~a~arneLers Se ~ Dose i ay r D o ; ~~mD ~ ~:esL Aianine ~spar ag~.~...
g%k~ j a11t1:1OL~3nSAe~a S2 SL_-35e llTt;nOL~atl t ~ ~ iU T_U
_ .-0.0 __ 0 ; 8.07 X1.20 33.33 ~3.:i0 j 7 I 9.07 Ø33 28.57 2.0 0 ~ 10.33 =1.76t 18.33 =..09 I , I 12 33 .3.52 0.00 x...03 I i i 5.0 ~ 0 ! 11.33 -'._56- - ''3,33~_.52 I 7 i 11.00 =3.05 20.07 =..06 0.0 0 10.33 :.-1.76 X3.00 =3.05 ~ 7 ~ 12.00 =2.04; 25.67 .4.'.'i M 2.0 I 0 9.00 -_1.00~ ;8.00 =..52 i ' 7 9.00 =0.57; 20.66 . .53 i 5.0 0 8.00 -_1.00I - 00 18.00 =1 7 11.33 -1.66; 23.00 :
=
~~ c~~~~~
s; . ~ CJ s;~ ry to 'a ., r.r L~ ~~i m.) s1 ~z«6z~6 ~~rr~z~~io~a~r TAB. 3 ACUTE TOXICITY. EFFECT OF ORALLY ADMINIST~t~
TTP ON THE I~riIATOLOGIC?.L PARAMETERS OF RABBTTS
Examined I
parameters Sex Dose Day of TTP Test Hemoglobin Hematooszt Leukocytes Erytzrocytesl I ~ ~ --glkg (mg%) (%) I x x 10 0.0 0 12.4=0.4438<3_1.857.32 =0.435.21=0.20 I
7 1 11.8=0.6337.7.1.206.30 x0.661 -_0.11 1 6.11 2.0 0 11.5._0.3136.7Ø885.69 0.531 _0.65 I I 5.d6;
r i 7 1 12.3_-0.4838.30.56 5.74 =0.76j --0.'.
i 5.69 j 0 112.3_0.1440.7--2.57.35 _0.991 Ø20 5.0 1 5.42 I ~ 111.6=0.2337.3Ø33, =1.791 -_0.19 ! 7 8.6a 5.08 0 ~ 0 X12.0=0.8637.3X1.20111.10_0.631 =0.26 0 -~~3 , I 7 111.9=0.6135.7.1.8510.80=0.941 =0.79 ' .~..i4 !
!9 0 112.3-_0.26X43.7-4.80~ =2.001 _0.65 ! 9.94 ~.tn 2.0 7 11.8=0.39134.%=2.02111.06_1.081 =0.50 ~.37 5.0 j 0 j12.7Ø6338.7_2.40j =1.361 :0.10 9.52 -.9 jl0.oX1.04,4Ø2.08I -_1..3! _0.6a 8.87 ..,_ '_~~B. -. C:IRONTC =~OXTCT'='<_'. ==~5C'.' OF ORALu..Y aDMTNIST°?c-0 T'.'? ON '.'HE MASd OF T4;; ORGANS OF ?ABBI'=S
Dose of TTP (mg/kg) I
i ' I ~ 0.0 ! 60 i 150 ~ 0.0 I 60 160 ;Sex ; .
(Organ ! Mass of Organ '_a ~ of Tocal Mass j t I I i jLung 0.72 X0.08 0.61 =0.1710.67 Ø0? 0.50 -0.05j0.54 _0.0910.1 =0.:.
I I
(Heart 0.23 Ø01 0.22 Ø02 0.25 _-0.03 0.24 -0.020.23 ~O.OtIO.?3 _'J.J2:
I I I
ISoleen 10.057=0.01 0.051=0.000.079=0.02 0.05 ~0.01~O.Oo =0.01i,0.J4 =O. J1 I i i ~ I, ;
jLiver 12.35 =0.1513.02 Ø08 3.22 -_0.37 2.32 =0.091'.77 _J.2813.12 -O.Jo.
1 ~ 1 !
:Cidney 0.59 =0.030.52 =0.0210.60 ~0.01I0.48 X0.0310.6_ _J.0110.49 -O. J3 ., Adrenal Gland 0.0160.01 0.015_0.01 0.020=0.01 0.013=0.01'0.013=0.02!0.012=0.0 Ovaries I I ~
with Uterus-0.58 _~0.04 0.39 =0.02 0.60 Ø04 - -TestiC
ales ~ - - - X0.30 -0.01 0.32 _0.0310.30 _0.02' ~U$s~s~U~~ ~~"~~~T
,~-.~;:
,a 3 S (.~ e. ~ ':9 ~,.t . 92/ 1 b21 b PCT/EP92/00491 Tpg, 5 CHRONIC TOX%CXTY. EFFECT OF Ok~ALLY ADMTPIISTERED
TTY ON THE H;rMATOLOGICAL FARAMETERS OF RABBITS
Examined parameters Sex Dose Week l TTP of Hemoglobin HematocritLeukoc yT~esrythsocytes E -' g/kg Test (mgt) (~ ) x , I 10- x 0 0 12 X0.05138.2.1.71i 9.33=0.7915.10Ø56 N! . 7 . =0.1540.7=0.331- 9.39=1.1816.34-_0.23 ~ 12.3 6 3.0 0.35141.7=1.30 9.38=1.1217.32=0.70 , i 12 13.6 =0.19'40.7~0.57j,11.23 _1.1015.30Ø46 SO ~ 0 12.9 ~O.d6135.7=1.821 7.62=0.7815.14;0.47 I
6 13.3 -u.35d1.0=:c.10~ 0.00=1. 0.3_".;.
";
12 13.7 =0.11j39.3=0.94 9.36=1.0?I6.19=0.19 i ' 0 X14.20 =0.2?36.7=1.811 8.19=0.1515.34.'J.;6 150 '.
I 3 11 25j 39.5=1.531 3.33=0.'815.6d=0.-10 05 =0 i ~ 6 . . =1.451 3.18=0.0610.34='J.10 (12 =0.53f37.3 .
I ~ 12 . Ø32!39.0 =0.63 8.24Ø0-110.~4=0.54 ~ 113.70 .
0 ~ 0 112.6 =0.=7160.2 :0.831 7.07=1.16i~.".,=0.17 j . ~ 3 ~i2.1 ~0.~3~39.3 -0.721 3. =1.011.,.'~=J.12 i6 j i ; 5 112.4 =0.86142_3 -1.20; 9.79=t.69~...36=0.?4 i ',~ j12.5 =0.87137.7 =2.181 10.?0=2.1614.00wJ.~9 i : 0 113.2=1.20135.0 =1.701 7.44=0.9615.47=~J.50 ; ;
t t I~ 6 113.0=0.55139.3 -1.201 7.25=0.96i7.08=0.?4 .
i 12 112.3=0.21/3'.. =4.331 9.31=0.35io.~'_'=0.32 y p '.'S.' _1.15138.3 =1.401 8.50=0.5416. =0.'J8 15p J9 i 3 '13.1 _1.10147.0 =2_901 3.10=0.76j5.35=~7 0 12 =0.92j41.0=1.15j 6.35-0.3115.61_~
~i I 12 . =0.701-'=1.'J=I.OOj 8.15=l.5di~.92='J.?3 13.1 ~IIBSTITUTE SH~~T
;~ ,:, ,r., d~ " t' 4 i a ,, r'-, i. C i c3 is ~. !.
1~(3 »/1~21G PC:l'/E~2/00~197 'TAB. 6 EFFECT OF TTP ON THE PHAGOCYTIC ACTIVITY OF
NEUTROPHTLS IId THE PERIPHERAL BLOOD OF RABBITS
Phagacytic Activity Neutrophil~s ~
Neutrophiles Group n (1000/1 NBT+ L.H L.W
mm ) U
Control 1 ml 6 1.9 2.3 2. 14.315.117.6154.560.659.81 7.:3'.511 ?3S 7.53 v .0 1 4 y4 ~4 O~t9.4 .4.3c6.8I~0.77 -_0.86=0.4 i 9 1 6 6 -3 . . __ _ . _ .
. . . _ .
TTD I, (Test *O *O *O * * * I
1) 5 mg/kg15 i.2 1.718.426.327.2 69.374.98.751U.39.38 2.0 59.2 _.v. I~0.9 _0.7.7.1.7.1Ø7_7.0~=0.7_-7.0=7.91-i.18-i.?S=1.0;
I
I TTP I ~ I
I ('='esta I xo *o *o *o ~c , _ z y .
15 mg/kgi 2.8 1.6 1.615.522.125.563.069.576.Si 3.5:3.:0 -.i6 ii. I =0.8_0.5'_1.0'_4.9_-7.0=9.11_7.0=7.1=3.91_0.33 -i.04.1.0 v.
.. Number of Test Animals ?9S ohosDhate bu=~er solution 0 3e.ore the administration o~ '~'='?
? 3 days a=ter the administration or TTP
6 6 days aster the administration _or TTP
* Statistically significant variation at ?<0.0S
in relation to the value before administration of ':
o Statistically signif'_cant variation at p<0.05 in comparison to the control group ~iJ~S~IT~ITE ~~I~~T
., ~~a ~.~ i i ~,:1 al ~.~ _~.
.92!16216 F~CT1~~°92JOii~191 '.CAB . 7 EFFECT OF TTP ON 'THE NUMBER OF LYMPFiOC~T~S ANn ON
~ PERCENTAGE OF ~ T-LYMEFiOCX'~5 (E-ROSETTES) pip OF B-LYMPHOCXTES ( EAC-e'2USET'I'ES ) 2N ~'FIE PERIPHERAL BL0017 OF RABBITS .
Lymphc~cyt~s ~
~ E-Rosettes ~~
EAC-Rosettes x I
/mm Group n 6 6 I
Control 1 ml PBS .6 .0 9.0 0.1 42.1 ~ 42.3 i.v. .2.0 .1.9 _.,.7~~.~ !
=6.3 ' =2.051 TTF? (':a5tI o 1 ) ~ 6.6 5.4 19.2 27.1 43.7 4'7.7 S mg/kg 15 -1.5 =1.4 a4.1 =3.2 .2.6 ~ -4.9 , I
TT? (best I
mg/kg 6.0 6.8 17.0 2) ~ 8 _1.0 I I ~
_.v. j =0.5 '_1.9248 I
41.5 I;
41.0 ~
.2 ;
_i.~
~, -2.1 n Number of Test Animals PBS ohosnhate buffer solution 0 3efore 'the administration oz TT?
6 6 says after the administration or TT?
Statistically significant variation at p<0.05 in relat'_on to the value before adminisLrarson cr 'L""' o Statistically significant variation at ?<O.OS
in cornDaraon to the control group T?,a. 3 NUMBER OF SPLF.:IOC'_'TES AF'='ER LONG-TER."'1 ~'vE?.A?v X11"- ~
~~~~°
ConL::ol~ TTP 1 601og~Kg."a:~
10 TT1 ' mg/kg.day n =
.rime v ?FCiI~~ SDlenOCyLeS
O
nmp_ in rla~kss4thDay 7th Day 4th Day Day 4th Day .~..Day 1 7th 3 I 367-_157 =41 1120=328* =240'! =236x'61=57 5 1 572.134 _47 11528_34'6* =62*j1239=280x232=90 7 I .45 133 .40 1 =136* =44 ~ =258'r141=63 9 I 1185 ;75 I .94 .36 ~ -_138167-62 466175 395 132 412 l 12 I =18 1S3 =30 1 .131 =39 ~ .130167=59 * Statistically significant variation at o<U.OS
in comparison to the control group ~t i'R~TiTI 1'T~ ~~~~~ly r:d' ~.~ CJ C7 1~ '.~ v ~Yi' '213~Zt6 PfT/~F92/~191 TAB. 9 CONOENTRATION OF THE ANTI-SFxBG (19S+7S) AhTTIBODIES
AT KONG-TERrS Z'F1X WI'IH TTP
Control TTP a0 TTP pg/kg~ay mg/kg.day n ~ 10 n = 10 Time ~
of Hemagglutinin, -log2 of Titer TTP
Doses in Weeks)4th Day 7th Day I 4th Day 7th 4th Day 7th Day Day I i 3 ~3.7St1.787.12~1.36I8.80t0.97* I8.20t0.40~6.90.1.37 9.2C1.46* 1 5 5.8 2 6.50ti.0017.70*0.84* 7.70v0.64*9.33~2.90T~~
1.40 9.10*2.02*
7 48 1010.83* 13.50=0.8313.>j=1.21 13 11% 4 i 0 40t1 60 74~4 20=0 . .
. .
= .
.
.
i 1 j3.2011.404.66~1.24I3.50=1.28 12.9011.226.33=1.52 9 4.0010.81 12 14.80 0.74 4.40~1.0115.11=1.09 6.60=1.51 15.11=1.19 5.1C=1.74 * Statistically significant variation at o<0.05 in _comparison to the cone=of group TAB. 10 CONCENT:2?TION OF TEE ANTI-SC'2BC (7S) ANTT30DIES
AT LONG-T°R~! THERAPX WITH TTP
Cons=of mg/k T'"P ;ng%kg 1 n = 10 n _- 10 Time ' of Hemagglutinin, log2 - of Titer TTP
Doses in Weeks)4th Pay 7th Day 4th Day 7th Day 4th Day 7th Day I I '~
.
3 1.75=0.606.37=0.8513.200.87* 5.9U11.703.80;0.87*4.90=0.741 I
5 12.50=0.706.12~1.76I2.20=0.97 5.70*_1.1012.30=1.415.33-0.74x1 7 (1.00*0.893.801.0910.60=0.80 2.50=1.11'"~0..55~0.832.55=0.68!
9 10.40=0.483.66=1.2410.3010.64 3.25=0.9610.00 4.331.24 12 0.80Ø973.80=0.7611.11=0.994.401.3511.55-1.494.30.1.25 * Statistically significant variation at p10.05 in comparison to the control group SUBSTITUTE ~HE~T
/aL~i.iBJ~~~,~.y.
VdCD 92/16216 P~"d'/EP92J00491 TAH . 1 1 zMP~ITJIdOM01?ULATORY EFFECT DF TTP ( ~SD }
Dase n PFC/10 ~ E-RosettesHemagglutininxHemagglutininxl TTP n Splenocytes Type 19S Type 7S
mg/kg I 4th Day 4th Day + 7S 7th Day .
4th Day Contzol30 469 13.6 5-4 82 _111 j d3.2 X1.1 -_1.3 x x 0.5 10 997 15.6 - 6.8 8.4 .139 .1.4 X1.0 ~1.1 j x x x . x i 2 10 839 20.3 6.9 I 9.7 . .177 .4. 1 =1.0 -1.4 i 5.0 10 746 23.2 7.2 9.8 .129 .4.7 I =O.o ;1.4 i x '~ I
x I
I 18.4 ' 8.3 ! 10.-:
10.0 j ~ 795 .0 .1.1 l .729 ~ .4.0 ~ -_1.3 I
j ! . ~ ~ :<
' . 3 '_5.0 ~ t 560 I 16.3 ~ 6 1 ;
, i ~ ~ =145 ~ _?.5 ., i -__1.' , ' ' 50.3'; j 100 14.' i 5.3 I 5.~
I I 57 j _-4.0 _-1.0 =1.9 ! x x ; x ! 100.010 375 11.3 ! 5.1 . .7 :
~ _ I
. 67 .2.'_ ; -0.3 .o , _.
"
x :L ~, .' 250.0 I 30S 9.5 j 4g ''3 -?.Q I -_1.i '1.7 _ 67 -x Statistically -significant variation at total comoar'_san O.OS
(Student t-Test) -log2 of titer _ . . .., ,-,-... a T, e-r 1"' C' L.1 C= ~T"
s 'i .
3_~ I_i r~
w~ ~~m6z~s ~~.-ri~~zioo~9~
TAg. 12 TTP AI7MTNTS'PER~I7 DAILY IN 3 DOS;rS
9th After 10th Day After Day Immunization Immunization with SRBC with SRBC
I
' Hemagglutinin Hemagglu tinin rP n 6 of titerI -log2 titer Dose T l of ~ E- PFC/10og2 ~ -RosettesSoleno- -7S~
oytes 19S
+ 7SI
'Control 15 13.7 469 5.7 0.3 ~ 9.8 3.4 I i ', l .3 .125 .1.3 .1.0 I .1.3 .?.5 1 0 ~
m .
x .
Control 15 I 15.3 579 6.1 0.5 ~ 9.0 3-5 I I ~ ~ ' ' 1 ml 0 .143 -_1.1 ~_0.9 ; -1.3 0 I =4 I ~ -_?.7 . .
x i 10.
..-~, mg% 1 ~ 1 ~ 364*7 . ~ I s~' . .
kg 7 9 . 4' .
4* i I
I1 x 0.3 .3.3 I .190.1.1 ~.1.? , _1.0 .'...
ml I I
~ 15 19.0'' 769* 7.1* 0.5 ' 9.5 ='.0 /k I ~
m ;' g ~
g _.
j ~ , 3 x 0.1 ~ ; _5.0 I .132=0.3 X0.3 i -1.0 __.0 ml , ~
~10 mg/kg ~ I i9.3*I 795*7.0* 0.3 ~, iU.4* ~.~*
13 I ~ !
3 ml I ~ .4 I -_102I Ø7 .i.0 I1 ? I -_1 .5 _' 0 .0 . .
x I10 mg/kg 15 17.5 ~ 650 7.3'~ 1.3 j 8.9 3 3 ~
~ I f 1 ml 7 I .1291.4 .1.. I .1.3 0 ~ .3 ~
3 ~
. ~ . _ x , SUSST1TUTE SE-l~~T
F:; i i ; 9j ;> ~..E .~.
9'YO 92/1~SZ16 PC~1'/EP92/00491 TAB. 13 EFFECT OF TTP ADMINISTERED ORALLY FOR 12 WEEKS ON THE
IMMUNOhOGICAL RESPONSE OF i'dICE IMMC3NIZED WITR SRBC
4th After 10th Day Day After Hemagglutinin Hemagglutinin I
Weeks E- Spleno--log2 % E- Spleno-~-log2 titer) of I of titer Rosettescytes Rosettestyres~-- , 19S 7S ~ I'19S
+ 7S1 I - 7Si 15.2 76~ 6.0 1.6 14.7 256 ~ 8.7 ~
7.9 ' 3 -3.8 -~6 _0.9 .1.3=3.4 =57 j .1.0 ~=1.0 j 7* 1033* 7.5* 2.9*23.3* 406* ~ i0.4*I
23 9.7*
. =177 -0.3 =0.2=3.7 -88 -_1,2 I=1.0 7.8 I I
' 13 422 5.1 0 . 12.7 209 7.1 ~
* Statistically significant variation at ?<0.0S
in relation to the value before administration of ':
o Statistically signif'_cant variation at p<0.05 in comparison to the control group ~iJ~S~IT~ITE ~~I~~T
., ~~a ~.~ i i ~,:1 al ~.~ _~.
.92!16216 F~CT1~~°92JOii~191 '.CAB . 7 EFFECT OF TTP ON 'THE NUMBER OF LYMPFiOC~T~S ANn ON
~ PERCENTAGE OF ~ T-LYMEFiOCX'~5 (E-ROSETTES) pip OF B-LYMPHOCXTES ( EAC-e'2USET'I'ES ) 2N ~'FIE PERIPHERAL BL0017 OF RABBITS .
Lymphc~cyt~s ~
~ E-Rosettes ~~
EAC-Rosettes x I
/mm Group n 6 6 I
Control 1 ml PBS .6 .0 9.0 0.1 42.1 ~ 42.3 i.v. .2.0 .1.9 _.,.7~~.~ !
=6.3 ' =2.051 TTF? (':a5tI o 1 ) ~ 6.6 5.4 19.2 27.1 43.7 4'7.7 S mg/kg 15 -1.5 =1.4 a4.1 =3.2 .2.6 ~ -4.9 , I
TT? (best I
mg/kg 6.0 6.8 17.0 2) ~ 8 _1.0 I I ~
_.v. j =0.5 '_1.9248 I
41.5 I;
41.0 ~
.2 ;
_i.~
~, -2.1 n Number of Test Animals PBS ohosnhate buffer solution 0 3efore 'the administration oz TT?
6 6 says after the administration or TT?
Statistically significant variation at p<0.05 in relat'_on to the value before adminisLrarson cr 'L""' o Statistically significant variation at ?<O.OS
in cornDaraon to the control group T?,a. 3 NUMBER OF SPLF.:IOC'_'TES AF'='ER LONG-TER."'1 ~'vE?.A?v X11"- ~
~~~~°
ConL::ol~ TTP 1 601og~Kg."a:~
10 TT1 ' mg/kg.day n =
.rime v ?FCiI~~ SDlenOCyLeS
O
nmp_ in rla~kss4thDay 7th Day 4th Day Day 4th Day .~..Day 1 7th 3 I 367-_157 =41 1120=328* =240'! =236x'61=57 5 1 572.134 _47 11528_34'6* =62*j1239=280x232=90 7 I .45 133 .40 1 =136* =44 ~ =258'r141=63 9 I 1185 ;75 I .94 .36 ~ -_138167-62 466175 395 132 412 l 12 I =18 1S3 =30 1 .131 =39 ~ .130167=59 * Statistically significant variation at o<U.OS
in comparison to the control group ~t i'R~TiTI 1'T~ ~~~~~ly r:d' ~.~ CJ C7 1~ '.~ v ~Yi' '213~Zt6 PfT/~F92/~191 TAB. 9 CONOENTRATION OF THE ANTI-SFxBG (19S+7S) AhTTIBODIES
AT KONG-TERrS Z'F1X WI'IH TTP
Control TTP a0 TTP pg/kg~ay mg/kg.day n ~ 10 n = 10 Time ~
of Hemagglutinin, -log2 of Titer TTP
Doses in Weeks)4th Day 7th Day I 4th Day 7th 4th Day 7th Day Day I i 3 ~3.7St1.787.12~1.36I8.80t0.97* I8.20t0.40~6.90.1.37 9.2C1.46* 1 5 5.8 2 6.50ti.0017.70*0.84* 7.70v0.64*9.33~2.90T~~
1.40 9.10*2.02*
7 48 1010.83* 13.50=0.8313.>j=1.21 13 11% 4 i 0 40t1 60 74~4 20=0 . .
. .
= .
.
.
i 1 j3.2011.404.66~1.24I3.50=1.28 12.9011.226.33=1.52 9 4.0010.81 12 14.80 0.74 4.40~1.0115.11=1.09 6.60=1.51 15.11=1.19 5.1C=1.74 * Statistically significant variation at o<0.05 in _comparison to the cone=of group TAB. 10 CONCENT:2?TION OF TEE ANTI-SC'2BC (7S) ANTT30DIES
AT LONG-T°R~! THERAPX WITH TTP
Cons=of mg/k T'"P ;ng%kg 1 n = 10 n _- 10 Time ' of Hemagglutinin, log2 - of Titer TTP
Doses in Weeks)4th Pay 7th Day 4th Day 7th Day 4th Day 7th Day I I '~
.
3 1.75=0.606.37=0.8513.200.87* 5.9U11.703.80;0.87*4.90=0.741 I
5 12.50=0.706.12~1.76I2.20=0.97 5.70*_1.1012.30=1.415.33-0.74x1 7 (1.00*0.893.801.0910.60=0.80 2.50=1.11'"~0..55~0.832.55=0.68!
9 10.40=0.483.66=1.2410.3010.64 3.25=0.9610.00 4.331.24 12 0.80Ø973.80=0.7611.11=0.994.401.3511.55-1.494.30.1.25 * Statistically significant variation at p10.05 in comparison to the control group SUBSTITUTE ~HE~T
/aL~i.iBJ~~~,~.y.
VdCD 92/16216 P~"d'/EP92J00491 TAH . 1 1 zMP~ITJIdOM01?ULATORY EFFECT DF TTP ( ~SD }
Dase n PFC/10 ~ E-RosettesHemagglutininxHemagglutininxl TTP n Splenocytes Type 19S Type 7S
mg/kg I 4th Day 4th Day + 7S 7th Day .
4th Day Contzol30 469 13.6 5-4 82 _111 j d3.2 X1.1 -_1.3 x x 0.5 10 997 15.6 - 6.8 8.4 .139 .1.4 X1.0 ~1.1 j x x x . x i 2 10 839 20.3 6.9 I 9.7 . .177 .4. 1 =1.0 -1.4 i 5.0 10 746 23.2 7.2 9.8 .129 .4.7 I =O.o ;1.4 i x '~ I
x I
I 18.4 ' 8.3 ! 10.-:
10.0 j ~ 795 .0 .1.1 l .729 ~ .4.0 ~ -_1.3 I
j ! . ~ ~ :<
' . 3 '_5.0 ~ t 560 I 16.3 ~ 6 1 ;
, i ~ ~ =145 ~ _?.5 ., i -__1.' , ' ' 50.3'; j 100 14.' i 5.3 I 5.~
I I 57 j _-4.0 _-1.0 =1.9 ! x x ; x ! 100.010 375 11.3 ! 5.1 . .7 :
~ _ I
. 67 .2.'_ ; -0.3 .o , _.
"
x :L ~, .' 250.0 I 30S 9.5 j 4g ''3 -?.Q I -_1.i '1.7 _ 67 -x Statistically -significant variation at total comoar'_san O.OS
(Student t-Test) -log2 of titer _ . . .., ,-,-... a T, e-r 1"' C' L.1 C= ~T"
s 'i .
3_~ I_i r~
w~ ~~m6z~s ~~.-ri~~zioo~9~
TAg. 12 TTP AI7MTNTS'PER~I7 DAILY IN 3 DOS;rS
9th After 10th Day After Day Immunization Immunization with SRBC with SRBC
I
' Hemagglutinin Hemagglu tinin rP n 6 of titerI -log2 titer Dose T l of ~ E- PFC/10og2 ~ -RosettesSoleno- -7S~
oytes 19S
+ 7SI
'Control 15 13.7 469 5.7 0.3 ~ 9.8 3.4 I i ', l .3 .125 .1.3 .1.0 I .1.3 .?.5 1 0 ~
m .
x .
Control 15 I 15.3 579 6.1 0.5 ~ 9.0 3-5 I I ~ ~ ' ' 1 ml 0 .143 -_1.1 ~_0.9 ; -1.3 0 I =4 I ~ -_?.7 . .
x i 10.
..-~, mg% 1 ~ 1 ~ 364*7 . ~ I s~' . .
kg 7 9 . 4' .
4* i I
I1 x 0.3 .3.3 I .190.1.1 ~.1.? , _1.0 .'...
ml I I
~ 15 19.0'' 769* 7.1* 0.5 ' 9.5 ='.0 /k I ~
m ;' g ~
g _.
j ~ , 3 x 0.1 ~ ; _5.0 I .132=0.3 X0.3 i -1.0 __.0 ml , ~
~10 mg/kg ~ I i9.3*I 795*7.0* 0.3 ~, iU.4* ~.~*
13 I ~ !
3 ml I ~ .4 I -_102I Ø7 .i.0 I1 ? I -_1 .5 _' 0 .0 . .
x I10 mg/kg 15 17.5 ~ 650 7.3'~ 1.3 j 8.9 3 3 ~
~ I f 1 ml 7 I .1291.4 .1.. I .1.3 0 ~ .3 ~
3 ~
. ~ . _ x , SUSST1TUTE SE-l~~T
F:; i i ; 9j ;> ~..E .~.
9'YO 92/1~SZ16 PC~1'/EP92/00491 TAB. 13 EFFECT OF TTP ADMINISTERED ORALLY FOR 12 WEEKS ON THE
IMMUNOhOGICAL RESPONSE OF i'dICE IMMC3NIZED WITR SRBC
4th After 10th Day Day After Hemagglutinin Hemagglutinin I
Weeks E- Spleno--log2 % E- Spleno-~-log2 titer) of I of titer Rosettescytes Rosettestyres~-- , 19S 7S ~ I'19S
+ 7S1 I - 7Si 15.2 76~ 6.0 1.6 14.7 256 ~ 8.7 ~
7.9 ' 3 -3.8 -~6 _0.9 .1.3=3.4 =57 j .1.0 ~=1.0 j 7* 1033* 7.5* 2.9*23.3* 406* ~ i0.4*I
23 9.7*
. =177 -0.3 =0.2=3.7 -88 -_1,2 I=1.0 7.8 I I
' 13 422 5.1 0 . 12.7 209 7.1 ~
8 ~ ~ ~ 6.7 . .53 .0 0 j -_2.i =41 ' _1.4 i_1.6 4 ~ 9 I ;
.2 . 933* . ~ I 20.* 430* I )).Ox*
23.0* 7.6* 2.7X: ~,'1.0 I, =5.1 X248 =1.5 ~,=i.3. ~2.7 -35 _ . .1.0 I ~
! 12.3 495 4.6 ~ . 14.0 245 . 3.9 i ~ 0.3 I
I -2.9 .85 .1.1 I=0.9' .5.1 .48 j -_1.6,=1.5 7 I j ' ;
20.3x 1120" 7.n* ''.lxj 26.9"', i, . 'J.3"
~ .?;
I I '_214 =0.6 '=1.5; _7.7 ', ' _ =0.-1 ~ X5.3 ~ -117 .0 I
' ; 15.0 . 531 I 6.2 ; ! 14.1 i 3.', ~
0.3 294 3.4 .
.2 . 933* . ~ I 20.* 430* I )).Ox*
23.0* 7.6* 2.7X: ~,'1.0 I, =5.1 X248 =1.5 ~,=i.3. ~2.7 -35 _ . .1.0 I ~
! 12.3 495 4.6 ~ . 14.0 245 . 3.9 i ~ 0.3 I
I -2.9 .85 .1.1 I=0.9' .5.1 .48 j -_1.6,=1.5 7 I j ' ;
20.3x 1120" 7.n* ''.lxj 26.9"', i, . 'J.3"
~ .?;
I I '_214 =0.6 '=1.5; _7.7 ', ' _ =0.-1 ~ X5.3 ~ -117 .0 I
' ; 15.0 . 531 I 6.2 ; ! 14.1 i 3.', ~
0.3 294 3.4 .
9 I -_2.1I -_67~ _0.8 ~~0.?I .2.3 I -1.3 ~=i.5 =53 I i8.6~'j 1049xI ,.7x I ~ 18.?x ; , .',.9xiiO.dW
1.9 id's ' .2.9 I .184~ Ø7 ~ ! -3.-1 I ; Ø3 .
-_1 -630 .1 .? .'J
I 15 ' 373 7.2 j ' 16.3 ! ~ 7.3 ' 0 1.s 256 12 . X143 I _-1.0I=1.2~ 2.5 ~ j -_1.1;_0.9 .2.6 -5c3 14.1 ~ 770*I '.2 I ! 19.0 ~ .1.3" !i0.6'~~
1.- 294 -_2,7 -_132 ~ =1.4 ',-_1.".j =4.i _63 ' _~J.=!.1.0 i JtatiStl.CallV si.gni~'_canc variation at p---0.05 in comparison ~o the control g:oup. ~n eac:~ groua, t.~.e.e we_e ~30 an~.mais.
r.~ tr,r~~rnrt tT~ '~NF=~T
G'J . h 's~ b.i° i,a 7 ~t ~~ L.'I e,~ t1 ,N ~~
W~ 'i~6216 PCT/E~2J0~0491 TAB..14 INE°LUENCE OF THE STORAGE CONDITIONS ON THE ACTTVITY OF
TTP IN VIEW OF THE ABILITY OF FORMING E-ROSETTES
$ E-Rosettes after two months storage Dose n Starting ;
Activity Temperature At Room ~ At Room * 4°C Temperature Temperature!
Light Admittedlin the Dar~i Control 8 I 13.0 .?.4 !~.o ~~.3 m .o -~.a I
0.1 mg/kg 8 I 15.8 .1.7 16.1 _~3.04 15.1 X2.9 j 16,0 =S.5 I
1 mg/kg 8 ~ 18.0 -0.8~' ~ 19.8 =4.1* 15.3 =2.8 ~ 19.8 =2.
_ ._ mg/kg $ ~ 20.4 =4.2* i8.0 =4.5x 16.8 -2.4* 10.9 -~
* Statist'_cally significant variation at a = 0.5 in compa;_son ~:~ t.._ control group TAB. 1.INFLUENCE OF T::E STORAG'c CJNDITT_ONS ON Tr-T.E AC'='=V='_"'_' O=
TTP IN VIEW OF THE NUMBER OF CvI.LS ?RODUC=NG
?~'C/106 SolenocyLes I i after two months storage Dose ~ ~ 5tar~ing I Activity I Temperature I At Room at 300:
-4°C ~ Temperature "='e:npe=s-~=°-:
I
I i I , Light Admittedl in t.._ ~ar::.
I Control a ~ 571 X59 ~ 514 -_128 ~ 514 -_128 5i4 =i_3 0.1 mg/kg 8 i 747 =144 ~ 1039 -325* i '718 -_135 '11002 =?'0't 1 mg/kg 8 1204 =155* 1026 =314* 793 _~186F 1046 =331 10 mg/kg I 8 1075 ~232'* 1070 _249* j, 869 ~160x ~~', 848 =15 * Statistically signi'icanc variation at c = 0.5 in comparisc.~. _~ _.._ control group ~UB~TiTU~T~ ~H~~ i L~ ~~ ~ :f v3 ~.
.1V~, ~Z/Ib~lb P~T/EP92/(~91 TAr3. 16 INFLUENCE OF THE STORAGE CONDITIONS ON THE ACTIVITY
OF TT° IN VIEW OF INFLUENCING THE FORMATION
OF ANTIBODIES OF THE TYPE ANfi I-SRBC 19Sø7S
-after two months storage ' "'' n S ring tar Activity TemperatureAt Room At Boom Dose +4 C ~ Temperature Temperature LightAdmit~edin Dark the Day 4th 10th 4th 10th4th 10th ~ 4th 10th I
Control 8 5.6 9.8 S.0 9.5 5.0 9.5 l 5.0 9.0 ~
=0.9i0.6 1.1 .2.1~1.1 .2.1 I=?.1 .2.1 0.1 mg/kg 8 7.4 11.4 7.8 10.5~.4 10.8 j 6.5 12.6 =0.4*_1.3*~_1.0*s1.7*=0.8 =0.7* j=0.7'~_2.2*i l 1 mg/kg ~ 6.8 12.2 I 6.7 ?3.85.? 13.2 I o.3 8 ii .3..
;5.3*=1.1*Is0.4*_2.1'~=~J.3* i=i.3*=t.3m =0.4*
mg/kg 8 5.4 11.0 5.2 12.04.8 10.5 ( 5.0 13.5 I
-1.2=1.4*-0.6 y2.3*l e2.a~ i~O.S -1.3m _1.3 R StatiStical'_y signi==cant ;variation at a = 0.5 i:~ comparison to the control group TAB. . 7 INFLUENCE OF THE STORAGE CONDITIONS ON '='HE
~1C':'T_'~T_'~°v OF '="='? IN VT_EW OF INFLUENCT_:1G THE F ORMATION
OF ANTI90DIES OF THE TVp~ ANTI-SRBC 7S
l l aster ~:~o months storage Dose ~ n Starting I
act_vity Temperature At Room at boom ;
-4C I Tert~oe=azure '~emperatur I l Light- 7arr.
l admittedlin ~
Dayi4th 1 4th 1 4th 1 Ot'.~.4tht Ot'.~.
Oth Oth ~
I ~
Control 8 0.4 9.2 0.3 8.1 0.3 8.1 0.38.?
t ~
0.8 ~ Ø7 .1.4~ .1.4 =0.'=1..1 Ø7 Ø7 l 0.1 mg/kg 8 0 10.8 ~ 0.7 9.3 1 10.0 O.i.1.3 0.8 ~i I
~1.U*=0.9 =0.9*=0.9 -O.o~' -_0.-1.4~'~
~
l 1 m /k 8 0.8 10.6 0.3 11.50.7 1U.3 0.711.0 9 g '-0.9=0.4*."Ø7=1.8~=0.9 =0.7~ i=0.9-1.0*i I
10 mg/kg 8 0 10.2 0.3 9.8 0.3 10.- i 10.3 I 0."
t1.3*=0.7 =1Ø0.7 =2.0~ ~=0.9=0.9~i * Statistically significant variation at a = 0.5 in compariso:i to the control group ~i IF~ST1TUT~ S~E~T
s;:
(.f ~i e7 t~ i.~
WO 1 b2 d 6 PCI'/~P9Z/0049 d TAB. 18 HEMATOLOGICAL PA.RAMIr"TERS FOR HEALTHY VOLUNTEERS BEFORE ANIJ
AFTER TWO~WEEFC ADMYNTSTRATIONS OF TTP AT A I70SE OF 1 MG/DAY
Placebon=6 TTP n=5 Parameter 0 14 0 14 days days Eryt:~rocytes I I 18 32;
x 10 4.44 .Ø464.32 ,0.454.32 -_0.32. .
/u1 .
Hemoglobin I
g/dl ~ 12.4 t2.5 11.9 ~.8 i2.8 Ø8 11.8=0.6 Hematoc~it I
38.1 .5.7 38 -6.9 38.7 _2.1 38.1=1.6 Leuk~cy~ces ' x 10 /u1 5.25 X0.59 6.27 =0.8 6.23 -_0.54.86-0.6 i Neut=oohils ~ ~
-i ~12 57.6 =11 ~ 64.2-2.6 66 -3.6 62.6 Lymphocytes 26 X7.5 30.4 -8.3 34 _4.1 32.4~
.3 '='hro~booytes i ~ ' x 10~/~.i.l ~ 220 =40.4 214 .35.8 I 221 -_38 214 _39.o j 3lood Sedime.~.c' mm/1h ~ 7.6 ~9 6.6 .1.9 ~i ,.3 -2.~ 6.9 .3.:
StISSTIT~JT~ SH~~T
4~' l l1 ~TC ch ~ i ~,! 'T
~~s 9,.y tJ C~ '~ ~,. .P
a ~z~mzi6 ~cr/~~>ziooa9~
TAB. 19 BIOCI3EMICAL PARAMETERS FOR HEALTHY VOZ.U~ITEERS BEFORE AND
AFTER TWO-Wi~EIC ADMIPdISTRATIONS OF TTP AT A DOSE OF 1 MG/DAY
Placebon=S p -Parameter 0 14 ays 0 14 ays Total 72.8 X1.76 68.42.3.2 73.8 _-1.9270 ;a.3 Protein 163.2X2.5 63.8~2.3 '63.5=1.8 54.4.2..
g/1 Albumina Globulinat I r0.4 3 =0.5 I =0.5 2.3 _0.-'.'.3~
~ 3.1 2.8 Globulina2 I X1.9 6.8 ~1.5 ~ =0.9 06.9Ø?
~ 5.95 0.93 Globulinp ~ 14..1-2.2 14.2_1.5 13.3 -1.9 '3.2_1.3 - -GlobulinY ~ 112.2.i.5 12.1=i.3 113.7-_0.9.2.3.i _ --IgG g/1 110.3.1.1 10.2~1 11', _'.o '1.'=i.-- r ---TgA g/1 i -_0.6 1.g _~.j I .j ..9 =0.3 t.8 gM g/1 ~ Ø3 1.4 _0.2 I -__0.21.; ='J.-1.4 i.1 .
i I Complementg/= I Ø1 1.1 -_0.1 ! -_0.2 0.. Ø.
C3 1.1 ,..
Complement g/1 j =0.5 9.3 =0.041 Ø0 0.? _O.
C4 0.2 0.2 J3;
i -Aianine 134.2 _0.05 0.3 X0.04130.?=9.'_ 3i.=
U/'_ =3.?
Asoaragine U/' ~32.d =4.4 40.2 =5.5 ~30.-~ .'... 39.x =5 ~U~STiTUT~. S~~~T
KM::Ir'>~h~$rr_~.
F:a ~.y ~~.~ a~ E ~ wa W( J16216 PCT/11'92141~91 TAB. 20 COMPARISON OF THE FREQUENCY OF ACUTE >:NFECTIONS OF THE
RESPIRATORY TRACTS DURING THE LAST QUARTERS OF THE YEARS
1989 AND 1990 IN RESPECT OF TWO GROUPS OF PATTENT5 ' TRF.ATEn WITH TTP AND PLACEBO RESP. DURING OCTOBER 1990 , TTP
applied during the 4th quarter i n 4th quarter4th quarterstatistical 1989 1990 signieicance (no TTP) (with ~~p"
TTP) average average number number o o infectionsinfections per patientper patient Cold 20~4.0 -__1.31.0 -4.5 < 0.01 ~
Sore 203.3 .1.3 0.8 -0.G < 0-01 throat Fever blisters 202.1 .2.2 0.3 -_0.4~ . 0.01 Caugh 201.4 _1.1 U.3 ~O.o j < 0.01 Bronchitis200.3 =0.5 0 ' ~i j PneumoniaI20~ 0.2 Ø90 Placebo applied during the 4th quarks= 1990 nl 4th quartez~ 4th quarter ~ scaL_s~ac~a~
1989 ~ 1990 evaluation o.-'.1 (no '='~'?) (with TTP) difference"c"~
average average number o~ number o~
infections infections per patient per patient . _ Cold 20~ 3.9 =1.4 3.1 ~1.> 0.05 Sore 20 2.3 .2.0 2.0 .1.7 > 0.05 throat Fever blisters 20 2.3 ._1.8 1.7 .2.1 > 0.05 Cough 20 1.5 -1.5 1.9 =2.0 > 0.05 Bronchitis 20 0.1 Ø3 0.1 Ø3 Pneumonia 20 0.1 Ø3 0.1 X0.3 ~~J~~T(TUT~ S~E~ T
y ~ z t~ 3_~ U ~.~ i~ ;:G _~.
WO . 4J 1 b216 ~ PCd'!EF'92JOU49 ~
TAB. 21 EFFECT OF INTRADERMAL INJECTION OF BACTERIAL ANTIGENS OR
PHA (PHYTOHEMAGGLUTININ) IN PATIENTS TREATED WITH TTP
Tuber- Strepto- Staphylo- PHA NaCl Patientculin lycin cocci/
Ser.(Init- RT~3 0 anatoxin No. ials) pre 3 3 pre 3 pre3 pre 3 (pre , I I I
I
1 I B.T. + + - ' ~ ' . ! , = I
_ 2 P.E. + _ ,. _ I _ _ _ _ - I
I + t + _ - -3.1 + + + ~
M.H. ~+ -I - I
-I
' 4.I - + ' ' W.H. - I
I - --., .. ~
! ~ ..
'~. - -. ~
-.
i , I
_ .
I
_ I
.
.
I
.
I
o.':
Z.N.
I--I=
+
I
_ -~
, I
I
I
I
t r 7.; I r I
P.J. - .~ I
I
i --I
r -_ _ I
-I
9.' S.M. ~- - !- ~Y ~~ i - j - i _ !
pre pretreatment 3 after 3 weeks TAB, z? EFFEC'~' OF INTRADERMAL IN~"EC';'ION OP' 9AC'~'ERIAL ANT;GENS
OR PHA IN UNTREATED (PLACEBO) °ATIENTS
Tube.- Strepco- PHA I
I Staphylo- NaC=
!
Patientculin lycin ~
l cocci/ I
Ser.I(Tnit-RTZ~ 0 anacoxin ;
No. ials) ' e re I I
I ~ I pre pz'-3 3 I .
~ ' pre ' pr p 1 M.M. I ~ ~
. - + -I _ T j -~' -, I ' , . r _ _ I i z. o.. ~ _ i I I
._ _ , , ! ~ + -3. O.H. I+ ~...+~I + _ j + i -i, -I
4. W.J. I _ _ - - -I - ~ - i ~ --K.C. ~ + + - p ~, _ _ - --I -b.~I M.J. + ++ ~+~. ~+ ~_ - I
I- -- i I
7. H.W. - + + - _ _ - + r -8. K.T. I
( _ _ + + + _ _ -I , ~ I ~
+ -i pre pretreatment 3 after 3 weeks ~IISSTI"I~IJT~ ~#~FFT
1 ~:' s.
~r .~ G.t e.'? 1 ~ ~..~ .,.
ii~C) 5 6216 PC.'T/~~92/0~~91 TAB. 23 xMMUNOGLOHULIIVS IN THE SEFtt3M OF PATIENTS WTTa:
UI~CUS CRURZS TtZEATEI? WSTH TTP
n Test Group Immunoglobulin in I ( mg before after Treatment Treatment I
1 I 1732.872207.327 ZgG 1732.87.207.52 Ig 1 1 G
8 A 2 IgA 304.25 _ 47.508 IgA 283.87 . 44.10 ~ I I I I
' I 3 ' 202.87 ~ 71.129 IgM ~ 189.75_- 75.15 IgM ' I
~
~ ~4 ~ 1 1924.371246.2510 IgG I 1972.00-_239.33 IgG ~ 1 8 g I I I 236.50_ 59.841 I j 285.50-_ 56.76 a IgA 11 IgA
6 ~ I 242.12. 56.95~ I I 532.7._ 65.;5 IgM i2 IgM
1 1 7 D O.OS ~ 4 i0 ? 0'05 StatlStlCS1 ~ VS > O.O~ 'IS >
Evaluac;on I 2 a D 0.05 I J 11 0.05 oz VS > VS 12 ~
3 9 p 5 ~
vs > vs I , ~ 7 i0 ~ O.JS
Di=?ezence 1 ; 4 ~ 0.05 vs >
Vs > 0.05 I ' o.os j 2 ; ~ 0.05- 1 a i1 ~ 0.05 VS > VS >
3 o a 9 12 ~
vs > ws >
SUSST1TUT~ S~~~T
1.9 id's ' .2.9 I .184~ Ø7 ~ ! -3.-1 I ; Ø3 .
-_1 -630 .1 .? .'J
I 15 ' 373 7.2 j ' 16.3 ! ~ 7.3 ' 0 1.s 256 12 . X143 I _-1.0I=1.2~ 2.5 ~ j -_1.1;_0.9 .2.6 -5c3 14.1 ~ 770*I '.2 I ! 19.0 ~ .1.3" !i0.6'~~
1.- 294 -_2,7 -_132 ~ =1.4 ',-_1.".j =4.i _63 ' _~J.=!.1.0 i JtatiStl.CallV si.gni~'_canc variation at p---0.05 in comparison ~o the control g:oup. ~n eac:~ groua, t.~.e.e we_e ~30 an~.mais.
r.~ tr,r~~rnrt tT~ '~NF=~T
G'J . h 's~ b.i° i,a 7 ~t ~~ L.'I e,~ t1 ,N ~~
W~ 'i~6216 PCT/E~2J0~0491 TAB..14 INE°LUENCE OF THE STORAGE CONDITIONS ON THE ACTTVITY OF
TTP IN VIEW OF THE ABILITY OF FORMING E-ROSETTES
$ E-Rosettes after two months storage Dose n Starting ;
Activity Temperature At Room ~ At Room * 4°C Temperature Temperature!
Light Admittedlin the Dar~i Control 8 I 13.0 .?.4 !~.o ~~.3 m .o -~.a I
0.1 mg/kg 8 I 15.8 .1.7 16.1 _~3.04 15.1 X2.9 j 16,0 =S.5 I
1 mg/kg 8 ~ 18.0 -0.8~' ~ 19.8 =4.1* 15.3 =2.8 ~ 19.8 =2.
_ ._ mg/kg $ ~ 20.4 =4.2* i8.0 =4.5x 16.8 -2.4* 10.9 -~
* Statist'_cally significant variation at a = 0.5 in compa;_son ~:~ t.._ control group TAB. 1.INFLUENCE OF T::E STORAG'c CJNDITT_ONS ON Tr-T.E AC'='=V='_"'_' O=
TTP IN VIEW OF THE NUMBER OF CvI.LS ?RODUC=NG
?~'C/106 SolenocyLes I i after two months storage Dose ~ ~ 5tar~ing I Activity I Temperature I At Room at 300:
-4°C ~ Temperature "='e:npe=s-~=°-:
I
I i I , Light Admittedl in t.._ ~ar::.
I Control a ~ 571 X59 ~ 514 -_128 ~ 514 -_128 5i4 =i_3 0.1 mg/kg 8 i 747 =144 ~ 1039 -325* i '718 -_135 '11002 =?'0't 1 mg/kg 8 1204 =155* 1026 =314* 793 _~186F 1046 =331 10 mg/kg I 8 1075 ~232'* 1070 _249* j, 869 ~160x ~~', 848 =15 * Statistically signi'icanc variation at c = 0.5 in comparisc.~. _~ _.._ control group ~UB~TiTU~T~ ~H~~ i L~ ~~ ~ :f v3 ~.
.1V~, ~Z/Ib~lb P~T/EP92/(~91 TAr3. 16 INFLUENCE OF THE STORAGE CONDITIONS ON THE ACTIVITY
OF TT° IN VIEW OF INFLUENCING THE FORMATION
OF ANTIBODIES OF THE TYPE ANfi I-SRBC 19Sø7S
-after two months storage ' "'' n S ring tar Activity TemperatureAt Room At Boom Dose +4 C ~ Temperature Temperature LightAdmit~edin Dark the Day 4th 10th 4th 10th4th 10th ~ 4th 10th I
Control 8 5.6 9.8 S.0 9.5 5.0 9.5 l 5.0 9.0 ~
=0.9i0.6 1.1 .2.1~1.1 .2.1 I=?.1 .2.1 0.1 mg/kg 8 7.4 11.4 7.8 10.5~.4 10.8 j 6.5 12.6 =0.4*_1.3*~_1.0*s1.7*=0.8 =0.7* j=0.7'~_2.2*i l 1 mg/kg ~ 6.8 12.2 I 6.7 ?3.85.? 13.2 I o.3 8 ii .3..
;5.3*=1.1*Is0.4*_2.1'~=~J.3* i=i.3*=t.3m =0.4*
mg/kg 8 5.4 11.0 5.2 12.04.8 10.5 ( 5.0 13.5 I
-1.2=1.4*-0.6 y2.3*l e2.a~ i~O.S -1.3m _1.3 R StatiStical'_y signi==cant ;variation at a = 0.5 i:~ comparison to the control group TAB. . 7 INFLUENCE OF THE STORAGE CONDITIONS ON '='HE
~1C':'T_'~T_'~°v OF '="='? IN VT_EW OF INFLUENCT_:1G THE F ORMATION
OF ANTI90DIES OF THE TVp~ ANTI-SRBC 7S
l l aster ~:~o months storage Dose ~ n Starting I
act_vity Temperature At Room at boom ;
-4C I Tert~oe=azure '~emperatur I l Light- 7arr.
l admittedlin ~
Dayi4th 1 4th 1 4th 1 Ot'.~.4tht Ot'.~.
Oth Oth ~
I ~
Control 8 0.4 9.2 0.3 8.1 0.3 8.1 0.38.?
t ~
0.8 ~ Ø7 .1.4~ .1.4 =0.'=1..1 Ø7 Ø7 l 0.1 mg/kg 8 0 10.8 ~ 0.7 9.3 1 10.0 O.i.1.3 0.8 ~i I
~1.U*=0.9 =0.9*=0.9 -O.o~' -_0.-1.4~'~
~
l 1 m /k 8 0.8 10.6 0.3 11.50.7 1U.3 0.711.0 9 g '-0.9=0.4*."Ø7=1.8~=0.9 =0.7~ i=0.9-1.0*i I
10 mg/kg 8 0 10.2 0.3 9.8 0.3 10.- i 10.3 I 0."
t1.3*=0.7 =1Ø0.7 =2.0~ ~=0.9=0.9~i * Statistically significant variation at a = 0.5 in compariso:i to the control group ~i IF~ST1TUT~ S~E~T
s;:
(.f ~i e7 t~ i.~
WO 1 b2 d 6 PCI'/~P9Z/0049 d TAB. 18 HEMATOLOGICAL PA.RAMIr"TERS FOR HEALTHY VOLUNTEERS BEFORE ANIJ
AFTER TWO~WEEFC ADMYNTSTRATIONS OF TTP AT A I70SE OF 1 MG/DAY
Placebon=6 TTP n=5 Parameter 0 14 0 14 days days Eryt:~rocytes I I 18 32;
x 10 4.44 .Ø464.32 ,0.454.32 -_0.32. .
/u1 .
Hemoglobin I
g/dl ~ 12.4 t2.5 11.9 ~.8 i2.8 Ø8 11.8=0.6 Hematoc~it I
38.1 .5.7 38 -6.9 38.7 _2.1 38.1=1.6 Leuk~cy~ces ' x 10 /u1 5.25 X0.59 6.27 =0.8 6.23 -_0.54.86-0.6 i Neut=oohils ~ ~
-i ~12 57.6 =11 ~ 64.2-2.6 66 -3.6 62.6 Lymphocytes 26 X7.5 30.4 -8.3 34 _4.1 32.4~
.3 '='hro~booytes i ~ ' x 10~/~.i.l ~ 220 =40.4 214 .35.8 I 221 -_38 214 _39.o j 3lood Sedime.~.c' mm/1h ~ 7.6 ~9 6.6 .1.9 ~i ,.3 -2.~ 6.9 .3.:
StISSTIT~JT~ SH~~T
4~' l l1 ~TC ch ~ i ~,! 'T
~~s 9,.y tJ C~ '~ ~,. .P
a ~z~mzi6 ~cr/~~>ziooa9~
TAB. 19 BIOCI3EMICAL PARAMETERS FOR HEALTHY VOZ.U~ITEERS BEFORE AND
AFTER TWO-Wi~EIC ADMIPdISTRATIONS OF TTP AT A DOSE OF 1 MG/DAY
Placebon=S p -Parameter 0 14 ays 0 14 ays Total 72.8 X1.76 68.42.3.2 73.8 _-1.9270 ;a.3 Protein 163.2X2.5 63.8~2.3 '63.5=1.8 54.4.2..
g/1 Albumina Globulinat I r0.4 3 =0.5 I =0.5 2.3 _0.-'.'.3~
~ 3.1 2.8 Globulina2 I X1.9 6.8 ~1.5 ~ =0.9 06.9Ø?
~ 5.95 0.93 Globulinp ~ 14..1-2.2 14.2_1.5 13.3 -1.9 '3.2_1.3 - -GlobulinY ~ 112.2.i.5 12.1=i.3 113.7-_0.9.2.3.i _ --IgG g/1 110.3.1.1 10.2~1 11', _'.o '1.'=i.-- r ---TgA g/1 i -_0.6 1.g _~.j I .j ..9 =0.3 t.8 gM g/1 ~ Ø3 1.4 _0.2 I -__0.21.; ='J.-1.4 i.1 .
i I Complementg/= I Ø1 1.1 -_0.1 ! -_0.2 0.. Ø.
C3 1.1 ,..
Complement g/1 j =0.5 9.3 =0.041 Ø0 0.? _O.
C4 0.2 0.2 J3;
i -Aianine 134.2 _0.05 0.3 X0.04130.?=9.'_ 3i.=
U/'_ =3.?
Asoaragine U/' ~32.d =4.4 40.2 =5.5 ~30.-~ .'... 39.x =5 ~U~STiTUT~. S~~~T
KM::Ir'>~h~$rr_~.
F:a ~.y ~~.~ a~ E ~ wa W( J16216 PCT/11'92141~91 TAB. 20 COMPARISON OF THE FREQUENCY OF ACUTE >:NFECTIONS OF THE
RESPIRATORY TRACTS DURING THE LAST QUARTERS OF THE YEARS
1989 AND 1990 IN RESPECT OF TWO GROUPS OF PATTENT5 ' TRF.ATEn WITH TTP AND PLACEBO RESP. DURING OCTOBER 1990 , TTP
applied during the 4th quarter i n 4th quarter4th quarterstatistical 1989 1990 signieicance (no TTP) (with ~~p"
TTP) average average number number o o infectionsinfections per patientper patient Cold 20~4.0 -__1.31.0 -4.5 < 0.01 ~
Sore 203.3 .1.3 0.8 -0.G < 0-01 throat Fever blisters 202.1 .2.2 0.3 -_0.4~ . 0.01 Caugh 201.4 _1.1 U.3 ~O.o j < 0.01 Bronchitis200.3 =0.5 0 ' ~i j PneumoniaI20~ 0.2 Ø90 Placebo applied during the 4th quarks= 1990 nl 4th quartez~ 4th quarter ~ scaL_s~ac~a~
1989 ~ 1990 evaluation o.-'.1 (no '='~'?) (with TTP) difference"c"~
average average number o~ number o~
infections infections per patient per patient . _ Cold 20~ 3.9 =1.4 3.1 ~1.> 0.05 Sore 20 2.3 .2.0 2.0 .1.7 > 0.05 throat Fever blisters 20 2.3 ._1.8 1.7 .2.1 > 0.05 Cough 20 1.5 -1.5 1.9 =2.0 > 0.05 Bronchitis 20 0.1 Ø3 0.1 Ø3 Pneumonia 20 0.1 Ø3 0.1 X0.3 ~~J~~T(TUT~ S~E~ T
y ~ z t~ 3_~ U ~.~ i~ ;:G _~.
WO . 4J 1 b216 ~ PCd'!EF'92JOU49 ~
TAB. 21 EFFECT OF INTRADERMAL INJECTION OF BACTERIAL ANTIGENS OR
PHA (PHYTOHEMAGGLUTININ) IN PATIENTS TREATED WITH TTP
Tuber- Strepto- Staphylo- PHA NaCl Patientculin lycin cocci/
Ser.(Init- RT~3 0 anatoxin No. ials) pre 3 3 pre 3 pre3 pre 3 (pre , I I I
I
1 I B.T. + + - ' ~ ' . ! , = I
_ 2 P.E. + _ ,. _ I _ _ _ _ - I
I + t + _ - -3.1 + + + ~
M.H. ~+ -I - I
-I
' 4.I - + ' ' W.H. - I
I - --., .. ~
! ~ ..
'~. - -. ~
-.
i , I
_ .
I
_ I
.
.
I
.
I
o.':
Z.N.
I--I=
+
I
_ -~
, I
I
I
I
t r 7.; I r I
P.J. - .~ I
I
i --I
r -_ _ I
-I
9.' S.M. ~- - !- ~Y ~~ i - j - i _ !
pre pretreatment 3 after 3 weeks TAB, z? EFFEC'~' OF INTRADERMAL IN~"EC';'ION OP' 9AC'~'ERIAL ANT;GENS
OR PHA IN UNTREATED (PLACEBO) °ATIENTS
Tube.- Strepco- PHA I
I Staphylo- NaC=
!
Patientculin lycin ~
l cocci/ I
Ser.I(Tnit-RTZ~ 0 anacoxin ;
No. ials) ' e re I I
I ~ I pre pz'-3 3 I .
~ ' pre ' pr p 1 M.M. I ~ ~
. - + -I _ T j -~' -, I ' , . r _ _ I i z. o.. ~ _ i I I
._ _ , , ! ~ + -3. O.H. I+ ~...+~I + _ j + i -i, -I
4. W.J. I _ _ - - -I - ~ - i ~ --K.C. ~ + + - p ~, _ _ - --I -b.~I M.J. + ++ ~+~. ~+ ~_ - I
I- -- i I
7. H.W. - + + - _ _ - + r -8. K.T. I
( _ _ + + + _ _ -I , ~ I ~
+ -i pre pretreatment 3 after 3 weeks ~IISSTI"I~IJT~ ~#~FFT
1 ~:' s.
~r .~ G.t e.'? 1 ~ ~..~ .,.
ii~C) 5 6216 PC.'T/~~92/0~~91 TAB. 23 xMMUNOGLOHULIIVS IN THE SEFtt3M OF PATIENTS WTTa:
UI~CUS CRURZS TtZEATEI? WSTH TTP
n Test Group Immunoglobulin in I ( mg before after Treatment Treatment I
1 I 1732.872207.327 ZgG 1732.87.207.52 Ig 1 1 G
8 A 2 IgA 304.25 _ 47.508 IgA 283.87 . 44.10 ~ I I I I
' I 3 ' 202.87 ~ 71.129 IgM ~ 189.75_- 75.15 IgM ' I
~
~ ~4 ~ 1 1924.371246.2510 IgG I 1972.00-_239.33 IgG ~ 1 8 g I I I 236.50_ 59.841 I j 285.50-_ 56.76 a IgA 11 IgA
6 ~ I 242.12. 56.95~ I I 532.7._ 65.;5 IgM i2 IgM
1 1 7 D O.OS ~ 4 i0 ? 0'05 StatlStlCS1 ~ VS > O.O~ 'IS >
Evaluac;on I 2 a D 0.05 I J 11 0.05 oz VS > VS 12 ~
3 9 p 5 ~
vs > vs I , ~ 7 i0 ~ O.JS
Di=?ezence 1 ; 4 ~ 0.05 vs >
Vs > 0.05 I ' o.os j 2 ; ~ 0.05- 1 a i1 ~ 0.05 VS > VS >
3 o a 9 12 ~
vs > ws >
SUSST1TUT~ S~~~T
Claims (82)
1. A process for producing a peat-derived bioactive product, wherein a concentrated solution of a peat extract containing inorganic salts and peat-derived bioactive ingredients is subjected to processing steps comprising:
(a) a dilution step wherein the solution is diluted with demineralized water, (b) a subsequent desalination step wherein the diluted solution is subjected to reverse osmosis in order to remove inorganic salts from the solution, such that the desalinated solution contains no more than 70% by weight, based on dry solids, of the inorganic salts being removed, (c) a concentrating and purifying step wherein the desalinated solution is concentrated and clarified, and (d) optionally, a sterilizing and/or drying step wherein the concentrated and clarified solution is sterilized and/or spray-dried, the steps yielding the peat-derived bioactive product.
(a) a dilution step wherein the solution is diluted with demineralized water, (b) a subsequent desalination step wherein the diluted solution is subjected to reverse osmosis in order to remove inorganic salts from the solution, such that the desalinated solution contains no more than 70% by weight, based on dry solids, of the inorganic salts being removed, (c) a concentrating and purifying step wherein the desalinated solution is concentrated and clarified, and (d) optionally, a sterilizing and/or drying step wherein the concentrated and clarified solution is sterilized and/or spray-dried, the steps yielding the peat-derived bioactive product.
2. The process as claimed in claim 1, wherein the inorganic salts include sodium chloride.
3. The process as claimed in claim 1 or 2, wherein in the dilution step, the solution is diluted with demineralized water in a ratio ranging from about 1:5 to about 1:8.
4. The process as claimed in claim 1, 2, or 3, wherein in the desalination step, following reverse osmosis, the desalinated solution contains no more than 60% by weight, based on dry solids, of the inorganic salts.
5. A process as claimed in any one of claims 1 to 4, wherein the concentrated solution subjected to step (a) is prepared by a process comprising successive steps of (i) primary and secondary alkaline hydrolysis of an air-dried raw peat material, (ii) acidification of the hydrolysate, (iii) separation of insoluble parts and elimination of unwanted substances by extraction with an organic solvent, thereby yielding a post-extraction aqueous phase, (iv) expelling residual organic solvent from the post-extraction aqueous phase, and (v) filtering the remaining aqueous phase through a sintered ceramic filter under reduced pressure, thereby yielding a clear filtrate of the concentrated solution, for further processing according to steps (a) through (d).
6. The process as claimed in any one of claims 1 to 5, wherein the sterilization in step (d) is carried out by means of a membrane filter.
7. The process as claimed in any one of claims 1 to 6, wherein the solution in step (d) is spray-dried with an inlet temperature of about 180°C and an outflow temperature of about 90°C.
8. A peat-derived bioactive product obtainable by desalination of a concentrated aqueous solution of peat extract, which solution contains inorganic salts and water-soluble peat-derived bioactive ingredients and originates from an extraction process comprising alkaline hydrolysis of raw peat material, the bioactive product containing not more than 70%, by weight of inorganic salts, based on dry solids.
9. The peat-derived bioactive product according to claim 8, wherein the bioactive product contains no more than 60% by weight of inorganic salts, based on dry solids.
10. A peat-derived bioactive product according to claim 8 or 9, wherein the product is present in the form of a concentrated solution or a dry powder.
11. The peat-derived product according to claim 10, wherein the product is present in the form of a concentrated solution and is a dark-brown liquid with a specific gravity of 1.02 to 1.09 g/ml and has a dry solids content of not less than 5% by weight.
12. The peat-derived product according to any one of claims 8 to 11, wherein the product has a pH of 5.0 to 6.5, when prepared as a 1% aqueous solution.
13. The peat-derived product according to any one of claims 8 to 11, wherein the product has a pH of about 6, when prepared as a 1% aqueous solution.
14. A peat-derived bioactive product according to any one of claims 8 to 13, obtained by a process according to any one of claims 1 to 7.
15. The peat-derived bioactive product according to any one of claims 8 to 14 for medical or cosmetic use.
16. The peat-derived product according to claim 15, for immunomodulation comprising non-specific immunostimulation and/or induction of cytokines.
17. A pharmaceutical composition comprising the peat-derived bioactive product according to any one of claims 8 to 16 and a pharmaceutically-acceptable carrier material.
18. The pharmaceutical composition according to claim 17 wherein the composition is formulated for oral or topical application.
19. The pharmaceutical composition according to claim 17 or 18, wherein the carrier material is a mixture of lactose and a lubricating substance.
20. The pharmaceutical composition according to claim 17, 18, or 19, wherein the weight ratio of the bioactive product to the carrier material is about 1:5 to 1:25.
21. The pharmaceutical composition according to claim 20, wherein the weight ratio of the bioactive product to the carrier material is about 1:9 to 1:19.
22. The pharmaceutical composition according to any one of claims 17 to 21, in the form of a tablet or a capsule.
23. Use of the peat-derived bioactive product according to any one of claims 8 to 16 or of the pharmaceutical composition according to any one of claims 17 to 22, for non-specific immunomodulation.
24. Use of the peat-derived bioactive product according to any one of claims 8 to 16 or of the pharmaceutical composition according to any one of claims 17 to 22, for the production of a medicament for non-specific immunomodulation.
25. Use of the peat-derived bioactive product according to any one of claims 8 to 16 or of the pharmaceutical composition according to any one of claims 17 to 22, for non-specific immunostimulation.
26. Use of the peat-derived bioactive product according to any one of claims 8 to 16 or of the pharmaceutical composition according to any one of claims 17 to 22, for the production of a medicament for non-specific immunostimulation.
27. Use of the peat-derived bioactive product according to any one of claims 8 to 16 or of the pharmaceutical composition according to any one of claims 17 to 22, for induction of cytokines.
28. Use of the peat-derived bioactive product according to any one of claims 8 to 16 or of the pharmaceutical composition according to any one of claims 17 to 22, for the production of a medicament for induction of cytokines.
29. A cosmetic preparation for treating skin, hair and/or teeth, comprising 0.01-10% by weight of the peat-derived bioactive product as claimed in any one of claims 8 to 16, and a carrier and/or another additive suitable for cosmetic preparations.
30. The cosmetic preparation according to claim 29, wherein the carrier is selected from the group consisting of aqueous solutions of alcohols, emulsions, gels, foaming compositions, and fatty carriers.
31. The cosmetic preparation according to claim 29 or 30, wherein the peat-derived bioactive product is present in a quantity of 0.05 to 1% by weight.
32. The cosmetic preparation according to claim 29 or 30, wherein the peat-derived bioactive product is present in a quantity of 0.05 to 0.10% by weight.
33. The cosmetic preparation as claimed in claim 29, 30, 31, or 32, further comprising at least one herb extract and/or at least one fragrance.
34. The cosmetic preparation according to any one of claims 29 to 33, in the form of a gel, and further comprising:
a sterile alcoholic herb extract, a sterile solution of a flavouring compound, selected from the group comprising menthol, thymol, mint, lemon and eucalyptus, and a gel-forming agent.
a sterile alcoholic herb extract, a sterile solution of a flavouring compound, selected from the group comprising menthol, thymol, mint, lemon and eucalyptus, and a gel-forming agent.
35. The cosmetic preparation according to claim 34, wherein the gel-forming agent is sterile glycerol with colloidal silica.
36. The cosmetic preparation according to claim 35, wherein the weight ratio of liquid components in the cosmetic preparation to silica is from 90:10 to 94:6.
37. A cosmetic preparation according to any one of claims 29 to 33, wherein the composition is in the form of an ointment and further comprises:
a sterile herb extract, a gel-forming agent, and a sterilized mixture of fatty components.
a sterile herb extract, a gel-forming agent, and a sterilized mixture of fatty components.
38. The cosmetic preparation according to claim 37, wherein the gel-forming agent is colloidal silica.
39. The cosmetic preparation according to claim 38, wherein the weight ratio of liquid components in the cosmetic preparation to silica is about 30:2.
40. The cosmetic preparation according to any one of claims 37 to 39, wherein the mixture of fatty components is eucerine and petrolatum.
41. The cosmetic preparation according to any one of claims 37 to 40, wherein the weight ratio of fatty components to the total remaining components in the cosmetic preparation is between 66:34 to 68:32.
42. A water soluble peat-derived bioactive product, containing not more than 70% by weight sodium chloride, based on dry mass.
43. A peat-derived bioactive product according to claim 42, containing not more than 60% by weight sodium chloride, based on dry mass.
44. A peat-derived bioactive product according to claim 42 or 43 obtained by a process, wherein a highly concentrated aqueous sodium chloride solution containing water soluble peat-derived bioactive ingredients is diluted with demineralized water and subjected to reverse osmosis in order to desalinate the solution, inorganic salts being removed, and wherein the resulting solution is concentrated and clarified.
45. A peat-derived bioactive product according to claim 42 or 43, obtained by a process, wherein a highly concentrated aqueous sodium chloride solution containing water soluble peat-derived bioactive ingredients, said solution being obtained by primary and secondary alkaline hydrolysis of air-dried raw peat material, followed by acidification of the hydrolysate, separation of insoluble parts and elimination of unwanted substances by extraction with organic solvents, and removal of the residual organic solvents from the post-extraction aqueous phase, is filtered through a sintered ceramic filter under reduced pressure, wherein the thus obtained clear filtrate is diluted with demineralized water and subjected to reverse osmosis in order to desalinate the solution, inorganic salt being removed, and wherein the resulting solution is concentrated and clarified.
46. A peat-derived bioactive product according to claim 44 or 45, obtainable by a process comprising at least one of the further steps of sterilizing and spray-drying said resulting solution.
47. A pharmaceutical composition containing as active ingredient a peat-derived bioactive product according to any one of claims 42 to 46.
48. A pharmaceutical composition containing as active ingredient a peat-derived bioactive product as claimed in any one of claims 42 to 46, and a pharmaceutically acceptable carrier material in a weight ratio of between about 1:5 and 1:25.
49. A pharmaceutical composition according to claim 48, wherein the weight ratio is between 1:9 and 1:19.
50. A cosmetic preparation containing as active ingredient a peat-derived bioactive product according to any one of claims 42 to 46, in a quantity of 0.01-10% by weight.
51. A cosmetic preparation according to claim 50, wherein the quantity is between 0.05 and 1% by weight.
52. A cosmetic preparation according to claim 50 or 51, containing, in addition, at least one herbal extract and at least one fragrant.
53. A peat-derived bioactive product containing not more than 70o by weight of inorganic salts, based on dry solids.
54. A peat-derived bioactive product according to claim 53, containing not more than 60% by weight of inorganic salts, based on dry solids.
55. A peat-derived bioactive product obtainable by a process wherein a highly concentrated aqueous solution of inorganic salts, containing peat-derived bioactive ingredients, is diluted with demineralized water and subjected to reverse osmosis in order to desalinate the solution, inorganic salts being removed, and the resulting solution is concentrated and clarified to provide a bioactive product in which inorganic salts are present at a concentration of less than 70% by weight, based on dry solids.
56. The peat-derived product according to claim 55, wherein the concentrated and clarified solution is sterilized and/or spray-dried.
57. A peat-derived bioactive product obtainable by a process wherein a highly concentrated aqueous solution of sodium chloride containing peat-derived bioactive ingredients, said solution being obtainable by primary and secondary alkaline hydrolysis of air-dried raw peat material, followed by acidification of the hydrosylate, separation of insoluble parts and elimination of unwanted organic substances by extraction with organic solvents and removal of residual organic solvents from a post-extraction aqueous phase, is filtered through a sintered ceramic filter under reduced pressure, is diluted with demineralized water, is subjected to reverse osmosis in order to desalinate the solution, and is concentrated and clarified, to provide a bioactive product in which inorganic salts are present at a concentration of less than 70% by weight, based on dry solids.
58. A process for producing a peat-derived bioactive products from a highly concentrated aqueous solution of inorganic salts containing peat-derived bioactive ingredients, wherein said solution is diluted with demineralized water, the dilution being effected with water volumes several times the volume of the solution to be diluted, the diluted solution is subjected to reverse osmosis in order to desalinate the solution, inorganic salts being removed, and the desalinated solution is concentrated and clarified, to provide a bioactive product in which inorganic salts are present at a concentration of less than 70% by weight, based on dry solids.
59. The process according to claim 56, wherein the concentrated and clarified solution is sterilized and/or spray-dried.
60. A process as claimed in claim 59, wherein the spray-drying is performed with an inlet temperature of about 180°C
and an outflow temperature of about 90°C.
and an outflow temperature of about 90°C.
61. A process according to claim 58, 59, or 60, wherein the concentrated aqueous solution containing peat-derived bioactive ingredients, is obtainable by primary and secondary alkaline hydrolysis of air-dried raw peat material, acidification of the hydrolysate, separation of insoluble parts, and elimination of unwanted organic substances by extraction with organic solvents.
62. A process as claimed in claim 61, wherein, after filtering, the solution is sterilized by means of a membrane filter.
63. A peat-derived bioactive product, obtained by a process according to any one of claims 58 to 62.
64. A peat-derived bioactive product according to claim 63, in powder form.
65. A process for preparing a pharmaceutical formulation containing a peat-derived bioactive product, wherein a sterile aqueous solution of the powdered peat-derived bioactive product according to claim 64 is combined with a suitable carrier and suitable additives.
66. A process according to claim 65, wherein the pharmaceutical formulation is prepared in the form of a gel, wherein a sterile alcoholic herb extract and a sterile solution of a flavouring compound are employed as the suitable additives, while a gel-forming composition is used as the suitable carrier to convert the liquid composition into gel form.
67. A process according to claim 66, wherein the flavouring compound is selected from the group consisting of menthol, thymol, mint, lemon and eucalyptus.
68. A process according to claims 66 or 67, wherein the gel-forming composition is sterile glycerol with colloidal silica.
69. A process according to claim 68, wherein the weight ratio of liquid mixture to silica is between 90:10 to 94:6.
70. A process according to claim 65, wherein the pharmaceutical formulation is prepared in the form of an ointment, wherein a sterile herb extract is employed as an additive and a gel-forming ingredient is added as the suitable carrier, whereafter the gel thus obtained is triturated with a previously sterilized mixture of fatty components.
71. A process according to claim 70, wherein the gel-forming ingredient is colloidal silica.
72. A process according to claim 70 or 71, wherein the mixture of fatty components is eucerine or petrolatum.
73. A process according to claim 71, wherein the weight ratio of liquid components to silica is about 30:2.
74. A process according to any one of claims 70 to 73, wherein the ratio of gel to fatty components is between 32:68 to 34:66.
75. A process as claimed in any one of claims 65 to 74, wherein the peat-derived bioactive product is as claimed in any one of claims 53 to 57, 63, and 64.
76. A pharmaceutical composition containing as active ingredient a peat-derived bioactive product as claimed in any one of claims 53 to 57, 63, and 64.
77. A pharmaceutical composition according to claim 76, wherein the weight ratio of the active ingredient to the pharmaceutically-acceptable carrier material is between 1:5 and 1:25.
78. A pharmaceutical composition according to claim 76, wherein the weight ratio of the active ingredient to the pharmaceutically-acceptable carrier material is between 1:9 and 1:19.
79. A cosmetic preparation containing as active ingredient a peat-derived bioactive product as claimed in any one of claims 53 to 57, 63, and 64 in a quantity of 0.01-10% by weight.
80. A cosmetic preparation according to claim 79, wherein the active ingredient is present in the cosmetic preparation in a weight ratio of 0.05 to 10%.
81. A cosmetic preparation according to claim 79, wherein the active ingredient is present in the cosmetic preparation in a weight ratio of 0.05 to 1%.
82. A cosmetic preparation as claimed in claim 79, 80, or 81, containing, in addition, at least one herb extract and/or at least
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| EP91104098 | 1991-03-16 | ||
| EP91104098.8 | 1991-03-16 | ||
| PCT/EP1992/000491 WO1992016216A1 (en) | 1991-03-16 | 1992-03-04 | Peat-derived bioactive products and pharmaceutical and cosmetic compositions containing them |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CA2083061A1 CA2083061A1 (en) | 1992-09-17 |
| CA2083061C true CA2083061C (en) | 2002-12-17 |
Family
ID=8206528
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002083061A Expired - Fee Related CA2083061C (en) | 1991-03-16 | 1992-03-04 | Peat-derived bioactive products and pharmaceutical and cosmetic compositions containing them |
Country Status (5)
| Country | Link |
|---|---|
| BG (1) | BG61501B1 (en) |
| CA (1) | CA2083061C (en) |
| NO (1) | NO307032B1 (en) |
| RO (1) | RO114867B1 (en) |
| ZA (1) | ZA921919B (en) |
-
1992
- 1992-03-04 CA CA002083061A patent/CA2083061C/en not_active Expired - Fee Related
- 1992-03-04 RO RO92-01429A patent/RO114867B1/en unknown
- 1992-03-16 ZA ZA921919A patent/ZA921919B/en unknown
- 1992-11-16 BG BG97085A patent/BG61501B1/en unknown
- 1992-11-16 NO NO924409A patent/NO307032B1/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| NO924409L (en) | 1993-01-12 |
| NO924409D0 (en) | 1992-11-16 |
| BG61501B1 (en) | 1997-10-31 |
| BG97085A (en) | 1994-03-24 |
| RO114867B1 (en) | 1999-08-30 |
| NO307032B1 (en) | 2000-01-31 |
| ZA921919B (en) | 1992-12-30 |
| CA2083061A1 (en) | 1992-09-17 |
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